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SENOLYTIC – Zombie Cell Killer

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SENOLYTIC

ZOMBIE CELL KILLER!

200:1 Concentration

Featuring:

  • ALANTOLACTONE (ELECAMPANE INULA ROOT EXTRACT)
  • ALPHA-MANGOSTIN (MANGOSTEEN EXTRACT)
  • Alpine Rose (Rhododendron ferrugineum L.) Leaf Extract
  • ANDROGRAPHOLIDE (ANDROGRAPHIS PANICULATA EXTRACT)
  • ANTRODIA CAMPHORATA (CINNAMO-MUM KANEHIRAI)
  • APIGENIN (CELERY SEED)
  • ARTEMISININ (ARTEMESIA ANNUA – WORMWOOD)
  • ARTOCARPIN (ARTOCARPUS INCISUS)
  • ASPALATHIN (ASPALATHUS LINEARIS)
  • ASTRAGALUS COMPLANATUS (Bei Bian Huang Qi)
  • AVICIN D (PLANT TRIPERNOID)
  • BAICALEIN (SCUTELLARIA BAICALENSIS EXTRACT)
  • BAVACHIN (PSORALEA CORYLIFOLIA)
  • BERBERINE (BERBERIS ARISTATA EXTRACT)
  • BETULINIC ACID (BERBERIS ARISTATA EXTRACT)
  • BOSWELLIC ACID (BOSWELLIA SERRATA GUM EXTRACT)
  • BREVILIN A (SESQUITERPENE LACTONE EXTRACTED FROM CENTIPEDA MINIMA)
  • BUTEIN (SOPHORA JAPONICA EXTRACT)
  • CAMPESTEROL (RADIX REHMANNIAE EXTRACT)
  • CARNOSIC ACID (ROSEMARY EXTRACT)
  • CARNOSINE (BETA-ALANYL-L-HISTIDINE)
  • CASTICIN (CHASTE TREE EXTRACT)
  • CELASTROL (THUNDER GOD VINE EXTRACT)
  • CHICORIC ACID (ECHINACEA EXTRACT POWDER)
  • CHLOROGENIC ACID (GREEN COFFEE BEAN EXTRACT)
  • CHLOROPHYLL A (LUDWIGIA OCTOVALVIS EXTRACT)
  • COSTUNOLIDE (COSTUS ROOT EXTRACT)
  • CUCURBITACIN D (CUCURBITACEAE LAGENARIA SICERARIA)
  • CURCUMIN (TURMERIC ROOT EXTRACT)
  • CYANIDIN (BLUEBERRY EXTRACT)
  • CYCLOASTRAGENOL (ASTRAGALUS ROOT EXTRACT)
  • DAIDZEIN (SOYBEAN EXTRACT)
  • DECURSIN (ANGELICA DECURSIVA)
  • DELPHINIDIN(CONSOLIDA AJACIS)
  • DIGITALIS PURPUREA EXTRACT (DIGOXIN)
  • DIOSMIN (CITRUS AURANTIUM EXTRACT
  • DX-9386 (GINSENG EXTRACT, ACORUS EXTRACT, POLYGALA EXTRACT AND HOELEN EXTRACT)
  • EF24 (TURMERIC ROOT EXTRACT)
  • EGCG (GREEN TEA EXTRACT)
  • EMBELIN (ARDISIA JAPONICA)
  • EMODIN (ALOE VERA EXTRACT)
  • ESCULETIN (HORSE CHESTNUT EXTRACT)
  • EVODIAMINE (EVODIA RUTAECARPA EXTRACT)
  • FISETIN (SMOKETREE EXTRACT)
  • FOXO4D-DRI (Forkhead box protein O4 RETRO-INVERSO PEPTIDE)
  • FUCOIDAN (KELP EXTRACT)
  • GALLIC ACID (GALLNUTS EXTRACT)
  • GAMBOGIC ACID (GARCINIA HANBURYI HOOK)
  • GANODERMA LUCIDUM (REISHI MUSHROOM EXTRACT)
  • GENISTEIN(LYCIUM BARBARUM L.)
  • gingerenone A
  • GINSENOSIDE RG3 (PANAX GINSENG EXTRACT)
  • GLYCITEIN (SOYBEAN EXTRACT)
  • GLYCYRRHIZIN (LICORICE ROOT EXTRACT)
  • GOSSYPOL (COTTON SEED EXTRACT)
  • GS25 (PANAX NOTOGINSENG)
  • GUGGULSTERONE (GUGGUL EXTRACT)
  • HESPERIDIN (CITRUS AURANTIUM EXTRACT)
  • HONOKIOL (MAGNOLIA BARK EXTRACT)
  • HYPERFORIN (ST.JOHN’S WORT EXTRACT)
  • ICARIIN (EPIMEDIUM EXTRACT)
  • INDIRUBIN (INDIGO BLUE EXTRACT)
  • INDOLE-3-CARBINOL (CABBAGE)
  • ISOALANTOLACTONE (INULA HELENIUM L)
  • ISOORIENTIN (BAMBOO LEAF EXTRACT)
  • ISORHAMNETIN (SEA BUCKTHORN FRUIT EXTRACT)
  • ISOVITEXIN (PASSION FLOWER EXTRACT)
  • JACEOSIDIN (WORMWOOD LEAF EXTRACT)
  • JUGLANIN (APPLE FLOWER)
  • KAEMPFEROL (GALANGA EXTRACT)
  • KURARINONE (SOPHORA FLAVESCENS EXTRACT)
  • LICORICIDIN (LICORICE EXTRACT)
  • LIPOIC ACID (ETHYL 6,8-DICHLOROCAPRYLATE)
  • LUPEOL (STRAWBERRY EXTRACT)
  • LUTEOLIN (PEANUT SHELL EXTRACT)
  • LYCOPENE (TOMATO EXTRACT)
  • MAGNOLOL (MAGNOLIA OFFICINALIS EXTRACT)
  • MULBERRY(MORUS ALBA)
  • MYRICETIN (BAYBERRY BARK EXTRACT)
  • NARINGENIN(GRAPEFRUIT EXTRACT)
  • NEEM LEAF(AZADIRACHTA INDICA)
  • O-COUMARIC ACID (O-HYDROXYCINNAMIC ACID)
  • OLEACEIN (OLEA EUROPAEA)
  • OLEANOLIC ACID(LIGUSTRUM LUCIDUM EXTRACT)
  • OLEUROPEIN(OLIVE LEAF EXTRACT)
  • ORIDONIN (RABDOSIA RUBESCENS EXTRACT)
  • OROXYLIN A (OROXYLUM INDICUM)
  • ORTHO-VANILLIN(VANILLA BEAN,PINEOSIDE, EUGENOL AND SAFROLE)
  • PARTHENOLIDE (FEVERFEW EXTRACT)
  • PATRINIA SCABIOSAEFOLIA FISCH (GOLDEN LACE)
  • PHLORETIN (APPLE PEEL EXTRACT)
  • PHLOROGLUCINOL (1,3,5-TRIISOPROPYLBENZENE)
  • PHOSPHATIDYLCHOLINE (SOYA BEAN)
  • PHYLLANTHUS EMLICA FRUIT EXTRACT (INDIAN GOOSEBERRY)
  • PICEATANNOL (GIANT KNOTWEED EXTRACT)
  • PIPERLONGUMINE(PIPER LONGUM)
  • POMEGRANATE PEEL (PUNICA GRANATUM)
  • PRISTIMERIN (CELASTRUS ORBICULATUS)
  • PROANTHOCYANIDINS (GRAPE SEED EXTRACT)
  • PROCYANIDIN B3 (GRAPE SEED EXTRACT)
  • PSEUDOLARIC ACID B (CORTEX HIBISCUS EXTRACT)
  • PTEROSTILBENE (BLUEBERRIES EXTRACT)
  • QUERCETAGEtin (TAGETES PATULA)
  • QUERCETIN (SOPHORA JAPONICA EXTRACT)
  • RESVERATROL (POLYGONUM CUSPIDATUM EXTRACT)
  • ROTTLERIN (MALLOTUS PHILIPPINENSIS EXTRACT)
  • RUTIN (SOPHORA JAPONICA EXTRACT)
  • SALVIA MILTIORRHIZAE (RED SAGE)
  • SANSALVAMIDE A CYCLOPEPTIDE SEPARATED FROM MARINE FUNGI (FUSARIUM)
  • SCUTELLARIN (SCUTELLARIA ALTISSIMA L LEAF EXTRACT)
  • SILYBIN (MILK THISTLE EXTRACT)
  • SINAPINIC ACID (SINAPIS ALBA L. SEED EXTRACT)
  • SOLIDAGO VIRGAUREA (GOLDEN ROD FLOWER EXTRACT)
  • SULFORAPHANE (BROCCOLI SPROUT EXTRACT)
  • TANGERETIN (TANGERINE PEEL EXTRACT)
  • TARAXASTEROL (DANDELION EXTRACT)
  • THYMOQUINONE (BLACK SEED EXTRACT)
  • TRICHOSTATIN A (STREPTOMYCES HYGROSCOPICUS)
  • UROLITHIN A (3,8-DIHYDROXYBENZO[C]CHROMEN-6-ONE)
  • URSOLIC ACID (LOQUAT LEAF EXTRACT)
  • WEDELOLACTONE (ECLIPTA ALBA EXTRACT)
  • WITHAFERIN A (ASHWAGANDHA EXTRACT)
  • WOGONIN (SCUTELLARIA BAICALENSIS ROOT EXTRACT)
  • XANTHOHUMOL (HOPS EXTRACT)
  • ZERUMBONE (ZINGIBER ZERUMBET EXTRACT)
  • 2-DG(2-DEOXY-D-GLUCOSE

“What are ‘zombie‘ cells?”


“They’re called zombie cells, because they are damaged and refuse to die.”

“As we age, these damaged cells start to accumulate and cause sterile inflammation which can alter metabolism and stem cell function, promoting aging and the conditions that are often associated with it, like Alzheimer’s disease. These zombie cells are formally called, senescent cells. When the cells get to a certain level of damage, they go through an aging process of their own called cellular senescence. When cells become damaged or if they replicate too many times, they undergo a process of irreversible removal from the cell cycle and start releasing inflammatory factors that stimulate the immune response to clear the damaged cells. A younger person’s immune system is healthy and is able to clear the damaged cells, but as people age, they aren’t cleared as effectively and they accumulate causing potential problems.”


Connecting Senescent Cells to Obesity and Anxiety

Senescent cells display a “zombie”-like behavior known as a senescence-associated secretory phenotype (SASP). In this death-defying, zombie-like state, the cells ramp up their release of proteins, bioactive lipids, DNA, and other factors that, like a zombie virus, induce nearby healthy cells to join in the dysfunction.”

“Could killing off these ‘zombie‘ cells in the mice delay their premature descent into old age? The answer was yes. In a 2011 study, the team found that eliminating these ‘senescent‘ cells forestalled many of the ravages of age. The discovery set off a spate of similar findings. In the seven years since, dozens of experiments have confirmed that senescent cells accumulate in ageing organs, and that eliminating them can alleviate, or even prevent, certain illnesses (see ‘Becoming undead’). This year alone, clearing the cells in mice has been shown to restore fitness, fur density and kidney function. It has also improved lung disease and even mended damaged cartilage. And in a 2016 study, it seemed to extend the lifespan of normally aging mice.”

Want to live for ever? Flush out your zombie cells

“To date about a dozen drugs have been reported that can mop up zombie cells. Clearance of the cells in mice has been shown to delay or alleviate everything from frailty to cardiovascular dysfunction to osteoporosis to, most recently, neurological disorders – though whether killing senescent cells extends life is complicated. Most of the benefit seen in mice seems to be in extending healthspan, the time free of frailty or disease, and as a result median lifespan (being sick, after all, is risky). True longevity – the maximum time the animals remain alive for – remains relatively unchanged, though studies published in July and September 2018 show an extension of remaining lifespan in mice that were treated when they were very old.”

Zombie cells found in brains of mice prior to cognitive loss

Zombie cells are the ones that can’t die but are equally unable to perform the functions of a normal cell. These zombie, or senescent, cells are implicated in a number of age-related diseases. “Senescent cells are known to accumulate with advancing natural age and at sites related to diseases of aging, including osteoarthritis; atherosclerosis; and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s,” says Darren Baker, Ph.D., a Mayo Clinic molecular biologist and senior author of the paper. “In prior studies, we have found that elimination of senescent cells from naturally aged mice extends their healthy life span.”

Removal of ‘zombie cells’ alleviates causes of diabetes in obese mice

Mayo Clinic researchers and their collaborators have shown that when senescent cells — also known as “zombie cells” — are removed from fat tissue in obese mice, severity of diabetes and a range of its causes or consequences decline or disappear.

Inflammation and dysfunction of fat tissue cause some of the insulin resistance in obese people. In many cases, that dysfunction is caused by zombie cells that already have been shown to be responsible for conditions related to aging and illness, including osteoporosis, muscle weakness, nerve degeneration and heart disease. These cells also accumulate in the fat tissues of obese and diabetic people and mice.

In this study, the researchers, using genetically modified mice and wild-type (normal) mice, removed zombie cells two ways: by causing genetically-mediated cell death and by administering a combination of senolytic drugs. Senolytic drugs selectively kill senescent cells but not normal cells. The result: Glucose levels and insulin sensitivity improved. The mice also showed a decline in inflammatory factors and a return to normal fat cell function.

Zombie” Cells and Clinical Biomarkers of Aging

Briefly, cellular senescence is a process whereby cells stop dividing and go through phenotypic changes, such as secretome and chromatin changes in addition to tumor-suppressor activation. Senescent cells accumulate in several organs as we grow older, are involved in tissue dysfunction and implicated in numerous pathologies such as cancer. Therefore, they are generally considered to be a “hallmark” of aging and have earned the nickname “zombie” cells.

Zombie’ cells signal death of tissue in Alzheimer’s

Cellular senescence allows the stressed cell to survive, but the cell may become like a zombie, functioning abnormally and secreting substances that kill cells around it. “When cells enter this stage, they change their genetic programming and become pro-inflammatory and toxic,” said study senior author Miranda E. Orr, Ph.D. She is a VA research health scientist at the South Texas Veterans Health Care System, faculty member of the Sam and Ann Barshop Institute for Longevity and Aging Studies, and instructor of pharmacology at UT Health San Antonio. “Their existence means the death of surrounding tissue.” The team reported the discovery in the journal Aging Cell. To clear senescent cells from the brains of middle-aged mice with advanced brain disease, researchers used a combination of drugs called senolytics.

SCIENTISTS: DRUGS THAT KILL “ZOMBIE CELLS” COULD PREVENT AGING

Doctors trying to eradicate age-related diseases have a new target: “zombified” senescent cells that can build up in people’s brains after an infection or stress. Most of the research that links senescent cells to conditions like Alzheimer’s disease, diabetes, and osteoporosis was conducted in mice, according to the Associated Press. But in February, a new experiment suggested that clearing senescent cells from humans with a fatal lung condition improved their well-being — a finding that gives anti-aging scientists and advocates new hope on their quest to keep people alive and healthy longer than ever before.

Scientists hunt zombie cells: ‘The grand challenge’

Zombie cells, labelled “senescent” by scientists because of their sleep-like state, are very resistant to dying. In a lab dish, they slumber for years under conditions that would kill ordinary cells within hours. They have been linked to diabetes, heart disease, osteoarthritis, lung fibrosis and Alzheimer’s.

SENESCENT CELLS

If one were to personify a senescent cell, it would be the grumpy, old employee who portrays a constant negative outlook. This guy was a great worker in his day, but now, not only does he not do his own job, he poisons the minds of his fellow employees. Furthermore, his morphology also changes, as his once trim and active physique has morphed into one of obesity and dysfunction. Others like to refer to these cells as zombie cells, but I prefer my analogy. Rather, these cells become dysfunctional, alter their morphology and are disruptive to their surroundings.


Deadly Impact of Zombie-like Cells

What this Mayo Clinic discovery further revealed is the degree of toxicity inflicted by senescent cells: If only one in 7,000 to 15,000 cells are senescent, then age-related deterioration starts to occur in laboratory mice.


“What is a ‘SENOLYTIC‘?”


Senolytics decrease senescent cells in humans

By definition, the target of senolytics is senescent cells, not a molecule or a single biochemical pathway.

Asenolytic(from the words senescence and -lytic, “destroying”) is among a class of small molecules under basic research to determine if they can selectively induce death of senescent cells and improve health in humans.[1] A goal of this research is to discover or develop agents to delay, prevent, alleviate, or reverse age-related diseases.[2][3] A related concept is “senostatic“, which means to suppress senescence. – WIKI

The Clinical Potential of Senolytic Drugs

Senolytic drugs are agents that selectively induce apoptosis of senescent cells. These cells accumulate in many tissues with aging and at sites of pathology in multiple chronic diseases. In studies in animals, targeting senescent cells using genetic or pharmacological approaches delays, prevents, or alleviates multiple age-related phenotypes, chronic diseases, geriatric syndromes, and loss of physiological resilience. Among the chronic conditions successfully treated by depleting senescent cells in preclinical studies are frailty, cardiac dysfunction, vascular hyporeactivity and calcification, diabetes mellitus, liver steatosis, osteoporosis, vertebral disk degeneration, pulmonary fibrosis, and radiation-induced damage. Senolytic agents are being tested in proof-of-concept clinical trials. To do so, new clinical trial paradigms for testing senolytics and other agents that target fundamental aging mechanisms are being developed, because use of long-term endpoints such as lifespan or healthspan is not feasible.

These strategies include testing effects on multimorbidity, accelerated aging-like conditions, diseases with localized accumulation of senescent cells, potentially fatal diseases associated with senescent cell accumulation, age-related loss of physiological resilience, and frailty. If senolytics or other interventions that target fundamental aging processes prove to be effective and safe in clinical trials, they could transform geriatric medicine by enabling prevention or treatment of multiple diseases and functional deficits in parallel, instead of one at a time.

Senolytic therapies for healthy longevity

The estimated “natural” life span of humans is ∼30 years, but improvements in working conditions, housing, sanitation, and medicine have extended this to ∼80 years in most developed countries. However, much of the population now experiences aging-associated tissue deterioration. Healthy aging is limited by a lack of natural selection, which favors genetic programs that confer fitness early in life to maximize reproductive output. There is no selection for whether these alterations have detrimental effects later in life. One such program is cellular senescence, whereby cells become unable to divide.

Cellular senescence enhances reproductive success by blocking cancer cell proliferation, but it decreases the health of the old by littering tissues with dysfunctional senescent cells (SNCs). In mice, the selective elimination of SNCs (senolysis) extends median life span and prevents or attenuates age-associated diseases (1, 2). This has inspired the development of targeted senolytic drugs to eliminate the SNCs that drive age-associated disease in humans.

Senescent Cells and Senolytics

A brief history of senolytics

The health and lifespan of mice have been demonstrated to improve by the removal of senescent cells using a transgenic suicide gene [3], and additional experiments showed that the same could be achieved using small molecules.Senescent cells comprise a small number of total cells in the body, but they secrete pro-inflammatory cytokines, chemokines, and extracellular matrix proteases, which, together, form the senescence-associated secretory phenotype, or SASP. The SASP is thought to significantly contribute to aging [4] and cancer [5]; thus, senolytics and the removal of the SASP are a potential strategy for promoting health and longevity.

It was discovered through transcript analysis that senescent cells have increased expression of pro-survival genes consistent with their resistance to apoptosis [6]. Drugs targeting these pro-survival factors selectively killed senescent cells. Two such drugs were dasatinib and quercetin, which were both able to remove senescent cells but were better in different tissue types. However, it was discovered that a combination of the two drugs formed a synergy that was significantly more effective at removing some senescent cell types [7].In other studies, removing only thirty percent of senescent cells was sufficient to slow down age-related decline. These results suggest the feasibility of selectively ablating senescent cells and the efficacy of senolytics in alleviating the diseases of aging and promoting healthy longevity [8, 9, 10].

Further confirming the potential of senolytics to treat age-related disease, a recent study demonstrated the benefits of senolytics for certain aspects of vascular aging [11]. This was the first study to show that clearance of senescent cells improves aspects of vascular aging and chronic hypercholesterolemia, thus making senolytics a possible viable method of reducing morbidity and mortality from cardiovascular diseases. Even more recently, progress has been made in treating atherosclerosis using senolytics to address the “foam cells” that contribute to this disease [12]. There has also been progress in ways to treat type 2 diabetes using senescent cell removal [13]. Senolytics also have the potential for slowing skin aging [14] and treating osteoarthritis [15].

Senescent cells, however, are not all bad, and evidence shows that they play a role in cellular reprogramming [16] and wound healing. Like all things in biology, it is therefore clearly a question of balance: too much clearance of senescent cells would be bad for wound healing and cellular reprogramming, but too many senescent cells lead to damage [17, 18]. Therefore, the key to developing effective senolytic therapies that combat the diseases of aging is the creation of even more accurate biomarkers to measure senescent cell numbers in tissue [19] combined with effective delivery methods for the selective removal of senescent cells.


Senolytic Therapies And The Quest To Cure Aging

Though the term “senolytic therapies” may be a bit foreign to the general public, the agents themselves are nothing new to scientists who have been looking to them to find treatments for a host of ailments that cause human beings to age. “Senolytic compounds are those that preferentially destroy senescent cells. Since these cells are one of the root causes of aging, there is considerable interest in finding and then quantifying the effectiveness of senolytic compounds…


The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs


The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age‐related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro‐survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL‐xL, or plasminogen‐activated inhibitor‐2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM‐MSCs.

The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation‐exposed, and progeroid Ercc1−/Δ mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1−/∆ mice, delaying age‐related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans.

These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.


senolytics and other anti-aging drugs may have a prominent role in preventing the transmission of the virus, as well as aid in its treatment”

What is the relationship between COVID-19 and advanced chronological age?
“Here, we suggest that the COVID-19 corona virus preferentially targets senescent lung cells, resulting in increased morbidity and mortality in the aging population. One possible solution for prevention/treatment would be the use of senolytics or other anti-aging drugs.”


“What is THE “SASP“?”


“Social Life” of Senescent Cells: What Is SASP and Why Study It?

Cellular senescence was first described as a failure of normal human cells to divide indefinitely in culture. Until recently, the emphasis in the study of cell senescence has been focused on the accompanying intracellular processes. The focus of the attention has been on the irreversible growth arrest and two important physiological functions that rely on it: suppression of carcinogenesis due to the proliferation loss of damaged cells, and the acceleration of organism aging due to the deterioration of the tissue repair mechanism with age. However, the advances of the past years have revealed that senescent cells can impact the surrounding tissue microenvironment, and, thus, that the main consequences of senescence are not solely mediated by intracellular alterations.

Recent studies have provided evidence that a pool of molecules secreted by senescent cells, including cytokines, chemokines, proteases and growth factors, termed the senescence-associated secretory phenotype (SASP), via autocrine/paracrine pathways can affect neighboring cells. Today it is clear that SASP functionally links cell senescence to various biological processes, such as tissue regeneration and remodeling, embryonic development, inflammation, and tumorigenesis. The present article aims to describe the “social” life of senescent cells: basically, SASP constitution, molecular mechanisms of its regulation, and its functional role.


The Senescence-Associated Secretory Phenotype: The Dark Side of Tumor Suppression

Cellular senescence is a tumor-suppressive mechanism that permanently arrests cells at risk for malignant transformation. However, accumulating evidence shows that senescent cells can have deleterious effects on the tissue microenvironment. The most significant of these effects is the acquisition of a senescence-associated secretory phenotype (SASP) that turns senescent fibroblasts into proinflammatory cells that have the ability to promote tumor progression.

THE SECRETORY PHENOTYPE OF SENESCENT CELLS

The senescent phenotype is not limited to an arrest of cell proliferation. In fact, a senescent cell is a potentially persisting cell that is metabolically active and has undergone widespread changes in protein expression and secretion, ultimately developing the SASP. This phenotype has also been termed the senescence-messaging secretome (). We recently provided a large-scale characterization of the SASP, using antibody arrays to quantitatively measure factors secreted by human fibroblasts and epithelial cells (), as well as mouse fibroblasts (J.P. Coppé & J. Campisi, unpublished data). The potential existence of the SASP was already suggested by large-scale comparative gene (mRNA) expression studies performed on fibroblasts from different-aged donors and different tissues of origin (). Among the cells that have been shown to senesce and secrete biologically active molecules are liver stellate cells (), endothelial cells (, ), and epithelial cells of the retinal pigment, mammary gland, colon, lung, pancreas, and prostate (, , , , ).

Senescence-associated changes in gene expression are specific and mostly conserved within individual cell types. Most differences between the molecular signatures of presenescent and senescent cells entail cell-cycle- and metabolism-related genes, as well as genes encoding the secretory proteins that constitute the SASP. The SASP includes several families of soluble and insoluble factors (see Table 1). These factors can affect surrounding cells by activating various cell-surface receptors and corresponding signal transduction pathways that may lead to multiple pathologies, including cancer. SASP factors can be globally divided into the following major categories: soluble signaling factors (interleukins, chemokines, and growth factors), secreted proteases, and secreted insoluble proteins/extracellular matrix (ECM) components. SASP proteases can have three major effects: (a) shedding of membrane-associated proteins, resulting in soluble versions of membrane-bound receptors, (b) cleavage/degradation of signaling molecules, and/or (c) degradation or processing of the ECM. These activities provide potent mechanisms by which senescent cells can modify the tissue microenvironment. In the following sections, we discuss these SASP subsets and some of their known paracrine effects on nearby cells, with an emphasis on their ability to facilitate cancer progression.

Overall, senescence is a molecular program with a unique phenotypic outcome. How its extracellular molecular signature is activated and maintained and the extent to which it influences the tissue milieu in healthy tissues, aged tissues, and diseased tissues are some of the many questions that remain unanswered. However, even with our currently limited knowledge of the SASP and its potential effects on carcinogenesis, promising new strategies for cancer therapies are possible. For example, restoring the activity of tumor-suppressor proteins is an attractive, potentially powerful therapeutic approach. Taking into account our present understanding of the cell-nonautonomous effects of tumor-suppressor genes such as p53, small chemicals that can pharmacologically restore their normal function would help reestablish the proper tissue and cell signals, thereby stimulating cancer regression (). Such approaches could stimulate cancer elimination for two reasons: First, they would limit inflammation and thus possibly allow proper tissue repair; second, they would directly promote the immune-mediated clearance of cells that drive cancer progression.


Senescence-associated secretory phenotype

Senescence-associated secretory phenotype (SASP) is a phenotype associated with senescent cells wherein those cells secrete high levels of inflammatory cytokines, immune modulators, growth factors, and proteases.[1][2] SASP is one of the three main features of senescent cells, the other two features being arrested cell growth, and resistance to apoptosis.[3]

The concept and abbreviation of SASP originated with Judith Campisi, who first published on the subject in 2008.[1]

SASP expression is induced by a number of transcription factors, the most important of which is NF-κB.[4] SASP factors induce insulin resistance.[5]

SASP disrupts normal tissue function by producing chronic inflammation, induction of fibrosis and inhibition of stem cells.[6] Chronic inflammation associated with aging has been termed inflammaging, although SASP may be only one of the possible causes of that condition.[7] SASP factors stimulate the immune system to eliminate senescent cells.[8]

SASP factors from senescent cells reduce nicotinamide adenine dinucleotide in non-senescent cells,[9] thereby reducing the capacity for DNA repair and sirtuin activity in the non-senescent cells.[10]

Despite the fact that cellular senescence probably evolved as means of protecting against cancer early in life, SASP promotes the development of late-life cancers.[6][4] Cancer invasiveness is promoted primarily though the actions of the SASP factors interleukin 6 (IL-6) and interleukin 8 (IL-8).[1] In fact, SASP from senescent cells is associated with many aging-associated diseases, including not only cancer, but atherosclerosis and osteoarthritis.[2] For this reason, senolytic therapy has been proposed as a generalized treatment for these and many other diseases.[2]

SASP can also play a beneficial role, however, by promoting wound healing.[11] But in contrast to the persistent character of SASP in chronic inflammation, beneficial SASP in wound healing is transitory.[11] -Wiki


IN SHORT: SASP IS VERY BAD; IT POISONS YOUR SYSTEM WITH TOXIC INFLAMMATORY CYTOKINES FROM ZOMBIE SENESCENT CELLS AND PREMATURELY AGES YOU. THE SASP MUST GO!


THE KEY TO ELIMINATING ZOMBIE SENESCENT CELLS CONSISTS OF TWO PARTS:

  1. DOWN-REGULATING ANT-APOPTOTIC PATHWAYS THAT PROTECT THEM
  2. UP-REGULATING PRO-APOPTOTIC PATHWAYS THAT CAN KILL THEM

WHICH PATHWAYS?

HERE’S AN EXAMPLE OF SOME OF THEM:



ATTENTION: THIS IS VERY IMPORTANT TO UNDERSTAND IF YOU WANT TO BECOME A ZOMBIE KILLING MACHINE

INSULIN IS ANTI-APOPTOTIC

WHICH MEANS IT

PROTECTS ZOMBIE CELLS FROM APOPTOSIS.



THIS IS WHY YOU CAN’T KILL THAT STUBBORN FAT NO MATTER WHAT YOU SEEM TO DO. INSULIN IS BLOCKING YOU FROM doing so.

  • FOR MAXIMUM BENEFIT IT IS ADVISED A BLACK COFFEE FAST (NO CALORIES / NO INSULIN) WITH ZOMBIE CELL KILLER FOR A 3-7 DAY DURATION; THE LONGER THE BETTER! IN THE PRESENCE OF INSULIN THE BLEND NEEDS TO WORK THAT MUCH HARDER SO WHEN YOU DO EAT KEEP IT LOW INSULIN; PALEO KETOGENIC DIET IS BEST.

IMPORTANT AND RELEVANT KEYWORDS ARE HIGHLIGHTED IN GREEN; FOR MORE IN DEPTH STUDIES AND EXPLANATIONS OF EACH GO TO BOTTOM OF PAGE.


INGREDIENTS:


alantolactonE

Bax/Bcl-2↑, caspase-3↑, STAT3⊥, caspase-8, MMP↓, Bid↑, NF-κB/p65↓, p53↑, Bax↑, Bcl-2↓, caspase-9↑, caspase-3↑, ADP-ribose↓, NF-κB⊥, ROS↑, activin/SMAD3 signaling↑, Cripto-1/ActRII⊥, ROS↑, cytochrome-c↑, Bax↑, PARP↓, ADP-ribose↓, NF-κB⊥, DNA-binding↓, IκBα phosphorylation↓, p21↑, Bcr/Abl↓, P-glycoprotein↓, cyclin B1↓, cyclin-dependent protein kinase-1↓


induces apoptosis via the p38 MAPK, NF-κB and Nrf2 signaling pathways


Human breast cancer is a malignant type of cancer with high prevalence. In the present study, the anticancer effects of alantolactone, a sesquiterpene lactone, on the human breast cancer cell line MF-7 were investigated in vitro. The MCF-7 cell morphology changed from diamond to round subsequent to treatment with alantolactone, and the cell viability reduced significantly compared with that of the control cells. Alantolactone induced apoptosis of MCF-7 cells by regulating the protein expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein, p53, caspase-3 and caspase-12, which are associated with the apoptotic pathway, and suppressed colony formation and migration by regulating the protein expression of matrix metalloproteinase (MMP)-2, MMP-7 and MMP-9.

Cell signaling pathway analysis confirmed that alantolactone increased the phosphorylation of p38, and decreased the nuclear expression levels of p65 and nuclear factor erythroid 2-related factor 2 (Nrf2), suggesting that the apoptosis-promoting and migration-suppressing effect of alantolactone may partially depend on regulating the p38 MAPK, NF-κB and Nrf2 pathways. These results also suggested that alantolactone may become a potential therapeutic strategy for treating breast cancer.


Caspase-3 & Bax Activator


In this study, we investigated the anti-tumor effects and the underlying mechanisms of ATL, a natural sesquiterpene lactone, in human SW480 and SW1116 colorectal cancer cell lines. We found that ATL potently suppressed the growth and proliferation of colorectal cancer cells, while the growth of the non-cancer BEAS-2B and L-O2 cells was not affected. ATL treatment acutely increased cellular ROS levels (within 15 min of ATL treatment), and elevated ROS resulted in a dramatic increase in cellular levels of 8-oxoG, the number of DNA double-strand breaks and cells with bright 53BP1 foci, indicating induction of extensive oxidative DNA damages (within 1 h of ATL treatment).

Consequently, the G1/S-CDK suppressor p21 and pro-apoptotic Bax and active caspase-3were upregulated (within 3 h of ATL treatment), and dissipation of mitochondrial membrane potential was observed within 6 h of ATL treatment, which were followed by cell cycle arrest at G1 and activation of the intrinsic apoptosis pathway (within 12 h of ATL treatment). Suppression of DNA damage and apoptosis by NAC validates the critical role of ROS in ATL-induced cancer cell death.

These studies provide further evidence showing that ATL has potent and selective anticancer activities that are related to induction of ROS overload and oxidative DNA damages. In addition, the results support promoting ROS overload as an important strategy for the development of new anticancer drugs.


Activates p53


It has also been documented that alantolactone significantly increased the expression of p53 in HepG2 cells [49, 50] with concomitant increase of its downstream target genes, mainly cyclin-dependent kinase inhibitor p21 in adriamycin (ADR)-resistant human erythroleukemia cell line K562/ADR [51]. Alantolactone induce p53-independent apoptosis in prostate cancer PC-3 cells [52].


Induces Apoptosis through GSH Depletion, Inhibition of STAT3 Activation, and Mitochondrial Dysfunction


Signal transducer and activator of transcription 3 (STAT3) constitutively expresses in human liver cancer cells and has been implicated in apoptosis resistance and tumorigenesis. Alantolactone, a sesquiterpene lactone, has been shown to possess anticancer activities in various cancer cell lines. In our previous report, we showed that alantolactone induced apoptosis in U87 glioblastoma cells via GSH depletion and ROS generation. However, the molecular mechanism of GSH depletion remained unexplored. The present study was conducted to envisage the molecular mechanism of alantolactone-induced apoptosis in HepG2 cells by focusing on the molecular mechanism of GSH depletion and its effect on STAT3 activation. We found that alantolactone induced apoptosis in HepG2 cells in a dose-dependent manner.

This alantolactone-induced apoptosis was found to be associated with GSH depletion, inhibition of STAT3 activation, ROS generation, mitochondrial transmembrane potential dissipation, and increased Bax/Bcl-2 ratio and caspase-3 activation. This alantolactone-induced apoptosis and GSH depletion were effectively inhibited or abrogated by a thiol antioxidant, N-acetyl-L-cysteine (NAC). The data demonstrate clearly that intracellular GSH plays a central role in alantolactone-induced apoptosis in HepG2 cells. Thus, alantolactone may become a lead chemotherapeutic candidate for the treatment of liver cancer.



α-Mangostin

TGF-β Supressor

TGF-β acts as a gateway in intracellular signaling. Thus, there is a need to develop drugs to inhibit the intracellular activity of TGF-β. The results of this study confirmed that α-MG not only inhibited the proliferation of HSCs but was also an effective marker of fibrogenesis through the TGF-β pathway. Therefore, α-MG should be further investigated as a potential target for the treatment of liver fibrosis.

α-Mangostin (α-MG), one of the active substances in Garcinia mangostana, has been shown to exhibit anti-cancer effects in various cancer cell types. α-MG treatment induces G1 arrest in cancer cell models through the induction of cyclin-dependent kinase inhibitors (CDKIs) and the subsequent loss of CDK activity. However, outside its role in the p53p21CIP1 axis, the precise molecular mechanisms underlying the effect of α-MG on cell cycle arrest remain unclear. In this study, we observed that α-MG inhibits the proliferation of HCT116 cells in a dose-dependent manner. Interestingly, although the loss of p53 rescued the α-MG effect on cell cycle arrest, in agreement with previous reports, p21Cip1 expression was only marginally delayed in the absence of p53 after α-MG treatment.

Instead, we found that the activation of p38 mitogen activated protein kinase (MAPK) and the subsequent downregulation of Bmi-1 also contributed to the induction of p16Ink4a, which is responsible for G1 arrest upon α-MG treatment. These findings indicate that α-MG exerts cytostatic effects on colon cancer cells by inducing G1 arrest via the p38MAPK-p16INK4a axis.


Protective Effects of α-Mangostin on UVB-induced Oxidative Stress and Cellular Senescence


One of subtypes in ultraviolet (UV), UVB, has been reported that the most powerful factors causing photoaging and DNA lesions. Finding and developing both cost-effective and efficient materials are the urgent issues to protect the skin from UVB-induced damages. Alpha-mangostin (α-mangostin), the first xanthone isolated from Garcinia mangostana (mangosteen), has been studied for anti-inflammation and antioxidant properties.

The purpose of this study to investigate effects of α-mangostin in HaCaT, keratinocyte against UVB radiation. To evaluate effects of α-mangostin upon UVB-induced cytotoxic damages, watersoluble tetrazolium salt (WST-1) assay, flow Cytometry analysis, qunatitative real-time PCR (qRT-PCR), and senescence associated β-galactosidase assay (SA-β-gal assay) were performed. Pretreatment of α-mangostin was verified protective effects on UVB-induced proliferative restrain, apoptotic cell death and senescence, in HaCaT keratinocytes. These results indicated the protective effects of α-mangostin against UVB, and suggest as a cosmeceutical ingredient.


Alpha-mangostin decreased cellular senescence in human umbilical vein endothelial cells


These results show that alpha-mangostin, similar to metformin, has anti-senescence effects in high-glucose conditions, which is probably due to its antioxidant activity through the SIRT1 pathway. Alpha-mangostin has previously shown anti-inflammatory effects and metabolic status improvement in animal and clinical studies. Therefore, this natural agent can be considered as a supplement to prevent vascular complications caused by high glucose in patients with diabetes.



Alpine Rose (Rhododendron ferrugineum L.) Leaf Extract


Deciphering the Phytochemical Profile of an Alpine Rose (Rhododendron ferrugineum L.) Leaf Extract for a Better Understanding of Its Senolytic and Skin-Rejuvenation Effects


Rhododendron ferrugineum, commonly named Alpine rose, is an emblematic medicinal plant of European mountains. In this study, the chemical profile of a glycerol/water extract developed from this plant as a cosmetic ingredient is investigated to understand the extract constituent(s) that could mostly contribute to its senolytic activity and skin-rejuvenation effects. For this purpose, the dereplication method “CARAMEL”, which combines Centrifugal Partition Chromatography to Nuclear Magnetic Resonance data interpretation, was directly applied to the hydro-glycerinated extract, leading to the unambiguous identification of fourteen Alpine rose metabolites, despite the strong presence of the heavy solvent glycerol.

Flavonoids derived from taxifolin, quercetin, and (+)-catechin were identified as significant constituents of the extract, followed by flavanones, orcinol derivatives, phloroacetophenone, and phenolic acids, as well as the pentacyclic triterpene lupeol. Given that senolytic molecules are known to selectively induce the death of senescent cells without affecting healthy proliferating cells, which can be achieved by the selective inhibition or downregulation of the anti-apoptotic Bcl-2 protein, and considering the well-recognized pro-apoptotic activity of hyperoside, taxifolin, naringenin and farrerol, the senolytic activity of the glycerol/water Alpine rose extract can be explained by the abundance of flavonoids present in the extract.


andrographolide

(from Andrographis Paniculata aka “Creat” or “Green Chireta”)

BCL-2 Inhibitor


Cancer is a disorder characterized by uncontrolled proliferation and reduced apoptosis. Inducing apoptosis is an efficient method of treating cancers. In this study, we investigated the effect of andrographolide on the induction of apoptosis as well as its regulatory effect on the activation of transcription factors in B16F-10 melanoma cells. Treatment of B16F-10 cells with nontoxic concentration of andrographolide showed the presence of apoptotic bodies and induced DNA fragmentation in a dose-dependent manner. Cell cycle analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays also confirmed the observation. The proapoptotic genes p53, Bax, caspase-9, and caspase-3 were found upregulatedin andrographolide-treated cells, whereas the antiapoptotic gene bcl-2 was downregulated.

This study also reveals that andrographolide treatment could alter the production and expression of proinflammatory cytokines and could inhibit the activation and nuclear translocation of p65, p50, and c-Rel subunits of nuclear factor-κB (NF-κB), and other transcription factors such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element–binding protein in B16F-10 melanoma cells.

These results suggest that andrographolide induces apoptosis via inhibiting NF-κB-induced bcl-2-mediated survival signaling and modulating p53-induced caspase-3-mediated proapoptotic signaling.


HSP90 INHIBITOR


Andrographolide is a diterpenoid compound isolated from Andrographis paniculata that exhibits anticancer activity. We previously reported that andrographolide suppressed v-Src-mediated cellular transformation by promoting the degradation of Src. In the present study, we demonstrated the involvement of Hsp90 in the andrographolide-mediated inhibition of Src oncogenic activity. Using a proteomics approach, a cleavage fragment of Hsp90α was identified in andrographolide-treated cells. The concentration- and time-dependent induction of Hsp90 cleavage that accompanied the reduction in Src was validated in RK3E cells transformed with either v-Src or a human truncated c-Src variant and treated with andrographolide. In cancer cells, the induction of Hsp90 cleavage by andrographolide and its structural derivatives correlated well with decreased Src levels, the suppression of transformation, and the induction of apoptosis.

Moreover, the andrographolide-induced Hsp90 cleavage, Src degradation, inhibition of transformation, and induction of apoptosis were abolished by a ROS inhibitor, N-acetyl-cysteine. Notably, Hsp90 cleavage, decreased levels of Bcr-Abl (another known Hsp90 client protein), and the induction of apoptosis were also observed in human K562 leukemia cells treated with andrographolide or its active derivatives.

Together, we demonstrated a novel mechanism by which andrographolide suppressed cancer malignancy that involved inhibiting Hsp90 function and reducing the levels of Hsp90 client proteins. Our results broaden the molecular basis of andrographolide-mediated anticancer activity.


Downregulates PI3k/AKT, TNF-A


Andrographolide (1), an active constituent of Andrographis paniculata, decreased tumor necrosis factor-α (TNF-α)-induced intercellular adhesion molecule-1 (ICAM-1) expression and adhesion of HL-60 cells onto human umbilical vein endothelial cells (HUVEC), which are associated with inflammatory diseases. Moreover, 1 abolished TNF-α-induced Akt phosphorylation. Transfection of an activated Akt1 cDNA vector increased Akt phosphorylation and ICAM-1 expression like TNF-α. In addition, 1 and LY294002 blocked TNF-α-induced IκB-α degradation and nuclear p65 protein accumulation, as well as the DNA-binding activity of NF-κB. Compound 1 exhibits anti-inflammatory properties through the inhibition of TNF-α-induced ICAM-1 expression.

The anti-inflammatory activity of 1 may be associated with the inhibition of the PI3K/Akt pathway and downstream target NF-κB activation in HUVEC cells.


potent inhibitory effect on the JAK-STAT3 pathwaY


Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess potent anti-inflammatory and anticancer properties. In this study, we sought to examine the effect of Andro on signal transducer and activator of transcription 3 (STAT3) pathway and evaluate whether suppression of STAT3 activity by Andro could sensitize cancer cells to a chemotherapeutic drug doxorubicin. First, we demonstrated that Andro is able to significantly suppress both constitutively activated and IL-6-induced STAT3 phosphorylation and subsequent nuclear translocation in cancer cells. Such inhibition is found to be achieved through suppression of Janus-activated kinase (JAK)1/2 and interaction between STAT3and gp130. For understanding the biological significance of the inhibitory effect of Andro on STAT3, we next investigated the effect of Andro on doxorubicin-induced apoptosis in human cancer cells.

In our study the constitutive activation level of STAT3 was found to be correlated to the resistance of cancer cells to doxorubicin-induced apoptosis. Both the short-term MTT assay and the long-term colony formation assay showed that Andro dramatically promoted doxorubicin-induced cell death in cancer cells, indicating that Andro enhances the sensitivity of cancer cells to doxorubicin mainly via STAT3 suppression.

These observations thus reveal a novel anticancer function of Andro and suggest a potential therapeutic strategy of using Andro in combination with chemotherapeutic agents for treatment of cancer.


INBITS HIF-1A & VEGF


Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro) was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10−7 mol/L), by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway.

It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF)gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice.

In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future.


andrographolide attenuates MMP-9 expression


There is much evidence indicating that human leukemic cells and monocytes/macrophages synthesize, and secrete, several matrix metalloproteinases (MMPs), and participate in the degradation of extracellular matrix components in tissue lesions. In this study, we investigated the effects and mechanisms of andrographolide, extracted from the herb Andrographis paniculata, on human monocytic MMPs expression and activation. Andrographolide (1-50 μM) exhibited concentration-dependent inhibition of MMP-9 activation, induced by either tumor necrosis factor-α (TNF-α), or lipopolysaccharide (LPS), in THP-1cells. In addition, andrographolide did not present an inhibitory effect on MMP-9 enzymatic activity at a concentration of 50 μM. By contrast, enzyme-linked immunosorbent assay (ELISA) showed that andrographolide partially affect TIMP-1 levels. Western blot analysis showed that both TNF-α, and LPS stimulators attenuated MMP-9 protein expression in a concentration-dependent manner. Using reverse transcription polymerase chain reaction (RT-PCR), we found that andrographolide suppressed expression of MMP-9 messenger RNA. Furthermore, we also found that andrographolide could significantly inhibit the degradation of inhibitor-κB-α (IκB-α) induced by TNF-α.

We used electrophoretic mobility shift assay and reporter gene detection to show that andrographolide also markedly inhibited NF-κB signaling, anti-translocation and anti-activation. In conclusion, we demonstrate that andrographolide attenuates MMP-9 expression, and its main mechanism might involve the NF-κB signal pathway. These results provide new opportunities for the development of new anti-inflammatory and leukemic therapies.


Antrodia cinnamomea

Antrodia camphorata

JAK2/STAT3 INHIBITOR


Lung cancer is the leading cause of cancer deaths worldwide and current therapies fail to treat this disease in majority of cases. Antrodia camphorata is a medicinal mushroom being widely used as food dietary supplement for cancer prevention. The sesquiterpene lactone antrocin is the most potent among >100 secondary metabolites isolated from A. camphorata . However, the molecular mechanisms of antrocin-mediated anticancer effects remain unclear. In this study, we found that antrocin inhibited cell proliferation in two non-small-cell lung cancer cells, namely H441 (wild-type epidermal growth factor receptor, IC 50 = 0.75 μM) and H1975 (gefitnib-resistant mutant T790M, IC 50 = 0.83 μM). Antrocin dose dependently suppressed colony formation and induced apoptosis as evidenced by activated caspase-3 and increased Bax/Bcl2 ratio. Gene profiling studies indicated that antrocin downregulated Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway.

We further demonstrated that antrocin suppressed both constitutively activated and interleukin 6-induced STAT3 phosphorylation and its subsequent nuclear translocation. Such inhibition is found to be achieved through the suppression of JAK2 and interaction between STAT3 and extracellular signal-regulated kinase. Additionally, antrocin increased microRNA let-7c expression and suppressed STAT signaling. The combination of antrocin and JAK2/STAT3 gene silencing significantly increased apoptosis in H441 cells. Such dual interruption of JAK2 and STAT3 pathways also induced downregulation of antiapoptotic protein mcl-1 and increased caspase-3 expression. In vivo intraperitoneal administration of antrocin significantly suppressed the growth of lung cancer tumor xenografts. Our results indicate that antrocin may be a potential therapeutic agent for human lung cancer cells through constitutive inhibition of JAK2/STAT3 pathway.


BCL-XL, NF-kB Inhibitor

The fruiting body of Antrodia camphorata is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. camphorata (EAC) fruiting bodies in two human liver cancer cell lines, Hep G2 and PLC/PRF/5. Treatment with EAC decreased the cell growth of Hep G2 and PLC/PRF/5 cells in a dose dependent manner. In Fas/APO-1 positive-Hep G2 cells, EAC increased the expression level of Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), in a p53-indenpendent manner. In addition, EAC also initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 both in Hep G2 and PLC/PRF/5 cells.

Furthermore, EAC also inhibited the cell survival signaling by enhancing the amount of IκBα in cytoplasm and reducing the level and activity ofNF-κBin the nucleus, and subsequently attenuated the expression of Bcl-XL in Hep G2 and PLC/PRF/5 cells. EAC therefore decreased the cell growth and induced apoptosis both in Hep G2 and PLC/PRF/5 cells.


Antroquinonol, a natural ubiquinone derivative of Antrodia camphorata, induces a cross talk between apoptosis, autophagy and senescence in human pancreatic carcinoma cells


Pancreatic cancer is a malignant neoplasm of the pancreas. A mutation and constitutive activation of K-ras occurs in more than 90% of pancreatic adenocarcinomas. A successful approach for the treatment of pancreatic cancers is urgent. Antroquinonol, a ubiquinone derivative isolated from a camphor tree mushroom, Antrodia camphorata, induced a concentration-dependent inhibition of cell proliferation in pancreatic cancer PANC-1 and AsPC-1 cells. Flow cytometric analysis of DNA content by propidium iodide staining showed that antroquinonol induced G1 arrest of the cell cycle and a subsequent apoptosis. Antroquinonol inhibited Akt phosphorylation at Ser473, the phosphorylation site critical for Akt kinase activity, and blocked the mammalian target of rapamycin (mTOR) phosphorylation at Ser2448, a site dependent on mTOR activity. Several signals responsible for mTOR/p70S6K/4E-BP1 signaling cascades have also been examined to validate the pathway. Moreover, antroquinonol induced the down-regulation of several cell cycle regulators and mitochondrial antiapoptotic proteins. In contrast, the expressions of K-ras and its phosphorylation were significantly increased. The coimmunoprecipitation assay showed that the association of K-ras and Bcl-xL was dramatically augmented, which was indicative of apoptotic cell death.

Antroquinonol also induced the cross talk between apoptosis, autophagic cell death and accelerated senescence, which was, at least partly, explained by the up-regulation of p21Waf1/Cip1 and K-ras. In summary, the data suggest that antroquinonol induces anticancer activity in human pancreatic cancers through an inhibitory effect on PI3-kinase/Akt/mTOR pathways that in turn down-regulates cell cycle regulators. The translational inhibition causes G1 arrest of the cell cycle and an ultimate mitochondria-dependent apoptosis. Moreover, autophagic cell death and accelerated senescence also explain antroquinonol-mediated anticancer effect.


APIGENIN

SASP INHIBITOR


During senescence, cells express molecules called senescence-associated secretory phenotype (SASP), including growth factors, proinflammatory cytokines, chemokines, and proteases. The SASP induces a chronic low-grade inflammation adjacent to cells and tissues, leading to degenerative diseases. The anti- inflammatory activity of flavonoids was investigated on SASP expression in senescent fibroblasts. Effects of flavonoids on SASP expression such as IL-1a, IL-1b, IL-6, IL-8, GM-CSF, CXCL1, MCP-2 and MMP-3 and signaling molecules were examined in bleomycin-induced senescent BJ cells. In vivo activity of apigenin on SASP suppression was identified in the kidney of aged rats. Among the five naturally-occurring flavonoids initially tested, apigenin and kaempferol strongly inhibited the expression of SASP. These flavonoids inhibited NF-kB p65 activity via the IRAK1/IkBa signaling pathway and expression of IkBz. Blocking IkBz expression especially reduced the expression of SASP. A structure-activity relationship study using some synthetic flavones demonstrated that hydroxyl substitutions at C-20,30,40,5 and 7 were important in inhibiting SASP production.

Finally, these results were verified by results showing that the oral administration of apigenin significantly reduced elevated levels of SASP and IkBz mRNA in the kidneys of aged rats. This study is the first to show that certain flavonoids are inhibitors of SASP production, partially related to NF-kB p65 and IkBz signaling pathway, and may effectively protect or alleviate chronic low-grade inflammation in degenerative diseases such as cardiovascular diseases and late-stage cancer. Inhibitory activity of apigenin on IL-6, IL-8, and IL-1b was the most potent among the five flavonoids that were tested (86.5%, 60.9%, and 94.9% at 10 mM, respectively).

HSP90 Inhibitor


Natural plant flavonoid apigenin directly disrupts Hsp90/Cdc37 complex and inhibits pancreatic cancer cell growth and migration •Apigenin can be digested, released and absorbed into blood circulation to accumulate. •Apigenin directly inhibited Hsp90/Cdc37 interaction with structural specificity. •The effect of apigenin on Hsp90/Cdc37 did not rely on CK2 activity. •Apigenin induced downstream kinase client protein degradation. •Apigenin induced ROS accumulation, inhibited cell proliferation and migration.


Apigenin is a common dietary plant flavonoid widely distributed in vegetables and fruits. It exhibits chemopreventive activity against various cancer cells. In this study, we demonstrated that apigenin directly blocked heat shock protein 90 (Hsp90)and cell division cycle protein 37 (Cdc37) interaction using split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) assay. Apigenin inhibited complemented Renilla luciferase (RL) activity of NRL-Hsp90/Cdc37-CRL, while its analogues did not. Apigenin also inhibited NRL-Hsp90 and Cdc37(Ser13Ala)-CRL complementation.

In addition, casein kinase II (CK2) specific inhibitor 4, 5, 6, 7-tetrabromobenzotriazole (TBB) did not affect NRL-Hsp90/Cdc37-CRL complementation, indicating that the inhibitory effect of apigenin on Hsp90/Cdc37 did not rely on CK2 activity. Moreover, apigenin blocked Hsp90/Cdc37 complex and induced kinase clients protein kinase B (Akt), cyclin-dependent-kinase 4 (CDK4) and matrix metalloproteinase-9 (MMP-9) degradation and, as a consequence, induced intracellular reactive oxygen species (ROS) accumulation and inhibited cell proliferation and migration in pancreatic cancer cells.


INHIBITS C-FLIP


Apigenin has been shown to induce apoptosis in different types of cells [, , , ]. In human keratinocytes and organotypic keratinocyte cultures, apigenin treatment enhanced UVB-induced apoptosis more than 2-fold. In addition, apigenin stimulated changes in Bax localization, and increased the release of cytochrome c from the mitochondria. Overexpression of the antiapoptotic protein Bcl-2 and expression of a dominant-negative form of Fas-associated death domain led to a reduction in apigenin-induced apoptosis, demonstrating that enhancement of UVB-induced apoptosis by apigenin treatment involves both the intrinsic and extrinsic apoptotic pathways []. In human prostate cancer cells, apigenin treatment has been shown to alter the Bax/Bcl-2 ratio in favor of apoptosis [].

In human promyelocytic leukemia HL-60 cells, apigenin induced caspase-3 activity and cleavage of poly-(ADP-ribose) polymerase (PARP), reduced mitochondrial transmembrane potential, released mitochondrial cytochrome c into the cytosol, and subsequently induced procaspase-9 processing []. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that kills various tumor cells without damaging normal tissues. However, many cancers remain resistant to TRAIL. Apigenin breaks TRAIL resistance by transcriptional down-regulation of c-FLIP, a key inhibitor of death receptor signaling, and by up-regulation of TRAIL receptor 2 [].


CDC2 CDK1 CYCLIN D1 INHIBITOR


Exposure of a wide array of malignant cells, including epidermal cells and fibroblasts to apigenin induces a reversible G2/M and G0/G1 arrest by inhibiting p34 (cdc2) kinase activity, accompanied by increased p53 protein stability (, ). Apigenin has also been shown to induce WAF1/p21 levels resulting in cell cycle arrest and apoptosis in androgen-responsive human prostate cancer, LNCaP cells and androgen-refractory DU145 cells, regardless of the Rb status and p53-dependence or p53 independence (, ). In addition, apigenin has been shown to induce apoptosis in a wide range of malignant cells (). Apigenin treatment has been shown to alter the Bax/Bcl-2 ratio in favor of apoptosis, associated with release of cytochrome c and induction of Apaf-1, which leads to caspase activation and PARP-cleavage ().

Apigenin-mediated cell growth inhibition along with G2/M arrest was accompanied by significant decrease in cyclin B1 and CDK1 protein levels, resulting in a marked inhibition of CDK1 kinase activity. Furthermore, apigenin treatment reduced the protein levels of CDK4, cyclin D1 and A, inhibited Rb-phosphorylation but did not affect the protein levels of cyclin E, CDK2 or CDK6. Recently, studies have shown that apigenin induces G ()/M phase cell cycle arrest in SK-BR-3 cells which is via regulation of CDK1 and p21 (Cip1) pathway. In addition, apigenin treatment resulted in ERK MAP kinase phosphorylation and activation in MDA-MB-468 cells ().


apigenin treatment significantly inhibited cell senescence and increased telomerase activity

Effect of Luteolin and Apigenin on the Cell Senescence and Telomerase Activity of DPCs at Various Passages. Senescenceassociated b-galactosidase is caused by upregulated lysosomal activities and altered cytosolic pH, which are upregulated with senescence and aging. To elucidate the effect of luteolin and apigenin on replicative senescence state of DPCs, the senescence-associated b-galactosidase activity (SA-b-gal) was evaluated. DPCs from passages 1, 3, 5, and 7 with/without luteolin/apigenin treatment were detected, albeit only the representative results of passages 3 and 7 were presented. The result revealed that DPCs at passage 3 with luteolin/apigenin induction and the control group did not show any obvious blue staining. DPCs at passage 7 without induction showed intense blue color, albeit DPCs at passage 7 with luteolin or apigenin induction revealed weak blue staining, not as intense as the control group at passage 7.

Similarly, there is no difference of the telomerase activity of DPCs at passage 3 with/without luteolin or apigenin induction albeit DPCs at passage 7 with luteolin or apigenin induction showed significantly higher telomerase activity than the control group at passage 7 (∗ 𝑃 < 0.05), which agreed with the result of b-galactosidase assay mentioned above. This result implied that luteolin and apigenin treatment significantly inhibited cell senescence and increased telomerase activity of DPCs, especially at late passages. Thus, luteolin and apigenin might be able to maintain DPCs in an undifferentiated and presenescent state.


Apigenin induces cell cycle arrest and p21/WAF1 expression in a p53-independent pathway.


Apigenin, a common dietary flavonoid, has been shown to induce cell growth-inhibition and cell cycle arrest in many cancer cell lines. One important effect of apigenin is to increase the stability of the tumor suppressor p53 in normal cells. Therefore, apigenin is expected to play a large role in cancer prevention by modifying the effects of p53 protein. However, the mechanisms of apigenin’s effects on p53-mutant cancer cells have not been revealed yet. We assessed the influence of apigenin on cell growth and the cell cycle in p53-mutant cell lines. Treatment with apigenin resulted in growth-inhibition and G2/M phase arrest in two p53-mutant cancer cell lines, HT-29 and MG63.

These effects were associated with a marked increase in the protein expression of p21/WAF1. We have shown that p21/WAF1 mRNA expression was also markedly increased by treatment with apigenin in a dose- and time-dependent manner. However, we could not detect p21/WAF1 promoter activity following treatment with apigenin. Similarly, promoter activity from pG13-Luc, a p53-responsive promoter plasmid, was not activated by treatment with apigenin with or without p53 protein expression.

These results suggest that there is a p53-independent pathway for apigenin in p53-mutant cell lines, which induces p21/WAF1 expression and growth-inhibition. Apigenin may be a useful chemopreventive agent not only in wild-type p53 status, but also in cancer with mutant p53.


Induction of p21/WAF1 and G1 cell-cycle arrest


Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanism by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes.

More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin-induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 microM apigenin resulted in dose-dependent cell-cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later.

A dose of apigenin as low as 10 microM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin.

Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 microM apigenin-treated cells.

In conclusion, apigenin treatment produced a G1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.


Artemisinin

Artemisinin induces apoptosis in human cancer cells.

Background: Artemisinin is a chemical compound extracted from the wormwood plant, Artemisia annua L. It has been shown to selectively kill cancer cells in vitro and retard the growth of implanted fibrosarcoma tumors in rats. In the present research, we investigated its mechanism of cytotoxicity to cancer cells.

Materials & Methods: Molt-4 cells, in complete RPMI-1640 medium, were first incubated with 12 microM of human holotransferrin at 37 degrees C in a humid atmosphere of 5% CO2 for one hour. This enhanced the iron supply to the cells. The cells were then pelleted and transferred to a complete RPMI-1640 containing 200 microM of an analog dihydroartemisinin (DHA) and incubation was started (0 h). In addition, some culture samples were treated with holotransferrin alone and some (controls) were assayed without neither holotransferrin nor DHA treatment. Cells were counted and DNA diffusion assay was used to evaluate apoptosis and necrosis in each sample at 0 h and at 1, 2, 4 and 8 h of incubation.

Results: DHA treatment significantly decreased cell counts and increased the proportion of apoptosis in cancer cells compared to controls (chi2=4.5, df=1, p<0.035). Addition of holotransferrin significantly further decreased cell counts (chi2=4.5, df=1, p<0.035) and increased apoptosis (chi2=4.5, df=1, p<0.035). No necrotic cells were observed.

Conclusion: This rapid induction of apoptosis in cancer cells after treatment with DHA indicates that artemisinin and its analogs may be inexpensive and effective cancer agents.


CDK4 INHIBITOR

Neuroblastoma is a malignant pediatric tumor with a wide range of stages, requiring a wide range of therapeutic options. However, successful therapeutic options remain limited. Therefore, it is urgently necessary to identify additional chemotherapeutic agents to target this disease. It has been reported that artemisinin has multiple anti-proliferative activity, including cell growth suppression (26,27), apoptosis induction (28), angiogenesis inhibition, cell migration disruption (2931), and modulation of nuclear receptor responsiveness (32,33). Yet, the effect of artemisinin on neuroblastoma remains unclear.

The results presented in the present study demonstrated that artemisinin led to significantly decreased cell growth and cell proliferation, and increased apoptosis in neuroblastoma cells. We first demonstrated that artemisinin treatment suppressed the ability of colony formation in vitro and tumorigenicity of neuroblastoma cells in vivo.

Artemisinin has been suggested to promote cytostasis by G0/G1-phase arrest and to decrease the expression level of cyclinB1, CDK2 and CDC25A in colon cancer (34), and to inhibit the promoter activity of CDK4 in prostate cancer (16). Artemisinin also induced G2/M phase arrest in osteosarcoma cells (35). Our data indicated that artemisinin induced cell cycle arrest at the G1 phase, together with a decrease in the expression levels of cyclinD1, CDK4 and cyclinE2 in all four neuroblastoma cell lines. cyclinB1 was downregulated only in the BE(2)-C and SHEP1 cells, but no significant difference was noted in the SK-N-AS and SK-N-DZ cells.

Previous studies have shown that artemisinin induced apoptosis in pancreatic tumor cells (36), lung adenocarcinoma cells (37), liver cancer cells (38), and non-small cell lung cancer cells (39). In the present study, we first demonstrated that artemisinin induced the cell death and apoptosis of neuroblastoma cells at a lower dose than that for clinical usage. Next, we investigated the mechanism and the expression of apoptotic relevant proteins.

Collectively, our results revealed that artemisinin inhibited cell proliferation and tumor growth, with cell cycle arrest and apoptosis induction in neuroblastoma cells. Since artemisinin has been used for the treatment of malaria for an extensive period of time, a large body of data regarding clinical tests and adverse drug reactions in patients are available. Therefore, artemisinin may serve as a potential new therapeutic agent for the treatment of neuroblastoma.


Artocarpin


NOXA ACTIVATOR


In conclusion, our results demonstrated that whereas artocarpin induces cytotoxic effects in human NSCLC cells, it may exert protective effects in normal HPAEpiCs. In addition, artocarpin may serve as a pro-oxidant only in human NSCLC cells, but not in normal HPAEpiCs. Previously, various flavonoids (including flavone acetic acid, quercetin, and flavopiridol) have entered human clinical trials, and have shown promising anticancer effects clinically []. We propose that cell apoptosis caused by artocarpin-induced oxidative stress and ROS generation can be an important mechanism for cancer prevention and therapy.

Additionally, this study is the first to demonstrate that artocarpin-induced apoptosis is mediated through activation of the Nox2/p47phox pathway leading to enhanced ROS production, which then induces the activation of two distinct signaling cascades, including ERK MAPK/ p38 MAPK/p53-dependent activation of PUMA/Cytochrome C/ Apaf-1/ caspase 3 pathway in A549 cells and PI3K/ Akts473/ p53-independent activation of NF-kB/ c-Myc/Noxa pathway in both A549 and H1299 cells


P53 Activator


The transcription factor NF-kB is a major PI3K/Akt downstream effector, and plays a dual role as an attenuator or promoter of apoptosis. It regulates the transcription of DNA, and mediates apoptosis in response to oxidative stress []. Recent studies have demonstrated that ROS-dependent NF-kB activation induced the protein expression of c-Myc and Noxa in p53-independent human NSCLC cell death []. On the contrary, NF-kB activation was involved in resistance to oxidative stress and p53-mediated programmed cell death []. Therefore, we investigated whether this transcription factor may play a role in artocarpin-induced apoptosis. In the current study, we observed that artocarpin induced the activation of NF-kB via a Nox2/ROS/PI3K/Akt dependent signaling pathway. Correspondingly, amelioration of Nox2/ROS/PI3K/Akt pathway significantly attenuated artocarpin-induced translocation ofNF-kB and up-regulation of c-Myc and Noxa proteins.

These results demonstrated that activation of the p53-independent ROS/NF-kB/c-Myc/Noxa signaling pathway by artocarpin plays a critical role in inducing apoptosis in A549 and H1299 cells.


Aspalathin

NRF2 Activator


Aspalathin (ASP) can protect H9c2 cardiomyocytes against high glucose (HG)-induced shifts in myocardial substrate preference, oxidative stress, and apoptosis. The protective mechanism of ASP remains unknown. However, as one of possible, it is well known that phytochemical flavonoids reduce oxidative stress via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation resulting in up-regulation of antioxidant genes and enzymes. Therefore, we hypothesized that ASP protects the myocardium against HG- and hyperglycemia-induced oxidative damage by up-regulating Nrf2expression in H9c2 cardiomyocytes and diabetic (db/db) mice, respectively.

Using an oxidative stress RT2 Profiler PCR array, ASP at a dose of 1 μM was demonstrated to protect H9c2 cardiomyocytes against HG-induced oxidative stress, but silencing of Nrf2 abolished this protective response of ASP and exacerbated cardiomyocyte apoptosis. Db/db mice and their non-diabetic (db/+) littermate controls were subsequently treated daily for six weeks with either a low (13 mg/kg) or high (130 mg/kg) ASP dose. Compared to nondiabetic mice the db/db mice presented increased cardiac remodeling and enlarged left ventricular wall that occurred concomitant to enhanced oxidative stress.

Daily treatment of mice with ASP at a dose of 130 mg/kg for six weeks was more effective at reversing complications than both a low dose ASP or metformin, eliciting enhanced expression of Nrf2 and its downstream antioxidant genes. These results indicate that ASP maintains cellular homeostasis and protects the myocardium against hyperglycemia-induced oxidative stress through activation of Nrf2and its downstream target genes.


Astragalus complanatus seed extract


Bax, P21, P27 levels increased / cyclinD1, CDK1, and CDK4 levels decreased.


Aim of the study: Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids fromAstragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells.

Materials and methods: Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC- 7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis.

Results: The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased.

Conclusions: These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor- dependent apoptotic pathways.


Avicin D

StAT3, BCL-2, VEGF Inhibitor


Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a) the cross-talk that occurs between redox and phosphorylation processes, and (b) the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3.

The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors.

Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways.


NFKB INHIBITOR


Avicins, a family of apoptotic triterpene electrophiles, are known to regulate cellular metabolism and energy homeostasis, by targeting the mitochondria. Having evolved from “ancient hopanoids,” avicins bear a structural resemblance with glucocorticoids (GCs), which are the endogenous regulators of metabolism and energy balance. These structural and functional similarities prompted us to compare the mode of action of avicin D with dexamethasone (Dex), a prototypical GC. Using cold competition assay, we show that Avicin D competes with Dex for binding to the GC receptor (GR), leading to its nuclear translocation.

In contrast to Dex, avicin-induced nuclear translocation of GR does not result in transcriptional activation of GC-dependent genes. Instead we observe a decrease in the expression of GC-dependent metabolic proteins such as PEPCK and FASN. However, like Dex, avicin D treatment does induce a transrepressive effect on the pro-inflammatory transcription factor NF-κB. While avicin’s ability to inhibit NF-κB and its downstream targets appear to be GR-dependent, its pro-apoptotic effects were independent of GR expression. Using various deletion mutants of GR, we demonstrate the requirement of both the DNA and ligand binding domains of GR in mediating avicin D’s transrepressive effects.

Modeling of avicin-GR interaction revealed that avicin molecule binds only to the antagonist confirmation of GR. These findings suggest that avicin D has properties of being a selective GR modulator that separates transactivation from transrepression. Since the gene-activating properties of GR are mainly linked to its metabolic effects, and the negative interference with the activity of transcription factors to its anti-inflammatory and immune suppressive effects, the identification of such a dissociated GR ligand could have great potential for therapeutic use.



Increases caspase-3 and -8; inhibits PI3K/Akt, NFƙB, iNOS, and MMP-2 and -9; attenuates Bcl-2; induces Bax


The inhibitory effect of baicalein, which is present in Indian trumpet flower and Chinese skullcap, or Scutellaria baicalensis, on human colon cancer was studied in vitro and in vivo . Research indicated that baicalein had a significant inhibitory effect on HCT-116 cells. The mechanisms of effect of baicalein occur through three pathways: i) the extrinsic pathway of apoptosis, ii) by decreasing the incidence of inflammation, and iii) by impairment of tumor formation through inactivation of the PI3K/Akt pathway. Baicalein increased the expression of caspase-3 and -8, which are involved in apoptosis.

The expression of NF-ƙB was inhibited, resulting in inhibition of iNOS, MMP-9, and MMP-2 genes, all of which are involved in inflammation , . The effect of baicalein on HT-29 cells was also investigated. The data indicated that baicalein had the ability to increase cell arrest in the G1 phase. Baicalein attenuated the expression of Bcl-2, whereas the expression of Bax was augmented. Moreover, induction of apoptosis was achieved by inactivation of the PI3K/Akt pathway .

Further studies on cancerous Institute for Cancer Research (ICR) mice induced by AOM supported the preventive effect of baicalein .



Scutellaria baicalensis (SB) and SB-derived polyphenols possess anti-proliferative activities in pancreatic cancer. Baicalein decreased mRNA and protein expression of the anti-apoptotic Bcl-2 family protein Mcl-1 and induced apoptosis. Mcl-1 knock-down induced apoptosis through caspase cascade, but Bcl-2 or Bcl-xL knock-down had no or only a slight effect. The effect of baicalein on apoptosis was significantly attenuated by Mcl-1 over-expression.


BAVACHIN

( from Psoralea corylifolia)

Bavachin induces the apoptosis of multiple myeloma cell lines by inhibiting the activation of nuclear factor kappa B and signal transducer and activator of transcription 3

Bavachin is a phytoestrogen purified from natural herbal plants such as Psoralea corylifolia. In this study, we examined the effect of bavachin in multiple myeloma(MM) cell lines. We found that bavachin decreased the viability of MM cell lines, but was not cytotoxic towards normal cells. It inhibited the activation of nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3). Furthermore, bavachin increased the expression of p53 and NOXA, and decreased the expression of X-linked inhibitor of apoptosis protein (XIAP), survivin, B cell lymphoma-extra large (Bcl-xL), and Bcl-2.

Additionally, bavachin induced apoptosis by the activation of caspase-3 and caspase-9, implicating the involvement of the mitochondrial pathway. Our results suggest that bavachin induces apoptosis through the inhibition of NF-κB and STAT3 activation in MM cell lines. Most importantly, few NF-κB and STAT3 inhibitors with high efficiency, specificity, and safety are currently available for clinical cancer therapy. Hence, bavachin, which targets NF-κB and STAT3, is a potential anticancer agent for the treatment of MM.


Berberine

(from Berberis Aristata aka “Indian Barberry”, “Chutro” and “Tree Turmeric”)

Key Functions:

Inhibits HIF-1a


These data indicated that HIF-1α repression is a critical step in the inhibitory effect of berberine on tumor-induced angiogenesis. Northern blot analyses plus pulse-chase assays revealed that berberine did not down-regulate HIF-1α mRNA but destabilized HIF-1α protein. We found that berberine-induced HIF-1α degradation was blocked by a 26S proteasome inhibitor.

Moreover, immunoprecipitation and Western blot analyses showed that berberine increased the lysine-acetylated HIF-1α in hypoxic SC-M1 cultures. These data indicated that a proteasomal proteolytic pathway and lysine acetylation were involved in berberine-triggered HIF-1α degradation. In conclusion, our data provided molecular evidence to support berberine as a potent antiangiogenic agent in cancer therapy


Attenuation of premature cellular senescence


Aging is the greatest risk factor for human diseases, as it results in cellular growth arrest, impaired tissue function and metabolism, ultimately impacting life span. Two different mechanisms are thought to be primary causes of aging. One is cumulative DNA damage induced by a perpetuating cycle of oxidative stress; the other is nutrient-sensing adenosine monophosphate-activated protein kinase (AMPK) and rapamycin (mTOR)/ ribosomal protein S6 (rpS6) pathways. As the main bioactive component of natural Chinese medicine rhizoma coptidis (RC), berberine has recently been reported to expand life span in Drosophila melanogaster, and attenuate premature cellular senescence.

Most components of RCincluding berberine, coptisine, palmatine, and jatrorrhizine have been found to have beneficial effects on hyperlipidemia, hyperglycemia and hypertension aging-related diseases. The mechanism of these effects involves multiple cellular kinase and signaling pathways, including anti-oxidation, activation of AMPK signaling and its downstream targets, including mTOR/rpS6, Sirtuin1/ forkhead box transcription factor O3 (FOXO3), nuclear factor erythroid-2 related factor-2 (Nrf2), nicotinamide adenine dinucleotide (NAD+) and nuclear factor-κB (NF-κB) pathways. Most of these mechanisms converge on AMPK regulation on mitochondrial oxidative stress.

Therefore, such evidence supports the possibility that rhizoma coptidis, in particular berberine, is a promising anti-aging natural product, and has pharmaceutical potential in combating aging-related diseases via anti-oxidation and AMPK cellular kinase activation.


Suppresses Activation of pi3k/AKT Signaling


Berberine (BBR), an isoquinoline alkaloid originally isolated from the Chinese herb Coptis chinensis (Huanglian), exhibits anti‑inflammatory and immunosuppressive properties. Since myocardial ischemia/reperfusion (I/R) injury is associated with an excessive immune response, the current study was conducted to investigate the impact of BBR on myocardial I/R injury, a common disorder in clinical settings. Preconditioning of Sprague‑Dawley rats with BBR (100 mg/kg/day, by gavage) for 14 days prior to the induction of I/R significantly attenuated myocardial I/R injury as manifested by a reduction in the incidence of ventricular arrhythmia and the amelioration of myocardial histological changes.

These effects were found to be associated with the suppression of the phosphoinositide 3‑kinase/AKT signaling pathway and the subsequent reduction of the expression of interleukin (IL)‑6, IL‑1β, and tumor necrosis factor‑α in the serum and myocardial tissue. These results indicate that BBR has the potential be an effective alternative therapy for the prevention and treatment of myocardial I/R injury in clinical practice.

Upregulates SIRT1


With a long history of application in Chinese traditional medicine, berberine (BBR) was reported to exhibit healthspan-extending properties in some age-related diseases, such as type 2 diabetes and atherosclerosis. However, the antiaging mechanism of BBR is not completely clear. By means of hydrogen peroxide- (H2O2-) induced premature cellular senescence model, we found that a low-concentration preconditioning of BBR could resist premature senescence in human diploid fibroblasts (HDFs) measured by senescence-associated β-galactosidase (SA-β-gal), accompanied by a decrease in loss of mitochondrial membrane potential and production of intracellular reactive oxygen species (ROS). Moreover, the low-concentration preconditioning of BBR could make cells less susceptible to subsequent H2O2-induced cell cycle arrest and growth inhibition. Experimental results further showed that the low concentration of BBR could induce a slight increase of ROS and upregulate the expression level of sirtuin 1 (SIRT1), an important longevity regulator.H2O2-induced activation of checkpoint kinase 2 (Chk2) was significantly attenuated after the preconditioning of BBR. The present findings implied that the low-concentration preconditioning of BBR could have a mitohormetic effect against cellular senescencetriggered by oxidative stress in some age-related diseases through the regulation of SIRT1.


MDM2 INHIBITION


MDM2 inhibition-mediated autophagy contributes to the pro-apoptotic effect of berberine in p53-null leukemic cells

Significance: MDM2 inhibits autophagy and apoptosis in leukemic cells in a p53-independent manner. BBR induces autophagy in p53-null leukemic cells through downregulating MDM2 expression at both transcriptional and post-transcriptional levels, which may contribute to the anti-cancer effect of BBR in leukemia.


EPHRIN-B2 & VEGFR2 INHIBITOR


Background: Berberine, a plant-derived compound isolated from Coptis chinensis used in traditional Chinese medicine, has been shown to possess anti-cancer properties. However, no study has shown that berberine could target ephrin-B2, which plays a critical role in cell proliferation and migration.

Purpose: The aim of this study is to investigate the effect of berberine on cancer cell growth and migration, through the regulation of ephrin-B2 and downstream signaling molecules.

Methods: In this study, a high ephrin-B2-expressing cell membrane chromatography method was developed to investigate 48 crude extracts from traditional Chinese medicine that could act on ephrin-B2. Cell proliferative and wound-healing assays were used to study the effect of berberine on cancer cell growth and migration. The mechanism of berberine was investigated using western blot.

Results: Berberine was isolated from C. chinensis extracts and showed activity on the HEK293/ephrin-B2 cell membrane chromatography column. Berberine showed a greater inhibitory effect in high-expressing ephrin-B2 cells (HEK293/ephrin-B2 cells) than in normal HEK293 cells, and decreased the expression of ephrin-B2 and its PDZ binding proteins, which indicates that ephrin-B2 is a target of berberine.Furthermore, berberine downregulates the phosphorylation of VEGFR2and downstream signaling members (AKT and Erk1/2), which in turn downregulates the expression of MMP2 and MMP9.

Conclusion: The above data confirm the inhibitory effects of berberine on ZR-75-30 cell proliferation and cell migration. Overall, our studies demonstrate that berberine inhibits cell growth and migration by targeting ephrin-B2.

In conclusion, results presented in this work demonstrate that berberine isolated from C. chinensis can interact with ephrin-B2, decrease the expression of ephrin-B2 and its PDZ binding proteins, and downregulate the phosphorylation of VEGFR2and downstream signaling members (AKT and Erk1/2), which in turn downregulates the expression of MMP2 and MMP9. All of these contribute to the inhibition of cell growth and cell migration by berberine. These data support the development of berberine as an ephrin-B2- mediated inhibitor of breast cancer ZR-75-30 cell growth.


EPHRIN-B2 IS ENHANCED IN SCAPS


Hypoxic conditions induced upregulated HIF-1α and VEGF expression in HUVECs, whereas ephrin-B2 gene was enhanced in SCAPs. Notably, this gene plays a central role in heart morphogenesis and angiogenesis by regulating cell adhesion and cell migration. Additionally, synergistic effects between HIF-1, VEGF, and ephrin-B2 led to an increase in the endothelial tubule number, vessel length, and branching points [92].


CDK1 INHIBITOR

Alkaloids, berberine and palmatine, isolated from the extracts of Berberis lycium Royle (Berberidacea) were found to inhibit proliferation of human promyelocytic HL-60 cells by the cell cycle arrest in S phase, as described in []. The compounds were shown to activate CHEK2, and degradation of CDC25A, and the subsequent inactivation of CDK1, as indicated in []. Berberine also downregulated the cyclin D1 expression, and induced the acetylation of α-tubulin [].


Boswellic Acid

(from the fragrant gum resin of the Boswellia serrata tree)


Acetyl-11-keto-β-boswellic acid suppresses docetaxel-resistant prostate cancer cells in vitro and in vivo by blocking Akt and Stat3 signaling, thus suppressing chemoresistant stem cell-like properties

Acquired docetaxel-resistance of prostate cancer (PCa) remains a clinical obstacle due to the lack of effective therapies. Acetyl-11-keto-β-boswellic acid (AKBA) is a pentacyclic triterpenic acid isolated from the fragrant gum resin of the Boswellia serrata tree, which has shown intriguing antitumor activity against human cell lines established from PCa, colon cancer, malignant glioma, and leukemia. In this study, we examined the effects of AKBA against docetaxel-resistant PCa in vitro and in vivo as well as its anticancer mechanisms. We showed that AKBA dose-dependently inhibited cell proliferation and induced cell apoptosis in docetaxel-resistant PC3/Doc cells; its IC50 value in anti-proliferation was 17 μM.

Furthermore, AKBA dose-dependently suppressed the chemoresistant stem cell-like properties of PC3/Doc cells, evidenced by significant decrease in the ability of mammosphere formation and down-regulated expression of a number of stemness-associated genes. The activation of Akt and Stat3 signaling pathways was remarkably enhanced in PC3/Doc cells, which contributed to their chemoresistant stem-like phenotype. AKBA (10–30 μM) dose-dependently suppressed the activation of Akt and Stat3 signaling pathways in PC3/Doc cells.

In contrast, overexpression of Akt and Stat3 significantly attenuated the inhibition of AKBA on PC3/Doc cell proliferation. In docetaxel-resistant PCa homograft mice, treatment with AKBA significantly suppresses the growth of homograft RM-1/Doc, equivalent to its human PC3/Doc, but did not decrease their body weight. In summary, we demonstrate that AKBA inhibits the growth inhibition of docetaxel-resistant PCa cells in vitro and in vivo via blocking Akt and Stat3 signaling, thus suppressing their cancer stem cell-like properties.


Inhibition of Cyclooxygenase-2 and Mcl-1 Expression


Background: We have previously shown that berberine exerts its anti-inflammatory effects through inhibition of cyclooxygenase-2 (COX-2) expression. In this study, we explored the biochemical influence of berberine-induced COX-2 reduction and apoptosis.

Materials and Methods: KB cells were treated with berberine, and the apoptosis was measured by morphology and caspase-3 activity. The effects of prostaglandin E2 (PGE2) on berberine-mediated cell growth were also determined. The expression of COX-2, Bcl-2, Mcl-1, Akt and phosphorylated Akt in berberine-treated KB cells, with or without PGE2, were assessed by Western blots.

Results: Berberine induced apoptosis in KB cells, and was partially reversed by incorporation of PGE2. Berberine treatment inhibited COX-2 and Mcl-1 expression dose-dependently, but not Bcl-2. PGE2 induced COX-2 and Mcl-1 expression and reversed the repressive effect of berberine on Mcl-1. In addition, PGE2 had no effect on total Akt, but slightly reversed the phosphorylated Akt, which was decreased by berberine.

Conclusion: These results suggest that berberine-induced apoptosis might be COX-2-dependent and is related to decreased Akt phosphorylation and Mcl-1 expression.



Betulinic acid


decreases expression of bcl-2, BCLw, MCL-1 and cyclin D1 / Increases BAX

Betulinic acid (BA) is a pentacyclic triterpene found in many plant species, among others in the bark of white birch Betula alba. BA was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial and anti-inflammatory activities, and in particular to inhibit growth of cancer cells. The aim of the study was further in vitro characterization of BA anticancer activity.

In this study, we demonstrated a remarkable antiproliferative effect of BA in all tested tumor cell cultures including neuroblastoma, rabdomyosarcoma-medulloblastoma, glioma, thyroid, breast, lung and colon carcinoma, leukemia and multiple myeloma, as well as in primary cultures isolated from ovarian carcinoma, cervical carcinoma and glioblastoma multiforme. Furthermore, we have shown that BA decreased cancer cell motility and induced apoptotic cell death.

We also observed decrease of bcl2 and cyclin D1genes expression, and increase of baxgene expression after betulinic acid treatment. These findings demonstrate the anticancer potential of betulinic acid and suggest that it may be taken into account as a supportive agent in the treatment of cancers with different tissue origin.



Brevilin A

(Sesquiterpene lactone from Centipeda Minima aka “Nakchhikni”)

BCL-XL Inhibitor


To get better insight into Brevilin A-induced apoptosis in U87 glioblastoma cells, we measured the expression of bcl-2 family proteins by Western blotting analysis. The data demonstrated that Brevilin A decreased the expression of anti-apoptotic Bcl-xL protein whereas it increased the expression of pro-apoptotic Bak protein. However, no change in the expression of anti-apoptotic Bcl-2 protein and pro-apoptotic Bax protein was observed. To support our data, we further measured the expression of cytochrome c in cytosolic fractions.

The data showed that Brevilin A treatment induced release of cytochrome c from the mitochondria into the cytosol. Next, we determined mitochondrial membrane potential in U87 glioblastoma cells by using the JC-1 fluorescent probe. In healthy cells, JC-1 forms complexes known as J-aggregates, which show intense red fluorescence; however, in cells with disrupted MMP, JC-1 remains in monomeric form and manifests green fluorescence. Thus, MMP is calculated by a decrease in red/green fluorescence ratio.

The data demonstrated that Brevilin A decreased red/green fluorescence ratio (MMP) in U87 glioblastoma cells in a dose-dependent manner. These findings clearly indicate that Brevilin A induces at least, in part, mitochondrial apoptosis in U87 glioblastoma cells.


potent Jak/stat inhibitor


Signal abnormalities in human cells usually cause unexpected consequences for individual health. We focus on these kinds of events involved in JAK-STAT signal pathways, especially the ones triggered by aberrant activated STAT3, an oncoprotein which participates in essential processes of cell survival, growth and proliferation in many types of tumors, as well as immune diseases. By establishing a STAT3 signal based high-throughput drug screening system in human lung cancer A549 cells, we have screened a library from natural products which contained purified compounds from medicinal herbs. One compound, named Brevilin A, exhibited both strong STAT3 signal inhibition and STAT3 signal dependent cell growth inhibition.

Further investigations revealed that Brevilin A not only inhibits STAT3 signaling but also STAT1 signaling for cytokines induced phosphorylation of STAT3 and STAT1 as well as the expression of their target genes. In addition, we found Brevilin A could attenuate the JAKs activity by blocking the JAKs tyrosine kinase domain JH1.

The levels of cytokine induced phosphorylation of STATs and other substrates were dramatically reduced by treatment of Brevilin A. The roles of Brevilin A targeting on JAKs activity indicate that Brevilin A may not only be used as a STAT3 inhibitor but also a compound blocking other JAK-STAT hyperactivation. Thus, these findings provided a strong impetus for the development of selective JAK-STAT inhibitors and therapeutic drugs in order to improve survival of patients with hyperactivated JAKs and STATs.


Inactivates PI3K/AKT/mTOR


Brevilin A is a sesquiterpene lactone isolated from Centipeda minima and possesses inhibitory effects on proliferation of various tumor cells. In this study, Brevilin A inhibitory effect on proliferation and its molecular mechanism of action were investigated both in vivo and in vitro in colon adenocarcinoma CT26 cells. The results indicated that the inhibitory effect of Brevilin A in CT26 proliferation was dose-dependent and this effect was due to apoptosis.

Furthermore, Brevilin A increased ROS levels, decreased mitochondrial membrane potential (MMP) and induced apoptosis of CT26 cell in a dose-dependent manner. Apoptosis induced by Brevilin A was higher than that induced by adriamycin under the same dose. Cleaved-caspase-8, cleaved-caspase-9 and cleaved-caspase-3 were up-regulated after Brevilin A treatment, together with an increase of Bax protein expression, while Bcl-2 was reduced.

Further investigation revealed that Brevilin A inhibited the phosphorylation of PI3K, AKT and mTOR and promoted the expressions of autophagy-related proteins LC3-II, Beclin1 and Atg5 and consequent formation of autophagosomes, whereas 3-methyladenine (3-MA), a type III PI3K inhibitor, inhibited autophagosomes formation induced by Brevilin A. In vivoinvestigation suggested that Brevilin A significantly inhibited the growth of CT26 tumor compared to adriamycin and concurrently promoted the expressions of LC3-II and cleaved-caspase-3 in tumor tissues.

Our results demonstrated that the anti-tumor activity of Brevilin A was mainly achieved by the induction of cell apoptosis and autophagy, suggesting a promising potential as antitumor drug against colon adenocarcinoma.


Butein
Toxicodendron vernicifluum

Butein inhibits the growth of drug-resistant cancer cells (A2780cis and H1975) with modest potency. Butein induces a significant degradation of Hsp90 client proteins in A2780cis and H1975 cell lines. The biochemical and cellular studies demonstrates that butein inhibits the Hsp90 folding machinery. The result suggests that butein would be a potential therapeutic lead to overcome the drug resistance of cancer.



Taken together, these results indicate that butein could possibly sensitize to TRAIL-induced apoptosis by inhibiting the activation of NF-κB, including Rel-A. In contrast, recently important findings showed that the activation status of NF-κB is not sufficient to determine the fate of a cell with respect to TRAIL-induced apoptosis in hepatocellular carcinoma (10). Braeuer et al. (39) also reported that constitutively activated NF-κB, but not induced NF-κB, leads to TRAIL resistance by upregulation of XIAP in human cancer cells.

Therefore, the effects of NF-κB will be investigated in TRAIL resistance. In addition, suppression of NF-κB activity by butein may also be involved in the stimulation of caspase-8 activity because the NF-κB–induced products, IAP-1, IAP-2, and XIAP, are known to cooperatively block caspase-8 activity (40).


Campesterol

down-regulates Bcl-2 and Bcl-xL / up-regulates Bax, Bad and Bak

This study demonstrates that Tb extract significantly inhibited A549 cell proliferation, while this extract affected the sensitivity of normal lung fibroblast cells to a lesser extent. Treating A549 cells with Tb extract led to a cell cycle arrest at the G2/M phase and induced apoptosis of A549 cells by down-regulating Bcl-2 and Bcl-xL protein expression and up-regulating Bax, Bad and Bak expression.

Additionally, dibutyl phthalate, 􏰁-linolenic acid, phytol, campesterol, stigmasterol and 􏰂-sitosterol were the major effective bioactive compounds in Tb extract. Further studies are needed to fully elucidate the mechanisms involved in cancer cell death and to ensure that this extract is safe for human consumption.

Deregulation of the normal cellular apoptotic function is a fundamental element in the etiology of most cancers and the anti-apoptotic B cell lymphoma 2 (BCL‑2) protein family is known to play crucial role in the regulation of this function. Overexpression of this protein family has been implicated in some cancers, such that agents that could inhibit their over-activity are now being explored for anticancer drug development. A number of studies have revealed the anticancer potential of Morinda lucida-derived extracts and compounds.

In search of more inhibitors of this anti-apoptotic protein family from plant resources, 47 compounds, identified in Morinda lucida Benth (Rubiaceae) were screened for their inhibitory activities against BCL-XL, BCL-2, and MCL-1 by molecular docking using BINDSURF, while binding interactions of the top compounds were viewed with PyMOL. Druglikeness and Absorption–Distribution–Metabolism–Excretion (ADME) parameters of the top 6 compounds from docking study were evaluated using SuperPred webserver.

Results revealed that out of the 47 compounds, 2 triterpenes (ursolic acid and oleanolic acid) and 4 phytosterols (cycloartenol, campesterol, stigmasterol, and β-sitosterol) have higher binding affinities for the selected BCL-2 proteins, compared to known standard inhibitors; these compounds also fulfill oral drugability of Lipinski rule of five.

Therefore, since these Morinda lucida-derived phytosterols and triterpenes show high binding affinity toward the selected anti-apoptotic proteins and exhibited good drugability characteristics, they qualify for further study on drug development against cancers characterized by overexpression of this family of protein.



Carnosic acid

down-regulation of c-FLIP and Bcl-2

Carnosic acid is a phenolic diterpene from rosmarinus officinalis, and has multiple functions, such as anti-inflammatory, anti-viral, and anti-tumor activity. In this study, we examined whether carnosic acid could sensitize TRAIL-mediated apoptosis in human renal carcinoma Caki cells. We found that carnosic acid markedly induced TRAIL-mediated apoptosis in human renal carcinoma (Caki, ACHN, and A498), and human hepatocellular carcinoma (SK-HEP-1), and human breast carcinoma (MDA-MB-231) cells, but not normal cells (TMCK-1 and HSF). Carnosic acid induced down-regulation of c-FLIP and Bcl-2 expression at the post-translational levels, and the over-expression of c-FLIP and Bcl-2 markedly blocked carnosic acid-induced TRAIL sensitization.

Furthermore, carnosic acid induced death receptor (DR)5, Bcl-2 interacting mediator of cell death (Bim), and p53 up-regulated modulator of apoptosis (PUMA) expression at the transcriptional levels via CCAAT/enhancer-binding protein-homologous protein (CHOP). Down-regulation of CHOP expression by siRNA inhibited DR5, Bim, and PUMA expression, and attenuated carnosic acid plus TRAIL-induced apoptosis.

Taken together, our study demonstrates that carnosic acid enhances sensitization against TRAIL-mediated apoptosis through the down-regulation of c-FLIP and Bcl-2expression, and up-regulation of ER stress-mediated DR5, Bim, and PUMA expression at the transcriptional levels.


CarNosine

(beta-alanyl-L-histidine)


DELAYS Cellular Senescence


Many diverse properties of the dipeptide carnosine, which are more completely described elsewhere in this volume, stimulated the idea that carnosine may have some useful therapeutic value, particularly with regard to old age. We were greatly inspired by the pioneering work of McFarland and Holliday [1], where it was shown and later confirmed [2] that the endogenous dipeptide carno- sine (β-alanyl-L-histidine) was able not only to rejuvenate human cells in cultures, but also influence the formation of long-lived clones “affecting earlier events during serial subculture”. The work of Kantha et al. who had also shown an anti-senescence effect of carnosine in vitro [3] encouraged us to test it on small mammals in order to obtain some in vivo data.

The Senescence Accelerated Mice line (hereafter SAM) was chosen for our initial experiments because of the short lifespan of the animals and the already large amount of literature that exists about this line [4]. In SAM animals, an over-production of free radicals occurring in their tissues may cause the accelerated senescence [5-8]. The full details of our exper- iments are described elsewhere [9, 10]. These experiments revealed that carnosine at a daily dose of 100 mg/kg of body weight was able to extend the mean lifespan of the mice by 20% but had no effect on the maximum lifespan. The polypotent effects of carnosine which are described throughout this journal and the wider literature make it an ideal candidate as a so-called “geropro- tector”—an agent which may delay or prevent some conditions intrinsic with old age.


MTor Inhibitor (acts like rapamyacin mimetic)


Anti-ageing mechanisms of carnosine include inhibition of mTOR and TGFβ/Smad3 pathways, and suppressing the effects of reactive carbonyl compounds. The causes of ageing are usually regarded as multifactorial; thus effective regulation might be achieved by intervention at multiple sites. It has been suggested that the endogenous dipeptide carnosine, also available as a food supplement, possesses anti-ageing activity and may achieve its reported age-alleviating effects via a number of mechanisms.

Carnosine’s possible anti-ageing mechanisms are therefore discussed; the evidence suggests that inhibition of the mechanistic target of rapamycin and carbonyl scavenging may be involved.


Carbonyl Scavenger


The metabolic syndrome is a risk factor that increases the risk for development of renal and vascular complications. This study addresses the effects of chronic administration of the endogenous dipeptide carnosine (β‐alanyl‐L‐histidine, L‐CAR) and of its enantiomer (β‐alanyl‐D‐histidine, D‐CAR) on hyperlipidaemia, hypertension, advanced glycation end products, advanced lipoxidation end products formation and development of nephropathy in the non‐diabetic, Zucker obese rat. The Zucker rats received a daily dose of L‐CAR or D‐CAR (30 mg/kg in drinking water) for 24 weeks. Systolic blood pressure was recorded monthly.

At the end of the treatment, plasma levels of triglycerides, total cholesterol, glucose, insulin, creatinine and urinary levels of total protein, albumin and creatinine were measured. Several indices of oxidative/carbonyl stress were also measured in plasma, urine and renal tissue. We found that both L‐ and D‐CAR greatly reduced obese‐related diseases in obese Zucker rat, by significantly restraining the development of dyslipidaemia, hypertension and renal injury, as demonstrated by both urinary parameters and electron microscopy examinations of renal tissue.

Because the protective effect elicited by L‐ and D‐CAR was almost superimposable, we conclude that the pharmacological action of L‐CAR is not due to a pro‐histaminic effect (D‐CAR is not a precursor of histidine, since it is stable to peptidic hydrolysis), and prompted us to propose that some of the biological effects can be mediated by a direct carbonyl quenching mechanism.


antiglycating Agent


During the process of glycation that happens with ageing, certain aldoses or aldehyde molecules attach to proteins (or to DNA) causing cross linking, that is, abnormal protein-to- protein or protein-to-DNA bonds. This process is facilitated by carbonyl groups. These abnormal proteins then may accumulate forming AGEs (Advance Glycation End-products) that may, in turn, react with free radicals to cause chronic degenerative diseases associated with aging.

Apart from its antioxidant activities discussed above, carnosine has been found to possess significant anti-glycating properties, interfering with the glycation processes at several steps.


On the enigma of carnosine’s anti-ageing actions


Carnosine (-alanyl-L-histidine) has described as a forgotten and enigmatic dipeptide. Carnosine’s enigma is particularly exemplified by its apparent anti-ageing actions; it suppresses cultured human fibroblast senescence and delays ageing in senescence-accelerated mice and Drosophila, but the mechanisms reponsible remain uncertain. In addition to carnosine’s well-documented anti-oxidant, anti-glycating, aldehyde-scavenging and toxic metal-ion chelating properties, its ability to influence the metabolism of altered polypeptides, whose accumulation characterises the senescent phenotype, should also be considered.

When added to cultured cells, carnosine was found in a recent study to suppress phosphorylation of the translational initiation factor eIF4E resulting in decreased translation frequency of certain mRNA species. Mutations in the gene coding for eIF4E in nemtodes extend organism lifespan, hence carnosine’s anti-ageing effects may be a consequence of decreased error-protein synthesis which in turn lowers formation of protein carbonyls and increases protease availability for degradation of polypeptides altered postsynthetically.

Other studies have revealed carnosine-induced upregulation of stress protein expression and nitric oxide synthesis, both of which may stimulate proteasomal elimination of altered proteins. Some anti-convulsants can enhance nematode longevity and suppress the effects of a protein repair defect in mice, and as carnosine exerts anti-convulsant effects in rodents, it is speculated that the dipeptide may participate in the repair of protein isoaspartyl groups. These new observations only add to the enigma of carnosine’s real in vivo functions. More experimentation is clearly required.


Caloric restriction mimetic


The long-term consumption of approximately 30–50% fewer calories than the amount consumed ad libitum (calorie restriction – CR), is perhaps the only widely accepted method of extending maximum lifespan in most animals studied so far. Not surprisingly, humans are not always willing to undergo a life-long CR diet, even if this may possibly mean an extension of the currently maximum human lifespan of around 110– 120 years. CR affects several genes, molecules, hormones, and other parameters, and during the past few years, there has been an attempt to identify compounds that may have biological activities similar to those of CR. Several such compounds have been identified and have been classified as Calorie Restriction Mimetics (CRM) [33].

It has been hypothesized [34] that CR may exert some of its benefits via suppression of glycolysis, thus reducing the formation of reactive oxygen species (ROS), and reducing the formation of glycating agents such as methyglyoxal (MG) [35]. As carnosine also reduces MG and ROS it may be considered as a CRM, mimicking several other physiological actions of CR itself [36].


Use of carnosine as a natural anti-senescence drug for human beings


Carnosine is an endogenous free-radical scavenger. The latest research has indicated that apart from the function of protecting cells from oxidation-induced stress damage, carnosine appears to be able to extend the lifespan of cultured cells, rejuvenate senescent cells, inhibit the toxic effects of amyloid peptide (A beta), malondialdehyde, and hypochlorite to cells, inhibit glycosylation of proteins and protein-DNA and protein-protein cross-linking, and maintain cellular homeostasis.

Also, carnosine seems to delay the impairment of eyesight with aging, effectively preventing and treating senile cataract and other age-related diseases. Therefore, carnosine may be applied to human being as a drug against aging.


Carnosine, a protective, anti-ageing peptide


Carnosine (β-alanyl-l-histidine) has protective functions additional to anti-oxidant and free-radical scavenging roles. It extends cultured human fibroblast life-span, kills transformed cells, protects cells against aldehydes and an amyloid peptide fragment and inhibits, in vitro, protein glycation (formation of cross-links, carbonyl groups and AGEs) and DNA/protein cross-linking.

Carnosine is an aldehyde scavenger, a likely lipofuscin (age pigment) precursor and possible modulator of diabetic complications, atherosclerosis and Alzheimer’s disease.


Effect of Carnosine on Age-Induced Changes in Senescence-Accelerated Mice


The effect of carnosine on the life span and several brain biochemical characteristics in senescence-accelerated mice—prone 1 (SAMP1) was investigated. A 50% survival rate of animals treated with carnosine increased by 20% as compared to controls. Moreover, the number of animals that lived to an old age significantly increased. The effect of carnosine on life span was accompanied by a decrease in the level of ′-tiobarbituric acid reactive substances (TBARS), monoamine oxidase b (MAO b), and Na/K-ATPase activity. There was also an increase in glutamate binding to N-methyl-D-aspartate receptors.

These observations are consistent with the conclusion that carnosine increases life span and quality of life by diminishing production of lipid peroxides and reducing the influence of reactive oxygen species (ROS) on membrane proteins


Carnosine, the Protective, Anti-aging Peptide


Carnosine attenuates the development of senile features when used as a supplement to a standard diet of senescence accelerated mice (SAM). Its effect is apparent on physical and behavioral parameters and on average life span. Carnosine has a similar effect on mice of the control strain, but this is less pronounced due to the non-accelerated character of their senescence processes.


Reaction of Carnosine with Aged Proteins


Cellular aging is often associated with an increase in protein carbo- nyl groups arising from oxidation- and glycation-related phenomena and suppressed proteasome activity. These “aged” polypeptides may either be degraded by 20S proteasomes or cross-link to form structures intractable to proteolysis and inhibitory to proteasome activity. Carnosine is present at surprisingly high levels (up to 20 mM) in muscle and nervous tissues in many animals, especially long-lived species. Carnosine can delay senescence in cultured human fibroblasts and reverse the senescent phenotype, restoring a more juvenile appearance. As better antioxidants/free-radical scav- engers than carnosine do not demonstrate these antisenescent effects, addition- al properties of carnosine must contribute to its antisenescent activity.

Having shown that carnosine can react with protein carbonyls, thereby generating “carnosinylated” polypeptides using model systems, we propose that similar adducts are generated in senescent cells exposed to carnosine. Polypeptide-car- nosine adducts have been recently detected in beef products that are relatively rich in carnosine, and carnosine’s reaction with carbonyl functions generated during amino acid deamidation has also been described. Growth of cultured human fibroblasts with carnosine stimulated proteolysis of long-labeled pro- teins as the cells approached their “Hayflick limit,” consistent with the idea that carnosine ameliorates the senescence-associated proteolytic decline. We also find that carnosine suppresses induction of heme-oxygenase-1 activity following exposure of human endothelial cells to a glycated protein.

The antisenescent activity of the spin-trap agent 􏰁-phenyl-N-t-butylnitrone (PBN) towards cultured human fibroblasts resides in N-t-butyl-hydroxylamine, its hydrolysis product. As hydroxylamines are reactive towards aldehydes and ketones, the antisenescent activity of N-t-butyl-hydroxylamine and other hydroxylamines may be mediated, at least in part, by reactivity towards macromolecular carbonyls, analogous to that proposed for carnosine.


Rejuvenates senescent cells


We have examined the effects of the naturally occurring dipeptide carnosine (β-alanyl-L-histidine) on the growth, morphology, and lifespan of cultured human diploid fibroblasts. With human foreskin cells, HFF-1, and fetal lung cells, MRC-5, we have shown that carnosine at high concentrations (20-50 mM) in standard medium retards senescence and rejuvenates senescent cultures. These late-passage cultures preserve a nonsenescent morphology in the presence of carnosine, in comparison to the senescent morphology first described by Hayflick and Moorhead. Transfer of these late-passage cells in medium containing carnosine to unsupplemented normal medium results in the appearance of the senescent phenotype.

The serial subculture of cells in the presence of carnosine does not prevent the Hayflick limit to growth, although the lifespan in population doublings as well as chronological age is often increased. This effect is obscured by the normal variability of human fibroblast lifespans, which we have confirmed. Transfer of cells approaching senescence in normal medium to medium supplemented with carnosine rejuvenates the cells but the extension in lifespan is variable. Neither D -carnosine, (β-alanyl-D-histidine), homocarnosine, anserine, nor β-alanine had the same effects as carnosine on human fibroblasts. Carnosine is an antioxidant, but it is more likely that it preserves cellular integrity by its effects on protein metabolism.


We investigated the effect of casticin on apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). We found that casticin potentiated TRAIL-induced apoptosis in human colon cancer cells. Casticin downregulated cell survival proteins including Bcl-xL, Bcl-2, survivin,XIAP and cFLIP, and induced death receptor 5 (DR5), but had no effect on DR4 and decoy receptors (DcR1 or DcR2). Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and casticin. In addition, casticin induced reactive oxygen species (ROS) generation in a dose-dependent manner. Collectively, the present study showed that casticin potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and induction of DR5 mediated by ROS.


Celastrol

(from Tripterygium Wilfordii aka “Thunder God Vine”)

Anti-Senescence effects


Reactive oxygen species (ROS) production has been implicated in the promotion of cellular senescence. Celastrol, a quinone methide triterpenoid isolated from the Celastraceae family, exerts antioxidant effects and enhances autophagy in various cell types. Since autophagy serves an important role in regulating ROS, it was hypothesized that the antioxidant effect of celastrol is via enhanced autophagy, thus inhibiting cell senescence. Therefore, the present study used a Senescence β‑Galactosidase Staining kit, western blot analysis and cell cycle analysis to investigate whether celastrol alleviates angiotensin (Ang) II‑induced cellular senescence by upregulating autophagy in vascular smooth muscle cells (VSMCs).

The results demonstrated that celastrol reduced Ang II‑induced senescence of VSMCs. Ang II‑induced generation of ROS and the subsequent VSMC senescence were counteracted by pretreatment with celastrol, determined by a ROS assay kit. Celastrol significantly upregulated VSMC autophagy, which reduced intracellular ROS and the subsequent cellular senescence induced by Ang II. Furthermore, celastrol markedly suppressed activity of the mechanistic target of rapamycin signaling pathway in VSMCs. In conclusion, the present study demonstrated that celastrol counteracts VSMC senescence probably by reducing ROS production via activation of autophagy, which may hold promise for the prevention and treatment of aging‑associated cardiovascular disorders such as atherosclerosis.


HSP90 Inhibitor


The molecular chaperone heat shock protein 90 (Hsp90) is required for the stabilization and conformational maturation of various oncogenic proteins in cancer. The loading of protein kinases to Hsp90 is actively mediated by the cochaperone Cdc37. The crucial role of the Hsp90-Cdc37 complex has made it an exciting target for cancer treatment. In this study, we characterize Hsp90 and Cdc37 interaction and drug disruption using a reconstituted protein system. The GST pull-down assay and ELISA assay show that Cdc37 binds to ADP-bound/nucleotide-free Hsp90 but not ATP-bound Hsp90. Celastrol disrupts Hsp90-Cdc37 complex formation, whereas the classical Hsp90 inhibitors (e.g. geldanamycin) have no effect.

Celastrol inhibits Hsp90 ATPase activity without blocking ATP binding. Proteolytic fingerprinting indicates celastrol binds to Hsp90 C-terminal domain to protect it from trypsin digestion. These data suggest that celastrol may represent a new class of Hsp90 inhibitor by modifying Hsp90 C terminus to allosterically regulate its chaperone activity and disrupt Hsp90-Cdc37 complex.


NF-KB Inhibitor


The NF-κB pathway plays an important role in chronic inflammatory and autoimmune diseases. Recently, NF-κB has also been suggested as an important mechanism linking obesity, inflammation, and metabolic disorders. However, there is no current evidence regarding the mechanism of action of NF-κB inhibition in insulin resistance and diabetic nephropathy in type 2 diabetic animal models. We investigated the effects of the NF-κB inhibitor celastrol in db/db mice. The treatment with celastrol for 2 months significantly lowered fasting plasma glucose (FPG), HbA1C and homeostasis model assessment index (HOMA-IR) levels. Celastrol also exhibited significant decreases in body weight, kidney/body weight and adiposity. Celastrol reduced insulin resistance and lipid abnormalities and led to higher plasma adiponectin levels. Celastrol treatment also significantly mitigated lipid accumulation and oxidative stress in organs including the kidney, liver and adipose tissue. The treated group also exhibited significantly lower creatinine levels and urinary albumin excretion was markedly reduced. Celastrol treatment significantly lowered mesangial expansion and suppressed type IV collagen, PAI-1 and TGFβ1 expressions in renal tissues.

Celastrol also improved abnormal lipid metabolism, oxidative stress and proinflammatory cytokine activity in the kidney. In cultured podocytes, celastrol treatment abolished saturated fatty acid-induced proinflammatory cytokine synthesis. Taken together, celastrol treatment not only improved insulin resistance, glycemic control and oxidative stress, but also improved renal functional and structural changes through both metabolic and anti-inflammatory effects in the kidney. These results suggest that targeted therapy for NF-κB may be a useful new therapeutic approach for the management of type II diabetes and diabetic nephropathy.


Celastrol induces apoptosis and autophagy via JNK ACTIVATION


Celastrol, a triterpene from traditional Chinese medicine, has been proved to possess potent anti-tumor effect on various cancers. However, the effect of celastrol on human osteosarcoma and the underlying mechanisms remains to be elucidated. We reported here that celastrol could inhibit cell proliferation by causing G2/M phase arrest. Exposure to celastrol resulted in the activation of caspase-3, -8, and -9, indicating that celastrol induced apoptosis through both extrinsic and intrinsic pathways. Autophagy occurred in celastrol-treated cells as evidenced by formation of autophagosome and accumulation of LC3B-II. The celastrol-induced cell death was remarkably restored by the combination of autophagy and apoptosis inhibitors. Furthermore, inhibition of apoptosis enhanced autophagy while suppression of autophagy diminished apoptosis.

Celastrol also induced JNK activation and ROS generation. The JNK inhibitor significantly attenuated celastrol-triggered apoptosis and autophagy while ROS scavenger could completely reverse them. The ROS scavenger also prevented G2/M phase arrest and phosphorylation of JNK. Importantly, we found that celastrol had the similar effects on primary osteosarcoma cells. Finally, in vivo, celastrol suppressed tumor growth in the mouse xenograft model. Taken together, our results revealed that celastrol caused G2/M phase arrest, induced apoptosis and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells. Celastrol is therefore a promising candidate for development of antitumor drugs targeting osteosarcoma.


Celastrol INHIBITS IAP1, IAP2, Bcl-2, Bcl-XL, c-FLIP, and survivin via NF-KB


Celastrol, a quinone methide triterpene derived from the medicinal plant Tripterygium wilfordii, has been used to treat chronic inflammatory and autoimmune diseases, but its mechanism is not well understood. Therefore, we investigated the effects of celastrol on cellular responses activated by TNF, a potent proinflammatory cytokine. Celastrol potentiated the apoptosis induced by TNF and chemotherapeutic agents and inhibited invasion, both regulated by NF-kappaB activation. We found that TNF induced the expression of gene products involved in antiapoptosis (IAP1, IAP2, Bcl-2, Bcl-XL, c-FLIP, and survivin), proliferation (cyclin D1 and COX-2), invasion (MMP-9), and angiogenesis (VEGF) and that celastrol treatment suppressed their expression.

Because these gene products are regulated by NF-kappaB, we postulated that celastrol mediates its effects by modulating the NF-kappaB pathway. We found that celastrol suppressed both inducible and constitutive NF-kappaB activation. Celastrol was found to inhibit the TNF-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 nuclear translocation and phosphorylation, and NF-kappaB-mediated reporter gene expression. Recent studies indicate that TNF-induced IKK activation requires activation of TAK1, and we indeed found that celastrol inhibited the TAK1-induced NF-kappaB activation. Overall, our results suggest that celastrol potentiates TNF-induced apoptosis and inhibits invasion through suppression of the NF-kappaB pathway.


Celastrol, a Triterpene, Enhances TRAIL-induced Apoptosis through the Down-regulation of Cell Survival Proteins and Up-regulation of Death Receptors*


Whether celastrol, a triterpene from traditional Chinese medicine, can modulate the anticancer effects of TRAIL, the cytokine that is currently in clinical trial, was investigated. As indicated by assays that measure plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3, celastrol potentiated the TRAIL-induced apoptosis in human breast cancer cells, and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that the triterpene down-regulated the expression of cell survival proteins including cFLIP, IAP-1, Bcl-2, Bcl-xL, survivin, and XIAP and up-regulated Bax expression. In addition, we found that celastrol induced the cell surface expression of both the TRAIL receptors DR4 and DR5.

This increase in receptors was noted in a wide variety of cancer cells including breast, lung, colorectal, prostate, esophageal, and pancreatic cancer cells, and myeloid and leukemia cells. Gene silencing of the death receptor abolished the effect of celastrol on TRAIL-induced apoptosis. Induction of the death receptor by the triterpenoid was found to be p53-independent but required the induction of CAAT/enhancer-binding protein homologous protein (CHOP), inasmuch as gene silencing of CHOP abolished the induction of DR5 expression by celastrol and associated enhancement of TRAIL-induced apoptosis. We found that celastrol also induced reactive oxygen species (ROS) generation, and ROS sequestration inhibited celastrol-induced expression of CHOP and DR5, and consequent sensitization to TRAIL. Overall, our results demonstrate that celastrol can potentiate the apoptotic effects of TRAIL through down-regulation of cell survival proteins and up-regulation of death receptors via the ROS-mediated up-regulation of CHOP pathway.


CHICORIC ACID

CASPASE 3 ACTIVATOR, induces apoptosis in 3T3-L1 preadipocytes


Chicoric acid has been reported to possess various bioactivities. However, the antiobesity effects of chicoric acid remain poorly understood. In this study, we investigated the effects of chicoric acid on 3T3-L1 preadipocytes and its molecular mechanisms of apoptosis. Chicoric acid inhibited cell viability and induced apoptosis in 3T3-L1 preadipocytes which was characterized by chromatin condensation and poly ADP-ribose-polymerase (PARP) cleavage. Mitochondrial membrane potential (MMP) loss, Bax/Bcl-2 dysregulation, cytochrome c release, and caspase-3 activationwere observed, indicating mitochondria-dependent apoptosis induced by chicoric acid. Furthermore, PI3K/Akt and MAPK (p38 MAPK, JNK, and ERK1/2) signaling pathways were involved in chicoric acid-induced apoptosis. The employment of protein kinase inhibitors LY294002, SB203580, SP600125, and U0126 revealed that PI3K/Akt signaling pathway interplayed with MAPK signaling pathways.

Moreover, chicoric acid induced reactive oxygen species (ROS) generation. Pretreatment with the antioxidant N-acetylcysteine (NAC) significantly blocked cell death and changes of Akt and MAPK signalings induced by chicoric acid. In addition, chicoric acid down regulated HO-1 and COX-2 via the PI3K/Akt pathway.


chlorogenic acid


strong matrix metalloproteinase-9 inhibitor


A phenolic compound responsible for anti-MMP-9, which is known to be involved in tumor cell invasion and metastasis, has been isolated from methanol extracts prepared from stem barks of Euonymus alatus by assay-guided fractionation. The compound has been identified as 5-caffeoylquinic acid (chlorogenic acid; CHA) by NMR and FAB-MS. CHA showed a strong inhibitory effect of matrix metalloproteinase (MMP)-9 activityin a concentration-dependent manner on zymography.

The purified CHA inhibited MMP-9 activity with the IC50 of 30–50 nM. Furthermore, the cytotoxic survival curve showed that CHA does not have cytotoxic effects on cellular proliferation, when Hep3B cells were treated with various concentrations of CHA and cell viability was measured using the XTT assay. The present data suggest a clue for possible mechanisms of cancer chemoprevention by CHA and other naturally occurring phenolic compounds. The results also imply that useful cancer chemopreventive agents can be further identified by combinations of in vitro (as a first screen) and in vivo studies.


HMGB1 Inhibitor


Chlorogenic Acid Attenuates High Mobility Group Box 1 (HMGB1) Sepsis is a complex, multifactorial, rapidly progressive disease characterized by an overwhelming activation of the immune system and the countervailing antiinflammatory response. In the current study in murine peritoneal macrophages, chlorogenic acid suppressed endotoxin-induced high mobility group box 1 (HMGB1) release in a concentration-dependent manner. Administration of chlorogenic acid also attenuated systemic HMGB1 accumulation in vivo and prevented mortality induced by endotoxemia and polymicrobial sepsis.

The mechanisms of action of chlorogenic acid included attenuation of the increase in toll-like receptor (TLR)-4 expression and suppression of sepsis-induced signaling pathways, such as c-Jun NH2-terminal kinase (JNK), p38 mitogenactivated protein kinase (MAPK) and nuclear factor (NF)-κB, which are critical for cytokine release. The protection conferred by chlorogenic acid was achieved through modulation of cytokine and chemokine release, suppression of immune cell apoptosis and augmentation of bacterial elimination. Chlorogenic acid warrants further evaluation as a potential therapeutic agent for the treatment of sepsis and other potentially fatal systemic inflammatory disorders.


Chlorophyll a (Ludwigia octovalvis)


activates the CD95 (APO-1/CD95) system and AMPK pathway in 3T3-L1 cells


Ludwigia octovalvis is an aquatic plant widely distributed in Taiwan. It is traditionally used as a diuretic and is consumed as health drink. In this study, we evaluated the anti-proliferative activity of extracts and active constituent (chlorophyll a; CHL-a) of L. octovalvis in 3T3-L1 adipocytes; its mode of action on apoptosis was also investigated. Results showed that, among the different extracts and fractions, the ethylacetate layer (EAL) possessed the most potent anti-proliferative activity. Activity guided fractionation of the EAL obtained the bioactive constituent CHL-a(IC50: 24.10 ± 0.83 nM). At concentrations 5–30 nM, CHL-a exhibited a dose-dependent accumulation of the Sub-G1 peak and caused cell cycle arrest at the G0/G1 phase. At 30 nM, it significantly reduced the cell viability, induced the appearance of DNA fragments, and enhanced the activation of caspase-3.

Western blot data revealed that CHL-a decreased the level of Bcl-2, and increased the expression of CD95 (APO-1/CD95) and Bax. Furthermore, CHL-a up-regulated the AMPK and p-AMPK levels, and down-regulated the expression of PPAR-γ. These results conclude that CHL-a possesses potent anti-proliferative activity, and its apoptotic effects on 3T3-L1 adipocytes are mediated through the activation of CD95 (APO-1/CD95) system and the AMPK signaling pathway.



Cianidanol


More than 23% of today’s population suffers from age-associated diseases such as arthritis, cancer, heart disease, and more. The ongoing economic impact of these diseases has been in the billions of dollars worldwide with no clear solution to date. This study addresses the underlying cause of these diseases by identifying the compounds that potentially eliminate senescent cells. Existing senolytic drugs are not abundantly found in nature, reducing accessibility. Hence, over 70,000 natural compounds available in the Canadian Food Database were used to screen penitential senolytic compounds that block PI3Kγ, reactivating apoptotic processes in senescent cells.
Molecular docking results revealed 23 natural compounds that blocks the PI3Kγ. Out of 23 compounds, Cianidanol, Ellagic acid, Eriodictyol, Kaempferol and Cyanidin were found abundantly in food sources range from 85 to 735 mg/100 g. These compounds are up to 46 times more abundant in foods than proven senolytic drug Fisetin. Further, molecular dynamics results showed ligand stability for 4 nanoseconds with PI3Kγ. The five compounds are proven to eliminate cancerous cells, have the potential to prevent age-related diseases, and could even slow down natural aging.

Costunolide induces apoptosis in platinum-resistant human ovarian cancer cells by generating reactive oxygen species

… production and apoptosis induced by costunolide in the platinum-resistant cancer cells as shown in Fig. 3D. Importantly, costunolide significantly inhibited the expression of Bcl2 (Fig. 4A). Bcl2 is unique among cellular genes because of its ability to block apoptotic cell death.

Curcumin

(from Curcuma longa aka Turmeric Root)

Senolytic activity


Curcumin and o-Vanillin cleared senescent intervertebral disc (IVD) cells and reduced the senescence-associated secretory phenotype (SASP) associated with inflammation and back pain. Cells from degenerate and non-mildly-degenerate human IVD were obtained from organ donors and from patients undergoing surgery for low back pain. Gene expression of senescence and SASP markers was evaluated by RT-qPCR in isolated cells, and protein expression of senescence, proliferation, and apoptotic markers was evaluated by immunocytochemistry (ICC). The expression levels of SASP factors were evaluated by enzyme-linked immunosorbent assay (ELISA). Matrix synthesis was verified with safranin-O staining and the Dimethyl-Methylene Blue Assay for proteoglycan content. Western blotting and ICC were used to determine the molecular pathways targeted by the drugs.

We found a 40% higher level of senescent cells in degenerate compared to non-mildly-degenerate discs from unrelated individuals and a 10% higher level in degenerate compared to non-mildly-degenerate discs from the same individual. Higher levels of senescence were associated with increased SASP. Both drugs cleared senescent cells, and treatment increased the number of proliferating as well as apoptotic cells in cultures from degenerate IVDs. The expression of SASP factors was decreased, and matrix synthesis increased following treatment. These effects were mediated through the Nrf2 and NFkB pathways.


Protective effect of curcumin against d-galactose-induced senescence in mice


Brain senescence plays an important role in cognitive dysfunction and neurodegenerative disorders. Curcumin was reported to have beneficial effect against several neurodegenerative disorders including Alzheimer’s disease. Therefore, the present study was conducted in order to explore the possible role of curcumin against d-galactose-induced cognitive dysfunction, oxidative damage, and mitochondrial dysfunction in mice. Chronic administration of d-galactose for 6 weeks significantly impaired cognitive function (both in Morris water maze and elevated plus maze), locomotor activity, oxidative defense (raised lipid peroxidation, nitrite concentration, depletion of reduced glutathione and catalase activity), and mitochondrial enzyme complex activities (I, II, and III) as compared to vehicle treated group.

Curcumin (15 and 30 mg/kg) and galantamine (5 mg/kg) treatment for 6 weeks significantly improved cognitive tasks, locomotor activity, oxidative defense, and restored mitochondrial enzyme complex activity as compared to control (d-galactose). Chronic d-galactose treatment also significantly increased acetylcholine esterase activity that was attenuated by curcumin (15 and 30 mg/kg) and galantamine (5 mg/kg) treatment. In conclusion, the present study highlights the therapeutic potential of curcumin against d-galactose induced senescence in mice.


Curcumin prevents mitochondrial dysfunction in brain of senescence-accelerated mouse


The aging brain suffers mitochondrial dysfunction and a reduced availability of energy in the form of ATP, which in turn may cause or promote the decline in cognitive, sensory, and motor function observed with advancing age. There is a need for animal models that display some of the pathological features of human brain aging in order to study their prevention by e.g. dietary factors. We thus investigated the suitability of the fast-aging senescence-accelerated mouse-prone 8 (SAMP8) strain and its normally aging control senescence-accelerated mouse-resistant 1 (SAMR1) as a model for the age-dependent changes in mitochondrial function in the brain. To this end, 2-months old male SAMR1 (n = 10) and SAMP8 mice (n = 7) were fed a Western type diet (control groups) for 5 months and one group of SAMP8 mice (n = 6) was fed an identical diet fortified with 500 mg curcumin per kg. Dissociated brain cells and brain tissue homogenates were analyzed for malondialdehyde, heme oxygenase-1 mRNA, mitochondrial membrane potential (MMP), ATP concentrations, protein levels of mitochondrial marker proteins for mitochondrial membranes (TIMM, TOMM), the mitochondrial permeability transition pore (ANT1, VDAC1, TSPO), respiration complexes, and fission and fusion (Fis, Opa1, Mfn1, Drp1). Dissociated brain cells isolated from SAMP8 mice showed significantly reduced MMP and ATP levels, probably due to significantly diminished complex V protein expression, and increased expression of TSPO. Fission and fusion marker proteins indicate enhanced mitochondrial fission in brains of SAMP8 mice.

Treatment of SAMP8 mice with curcumin improved MMP and ATP and restored mitochondrial fusion, probably by up-regulating nuclear factor PGC1α protein expression. In conclusion, SAMP8 compared to SAMR1 mice are a suitable model to study age-dependent changes in mitochondrial function and curcumin emerges as a promising nutraceutical for the prevention of neurodegenerative diseases that are accompanied or caused by mitochondrial dysfunction. Highlights ► Mitochondrial dysfunction and reduced energy production occur in the aging brain. ► We studied if SAMP8 vs. SAMR1 mice can be used as a model for mitochondrial function. ► Mitochondrial function was reduced and fission enhanced in SAMP8 vs. SAMR1 mice. ► Curcumin-intake restored mitochondrial function & fusion, perhaps by activating PGC1α. ► Age-dependent changes in brain mitochondrial function can be prevented by curcumin.

The Role of Curcumin in the Modulation of Ageing


It is believed that postponing aging is more effective and less expensive than the treatment of particular age-related diseases. Compounds which could delay symptoms of ageing, especially natural products present in a daily diet, are intensively studied. One of them is curcumin. It causes the elongation of the lifespan of model organisms, alleviates ageing symptoms and postpones the progression of age-related diseases in which cellular senescence is directly involved. It has been demonstrated that the elimination of senescent cells significantly improves the quality of life of mice. There is a continuous search for compounds, named senolytic drugs, that selectively eliminate senescent cells from organisms. In this paper, we endeavor to review the current knowledge about the anti-aging role of curcumin and discuss its senolytic potential.



Although treatment of Hodgkin’s lymphoma (HL) with a multi‐drug approach has been very successful, its toxicity becomes evident after several years as secondary malignancies and cardiovascular disease. Therefore, the current goal in HL treatment is to find new therapies that specifically target the deregulated signaling cascades, such as NF‐κB and STAT3, which cause Hodgkin and Reed‐Sternberg (H‐RS) cell proliferation and resistance of apoptosis. Based on the above information, we investigated the capacity of curcumin to inhibit NF‐κB and STAT3 in H‐RS cells, characterizing the functional consequences.

Curcumin is incorporated into H‐RS cells and acts inhibiting both NF‐κB and STAT3 activation, leading to a decreased expression of proteins involved in cell proliferation and apoptosis,e.g. Bcl‐2, Bcl‐xL, cFLIP, XIAP, c‐IAP1, survivin, c‐myc and cyclin D1. Interestingly, curcumin caused cell cycle arrest in G2‐M and a significant reduction (80–97%) in H‐RS cell viability. Furthermore, curcumin triggered cell death by apoptosis, as evidenced by the activation of caspase‐3 and caspase‐9, changes in nuclear morphology and phosphatidylserine translocation. The above findings provide a mechanistic rationale for the potential use of curcumin as a therapeutic agent for patients with HL.


INHIBITS Bcl-2 & BCL-XL


Curcumin, a natural, biologically active compound extracted from rhizomes of Curcuma species, has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. The mechanism by which curcumin initiates apoptosis remains poorly understood. In the present report we investigated the effect of curcumin on the activation of the apoptotic pathway in human renal Caki cells. Treatment of Caki cells with 50 microM curcumin resulted in the activation of caspase 3, cleavage of phospholipase C-gamma1 and DNA fragmentation. Curcumin-induced apoptosis is mediated through the activation of caspase, which is specifically inhibited by the caspase inhibitor, benzyloxycarbony-Val-Ala-Asp-fluoromethyl ketone.

Curcumin causes dose-dependent apoptosis and DNA fragmentation of Caki cells, which is preceded by the sequential dephosphorylation of Akt, down-regulation of the anti-apoptotic Bcl-2, Bcl-XL and IAP proteins, release of cytochrome c and activation of caspase 3. Cyclosporin A, as well as caspase inhibitor, specifically inhibit curcumin-induced apoptosis in Caki cells. Pre-treatment with N-acetyl-cysteine, markedly prevented dephosphorylation of Akt, and cytochrome c release, and cell death, suggesting a role for reactive oxygen species in this process. The data indicate that curcumin can cause cell damage by inactivating the Akt-related cell survival pathway and release of cytochrome c, providing a new mechanism for curcumin-induced cytotoxicity.


Curcumin induces cell-arrest and apoptosis in association with the inhibition of constitutively active NF-kappaB and STAT3 pathways in Hodgkin’s lymphoma cells.


Although treatment of Hodgkin’s lymphoma (HL) with a multi-drug approach has been very successful, its toxicity becomes evident after several years as secondary malignancies and cardiovascular disease. Therefore, the current goal in HL treatment is to find new therapies that specifically target the deregulated signaling cascades, such as NF-kappaB and STAT3, which cause Hodgkin and Reed-Sternberg (H-RS) cell proliferation and resistance of apoptosis. Based on the above information, we investigated the capacity of curcumin to inhibit NF-kappaB and STAT3 in H-RS cells, characterizing the functional consequences.

Curcumin is incorporated into H-RS cells and acts inhibiting both NF-kappaB and STAT3 activation, leading to a decreased expression of proteins involved in cell proliferation and apoptosis, e.g. Bcl-2, Bcl-xL, cFLIP, XIAP, c-IAP1, survivin, c-myc and cyclin D1. Interestingly, curcumin caused cell cycle arrest in G2-M and a significant reduction (80-97%) in H-RS cell viability. Furthermore, curcumin triggered cell death by apoptosis, as evidenced by the activation of caspase-3 and caspase-9, changes in nuclear morphology and phosphatidylserine translocation. The above findings provide a mechanistic rationale for the potential use of curcumin as a therapeutic agent for patients with HL.


MDM2 INHIBITOR

Curcumin, a Dietary Component, Has Anticancer, Chemosensitization, and Radiosensitization Effects by Down-regulating the MDM2Oncogene through the PI3K/mTOR/ETS2 Pathway

The oncoprotein MDM2, a major ubiquitin E3 ligase of tumor suppressor p53, has been suggested as a novel target for human cancer therapy based on its p53-dependent and p53- independent activities. We have identified curcumin, which has previously been shown to have anticancer activity, as an inhibitor of MDM2 expression.


Cucurbitacin D


Disruptor of the HSP90 Chaperone Machinery


Heat shock protein 90 (Hsp90) facilitates the maturation of many newly synthesized and unfolded proteins (clients) via the Hsp90 chaperone cycle, in which Hsp90 forms a heteroprotein complex and relies upon cochaperones, immunophilins, etc., for assistance in client folding. Hsp90 inhibition has emerged as a strategy for anticancer therapies due to the involvement of clients in many oncogenic pathways. Inhibition of chaperone function results in client ubiquitinylation and degradation via the proteasome, ultimately leading to tumor digression. Small molecule inhibitors perturb ATPase activity at the N-terminus and include derivatives of the natural product geldanamycin.

However, N-terminal inhibition also leads to induction of the pro-survival heat shock response (HSR), in which displacement of the Hsp90-bound transcription factor, heat shock factor-1, translocates to the nucleus and induces transcription of heat shock proteins, including Hsp90. An alternative strategy for Hsp90 inhibition is disruption of the Hsp90 heteroprotein complex. Disruption of the Hsp90 heteroprotein complex is an effective strategy to prevent client maturation without induction of the HSR. Cucurbitacin D, isolated from Cucurbita texana, and 3-epi-isocucurbitacin D prevented client maturation without induction of the HSR. Cucurbitacin D also disrupted interactions between Hsp90 and two cochaperones, Cdc37 and p23.


Cycloastragenol

STAT3 INHIBITOR

Cycloastragenol can negate constitutive STAT3 activation and promote paclitaxel-induced apoptosis in human gastric cancer cells

We observed that CAG exhibited cytotoxic activity against SNU-1 and SNU-16 cells to a greater extent as compared to normal GES-1 cells. CAG predominantly caused negative regulation of STAT3 phosphorylation at tyrosine 705 through the abrogation of Src and Janus-activated kinases (JAK1/2) activation. We noted that CAG impaired translocation of STAT3protein as well as its DNA binding activity. It further decreased cellular proliferation and mediated its anticancer effects predominantly by causing substantial apoptosis rather than autophagy. In addition, CAG potentiated paclitaxel-induced anti-oncogenic effects in gastric tumor cells.

Conclusions: Our results indicate that CAG can function to impede STAT3 activation in human gastric tumor cells and therefore it may be a suitable candidate agent for therapy of gastric cancer.


Telomerase activation and lengthening of telomeres

Cycloastragenol (CAG) is the aglycone derivative of astragaloside IV which has recently been demonstrated to activate telomerase and represents a potential drug candidate for the treatment of degenerative diseases. In the present study, intestinal absorption and metabolism of CAG were examined using the Caco-2 model and liver microsomes, respectively. The results showed that CAG could rapidly pass through the Caco-2 cell monolayer by passive diffusion. Four different glucuronide conjugates and two oxidized CAG metabolites were found in the apical and basolateral side of Caco-2 monolayer, suggesting that first-pass intestinal metabolism of CAG might occur upon passage through the intestinal epithelium.

CAG underwent extensive metabolism in rat and human liver microsomes with only 17.4% and 8.2%, respectively, of the starting amount of CAG remaining after 30 min of incubation. Monohydroxylation of the parent and oxidization of the hydroxylated CAG were found in the liver samples. The present study indicated that CAG can be efficiently absorbed through intestinal epithelium. However, extensive first-pass hepatic metabolism would limit the oral bioavailability of this compound.


ANTI-AGING EFFECTS

Cycloastragenol (CAG) is a triterpenoid saponin compound and a hydrolysis product of the main active ingredient in Astragalus membranaceus (Fisch.) Bunge. An increasing body of evidence has indicated that CAG has a wide spectrum of pharmacological functions, which are attracting attention in the research community. The aim of the present review paper was to review and elucidate the advanced study of CAG. The focus was on advanced studies of CAG in English and Chinese databases; the literature was collected and reviewed to summarize the latest efficacy, pharmacokinetics and adverse reactions of CAG. Extensive pharmacological effects have been attributed to CAG, including telomerase activation, telomere elongation, anti‑inflammatory and anti‑oxidative properties; CAG has also been reported to improve lipid metabolism.

Clinical research has demonstrated that CAG activates telomerase in humans and ameliorates various biomarkers. CAG is absorbed through the intestinal epithelium via passive diffusion and undergoes first‑pass hepatic metabolism. Within a certain dose range, oral CAG is relatively safe; however, underlying mechanisms associated with CAG are not clear, and thus, we should be aware of potential adverse reactions associated with CAG. According to existing studies and clinical trials, CAG is safe and has broad application prospects.


daidzein


BCL-2/BAX INHIBITOR


Daidzein induces apoptosis via the extrinsic receptor-mediated pathway, intrinsic mitochondrial pathway or endoplasmic reticulum stress pathway, depending on the type of tumor (21). For example, daidzein induces tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in prostate cancer cells (41), however activates the mitochondria-mediated pathway in breast cancer, gastric carcinoma and hepatic cancer (2224). Vilela et al (42) reported that bio-transformed soybean extract containing daidzein increases expression of TRAIL and its receptor DR4 in melanoma, resulting in cell apoptosis. Equol, which is a metabolite of daidzein, induces apoptosis in SMMC-7721 human hepatocellular carcinoma cells through the intrinsic and endoplasmic reticulum stress pathways (26). Caspase-9 and the Bcl-2/Bax ratio are commonly used activity markers of the mitochondrial apoptotic pathway (4345).

In the present study, western blotting was used to detect these markers, revealing that the cleavage of caspase-9 increased and the ratio of Bcl-2/Bax decreased in both cell lines in a dose-dependent manner, which indicated that daidzein-induced apoptosis was mediated via the mitochondrial apoptotic pathway. This process is similar to that in the human gastric carcinoma cell line BGC-823, as Tang et al (23) demonstrated that daidzein induces apoptosis via downregulation of Bcl-2/Bax and triggering of the mitochondrial pathway.


downregulates Bcl-2, Bcl-x and Baid proteins and upregulates Bim


In the present study, the in vitro cytotoxicity of daidzein was evaluated in human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines. BEL-7402 cells were sensitive to daidzein treatment, with an IC50 value of 59.7±8.1 µM. Daidzein showed no cytotoxic activity toward A549, HeLa, HepG-2 and MG-63 cells. Daidzein increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Morphological and comet assays showed that daidzein effectively induced apoptosis in BEL-7402 cells. Additionally, daidzein caused cell cycle arrest at the G2/M phase in the BEL-7402 cell line.

Daidzein downregulated the expression of Bcl-2, Bcl-x and Baid proteins and upregulated the levels of Bim protein in the BEL-7402 cells. The results demonstrated that daidzein induced BEL-7402 cell apoptosis through an ROS-mediated mitochondrial dysfunction pathway.


Decursin

Downregulates STAT3, BCL-2, BCL-XL, SURVIVIN & VEGF


Recent reports have indicated that decursin can induce apoptosis, suppress tumor growth, and inhibit angiogenesis. In this experiment, we investigated how decursin could potentiate the cytotoxic effects of bortezomib in human multiple myeloma cells. We found that decursin inhibited cell viability in U266, MM.1S and ARH77 cells, but not in peripheral blood mononuclear cells (PBMC). Decursin-induced apoptosis through the activation of caspase-8, -9, and -3 in U266 cells. This correlated with the down-regulating of cyclin D1, bcl-2, bcl-xL, survivin, and the vascular endothelial growth factor (VEGF), which are all regulated by the activation of signal transducers and the activator of transcription 3 (STAT3). Indeed, decursin inhibited constitutive STAT3 activation through inhibition of the activation of Janus-activated kinase 2 (JAK2) in U266 cells.

In addition, decursin inhibited interleukin-6-inducible STAT3 activation in a time-dependent manner in MM.1S cells. Interestingly, decursin significantly potentiated the apoptotic effects of bortezomib in U266 cells. These effects of decursin were correlated with the suppression of constitutive STAT3 activation in U266 cells. Overall, these results suggest that decursin is a novel blocker of STAT3 activation and it may be a potential candidate for overcoming chemo-resistance through suppression of this signaling.


Delphinidin

Downregulates BCL-2 and BCL-XL

Solar UV radiation, in particular its UVB component, is the primary cause of many adverse biological effects, the most damaging of which is skin cancer. Here, we assessed the photochemopreventive effect of delphinidin, a major anthocyanidin present in many pigmented fruits and vegetables, on UVB-mediated responses in human immortalized HaCaT keratinocytes and SKH-1 hairless mouse skin. We found that pretreatment of cells with delphinidin (1-20 microM for 24 hours) protected against UVB (15-30 mJ/cm2, 24 hours)-mediated (i) decrease in cell viability and (ii) induction of apoptosis. Furthermore, we found that pretreatment of HaCaT cells with delphinidin inhibited UVB-mediated (i) increase in lipid peroxidation; (ii) formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG); (iii) decrease in proliferating cell nuclear antigen expression; (iv) increase in poly(ADP-ribose) polymerase cleavage; (v) activation of caspases; (vi) increase in Bax;(vii) decrease in Bcl-2; (viii) upregulation of Bid and Bak; and (ix) downregulation of Bcl-xL.

Topical application of delphinidin (1 mg/0.1 ml DMSO/mouse) to SKH-1 hairless mouse skin inhibited UVB-mediated apoptosis and markers of DNA damage such as cyclobutane pyrimidine dimers and 8-OHdG. Taken together our results suggest that treatment of HaCaT cells and mouse skin with delphinidin inhibited UVB-mediated oxidative stress and reduced DNA damage, thereby protecting the cells from UVB-induced apoptosis.


Induces ROS accumulation; suppresses EGFR; damages DNA; downregulates HIF-1, p27, PI3K/Akt/mTOR, and NFƙB; induces caspase-3, -8, and -9


Delphinidin is one of the main compounds in blueberries, and an anticancer effect of delphinidin on a human colon cancer cell line (colo205) has been reported . The inhibitory effect of delphinidin on LoVo/ADR cell lines was also investigated. The data revealed that delphinidin inhibited metastatic CRC and that this may have been due to cellular ROS accumulation . Further investigation of delphinidin indicated that it inhibited HT-29 human tumor cells through the suppression of EGFR .

A recent study on delphinidin claimed that it had antioxidant activity against human CRC HTC-116 and HT-29 cells and could also induce DNA damage . In addition, delphinidin potently inhibited HTC-116 and HT-29 cell lines through the downregulation of HIF-1 and p27 by affecting the PI3K/Akt/mTOR signaling pathway . HCT-116 cells treated with delphinidin suppressed the NF-kappa B pathway and activated the expression of caspase-3, -8 and -9, resulting in cell cycle arrest in the G2/M phase, thereby leading to apoptosis .

Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity

SENOLYTIC EFFECT

Digoxin is senolyticin lung fibrosis. Next, we wanted to test the senolytic effect of Digoxin using another in vivo model of a non- tumor related disease. For this, we established a mouse model of lung fibrosis induced by intratracheal administration of senescent human cells. Normal proliferating or gamma-irradiated (g-IR) senescent human fibroblasts IMR90 were delivered into the lungs of immunodeficient mice. Cell death analysis of in vitro treatment with Digoxin of proliferating and g-IR IMR90 confirmed once again the senolytic effect of the CG . Three weeks after intratracheal instillation, these animals were subjected to Digoxin or vehicle treatment for ten days and their lungs were removed and analyzed. First, we measured the expression levels of CDKN2A (coding for p16INK4a) a typical marker of primary fibroblast senescence42. Lungs from animals injected with g-IR IMR90 showed high levels of this human gene but not of the mouse ortholog, Cdkn2a, indicative of the presence of senescent human cells in the mouse lungs . Similarly, human CDKN1A (coding for p21) was also detected in the lungs of mice receiving senescent human cells . Treatment with Digoxin caused a significant reduction in CDKN2A expression levels and of CDKN1A (although not reaching statistical significance for this gene), suggesting that Digoxin caused the clearance of the human senescent cells. Finally, we stained the lungs with Masson Trichrome, a well-established marker of fibrosis.

Lungs from animals injected with senescent g-IR IMR90 and treated with vehicle stained positive, indicative of fibrosis, while those treated with Digoxin had a reduced score of fibrosis. In addition, we determined hydroxyproline content in lungs as a measure of fibrosis and found that animals receiving senescent g-IR IMR90 cells had a significantly higher level of this marker compared to the ones injected with proliferating cells (Supple- mentary Fig. 5b). In line with our results using Masson Tri- chrome staining, mice treated with Digoxin showed a tendency to have reduced levels of hydroxyproline.

These results show the potential senolytic effect exerted by Digoxin in vivo using a model of a disease, other than cancer, caused by the accumulation of senescent cells.


Diosmin

Downregulates BCL-2


Reperfusion of ischemic tissue leads to the generation of oxygen derived free radicals which plays an important role in cellular damage. Objective of the current study is to evaluate the cardio-protective and antioxidant effect of diosmin on ischemia–reperfusion related cardiac dysfunction, oxidative stress and apoptosis. Diosmin (50 and 100 mg/kg body weight (bw)) was given every day to the rats orally throughout the experimental period. Ischemia/reperfusion protocol was carried out ex vivo using langendorff perfusion method and the cardiac functional recovery was assessed in terms of percentage rate pressure product. Coronary effluents of LDH and CK-MB activities, antioxidant enzyme activities, lipid peroxidation products, activity of TCA cycle enzymes were evaluated.

Moreover, in vitro superoxide anionand hydroxyl radical scavenging potential of diosmin was also quantified. Finally, quantitative real-time PCRwas used for assessing Bcl-2 mRNA expression in heart. Cardiac functional recovery was impaired after reperfusion compared with continuously perfused heart. It was significantly prevented by diosmin treatment. Impaired antioxidant enzyme activities and elevated lipid peroxidation products level were also significantly suppressed. The activity of TCA cycle enzymes was protected against reperfusion stress. Down regulated Bcl-2 was also significantly increased.This study concluded that diosmin pretreatment prevents all the impaired patterns including cardiac function, oxidative stress and apoptosis associated with reperfusion in control heart by its antioxidant role.


IL-6 / STAT3 Inhibitor


Diosmin is naturally found flavanoid in many citrus fruits known to have anti-inflammatory, antihyperglycemic, antioxidant and antimutagenic properties. Effects of Diosmin on IL-6/STAT-3 expression in hamster buccal pouch carcinogenesisremain unclear. Alterations in many genes encode crucial proteins, which regulate cell proliferation, differentiation and apoptosis have been implicated in oral cancer. In the present study, we investigated the effect of dietary Diosmin on IL-6/STAT-3 signaling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis by examining the protein expression of IL-6/STAT-3 and its related genes. Immunoblotting and immunohistochemical analyses revealed that Diosmin (100 mg/kg b.w) supplement inhibits key events in signaling especially STAT-3 phosphorylation and subsequent nuclear translocation.

Results revealed that inhibition of proliferation and angiogenesis is associated with regulation of the STAT-3 pathway; where Diosmin prevents phosphorylation of JAK-1 which was ascend by IL-6, thereby inhibiting STAT-3 phosphorylation. Consequently, an imbalance in the Bax/Bcl-2 ratio triggered the caspase cascade in favor of apoptosis. Transmission electron microscopic studies proved the effect of Diosmin on ultrastructural changes. Finally our results provide significant evidence that Diosmin prevents the development and progression of HBP carcinomas through the inhibition of IL-6/STAT-3 signaling and its downstream events. Thus, Diosmin functions as a potent inhibitor of tumor development and progression by targeting IL-6/STAT-3 signaling may be an ideal candidate for cancer chemoprevention.


 

 

 

 

 

 

 

 

 

 

 

DX-9386

(a traditional Chinese formula: ginseng extract, acorus extract, polygala extract and hoelen extract)

Anti-aging effect of DX-9386 in senescence accelerated mouse.


The effects of DX-9386, a traditional Chinese prescription (ginseng, acorus, polygala and hoelen) were studied on life span, the degree of senescence, motor activity and the antibody production response in senescence accelerated mouse (SAM). DX-9386-containing food was given to SAM for 13 consecutive months from 2 months of age. DX-9386 significantly prolonged the life spanof SAM, prevented body weight decrease with aging and tended to improve the senile syndrome.

The motor activity of SAMP8 was higher than that of SAMR1, and DX-9386 tended to increase the activity in SAMP8. The in vivo antibody production was markedly decreased in SAMP8 and DX-9386 showed no ameliorating effect on that. These results suggest that DX-9386 has anti-aging impact.


Ameliorative effects of chronic treatment using DX-9386, a traditional Chinese prescription, on learning performance and lipid peroxide content in senescence accelerated mouse.


DX-9386 is a traditional Chinese prescription consisting of ginseng, polygala, acorus and hoelen. The effect of chronic oral treatment with this preparation on learning behaviors and lipid peroxide concentration was studied in senescence accelerated mouse (SAM). SAM P8, a senescence prone of SAM, and SAM R1, a senescence resistant substrain of SAM, were started on a diet containing 1% of DX-9386 from the age of 2 months. All the experiments were performed at the age of 10 months. The prescription ameliorated the memory disorders of SAM P8, as evaluated in a step down test as well as a spatial memory test. The preparation, however, did not affect the learning behaviors in SAM R1. DX-9386 reduced the elevated levels of lipid peroxide in the serum and liver of SAM P8, while it did not alter that in SAM R1. These results suggested that DX-9386 slowed the aging process of SAM P8 in terms of learning behaviors and lipid peroxidation.


Ingredients of a Chinese Prescription DX-9386, Individually Promote Hippocampal Long-Term Potentiation in Vivo


DX-9386 is a traditional Chinese medicinal prescription consisting of ginseng (Panax Ginseng C. A. MEYER), polygala (Polygala Tenuifolia WILLDENEW), acorus (Acorus Gramineus SOLAND) and hoelen (Poria Cocos WOLF). We recently found that oral administration of the prescription at a dose of 500 mg/kg intensified the formation of long-term potentiation (LTP) in the dentate gyrus of anesthetized rats. To evaluate the individual contribution of separate ingredients in DX-9386 towards the observed biological activity, we investigated their direct influence upon LTP formation in vivo. A single oral administration of hoelen and ginseng (250 and 500 mg/kg) significantly increased the spike amplitude evoked by a subthreshold tetanic stimulation at time intervals up to 30 min after tetanus. Only minor effects of polygala (500 mg/kg) and no influence of acorus up to 500 mg/kg were observed. No drugs affected the basal spike amplitude induced by a test stimulus. In addition, we ascertained that DX-9386 was also active at a dose of 250 mg/kg. Taken together, these results indicate that hoelen and ginseng are the active components of DX-9386 with regard to the enhancement of hippocampal LTP.



(Commonly known as “Sweet Flag”)


Phenolic constituents from the rhizomes of Acorus gramineus and their biological evaluation on antitumor and anti-inflammatory activities


On the search for anti-cancer compounds from natural Korean medicinal sources, a bioassay-guided fractionation and chemical investigation of the MeOH extract from the rhizomes of Acorus gramineus resulted in the isolation and identification of thirteen phenolic derivatives (113) including two new 8-O-4′-neolignans, named surinamensinols A (1) and B (2) and a new phenolic compound, named acoramol (9). The structures of these new compounds were elucidated on the basis of 1D and 2D NMR spectroscopic data analyses as well as circular dichroism (CD) spectroscopy studies.

The cytotoxic activities of the isolates (113) were evaluated by determining their inhibitory effects on human tumor cell lines. The new 8-O-4′-neolignans, compounds 1 and 2, showed moderate antiproliferative activities against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines with IC50 values in the range of 4.17–26.18 μM. On the basis of the expanded understanding that inflammation is a crucial cause of tumor progression, anti-inflammatory activities of these compounds were determined by measuring nitric oxide (NO) levels in the medium using murine microglia BV-2 cells. Compounds 1, 2, 4, 7 and 10 inhibited NO production in BV-2 stimulated by lipopolysaccharide with IC50 values of 8.17–18.73 μM via NO scavenging, inhibition of iNOS activity, and/or suppression of iNOS expression.


Immuno-regulatory and Anti-cancer Effect of Acorus gramineus Solander


Methanol extracts of Acorus gramineus Solander(AGS) were found to exhibit immuno-regulatory action in BALB/c mice. Oral administration of AGS increased murine splenic T lymphocytes, especially and subpopulations were increased significantly. Treatment of AGS exerted strong cytotoxicity against U937 and HL60 human leukemia cells. Also, AGS induced apoptosis of U937 leukemia cells in a dose dependent manner. Nitric oxide(NO) production and iNOS gene expression were also increased in AGS-treated RAW264.7 cells. Treatment of AGS increased the expression of p53 gene and decreased the expression of PCNA protein in cultured U937 cells. These data suggest that AGS are effective on the immuno-regulatory action and anti-cancer properties.


Acetylcholinesterase-inhibitory and memory-enhancing effect


In Ayurveda, herbal medicines with rasayana. effects are believed to be restorative, to attain longevity, intelligence, and freedom from age-related disorders. AC is regarded in Ayurvedic medicine as promoting rasayana effects and has been used to treat memory loss (Kirtikar & Basu, 1954; Mukherjee & Wahile, 2006a). AC is used in Ayurvedic medicine on a regular basis for the treatment of memory loss and other mental disorders (Kirtikar & Basu, 1954; Howes & Houghton., 2003). AC extract has also been used as traditional Chinese prescription, and its beneficial effects on memory disorder, on learning performance, lipid peroxide content, and anti-aging effects in senescence have been reported (Nishiyama et al., 1994; Zhang et al., 1994). The in vitro. acetylcholinesterase (AChE) inhibitory effect of hydro- alcohol extract and essential oil of AC rhizomes was reported based on Ellman’s method in 96-well microplates using bovine erythrocytes. The essential oil showed stronger inhibition than the hydro-alcohol extract (Houghton et al., 2006a).

Methanol extracts of AC showed significant acetylcholinesterase enzyme inhibition at a concentration 200 µg/mL (Oh et al., 2004). Houghton et al. (2006b) reported the in vitro. acetylcholinesterase inhibitory effect of β.-asarone and α.-asarone. β.-Asarone isat least an order of magnitude more active than its trans.-isomer α.-asarone. The AChE-inhibitory activity of the oil can be ascribed to β.-asarone. Because cognitive performance and memory are related to acetylcholine levels, the AChE-inhibitory effect of the plant may account for its traditional use.

Suppresses NF-kB


We revealed that β-Asarone (found in Acorus) suppressed not only basal NF-κB activity (activated in aging) but also Tumor necrosis factor α (TNF-α) induced NF-κB activity. Moreover, blocking NF-κB signaling inactivation was critical for β-Asarone induced apoptosis and inhibition of proliferation.



Polgala Tenuifolia
INHIBTS TLR4 & NFKB


Anti-inflammatory effects of Polygala tenuifoliaroot through inhibition of NF-κB activationin lipopolysaccharide-induced BV2 microglial cells

The root of Polygala tenuifolia Willd is a well-known traditional Oriental medicine and has been prescribed for treatment of dysfunction in memorial systems and various brain inflammatory diseases. The present study was designed to validate the anti-inflammatory effects of the water extract of Polygala tenuifolia root (WEPT).

Materials and methods: The anti-inflammatory properties of WEPT were studied using lipopolysaccharide (LPS)-stimulated murine BV2 microglia model. As inflammatory parameters, the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β were evaluated. We also examined the extract’s effect on the activity of nuclear factor-kappaB (NF-κB), and toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (Myd-88) expression.

Results: WEPT suppressed LPS-induced production of NO, PGE2, and expression of iNOS and COX-2 in a dose-dependent manner, without causing cytotoxicity. It also significantly reduced generation of proinflammatory cytokines, including IL-1β and TNF-α. In addition, WEPT suppressed NF-κB translocation by blockade of IkappaB-α (IκB-α) degradation and inhibited TLR4 and Myd-88 expression in LPS-stimulated BV2 cells.

Conclusions: These results indicate that the inhibitory effects of WEPT on LPS-stimulated inflammatory mediator production in BV2 microglia are associated with suppression of the NF-κB and toll-like receptor signaling pathways. Therefore, Polygala tenuifolia extracts may be useful in treatment of neurodegenerative diseases by inhibition of inflammatory mediator production in activated microglia.



HOELEN (Poria Cocos)

downregulateS AKT & C-FLIP


Activated Akt signaling in cancer cells inhibits apoptosis by phosphorylation of downstream substrates, such as BAD and Bcl-2, which are involved in the regulation of the intrinsic apoptotic pathway (Vivanco and Sawyers 2002). Furthermore, Akt also inhibits the death receptor-mediated apoptosis pathway through up-regulation of c-FLIP expression (Panka et al. 2001). As an anti-apoptotic protein, c-FLIP blocks apoptosis induced by the oligomerization of the adapter protein and therefore functions as a caspase-8 dominant negative (Panka et al. 2001). Targeting the PI3-kinase/Akt signaling pathway has been reported to be an effective strategy for the treatment of lung cancer (Crowell and Steele 2003). In the experiments, PPAC treatment decreased activation of Akt and increased the stability of PTEN in A549 cells.

Based on these results, it was therefore postulated that the down-regulation of Akt activitymight decrease c-FLIP expression, which would in turn cause the activation of caspase-8. The suppression of Akt activity by PPAC might be one of the essential mechanisms of PPAC-induced apoptosis. However, it remains to be elucidated whether Akt inhibition would solely or in combination with other factors contribute towards PPAC-induced apoptosis.


EF24 (Tumeric Root)

(curcumin analog. More potent and bioavailable than curcumin, with 10-fold greater potency).

novel Senolytic agent

NF-KB Inhibitor

IKK inhibitor

Bioactivities of EF24, a Novel Curcumin Analog: A Review

Inhibition of NF-κB Signaling

Most studies suggest that EF24 impairs cell growth by inducing cell cycle arrest followed by induction of apoptosis, which is accompanied by caspase-3 activation. However, the cell signaling pathway mediating the EF24 effect was not elucidated until 2008 when Kasinski et al. first revealed that EF24 induced cell apoptosis via suppressing NF-κB signaling pathway through direct action on IkB kinase (IKK) (21). NF-κB regulates a wide variety of genes involved in cell proliferation, differentiation, cell cycle control (36), oncogenic activation (37) and metastasis (38). EF24 can inhibit the catalytic activity of IKK protein complex, which blocks IκB phosphorylation and subsequent degradation, and finally prevents the nuclear translocation of p65 subunit of NF-κB. The study provides a molecular explanation for the superior activity of EF24 over curcumin (with a potency about 10 times higher than that of curcumin).

Other groups further verified the involvement of EF24 in inhibiting NF-κB signaling pathway in cancer (3942). EF24 robustly conferred radiation-induced cell death mainly by inhibiting radiation-induced NF-κB signaling in breast cancer (43). In the same year, the same group found that EF24 can suppress the radiation-induced NF-κB-DNA binding activity/promoter activation in genetically varied human neuroblastoma (44). Inhibition of the NF-κB signaling extends the therapeutic application of EF24 to other NF-κB-dependent diseases, such as inflammatory diseases (described below).

Comparison of EF24 to Curcumin in Anti-cancer Activity and Bioavailability

Since the development of EF24, scientists have compared its activity with its parent compound curcumin. Most of the studies were focused on the anti-cancer activity. Adams et al. synthesized a series of curcumin analogs including EF24, and submitted them to the NCI anti-cancer cell line screen. The results showed that EF24 was effective against all of the cell lines. The mean panel GI50 (concentration at which the drug inhibits tumor cell growth by 50%) was 10-fold better than curcumin and cisplatin (18). The authors also submitted the analogs to an in vitro anti-angiogenesis screen and revealed that EF24 was more active than curcumin in the assay. Their in vivo experiment suggested that EF24 was well tolerated by mice and much safer than the chemotherapy drug cisplatin (18). Later, Subramaniam et al. further compared the potency of EF24 to curcumin in gastrointestinal cancer cells and demonstrated that EF24 was more potent than curcumin. For example, 1 μmol/L of EF24 significantly suppressed proliferation and colony formation of the colon and gastric cancer cell lines while at the same dose of curcumin had no effect (29). Consistently, EF24 exhibited IC50 values 10 to 20 times lower than that of curcumin in multiple cancer cell lines, including lung, ovarian, cervical, breast, prostate cancer cells and cholangiocarcinoma cells (21, 22, 28, 40, 48). Similarly, in human osteogenic sarcoma cells (Saos2), EF24 was 3-fold more potent than curcumin (31). This evidence collectively suggests that EF24 displays much more potent anti-cancer activity than its parent compound in vitro. Although EF24 shared many anti-cancer mechanisms with its parent compound curcumin, such as inhibiting NF-κB and HIF-1α, it exerts its effects in different ways, for example, in how it regulates HIF-1α activity (described above). In addition, curcumin efficiently inhibited proteasome activity. By contrast, EF24 was 20-fold less active than curcumin for proteasome inhibition (45). Likewise, curcumin can regulate the STAT3, while EF24 has no effect on STAT activation (64).

The in vivo activity of a compound relies on the bioavailability of the compound at the site of the tumor. Dietary curcumin is poorly absorbed through the intestinal tract, therefore curcumin does not have a therapeutic effect at low doses (29). By contrast, EF-24 has higher oral bioavailability (60%) in mice (66), explaining to some extent the improved in vivo activity of EF24 compared to curcumin.

In addition to the improvement of anti-cancer activity, EF24 shows low toxicity to normal cells. EF24 has been shown to induce apoptosis in cancer cells and inhibit the growth of human breast tumors in a mouse xenograft model but showed low toxicity (18). Subramaniam et al. reported that EF24 inhibited intestinal cancer cell proliferation, but did not affect the proliferation of normal mouse embryonic fibroblasts cells, suggesting that EF24 is not toxic to normal cells (29). Similarly, EF24 inhibits tumor growth in human cholangiocarcinoma while displaying low toxicity levels. As a sensitizer, co-treatment of EF24 and rapamycin selectively enhances the cytotoxicity in gastric cancer cells but not in normal cells (60). Above all, multiple molecular targets, wide-spectrum potency, enhanced bioavailability as well as low toxicity to normal cells confer EF24 a series of advantages in clinical applications.

Regulation of HIF-1α Expression

Another important role of EF24 is to regulate HIF-1α expression which is closely associated with the outcome of chemotherapy in cancer treatment. For example, Liang et al. reported that sorafenib therapy could induce drug resistance in the treatment of hepatocellular carcinoma, in which HIF-1α plays an important role (50). Anti-angiogenic effects of sustained sorafenib therapy caused intratumor hypoxia, which induced HIF-1α and protected cancer cells from sorafenib treatment. EF24 can inhibit HIF-1α by sequestering it in cytoplasm and can promote its degradation by upregulating Von Hippel-Lindau tumor suppressor (VHL). Combination of EF24 and sorafenib showed synergistic effects against metastasis both in vivo and in vitro (50), providing compelling evidence for the potential of clinical application of EF24. Similar to EF24, the parent compound curcumin can also regulate HIF-1α expression (51), albeit through distinct mechanisms. Curcumin inhibited HIF-1α gene transcription, while EF24 exerted the activity by inhibiting HIF-1α post-transcriptionally (22).

The main mechanisms of action for EF24 include inhibition of the NF-κB pathway and HIF-1α protein and regulation of the MAPK pathway and ROS production.


downregulates cFLIP

cFLIP expression is regulated by a broad spectrum of mechanisms, including histone acetylation [] DNA damage and ubiquitination [] miR-375 [] sonic hedgehog and myc signalling [, ]; and Hsp90 [, ]. Here we focussed on NF-κB-driven cFLIP expression because it is common in many cancers [, ] Indeed, the NF-κB inhibitor, EF24 elicited cFLIP downregulation in primary thymoma TECs and 1889 thymic carcinoma cells [] (Figure (Figure4A4A and Supplementary Figure 6A) and sensitized them to TNFα induced cell death (Figure (Figure4B4B and and4C4C and Supplementary Figure 6B and 6C). Like cFLIP knockdown, NF-κB blockade induced both apoptosis and autophagy in 1889c TC cells (Supplementary Figure 7 and Figure Figure7)7) and in primary thymoma-derived TECs (Figure (Figure4;4; Figure Figure55 and Figure Figure7).7). Whether NF-κB expression in neoplastic TECs is driven by oncogenic mechanisms [] or echoes the epithelial NF-κB signaling that is operative during early thymus development [] is unknown.


EGCG

Suppression of NFkB and STAT3


Persistent activation of signal transducer and activator of transcription (STAT-3) is a common feature of PCa (20,21) and increased resistance to apoptosis occurs due to constitutive activation of STAT-3. Western blot analysis was performed to look at the protein expression of STAT-3, we observed a consistent upregulation of the protein as the tumor progressed from not-detectable at 8 weeks to poorly-differentiated at 32 weeks in dorsolateral prostate of TRAMP mice. This upregulation was significantly inhibited in age matched mice with continuous GTP infusion (Fig. 4A). These results were further confirmed by immunohistochemical analysis of STAT-3 levels (Fig. 4B), indicating a significant decrease in STAT-3 protein expression in green tea polyphenol-fed TRAMP mice.

Taken together this study highlights the chemo-preventive effects of oral infusion of GTP in TRAMP mice. We suggest that the observed inhibition of NFκB signaling pathway along withSTAT3 inhibition may potentially be involved in repressing tumor progression in TRAMP mice.


EGCG-induced JNK activation


Polyphenols such as epigallocatechin-3-gallate (EGCG) from green tea extract can exert a growth-suppressive effect on human pancreatic cancer cells in vitro. In pursuit of our investigations to dissect the molecular mechanism of EGCG action on pancreatic cancer, we observed that the antiproliferative action of EGCG on pancreatic carcinoma is mediated through programmed cell death or apoptosis as evident from nuclear condensation, caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. EGCG-induced apoptosis of pancreatic cancer cells is accompanied by growth arrest at an earlier phase of the cell cycle. In addition, EGCG invokes Bax oligomerization and depolarization of mitochondrial membranes to facilitate cytochrome c release into cytosol. EGCG-induced downregulation of IAP family member X chromosome linked inhibitor of apoptosis protein (XIAP)might be helpful to facilitate cytochrome c mediated downstream caspase activation. On the other end, EGCG elicited the production of intracellular reactive oxygen species (ROS), as well as the c-Jun N-terminal kinase (JNK) activation in pancreatic carcinoma cells. Interestingly, inhibitor of JNK signaling pathway as well as antioxidant N-acetyl-L-cysteine (NAC) blocked EGCG-induced apoptosis. To summarize, our studies suggest that EGCG induces stress signals by damaging mitochondria and ROS-mediated JNK activation in MIA PaCa-2 pancreatic carcinoma cells.

The prevention of EGCG-mediated apoptosis by JNK inhibitor II and NAC suggests the involvement of ROS-mediated JNK activation in this pathway. The ROS comprise of singlet oxygen, hydroxyl radicals, superoxide, hydroperoxides and peroxides. In our studies, we have observed an increase in hydrogen peroxide level owing to EGCG exposure as noted in the case of lung tumor cell lines (21). Lei et al. (42) have indicated that JNK signaling is necessary for the stress-induced release of cytochrome c release and programmed cell death. JNK signaling and cytochrome c release may be interlinked to a pro-apoptotic member of Bcl-2 family because activated JNK is unable to evoke apoptosis in cells deficient of Bax. Our observation indicates that the concerted efforts of JNK activation and Bax oligomerization might play a pivotal role in the demise of pancreatic cancer cells.

Since increased ROS levels were observed to exert the activation of stress kinase JNK (40,41), we were interested to assess the activation status of JNK by immunocomplex kinase assay in the control and EGCG-exposed MIA PaCa-2 cells. Figure 10A clearly demonstrates the EGCG-induced JNK activation in MIA PaCa-2 cells. In order to understand whether JNK activation is necessary for EGCG-mediated cell killing, we next examined whether the blocking of JNK activity exerts any effect on EGCG-induced apoptosis.


BCL, Bcl-xL, Bcl-w INHIBITOR


high concentration of EGCG exerted a decrease in the expression of Bcl-xL and Bcl-w


MDM2 Inhibitor


Epigallocatechin gallate promotes p53 accumulation and activity via the inhibition of MDM2-mediated p53 ubiquitination in human lung cancer cells.

Epigallocatechin gallate (EGCG), which is derived from green tea, is well known for its chemopreventive activity. Several studies have shown that p53 plays an important role in the activity of EGCG; however, the mechanism by which EGCG regulates p53 requires further investigation. In the present study, we showed that EGCG inhibits anchorage-independent growth of human lung cancer cells by upregulating p53 expression. EGCG treatment can substantially increase p53 stability, promote nuclear localization of p53 and decrease nuclear accumulation of MDM2. We also found that EGCG increases the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity. Although EGCG promotes MDM2 expression in a p53-dependent manner, the interaction between MDM2 and p53 was significantly inhibited following EGCG treatment, which resulted in the inhibition of MDM2-mediated p53 ubiquitination. Thus, our results suggest that the stabilization and activation of p53 may partly contribute to the anticancer activity of EGCG.


SENOLYTIC ATTRIBUTES OF [EGCG] MATCHA


Senescence is defined the condition or process of deterioration with age [177]. Senescence plays a role in multiple biological events, such as embryogenesis, tissue regeneration and repair, ageing, cardiovascular, renal, liver diseases, and cancer [178,179]. EGCG has been implicated in senescence in different works. For example human mesenchymal stem cells (hMSCs) show the peculiar behavior of producing abundant ROS during culturing and this issue has been exploited with a natural anti‐ oxidant as EGCG [180]. The work explored the anti‐senescent effect of EGCG in H2O2‐exposed hMSCs and proved that EGCG 50 or 100 μM reversed oxidative stress by downregulating the p53–p21 signaling pathway and upregulating Nrf2 expression. Similarly, in primary cells, including rat vascular smooth muscle cells (RVSMCs), human dermal fibroblasts (HDFs) and human articular chondrocytes (HACs), the same concentrations of EGCG (50 or 100 μM) could prevent senescence and recover cell cycle progression, again impacting on the p53 pathway at least in HDFs cells [181].

Furthermore, in 3T3‐L1 preadipocytes, EGCG could inhibit stress‐induced senescence (H2O2‐ induced) by countering DNA damage and cell cycle arrest [182]. In this work, EGCG (same concentrations as above, 50 or 100 μM) modulated PI3K/Akt/mTOR and AMPK pathways by blocking ROS, iNOS, Cox‐2, NF‐kB, and p53, and increasing apoptosis by suppressing antiapoptotic protein Bcl‐2. Moreover, in WI‐38 fibroblasts at late‐stage (population doubling > 25) used as model of oxidative stress and inflammation, EGCG (25, 50, 100 μM) reduced TNF‐α, IL‐6, ROS, again acting on p53 [183]. ECGC also decreased retinoblastoma expressions, while enhancing E2F2 expressions and superoxide dismutase (SOD) 1 and 2, all being implicated in oxidation processes. EGCG has been shown to affect the development and severity of cellular senescence, but from a therapeutic perspective more work is needed to justify its implication in human consumption. 💥Senolytic attributes of EGCG are interesting, especially for attenuating age‐associated disorders, though clinical trails and health consequence still need to be proposed and discovered.


TLR4 INHIBITOR


TLR4 signaling inhibitory pathway induced by green tea polyphenol epigallocatechin-3-gallatethrough 67-kDa laminin receptor.

Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to downregulate inflammatory responses in macrophages; however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor that mediates the anticancer action of EGCG at physiologically relevant concentrations (0.1-1 microM). In this study, we show the molecular basis for the downregulation of TLR4 signal transduction by EGCG at 1 microM in macrophages. Anti-67LR Ab treatment or RNA interference-mediated silencing of 67LR resulted in abrogation of the inhibitory action of EGCG on LPS-induced activation of downstream signaling pathways and target gene expressions. Additionally, we found that EGCG reduced the TLR4 expression through 67LR.

Interestingly, EGCG induced a rapid upregulation of Toll-interacting protein (Tollip), a negative regulator of TLR signaling, and this EGCG action was prevented by 67LR silencing or anti-67LR Ab treatment. RNA interference-mediated silencing of Tollip impaired the TLR4 signaling inhibitory activity of EGCG. Taken together, these findings demonstrate that 67LR plays a critical role in mediating anti-inflammatory action of a physiologically relevant EGCG, and Tollip expression could be modulated through 67LR. These results provide a new insight into the understanding of negative regulatory mechanisms for the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.



Ellagic acid


Identification of novel senolytic compounds from natural food sources


More than 23% of today’s population suffers from age-associated diseases such as arthritis, cancer, heart disease, and more. The ongoing economic impact of these diseases has been in the billions of dollars worldwide with no clear solution to date. This study addresses the underlying cause of these diseases by identifying the compounds that potentially eliminate senescent cells. Existing senolytic drugs are not abundantly found in nature, reducing accessibility. Hence, over 70,000 natural compounds available in the Canadian Food Database were used to screen penitential senolytic compounds that block PI3Kγ, reactivating apoptotic processes in senescent cells.

Molecular docking results revealed 23 natural compounds that blocks the PI3Kγ. Out of 23 compounds, Cianidanol, Ellagic acid, Eriodictyol, Kaempferol and Cyanidin were found abundantly in food sources range from 85 to 735 mg/100 g. These compounds are up to 46 times more abundant in foods than proven senolytic drug Fisetin. Further, molecular dynamics results showed ligand stability for 4 nanoseconds with PI3Kγ. The five compounds are proven to eliminate cancerous cells, have the potential to prevent age-related diseases, and could even slow down natural aging.


EMBELIN

STAT3 INHIBITOR


Even though embelin, an inhibitor of the XIAP, is known to exhibit anti-inflammatory and anti-cancer activities, very little is known about its mechanism of action. Here, we investigated whether embelin mediates its effect through interference with the signal transducer and activator of transcription 3 (STAT3) pathway. We found that embelin inhibited constitutive STAT3 activation in a variety of human cancer cell lines such as U266, DU-145, and SCC4 cells. The suppression of STAT3 was mediated through inhibition of the activation of JAK2 and c-Src. Pervanadate treatment also reversed the embelin-induced down-regulation of STAT3, suggesting the involvement of a protein tyrosine phosphatase.

Indeed, we found that embelin-induced the expression of the tyrosine phosphatase PTEN and deletion of the PTEN gene by small interfering RNA abolished the ability of embelin to inhibit STAT3 activation. Besides, embelin failed to suppress STAT3 activation in PTEN-null PC3 cells, thus indicating that the inhibitory effect of embelin on STAT3 is PTEN-dependent. Embelin down-regulated the expression of STAT3-regulated gene products; this correlated with the suppression of cell proliferation and invasion, and the induction of apoptosis through the activation of caspase-3. Overall, our results indicate that the anti-inflammatory and anti-cancer activities previously assigned to embelin may be mediated in part through the suppression of the STAT3 pathway.

STAT3 plays an important role in carcinogenesis and metastasis of various human cancers. ► Embelin could suppress constitutive STAT3 activation through induction of PTEN. ► Embelin down-regulated the expression of STAT3-regulated gene products.


Embelin: a benzoquinone possesses therapeutic potential for the treatment of human cancer


Natural products have been gaining recognition and are becoming a significant part of research in the area of drug development and discovery. Phytochemicals derived from these sources have been comprehensively studied and have displayed a wide range of activities against many fatal diseases including cancer. One such product that has gained recognition from its pharmacological properties and nontoxic nature is embelin, obtained from Embelia ribes. Amid all the vivid pharmacological activities, embelin has gained its prominence in the area of cancer research. Embelin binds to the BIR3 domain of XIAP, preventing the association of XIAP and caspase-9 resulting in the suppression of cell growth, proliferation and migration of various types of cancer cells.

Furthermore, embelin modulates anti-apoptotic pathways by suppressing the activity of NF-κB, PI3-kinase/AKT, JAK/STAT pathway– among others. The present review summarizes the various reported effects of embelin on different types of cancer cells and highlights the cellular mechanisms of action.


Emodin

Emodin has a cytotoxic activity against human multiple myeloma as a JAK 2 inhibitor.


Emodin is an active component of a traditional Chinese and Japanese medicine isolated from the root and rhizomes of Rheum palmatum L. Here, we show that emodin significantly induces cytotoxicity in the human myeloma cells through the elimination of myeloid cell leukemia 1 (Mcl-1). Emodin inhibited interleukin-6-induced activation of Janus-activated kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription 3 (STAT3), followed by the decreased expression of Mcl-1. Activation of caspase-3 and caspase-9 was triggered by emodin, but the expression of other antiapoptotic Bcl-2 family members, except Mcl-1, did not change in the presence of emodin. To clarify the importance of Mcl-1 in emodin-induced apoptosis, the Mcl-1 expression vector was introduced into the human myeloma cells by electroporation.

Induction of apoptosis by emodin was almost abrogated in Mcl-1-overexpressing myeloma cells as the same level as in parental cells, which were not treated with emodin. In conclusion, emodin inhibits interleukin-6-induced JAK2/STAT3 pathway selectively and induces apoptosis in myeloma cells via down-regulation of Mcl-1, which is a good target for treating myeloma. Taken together, our results show emodin as a new potent anticancer agent for the treatment of multiple myeloma patients.


Emodin enhances cytotoxicity of chemotherapeutic drugs in prostate cancer cells: the mechanisms involve ROS-mediated suppression of multidrug resistance and hypoxia inducible factor-1.

The intrinsic or acquired resistance to multiple drugs (MDR) of cancer cells remains one of the main obstacles for chemotherapy. Development of small molecule targeting to hypoxia inducible factor-1 (HIF-1) has been recently proposed as strategy for treatments of drug-resistant solid tumors. In the present study, emodin, proven as a reactive oxygen species (ROS) generator by our previous work, was applied in combination with cisplatin and other chemotherapeutic drugs in the multidrug resistant prostate carcinoma cell line DU-145 and normal human dermal fibroblasts.

Results showed that emodin/cisplatin co-treatment remarkably elevated ROS level and enhanced chemosensitivity in DU-145 cells, compared with cisplatin-only treatment, but exerted little effect on non-tumor cells. The effect of co-treatment on MDR1 gene and its upstream regulator HIF-1 was then investigated in DU-145. Co-treatment downregulated MDR1 expression and promoted drug retention, and meanwhile suppressed transactivation of HIF-1 in response to hypoxia without changing expression of HIF-1 alpha. The experiments on tumor-bearing mice showed that co-treatment inhibited the tumor growth in vivo, owing to oxidative stress and MDR1 down-regulation within tumors. HIF-1 transactivation and clonegenesis were suppressed in cells isolated from the tumors. Finally, examinations for the body weight, the organ histology and the antioxidant capacity of serum suggested that no systemic toxicity related to co-treatment was discernable. In conclusions, emodin, as a novel small inhibitor of HIF-1, may be recognized an effective adjunctive to improve efficacy of cytotoxic drugs in prostate cancer cells with over-activated HIF-1 and potent MDR.


Matrix metalloproteinases (MMPs) are the proteases involved in the degradation of the extracellular matrix. MMP-1 is thought to be one of the key enzymes acting in fibrolysis, a process closely related to tissue remodeling. In this study, we found that emodin, an anthraquinone which has been isolated from the rhizome of Rheum palmatum, significantly inhibited TNF alpha-induced MMP-1 gene expressionin a concentration-dependent manner. Therefore, we have attempted to characterize the inhibitory mechanism of emodin in TNF alpha-induced MMP-1 expression.

Emodin was determined to inhibit TNF alpha-induced activation of AP-1 promoter, an important nuclear transcription factor in MMP-1 expression. Additionally, we detected that emodin suppressed the TNF alpha-induced phosphorylation of two mitogen-activated protein kinases, extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but it did not suppress the TNF alpha-induced phosphorylation of p38 kinase. In a consistent result, the TNF alpha-induced MMP-1 expression was inhibited by PD98059 (MEK/ERK inhibitor) and SP600125 (JNK inhibitor), but was not inhibited by SB203580, a p38 MAPK inhibitor. Taken together, these results show that emodin suppresses TNF alpha-induced MMP-1 expression through the inhibition of the AP-1 signaling pathway.


CASPASE 3 ACTIVATOR


Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active constituent of Rheum palmatum, and showed inhibitory activity on lipopolysaccharide-induced NO production in our previous study. However, the apoptosis-inducing activity of emodin has remained undefined. Among three structurally related anthraquinones, including emodin, physcion, and chrysophanol, emodin showed the most potent cytotoxic effects on HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells. Emodin at apoptosis-inducing concentrations causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity, according to cleavage of caspase 3 substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase 3 fragments being detected in emodin- but not physcion- or chrysophanol-treated HL-60 cells.

A decrease in the anti-apoptotic protein, Mcl-1, was detected in emodin-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bad remained unchanged. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated emodin-induced DNA ladders, associated with the blockage of PARP and D4-GDI cleavage. Free radical scavenging agents including NAC, catalase, SOD, ALL, DPI, l-NAME and PDTC showed no preventive effect on emodin-induced apoptotic responses, whereas NAC, CAT and PDTC prevented HL-60 cells from ROS (H2O2)-induced apoptosis through inhibition of caspase 3 cascades. Induction of catalase, but not SOD, activity was detected in emodin-treated HL-60 cells by in gel activity assays, and H2O2-induced intracellular peroxide level was significantly reduced by prior treatment of emodin in HL-60 cells. Our experiments provide evidence that emodin is an effective apoptosis inducer in HL-60 cells through activation of the caspase 3 cascade, but that it is independent of ROS production.



Eriodictyol


Identification of novel senolytic compounds from natural food sources


More than 23% of today’s population suffers from age-associated diseases such as arthritis, cancer, heart disease, and more. The ongoing economic impact of these diseases has been in the billions of dollars worldwide with no clear solution to date. This study addresses the underlying cause of these diseases by identifying the compounds that potentially eliminate senescent cells. Existing senolytic drugs are not abundantly found in nature, reducing accessibility. Hence, over 70,000 natural compounds available in the Canadian Food Database were used to screen penitential senolytic compounds that block PI3Kγ, reactivating apoptotic processes in senescent cells.

Molecular docking results revealed 23 natural compounds that blocks the PI3Kγ. Out of 23 compounds, Cianidanol, Ellagic acid, Eriodictyol, Kaempferol and Cyanidin were found abundantly in food sources range from 85 to 735 mg/100 g. These compounds are up to 46 times more abundant in foods than proven senolytic drug Fisetin. Further, molecular dynamics results showed ligand stability for 4 nanoseconds with PI3Kγ. The five compounds are proven to eliminate cancerous cells, have the potential to prevent age-related diseases, and could even slow down natural aging.


Esculetin


CASPASE 3 ACTIVATOR


In this study, we have investigated whether esculetin exerts anti-proliferative and apoptotic effects on human leukemia U937 cells. It was found that esculetin could inhibit cell viability in a time-dependent manner, which was associated with the induction of apoptotic cell death such as increased populations of apoptotic- sub G1 phase. Apoptosis of U937 cells by esculetin was associated with an inhibition of Bcl-2/Bax binding activity, formation of tBid, down-regulation of X-linked inhibitor of apoptotic protein (XIAP) expression, and up-regulation of death receptor 4 (DR4) and FasL expression. Esculetin treatment also induced the degradation of {beta}-catenin and DNA fragmentation factor 45/inhibitor of caspase-activated DNase (DFF45/ICAD). Furthermore, a caspase-3 specific inhibitor, z-DEVD-fmk, significantly inhibited sub-G1 phase DNA content, morphological changes and degradation of {beta}-catenin and DEE45/ICAD. These results indicated that a key regulator in esculetin-induced apoptosis was caspase-3 in human leukemia U937 cells.


Evodiamine (EVO) is an indoloquinazoline alkaloid that exerts its various anti-oncogenic actions by blocking phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), mitogen-activated protein kinase (MAPK), c-Met, and nuclear factor kappa B (NF-κB) signaling pathways, thus leading to apoptosis of tumor cells. We investigated the ability of EVO to affect hepatocyte growth factor (HGF)-induced c-Met/Src/STAT3 activation cascades in castration-resistant prostate cancer (CRPC). First, we noted that EVO showed cytotoxicity and anti-proliferation activities in PC-3 and DU145 cells. Next, we found that EVO markedly inhibited HGF-induced c-Met/Src/STAT3 phosphorylation and impaired the nuclear translocation of STAT3 protein.
Then, we noted that EVO arrested the cell cycle, caused apoptosis, and downregulated the expression of various carcinogenic markers such as B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), cyclin D1, cyclooxygenase 2 (COX-2), survivin, vascular endothelial growth factor (VEGF), and matrix metallopeptidases 9 (MMP-9). Moreover, it was observed that in cPC-3 and DU145 cells transfected with c-Met small interfering RNA (siRNA), Src/STAT3 activation was also mitigated and led to a decrease in EVO-induced apoptotic cell death. According to our results, EVO can abrogate the activation of the c-Met/Src/STAT3 signaling axis and thus plays a role as a robust suppressor of tumor cell survival, proliferation, and angiogenesis.

Fisetin

(from Cotinus coggygria Scop. aka “smoke tree”)

Fisetin is a senotherapeutic that extends health and lifespan.

BACKGROUND: Senescence is a tumor suppressor mechanism activated in stressed cells to prevent replication of damaged DNA. Senescent cells have been demonstrated to play a causal role in driving aging and age-related diseases using genetic and pharmacologic approaches. We previously demonstrated that the combination of dasatinib and the flavonoid quercetin is a potent senolytic improving numerous age-related conditions including frailty, osteoporosis and cardiovascular disease. The goal of this study was to identify flavonoids with more potent senolytic activity.

METHODS: A panel of flavonoid polyphenols was screened for senolytic activity using senescent murine and human fibroblasts, driven by oxidative and genotoxic stress, respectively. The top senotherapeutic flavonoid was tested in mice modeling a progeroid syndrome carrying a p16INK4a-luciferase reporter and aged wild-type mice to determine the effects of fisetin on senescence markers, age-related histopathology, disease markers, health span and lifespan. Human adipose tissue explants were used to determine if results translated.

FINDINGS: Of the 10 flavonoids tested, fisetin was the most potent senolytic. Acute or intermittent treatment of progeroid and old mice with fisetin reduced senescence markers in multiple tissues, consistent with a hit-and-run senolytic mechanism. Fisetin reduced senescence in a subset of cells in murine and human adipose tissue, demonstrating cell-type specificity. Administration of fisetin to wild-type mice late in life restored tissue homeostasis, reduced age-related pathology, and extended median and maximum lifespan.

INTERPRETATION: The natural product fisetin has senotherapeutic activity in mice and in human tissues. Late life intervention was sufficient to yield a potent health benefit. These characteristics suggest the feasibility to translation to human clinical studies.

New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and A1155463.


Senescent cells accumulate with aging and at sites of pathology in multiple chronic diseases. Senolytics are drugs that selectively promote apoptosis of senescent cells by temporarily disabling the pro-survival pathways that enable senescent cells to resist the pro-apoptotic, pro-inflammatory factors that they themselves secrete. Reducing senescent cell burden by genetic approaches or by administering senolytics delays or alleviates multiple age- and disease-related adverse phenotypes in preclinical models. Reported senolytics include dasatinib, quercetin, navitoclax (ABT263), and piperlongumine.

Here we report that fisetin, a naturally-occurring flavone with low toxicity, and A1331852 and A1155463, selective BCL-XL inhibitors that may have less hematological toxicity than the less specific BCL-2 family inhibitor navitoclax, are senolytic. Fisetin selectively induces apoptosis in senescent but not proliferating human umbilical vein endothelial cells (HUVECs). It is not senolytic in senescent IMR90 cells, a human lung fibroblast strain, or primary human preadipocytes.A1331852 and A1155463 are senolytic in HUVECs and IMR90 cells, but not preadipocytes. These agents may be better candidates for eventual translation into clinical interventions than some existing senolytics, such as navitoclax, which is associated with hematological toxicity.

Fisetin activates Hippo pathway and JNK/ERK/AP-1 signaling to inhibit proliferation and induce apoptosis of human osteosarcoma cells via ZAK overexpression.


Osteosarcoma (OS) is a tumor entity that can cause a large number of cancer-related deaths. Although chemotherapy can decrease proliferation and increase apoptosis of human OS cells, the clinical prognosis remains poor. Fisetin is a flavonol found in fruits and vegetables and is reported to inhibit cell growth in numerous cancers. But the molecular mechanism underlying fisetin in human OS cells is not clear. It is known that sterile-alpha motif and leucine zipper containing kinase (ZAK), a kinase in the MAP3K family, is involved in various cell processes, including proliferation and apoptosis. In our lab, we have demonstrated that overexpression of ZAK can induce apoptosis in human OS cells. In the previous studies, MAP4K, the upstream of MAP3K, can act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway. Turning on the Hippo pathway can decrease proliferation and otherwise cause cell apoptosis in cancer cells.

In this study, we found that fisetin can upregulate ZAK expression to induce the Hippo pathway and mediate the activation of JNK/ERK, the downstream of ZAK, to trigger cell apoptosis via AP-1 dependent manner in human OS cells. These findings reveal a novel molecular mechanism underlying fisetin effect on human OS cells.

MTOR INHIBITOR

Fisetin (3,7,3′,4′-tetrahydroxyflavone) belongs to the flavonol subgroup of flavonoids which includes quercetin, myricetin and kaempferol. Epidemiological and preclinical studies have shown that fisetin consumption affects various molecular targets which collectively slow the ageing process (George, 2016). Fisetin has been shown to inhibit the association between mTOR pathway signaling pathway constituents, Raptor, and Rictor. Fisetin also decreases PI3-K content, and enhances phosphorylation of Akt. Phosphorylated Akt in turn inhibits mTOR activity. Fisetin can also directly activate negative regulators of mTOR such as Tsc complex and AMPK as shown in Fig. 1 (Adhami et al., 2012). Moreover, fisetin has been shown to enhance cytotoxic effects when used in combination with other chemotherapeutic drugs (Haddad et al., 2010; Klimaszewska-Wisniewska et al., 2016).


inhibition of GSK3β


In summary, our definitive and novel findings obtained in this study indicate that fisetin confers cardioprotection against myocardial IRI, by bolstering the mitochondrial physiology, suppressing the oxidative stress, and augmenting the mitochondrial biogenesis, and these effects are mediated via inhibition of GSK3β activity. Since fisetin is well tolerated in human subjects and does not show toxic effects, this natural small molecule has bright prospects for further pharmaceutical development to be used against I/R-induced myocardial tissue injury and potentially for the treatment of cardiovascular diseases.


BCL-W INHIBITOR


This study aimed to investigate the effects of fisetin, a common dietary natural flavonoid, on apoptosis of Huh-7 cells. The MTT assay was used to evaluate the reduction of cell viability. In the DNA fragmentation assay and comet assay, fisetin-induced DNA damage was visible as a formation of DNA fragmentation and comet tails. Fisetin also induced intracellular accumulation of reactive oxygen species. Two-dimensional gel electrophoresis was performed to evaluate protein expression in fisetin-treated and control cells, and Baculoviral IAP repeat-containing protein 8 (BIRC8) and apoptosis regulator Bcl-W (Bcl2L2) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their expression was confirmed by western blotting. The anticancer effect of fisetin in Huh-7 cells may result from the down regulation of BIRC8 and the Bcl2L2.


enhancement of proapoptotic Bad and Bim


Although fisetin attenuates the COX-2 and MAPK pathways in HT-29 cells, the growth of HCT-116 cells was inhibited through the apoptosis mechanism. Several factors were involved in this mechanism, such as reduction of antiapoptotic Bcl-xL and Bcl-2 at the protein level and enhancement of proapoptotic Bad and Bim. Moreover, excitation of mitochondrial permeability and consequently activation of the caspase cascade, including caspase-3, -7, -8, and -9, and downstream factors such as cytochrome c are other means of promoting apoptosis. Activation of death receptors (Fas and tumor necrosis factor) by fisetin assisted in promoting apoptosis. In addition, fisetin increased p53 expression .


FOXO4 D-Retro-Inverso peptide


Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging


The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored.

Here, we identifyFOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide which perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized Doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred and in doing so tissue homeostasis can effectively be restored.

FOXO4-DRI has a strong preference for targeting high-SASP subpopulations ofsenescent cells, but it is unclear what causes heterogeneity in the SASP. It will be a major achievement to unravel those mechanisms and to steer these such that therapeutic targeting is most beneficial. In that sense, identification of senescence-driven pathologies that rely on SASP may help in optimizing candidates for therapy. XpdTTD/TTD is pleiotropic model for aging that can be effectively used as a basis for such research. It is a well-established model for osteoarthritis, especially in cohorts of older age than we used here (52w) (Botter et al., 2011) and for the unhealthy loss in muscle (sarcopenia) and fat mass(Wijnhoven et al., 2005).

Last, it is relevant to note that independent of aging and age-related diseases, FOXO4-DRI may be of use against the progression, stemness and migration of malignant cancer. Given that SASP factors influence these(Campisi, 2013), it will be particularly interesting to determine whether FOXO4-DRI affects those p53-wt cancer cells that have adopted a more migratory and stem-like state due to reprogramming by chronic SASP exposure. In any case, the here reported beneficial effects of FOXO4-DRI provide a wide range of possibilities for studying the potential of therapeutic removal of senescence against diseases for which few options are available.

Regulation of cellular senescencevia the FOXO4‐p53 axis


Forkhead box O (FOXO) and p53 proteins are transcription factors that regulate diverse signalling pathways to control cell cycle, apoptosis and metabolism. In the last decade both FOXO and p53 have been identified as key players in aging, and their misregulation is linked to numerous diseases including cancers. However, many of the underlying molecular mechanisms remain mysterious, including regulation of ageing by FOXOs and p53. Several activities appear to be shared between FOXOs and p53, including their central role in the regulation of cellular senescence. In this review, we will focus on the recent advances on the link between FOXOs and p53, with a particular focus on the FOXO4‐p53 axis and the role of FOXO4/p53 in cellular senescence. Moreover, we discuss potential strategies for targeting the FOXO4‐p53 interaction to modulate cellular senescence as a drug target in treatment of aging‐related diseases and morbidity.

Given is role in aging and age‐related diseases, cellular senescence and associated signaling pathway have been extensively studied the past years. A better understanding of the exact molecular mechanisms involved in senescence is a key step toward the discovery of new drug that can efficiently and selectively target senescent cells in order to counteract age‐related pathology or more “utopically” in normal aging. In this way, we and co‐workers designed a peptide that interfere with FOXO‐p53 mediated senescence that showed great potency and selectivity in targeting apoptosis of senescent cells in mice. Based on this work, one can speculate on many other targets that could be used for similar therapeutic approaches. Indeed, interfering with (a) FOXO4 activation via the ROS/JNK or ROS/MDM2 pathway (b) nuclear translocation of active p53 (c) FOXO4 and/or p53 DNA binding to the promoter region of p21, could possibly lead to similar outcome.

For this purpose, detailed structural information on binary complexes involved in the corresponding pathway are of great help in order to efficiently and selectively target one given interaction therefore limiting drug toxicity in vivo. Given the intricate networks in which p53 mediates the interaction with numerous binding partners, it remains to be studied how drug‐like molecules modulate binding of p53 to other p53 regulators. Therefore, atomic details of p53‐drug interactions are required in order to clarify the exact mechanisms leading to the clearance of senescent cells. Such structural and functional work will allow to optimize the binding properties, affinity/specificity of drug‐like molecules and to reduce potential off‐target effects.


Rejuvenation by Therapeutic Elimination of Senescent Cells

In this issue of Cell, Baar et al. show how FOXO4 protects senescent cell viability by keeping p53 sequestered in nuclear bodies, preventing it from inducing apoptosis. Disrupting this interaction with an all-D amino acid peptide (FOXO4-DRI) restores p53’s apoptotic role and ameliorates the consequences of senescence-associated loss of tissue homeostasis.

In 2016, Van Deursen and coworkers showed that prophylactic ablation of cells that express the p16Ink4a senescent marker mitigates tissue degeneration and extends the healthspan of mice (Baker et al., 2016). Although the Baker et al. study established the causative role of senescent cells in tissue degeneration, it did not reveal the underlying mechanisms responsible for their generation. This is now addressed in the article by Baar et al. (2017). The authors compared the expression profiles of normal human IMR90 cells with their radiation-induced senescent counterparts. Whereas they expected that the senescent cells would show reduced levels of pro-apoptotic and/or an increase in anti-apoptotic proteins, they observed the opposite, suggesting lack of an upstream trigger to activate the death program. Therefore, they searched for changes in the expression levels of transcription regulators that could underlie these changes.
One immediate candidate was FOXO4, a member of a transcription factor family previously implicated in aging and longevity (Martins et al., 2016), which appeared upregulated upon senescence induction. Therefore, FOXO4 was further scrutinized for its potential role in arresting cells in a senescent state. Indeed, inhibition of FOXO4 expression using a lentiviral shRNA made cells undergo apoptosis rather than become senescent upon irradiation. More importantly, FOXO4 inhibition in already senescent cells reduced their viability. This established that FOXO4 plays a critical role in consolidating the senescent state and that its inactivation causes senescent cells to undergo apoptosis.

FUCOIDAN

Caspase 3 Activator

Fucoidan, a sulfated polysaccharide in brown seaweed, was found to inhibit proliferation and induce apoptosis in human lymphoma HS-Sultan cell lines. Fucoidan-induced apop- tosis was accompanied by the activation of caspase-3and was partially prevented by pretreatment with a pan-caspase inhibitor, Z-VAD-FMK. The mitochondrial potential in HS-Sultan cells was decreased 24 hr after treatment with fucoidan, indicating that fucoi- dan induced apoptosis through a mitochondrial pathway. When HS-Sultan was treated with 100 mg/mL fucoidan for 24 hr, phosphorylation of ERK and GSK markedly decreased. In contrast, phosphorylation of p38 and Akt was not altered by treatment with fucoidan. L-Selectin and P-selectin are known to be receptors of fucoidan; however, as HS-Sultan does not express either of these selectins, it is unlikely that fucoidan induced apoptosis through them in HS-Sultan.

The neutralizing antibody, Dreg56, against human L-selectin did not prevent the inhibitory effect of fucoidan on the proliferation of IM9 and MOLT4 cells, both of which express L-selectin; thus it is possible fucoidan induced apoptosis though different receptors. These results demonstrate that fucoidan has direct anti- cancer effects on human HS-Sultan cells through caspase and ERK pathways.


Galangin

Alpinia officinarum

apoptosis-inducing factor (AIF) ACTIVATOR

Studies have showed that most flavonoids exhibit anti-proliferative effects against tumor derived cell lines including leukemia[13], melanoma[14], colon[15], breast carcinoma[16], lung, and prostate[17]. Some reports have demonstrated that galangin is a naturally occurring non-toxic flavonoid with chemopreventive and anti-proliferative effects[68,18,19].

In this study, we demonstrated that galangin inhibited the proliferation of HCC cells and induced apoptosis at concentrations as low as 46.25 μmol/L and in excess of 185 μmol/L, respectively. Galangin-induced apoptosis was characterized by analyzing the effects on caspase-3 activation, PARP cleavage, and DNA condensation in HCC cells.

The mitochondrial pathway is commonly involved in the death stimuli. There are primarily two major events involved in apoptosis via the mitochondrial pathway. The first event is a change in mitochondrial membrane permeability, which leads to decreased mitochondrial membrane potential. Our data demonstrated reduced mitochondrial membrane potential as indicated by rhodamine 123 staining following treatment with galangin at different concentrations. The second event in the mitochondria-induced apoptotic pathway is the release of cytochrome c and AIF from the intermembrane space of the mitochondria into the cytosol. Here, we also showed that galangin increased the release of cytochrome c and AIF in the cytosol. Thus, our data indicates that galangin-induced apoptosis of HCC cells occurs through the mitochondrial pathway.

The Bcl-2 family of proteins is involved in the mitochondrial apoptotic pathway by inducing the release of cytochrome c from the mitochondrial intermembrane space. Cytochrome c cooperates with Apaf-1 (Apoptotic protease activating factor 1) to induce caspase activation, leading to cell apoptosis[20]. The Bcl-2 family proteins are categorized into three groups based on the four Bcl-2 homology domains (BH1-4 domains). Bcl-2, Bcl-w, Bcl-xL and Mcl-1, which contain BH domains 1-4 and are localized to the outer mitochondrial membrane, are anti-apoptotic Bcl-2 proteins[21]. These proteins can directly bind and inhibit the proapoptotic Bcl-2 family in the mitochondria pathway of apoptosis. The proapoptotic proteins of Bcl-2 family members are functionally divided into two classes. One class is the effector molecule, which includes Bak and Bax, and permeabilizes the outer mitochondrial membrane to release cytochrome c into the cytosol. The other class is the BH3-only proteins including Bad, Bid, Bik, Bim, Bmf, bNip3, Hrk, Noxa and Puma, which promote cell apoptosis through protein-protein interactions with other Bcl-2 family members[21].

Our data indicates that galangin causes Bax translocation to the mitochondria in HCC cells. In non-apoptotic cells, Bax is located in the cytosol or loosely bound to the outer membrane of the mitochondria in monomeric forms. However, Bax translocates to the mitochondrial membrane and homodimerizes in the presence of a death signal. As a result, the outer mitochondrial membrane is permeable to release cytochrome c and AIF into the cytosol. The release of cytochrome c, which is an important protein in the electron transfer chain, can lead to reduced mitochondria membrane potential and adenosine triphosphate (ATP) synthesis. The results of our experiments showed that galangin induces cytochrome c release and decreases mitochondrial membrane potential.

In the cytosol, cytochrome c can bind to Apaf-1, which is a cytosolic protein. Apaf-1 undergoes a conformational change upon binding to dATP or ATP, leading to the formation of the apoptosome complex. The apoptosome recruits procaspase-9, resulting in caspase 9-caspase 3 activation. This caspase cascade is responsible for the hydrolysis of key cytoplasmic proteins and for the cleavage of genomic DNA nucleosomes into 180 bp fragments via caspase-activated DNase, such as PARP. Caspase-3 is an executioner of apoptosis that subsequently cleaves many important intracelluar substrates, leading to chromatin condensation, nucleosomal DNA fragmentation, nuclear membrane breakdown, externalization of phosphatidylserine, and formation of apoptotic bodies[22]. Our data also shows galangin-treated HCC cells did indeed cause caspase-9 and caspase-3 activation, and PARP cleavage.

In our study, overexpression of Bcl-2 could suppress the apoptotic effects of galangin on HCC cells. Bcl-2 is an important anti-apoptotic protein that suppresses different drug-induced activation of the mitochondria-apoptotic pathway, such as etoposide[23], berberine[24], safatoposide[25], epigallocatechin-3-gallate[26], curcumin[27] and anti-inflammatory drugs[28]. The Bcl-2 protein can block the oligomerization of Bax and Bak and inhibit the apoptotic program[29,30]. Moreover, our data also showed that Bcl-2 decrease could enhance HCC cell sensitivity to galangin. These results show that Bcl-2 can modulate the effects of galangin on HCC cells and indicate that galangin induces apoptosis viathe mitochondrial pathway.

We demonstrated that AIF is released from mitochondria into the cytosol in HCC cells upon galangin treatment. AIF migrates into the nucleus and induces high-molecular-mass DNA fragmentation and marginal chromatin condensation independent of caspases[31,32]. Therefore, HCC cells undergoing apoptosis may be due to a combination of caspase activation and AIF release.

In summary, we demonstrate that galangin induces HCC cell apoptosis via the mitochondrial pathway. Our data demonstrated that (1) galangin induces HCC cell apoptosis by triggering Bax translocation to the mitochondria; (2) galangin-treated HCC cells causes the release of AIF and cytochrome c into the cytosol from the mitochondria; and (3) overexpression of Bcl-2 attenuated galangin-induced HepG2 cells apoptosis, while down-regulated Bcl-2 expression enhanced galangin to induce cell apoptosis.



Our observation exhibits that GA treatments upregulated levels of p21 and p27, whereas downregulated levels of CDK4-cyclin D1 and cyclin E-CDK2 complexes. Taken together, it suggests that induction of G1 phase arrest of TNBC cells by GA treatments may attribute to upregulation of p21 and p27, and the consequent disrupting CDK4-cyclin D and CDK2-cyclin E complexes. Mounting evidences have indicated that cell-cycle arrest and apoptosis may be linked. CDK inhibitors have been suggested to be indirectly involved in apoptosis through regulation of CDKs. p21 can be promoted by both p53-dependent and p53-independent mechanisms following stress [42]. Moreover, overexpression of p21 has been reported to trigger apoptosis [43]. Our findings show that GA treatment increased ratio of sub-G1 phase and number of apoptotic cells in MDA-MB-231 cells, suggesting that GA treatment not only induced G1 phase arrest but also triggered apoptosis of MDA-MB-231 cells.Stress signals can activate p38 MAPK cascades and subsequently mediate cellular responses such as cellular apoptosis.

Conclusion The present study demonstrates that GA treatment significantly reduces cell viability of malignant human breast cancer cell line MDA-MB-231 via induction of both G1 phase arrest and apoptosis, which may attribute to the increase of p21 and p27 by activation of p53. These findings indicate that GA may provide potent


Gallic Acid Induces Apoptosis in 3T3-L1 Pre-adipocytes via a Fas- and Mitochondrial-Mediated Path


Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally abundant plant phenolic compound. Our previous studies have shown that some phenolic acids such as gallic acid inhibit cell growth and induce apoptosis in 3T3-L1 pre-adipocytes. However, the molecular mechanism of gallic acid in the induction of cell apoptosis is still unclear. In this study, we investigated the effect of gallic acid on the apoptotic pathway in 3T3-L1 pre-adipocytes. Western blot data revealed that gallic acid stimulated an increase in the protein expression of Fas, FasL, and p53. The ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members was changed by gallic acid treatment. Gallic acid released mitochondrial cytochrome c into the cytosol and subsequently induced the activation of caspase-9 and caspase-3, which were followed by the cleavage of poly(ADP-ribose) polymerase.

Pretreatment with a general caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) prevented gallic acid from inhibiting cell viability in 3T3-L1 pre-adipocytes. The data also indicated that treatment with gallic acid inhibited histone deacetylase activity in 3T3-L1 pre-adipocytes. These results demonstrate that gallic acid induces apoptosis in 3T3-L1 pre-adipocytes through the Fas and mitochondrial pathway. The induction of cell apoptosis by gallic acid may prove to be a pivotal mechanism for decreased pre-adipocyte proliferation.

Keywords: Gallic acid; apoptosis; 3T3-L1 pre-adipocytes

Gambogic Acid

(Garcinia hanburyi Hook. aka “Hanbury’s garcinia”)

matrix metalloproteinase (MMP) 2 and 9 suppression


Cancer cell invasion is one of the crucial events in local spreading, growth, and metastasis of tumors. The present study investigated the antiinvasive and antimetastatic action of gambogic acid (GA) in MDA-MB-435 human breast carcinoma cells. GA caused a concentration-dependent suppression of cell invasion through Matrigel and significantly inhibited lung metastases of the cells transplanted in vivo.

The potent effects of GA have been attributed to its ability to reduce the expression of matrix metalloproteinases (MMP) 2 and 9 in vitro and in vivo both at the protein and mRNA levels, which were associated with protein kinase C (PKC) signaling pathway as supported by the diminished antiinvasive effect of GA in the presence of specific activator of the pathway. Collectively, our data demonstrated that GA exhibited antiinvasion properties on highly invasive cancer cells via PKC mediated MMP-2/9 expression inhibition. This indicated that GA can be served as a potential novel therapeutic candidate for the treatment of cancer metastasis.

Gambogic acid, a potent inhibitor of survivin, reverses docetaxel resistance in gastric cancer cells.


Objective: Chemoresistance is a major obstacle to successful cancer chemotherapy. In this study, we examined the ability of gambogic acid (GA) to reverse docetaxel resistance in BGC-823/Doc gastric cancer cells.

Methods: The cytotoxic and apoptotic effect of drugs were evaluated by MTT assay and double staining with both Annexin-V-FITC and PI. Cell cycle analysis was determined by PI-stained flow cytometry. Expression of survivin and bcl-2 were evaluated by real-time quantitative RT-PCR.

Results: Treatment of BGC-823/Doc cells with gambogic acid at concentrations of 0.05 microM, 0.1 microM, and 0.2 microM, led to a dramatic increase in docetaxel-induced cytotoxicity without any cytotoxicity by itself. In parallel, gambogic acid treatment caused an increase in apoptotic cell death by docetaxel. Cell cycle analysis indicated that gambogic acid treatment potentiated docetaxel-induced G2/M arrest. Analysis of apoptotic associated gene revealed that gambogic acid singly or in combination with docetaxel significantly downregulate the mRNA expression of survivin, while with no effect on bcl-2.

Conclusion: Our results describe the potential role of gambogic acid to reverse docetaxel resistance though downregulation of survivin, which may make it an attractive new agent for the chemosensitization of cancer cells.


Ganoderma lucidum (Reishi)

Ganoderma Lucidum (Reishi) Inhibits Cancer Cell Growth and Expression of Key Molecules in Inflammatory Breast Cancer


Inflammatory breast cancer (IBC) is the most lethal and least understood form of advanced breast cancer. Its lethality originates from its nature of invading the lymphatic system and absence of a palpable tumor mass. Different from other metastatic breast cancer cells, IBC cells invade by forming tumor spheroids that retain E-cadherin-based cell–cell adhesions. Herein we describe the potential of the medicinal mushroom Ganoderma lucidum (Reishi) as an attractive candidate for anti-IBC therapy. Reishi contains biological compounds that are cytotoxic against cancer cells. We report the effects of Reishi on viability, apoptosis, invasion, and its mechanism of action in IBC cells (SUM-149).

Results show that Reishi selectively inhibits cancer cell viability although it does not affect the viability of noncancerous mammary epithelial cells. Apoptosis induction is consistent with decreased cell viability. Reishi inhibits cell invasion and disrupts the cell spheroids that are characteristic of the IBC invasive pathology. Reishi decreases the expression of genes involved in cancer cell survival and proliferation (BCL-2, TERT, PDGFB), and invasion and metastasis (MMP-9), whereas it increases the expression of IL8. Reishi reduces BCL-2, BCL-XL, E-cadherin, eIF4G, p120-catenin, and c-Myc protein expression and gelatinase activity. These findings suggest that Reishi is an effective anti-IBC therapeutic.



Genistein

(from Lycium barbarum L. aka “Chinese wolfberry” and “Himalayan /Tibetan goji”)

inhibits tyrosine kinase


Genistein, a natural isoflavonoid phytoestrogen, is a strong inhibitor of protein tyrosine kinases. We analyzed the effects of genistein on in vitro growth, cell-cycle progression and chromatin structure of Jurkat cells, a T-cell leukemia line with a constitutively increased tyrosine phosphorylation pattern. Exposure of in vitrocultured Jurkat cells to genistein resulted in a dose-dependent, growth inhibition. Cell-cycle analysis of genistein-treated cells revealed a G2/M arrest at low genistein concentrations (5–10 μg/ml), while at higher doses (20–30 μg/ml) there was also a perturbation in S-phase progression.

The derangements in cell-cycle control were followed by apoptosic death of genistein-treated cells. Immunocytochemical analysis of cells stained with a FITC-conjugated anti-phosphotyrosine monoclonal antibody showed that 30 μ/ml genistein effectively inhibit tyrosine kinase activityin cultured Jurkat cells. Our results indicate that the natural isoflavone genistein antagonizes tumor cell growth through both cell-cycle arrest and induction of apoptosis and suggest that it could be a promising new agent in cancer therapy.

Inhibits BCL-2


Genistein inactivates bcl-2, delays the G2/M phase of the cell cycle, and induces apoptosis of human breast adenocarcinoma MCF-7 cells.The aim of this study was to identify the molecular mechanism of action of the isoflavone, genistein. Genistein at 0.15 mM caused MCF-7 apoptotic cell death, which was accompanied by cell cycle delay in the G2/M phase. Twenty-four hours post-treatment, 47.3% of the MCF-7 cells accumulated at G2/M, compared with 19.9% in the untreated controls. At 0.15 mM, genistein caused an increase in the steady-state levels of the wild-type tumour suppressor p53, which was attributed to stabilising the tumour suppressor protein, since p53 mRNA levels did not increase.

Prior to the upregulation of p53, which became evident within 6 h of genistein treatment, there was increased bcl-2 phosphorylation at 30 min post-treatment. Although early changes (30-120 min) in the phosphotyrosine peptide patterns were not detected, after 24h, genistein inhibited phosphorylation of several peptides. These results suggest that genistein’s dual roles of protein tyrosine kinase inhibitor and topoisomerase II inhibitor are essential for the initiation of apoptosis.

Hsp90 INHIBITIOR


Genistein down-regulates androgen receptor by modulating HDAC6-Hsp90 chaperone function.Androgen receptor (AR) is a ligand-activated transcription factor belonging to the steroid hormone receptor family and is very important for the development and progression of prostate cancer. The soy isoflavone genistein has been shown previously to down-regulate AR in androgen-dependent prostate cancer cell lines such as LNCaP. However, the mechanism(s) by which AR is down-regulated by genistein is still not known fully. We show a new mechanism by which genistein inhibits AR protein levels. We show that genistein-treated LNCaP cells exhibit increased ubiquitination of AR, suggesting that AR protein is down-regulated via a proteasome-mediated pathway. AR is normally stabilized by the chaperone activity of the heat shock protein Hsp90.

The increased ubiquitination of AR after genistein treatment is attributed to decreased Hsp90 chaperone activity as assessed by its increased functionally inactive acetylated form. Consistent with this result, we find that HDAC6, which is a Hsp90 deacetylase, is inhibited by the antiestrogenic activity of genistein. Hence, in this study, we elucidate a novel mechanism of AR down-regulation by genistein through inhibition of HDAC6-Hsp90 cochaperone function required to stabilize AR protein. Our results suggest that genistein could be used as a potential chemopreventive agent for prostate cancers along with known inhibitors of HDAC6 and Hsp90.

Bcl-2, Bcl-XL & CDC2 downregulation

Genistein, biochanin-A, and daidzein, the predominant soy isoflavones, have been reported to lower the risk of cancer, but it is not known whether they protect against human hepatoma cancer. This study was designed to investigate their effects on cell growth, the cell cycle, and apoptosis induction in the human hepatoma cell lines, HepG2, Hep3B, Huh7, PLC, and HA22T. Genistein, biochanin-A, and daidzein inhibited growth of all five lines in a dose-dependent manner. DNA fragmentation studies and the TUNEL assay demonstrated that isoflavones caused tumor cell death by induction of apoptosis. Activation of caspase-3 and cleavage of the caspase-3 substrate, poly(ADP-ribose)polymerase, was seen in hepatoma cells after 24 hours’ exposure to isoflavones. In addition, isoflavone cytotoxicity correlated with downregulation of Bcl-2 and Bcl-XL expression.

Synergistic effects of the three isoflavones were observed on cell growth inhibition, apoptosis induction, and anti-apoptotic protein expression. Flow cytometry showed that genistein, but not biochanin-A or daidzein, induced progressive and sustained accumulation of hepatoma cancer cells in the G2/M phase as a result of inhibition of Cdc2 kinase activity. Coapplication of caffeine prevented this cell cycle arrest, but not apoptosis, showing that cell cycle arrest was not necessary for apoptosis. Furthermore, the isoflavones combination also had a significant tumor-suppressive effect in nude mice. These results suggest that isoflavones might be promising agents for the treatment of human hepatoma.


Inhibits HIF-1a & VEGF

Antiangiogenic activity of genistein in pancreatic carcinoma cells is mediated by the inhibition of hypoxia‐inducible factor‐1 and the down‐regulation of VEGF gene expression

Previous reports indicate that Genistein, a naturally occurring isoflavonoid, exhibits strong antiangiogenic activity. The underlying mechanism of inhibition, however, remains unclear. Among the biologic effects of Genistein are the inhibition of tyrosine kinases and the inhibition of hypoxic activation of hypoxia‐inducible factor‐1 (HIF‐1), one of the main regulators of VEGF gene expression.

METHODS: Hypoxic cell culture was performed in a modular incubator chamber. Vascular endothelial growth factor (VEGF) protein secretion was measured using the enzyme‐linked immunosorbent assay, binding of DNA by HIF‐1 was measured using the electrophoretic mobility shift assay, and mRNA quantification was performed using Northern blot analysis. Pancreatic carcinoma was studied in an orthotopic murine model. Angiogenesis in vivo was quantified by staining xenograft tumors for endothelial cell markers.

RESULTS: VEGF protein secretion was dose‐dependently suppressed with increasing doses of Genistein. Furthermore, treatment of pancreatic carcinoma cells with Genistein led to impaired activation of HIF‐1 under hypoxic culture conditions. Northern blot analysis indicated that VEGF mRNA expression decreased upon treatment with Genistein, both under normoxic and hypoxic culture conditions. In vivo, Genistein inhibited tumor growth for xenograft pancreatic carcinoma cells, whereas extensive hypoxia was observed in xenograft tumors and was not influenced by Genistein therapy. Similarly, decreased VEGF mRNA levels were observed in Genistein‐treated Capan‐1 xenograft tumors.

CONCLUSIONS: The current study indicates that the previously reported antiangiogenic activity of Genistein probably is mediated by the inhibition of HIF‐1, an important regulator ofVEGF gene homeostasis, particularly under low‐oxygen conditions. Therefore, this bioactive compound may well be beneficial to patients with pancreatic carcinoma.


genistein inhibits CDK2 & CDC2


Following genistein treatment of cells, an increased binding of p21 with Cdk2 and Cdc2 paralleled a significant decrease in Cdc2 and Cdk2kinase activity with no change in Cdk2 and Cdc2 expression. Genistein also induced the activation of a p21 promoter reporter construct, utilizing a sequence distinct from the p53-binding site. Analysis of deletion constructs of the p21 promoter indicated that the response to genistein could be localized to the 300 base pairs proximal to the transcription start site. These data suggest that genistein may exert a strong anticarcinogenic effect, and that this effect possibly involves an induction of p21, which inhibits the threshold kinase activities of Cdks and associated cyclins, leading to a G2/M arrest in the cell cycle progression.



gingerenone A


Identification of gingerenone A as a novel senolytic compound


Senescent cells accumulate with aging and have been shown to contribute to age-associated diseases and organ dysfunction. Eliminating senescent cells with senolytic drugs has been shown to improve age phenotypes in mouse models and there is some initial evidence that it may improve the health of persons with chronic diseases. In this study, we employed WI-38 human fibroblasts rendered senescent by exposure to ionizing radiation (IR) to screen several plant extracts for their potential senolytic and/or senomorphic activity. Of these, ginger extract (Zingiber officinale Rosc.) selectively caused the death of senescent cells without affecting proliferating cells.

Among the major individual components of ginger extract, gingerenone A and 6-shogaol showed promising senolytic properties, with gingerenone A selectively eliminating senescent cells. Similar to the senolytic cocktail dasatinib and quercetin (D+Q), gingerenone A and 6-shogaol elicited an apoptotic program. Additionally, both D+Q and gingerenone A had a pronounced effect on suppressing the senescence-associated secretory phenotype (SASP). Gingerenone A selectively promotes the death of senescent cells with no effect on non-senescent cells and these characteristics strongly support the idea that this natural compound may have therapeutic benefit in diseases characterized by senescent cell accumulation.


Ginsenoside Rg3

(a ginsenoside found in Panax ginseng and Panax japonicus var. major)

key function: Rg3 greatly suppresses the major components of SASP, IL-6 & IL-8.

Inhibition of NF-kappaB by ginsenoside Rg3

Ginsenoside Rg3, the main constituent isolated from Panax ginseng, has been of interest for use as a cancer preventive or therapeutic agent. We investigated here whether Rg3 can inhibit the activity of NF-kappaB, a key transcriptional factor constitutively activated in colon cancer that confers cancer cell resistance to chemotherapeutic agents. To investigate whether RG3 can suppress activation of NF-kappaB, and thus inhibit cancer cell growth, we examined the susceptibility of colon cancer cells (SW620 and HCT116) to treatment with Rg3 (25, 50, 75, 100 microM) and RG3-induced activation of NF-kappaB. RG3 dose-dependently inhibited cancer cell growth through induction of apoptosis and decreased NF-kappaB activity. In a further study of compounds in colon cancer, we used half of the IC(50) dose, values in combined treatments of Rg3 (50 microM) with conventional agents – docetaxel (5 nM), paclitaxel (10 nM) cisplatin (10 microM) and doxorubicin (2 microM). Compared to treatment with Rg3 or chemotherapy alone, combined treatment was more effective (i.e., there were synergistic effects) in the inhibition of cancer cell growth and induction of apoptosis and these effects were accompanied by significant inhibition of NF-kappaB activity.

NF-kappaB target gene expression of apoptotic cell death proteins (Bax, caspase-3, caspase-9) was significantly enhanced, but the expression of anti-apoptotic genes and cell proliferation marker genes (Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP), Cox-2, c-Fos, c-Jun and cyclin D1) was significantly inhibited by the combined treatment compared to Rg3 or docetaxel alone. These results indicate that ginsenoside Rg3 inhibits NF-kappaB, and enhances the susceptibility of colon cancer cells to docetaxel and other chemotherapeutics. Thus, ginsenoside Rg3 could be useful as an anti-cancer or adjuvant anti-cancer agent.


Some saponins, like ginsenosides, have been investigated for their anti-metastatic activity in human breast cancer (Nag et al., 2012). It has also been suggested that certain ginsenosides can prevent cartilage collagen matrix breakdown in patients with arthritis (Lee et al., 2014). Testing for their anti-senescence properties revealed that ginsenoside Rb1 was able to reverse the unfavorable effects of H2O2 treatment in HUVECs through a reduction in malondialdehyde concentrations, an increase in superoxide dismutase activity, a reduction in SA β-gal ac- tivity, and induction of SIRT1 expression (Liu et al., 2011; Song et al., 2014). It also restored normal conditions in human WI-38 diploid fi- broblasts after induction of premature senescence with tert-butyl hy- droperoxide (t-BHP). Senescent fibroblasts typically showed elevated p21 and p16 levels and reduced ATP synthesis associated with cell cycle arrest. Treatment with ginsenoside Rg1 attenuated these features and restarted the cell cycle towards the S phase, thereby delaying senes- cence (Chen et al., 2008).

In a study involving the D-galactose-induced mouse aging model, ginsenoside Rg1 also exerted neuroprotective ef- fects, since it i) protected hippocampal stem cells by raising SOX2 levels and glutathione peroxidase and superoxide dismutase activity, thus enhancing telomerase activity and promoting telomere elongation, and ii) reduced inflammation by reducing IL-1β, IL-6 and TNF-α levels and downregulating p53, p21Cip1/Waf1 and p19Arf gene expression, ultimately inducing a general improvement in cognitive ability and neurogenesis (Zhu et al., 2014). Two similar studies of D-galactose-induced mouse pancreas (Dong et al., 2017) and kidney (Fan et al., 2016) aging de- monstrated that ginsenoside Rg1 ameliorated aging-related conditions, reducing senescence markers and the number of damaged cells.


Anti-cancer natural products isolated from chinese medicinal herbs

Extracted from Panax ginseng C.A. Mey. (Renshen) and Panax quinquefolius L., Araliaceae (Xiyangshen), ginsenoside Rg3 (Figure 1N) is a biologically active component with both in vitro and in vivo anti-cancer activities [227, 228]. The anti-proliferative mechanism of ginsenoside Rg3 is associated with the inactivation of NF-κB [229, 230], modulation of MAPKs [231] and the down-regulation of Wnt/β-catenin signaling [232]. Ginsenoside Rg3 affects the ephrin receptor pathway in HCT-116 human colorectal cancer cells [233]. The anti-proliferative mechanism of ginsenoside Rg3 is also associated with the molecules of mitotic inhibition, DNA replication, repair, and growth factor signaling [234].

Ginsenoside Rg3 inhibits the proliferation of HUVEC and suppresses the capillary tube formation of HUVEC on a matrigel at nanomole scales in the presence or absence of VEGF. Ginsenoside Rg3 attenuates VEGF-induced chemo-invasion of HUVEC and ex vivo microvascular sprouting in rat aortic ring. bFGF-induced angiogenesis may be abolished by ginsenoside Rg3[227]. In lung metastasis models of ovarian cancer, ginsenoside Rg3 decreases the number of tumor colonies in the lung and vessels oriented toward the tumor mass [235]. This effect may be partially due to the inhibition of angiogenesis and the decrease in MMP9 expression [235].

Ginsenoside Rg3 increases the efficacy of cancer chemotherapy. Combined treatments with ginsenoside Rg3 enhance the susceptibility of colon cancer cells to docetaxel, paclitaxel, cisplatin and doxorubicin; the mechanism of such an enhancement is related to the inhibition of the constitutively activated NF-κB [229]. A similar phenomenon has been observed in prostate cancer cells, in which the combination of ginsenoside Rg3 and docetaxel more effectively induces apoptosis and G1 cell cycle arrest, accompanied by the inhibition of NF-κB activity [230]. Low-dose administration of cyclophosphamide (CTX) with ginsenoside Rg3 increases the efficacy of targeting the tumor microvasculature and the two-drug combination treatment results demonstrate the longest patient survival rates [236]. Ginsenoside Rg3 combined with gemcitabine not only enhances the efficacy of tumor growth suppression and survival prolongation, but also decreases VEGF expression and microvessel density in tumors [228].


GLYCITEIN


Stat3 NFKB INHIBITOR


Glycitein is an isoflavone that reportedly inhibits the proliferation of human breast cancer and prostate cancer cells. However, its anti‐cancer molecular mechanisms in human gastric cancer remain to be defined. This study evaluated the antitumor effects of glycitein on human gastric cancer cells and investigated the underlying mechanisms. We used MTT assay, flow cytometry and western blotting to investigate its molecular mechanisms with focus on reactive oxygen species (ROS) production. Our results showed that glycitein had significant cytotoxic effects on human gastric cancer cells. Glycitein markedly decreased mitochondrial transmembrane potential (ΔΨm) and increased AGS cells mitochondrial‐related apoptosis, and caused G0/G1 cell cycle arrest by regulating cycle‐related protein.

Mechanistically, accompanying ROS, glycitein can activate mitogen‐activated protein kinase (MAPK) and inhibited the signal transducer and activator of transcription 3 (STAT3) and nuclear factor‐kappaB (NF‐κB) signaling pathways. Furthermore, the MAPK signaling pathway regulated the expression levels of STAT3 and NF‐κB upon treatment with MAPK inhibitor and N‐acetyl‐L‐cysteine (NAC). These findings suggested that glycitein induced AGS cell apoptosis and G0/G1 phase cell cycle arrest via ROS‐related MAPK/STAT3/NF‐κB signaling pathways. Thus, glycitein has the potential to a novel targeted therapeutic agent for human gastric cancer.



Gossypol

Bcl-2 and Bcl-xl inhibitor

Induction of apoptosis and antitumor effects of a small molecule inhibitor of Bcl-2 and Bcl-xl, gossypol acetate, in multiple myeloma in vitro and in vivo

Gossypol is a naturally occurring polyphenolic compound extracted from cotton plants. Recent studies revealed that gossypol is a non-peptidic small molecule inhibitor of Bcl-2/Bcl-xl. The aim of the present study was to investigate the induction of apoptosis and antitumor effects of gossypol acetate in multiple myeloma and the possible mechanism(s) of action. Our results showed that gossypol acetate resulted in a dose- and time-dependent inhibition of multiple myeloma cell proliferation, with an IC50 value to both U266 and Wus1 cells at 2.4, 2.2 µM at 48 h after treatment. Gossypol acetate effectively induced the apoptosis of multiple myeloma cells as demonstrated by typical morphological changes, DNA ladder formation and increase in the percentage of cells in subdiploid peak.

Furthermore, colorimetric assays showed activation of both caspase-3 and caspase-9. Bcl-2 and Bcl-xl expression was decreased by 86.5±1.2% and 35.9±3.6%, respectively, after treatment with gossypol acetate at 25 µmol/l for 24 h. Preliminary studies in vivo showed that a growth inhibition (T/C) of 30.9% (gossypol acetate 40 mg/kg) was obtained in Balb/C mice bearing Wus1 cells. In addition, there was no body weight loss for the treated group in comparison with the vehicle mice. Our results demonstrated that the potent inhibitor of Bcl-2 and Bcl-xl gossypol acetate had significant antiproliferative and antiapoptotic effects on multiple myeloma cells in vitro and in vivo. Gossypol acetate may represent a promising new anticancer agent with a novel molecular mechanism and warrants further investigation as a single agent, or in combination with other chemotherapeutics, for human multiple myeloma with Bcl-2 overexpression.


In summary, gossypol is a dual inhibitor of MDM2 and VEGFthat disrupts the molecular interaction between MDM2 protein and VEGF mRNA, induces MDM2 self-ubiquitination and degradation, decreases VEGF mRNA stability and protein translation simultaneously, and therefore exerts anti-cancer effects through apoptotic and anti-angiogenesis pathways in human breast cancer in vitro and in vivo, regardless of the p53 status of the cancer cells. We believe development of these MDM2-VEGF inhibitors as potential anticancer drugs for clinical use is worthwhile and represents a novel strategy for improving cancer outcome.


Antiapoptotic members of the Bcl-2 family proteins are overexpressed in prostate cancer and are promising molecular targets for modulating chemoresistance of prostate cancer.(-)-Gossypol, a natural BH3 mimetic, is a small-molecule inhibitor of Bcl-2/Bcl-xL/Mcl-1 currentlyA natural BH3 mimeticinduces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2–Beclin1 interaction at endoplasmic reticulum A natural BH3-mimetic, small-molecule inhibitor of Bcl-2, (−)-gossypol, shows promise in ongoing phase II and III clinical trials for human prostate cancer. In this study we show that (−)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent (AD) cells with low Bcl-2 and sensitive to apoptosis.
The Bcl-2 inhibitor induces autophagy through blocking Bcl-2–Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1, and activating the autophagic pathway. The (−)-gossypol-induced autophagy is dependent on Beclin1 and Atg5. Our results show for the first time that (−)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (−)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which will facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.


Objective: To determine the effects of guggulsterone (GS), the active substance in guggulipid, on apoptosis, adipogenesis, and lipolysis using 3T3‐L1 cells.

Methods and Procedures: For apoptosis and lipolysis experiments, mature adipocytes were treated with GS isomers. Viability, apoptosis, and caspase 3/7 activation were quantified using MTS, enzyme‐linked immunosorbent assay (ELISA), caspase‐Glo 3/7 activity assay, respectively. The expression of cytochrome c was demonstrated by western blot. Lipolysis was quantified by measuring the release of glycerol. For adipogenesis experiments, postconfluent preadipocytes were incubated with GS isomers for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye. Western blot was also used to demonstrate the adipocyte‐specific transcription factors peroxisome proliferator–activated receptor γ2 (PPARγ2), CCAAT/enhancer binding protein α (C/EBPα), and C/EBPβ.

Results: In mature adipocytes cis‐GS decreased viability, whereas the trans‐GS isomer had little effect. Both isomers caused dose‐dependent increases in apoptosis and cis‐GS was more effective than trans‐GS in inducing apoptosis. cis‐ and trans‐GS also increased caspase‐3 activity and release of cytochrome c from mitochondria. In maturing preadipocytes, both isomers were equally effective in reducing lipid content. The adipocyte‐specific transcription factors PPARγ2, C/EBPα, and C/EBPβ were downregulated after treatment with cis‐GS during the maturation period. Furthermore, cis‐GS increased basal lipolysis of mature adipocytes, but trans‐GS had no effect.

Discussion: These results indicate that GS isomers may exert antiobesity effects by inhibiting differentiation of preadipocytes, and by inducing apoptosis and promoting lipolysis of mature adipocytes. The cis‐GS isomer was more potent than the trans‐GS isomer in inducing apoptosis and lipolysis in mature adipocytes.


Enhanced pro-apoptotic and anti-adipogenic effects of genistein plus guggulsterone in 3T3-L1 adipocytes



Panax notoginseng extract

GS25 (PANAX NOTOGINSENG)


Prevention of prostate cancer by natural product MDM2 inhibitor GS25: in vitro and in vivo activities and molecular mechanisms


Prostate cancer remains a major health problem in the USA and worldwide. There is an urgent need to develop novel approaches to preventing primary and metastatic prostate cancer. We have identified 25-OCH3-protopanaxadiol (GS25), the most active ginsenoside that has been identified so far; it has potent activity against human cancers, including prostate cancer. However, it has not been proven if GS25 could be a safe and effective agent for cancer prevention. In this study, we used the TRAMP model and clearly demonstrated that GS25 inhibited prostate tumorigenesis and metastasis with minimal host toxicity.
Mechanistically, GS25 directly bound to the RING domain of MDM2, disrupted MDM2–MDMX binding and induced MDM2 protein degradation, resulting in strong inhibition of prostate cancer cell growth and metastasis, independent of p53 and androgen receptor status. In conclusion, our in vitro and in vivo data support the potential use of GS25 in prevention of primary and metastatic prostate cancer.

Therapeutic Potential and Cellular Mechanisms of Panax Notoginseng on Prevention of Aging and Cell Senescence-Associated Diseases


Owing to a dramatic increase in average life expectancy, most countries in the world are rapidly entering an aging society. Therefore, extending health span with pharmacological agents targeting aging-related pathological changes, are now in the spotlight of gerosciences. Panax notoginseng (Burk.) F. H. Chen, a species of the genus Panax, has been called the “Miracle Root for the Preservation of Life,” and has long been used as a Chinese herb with magical medicinal value. Panax notoginseng has been extensively employed in China to treat microcirculatory disturbances, inflammation, trauma, internal and external bleeding due to injury, and as a tonic.

In recent years, with the deepening of the research pharmacologically, many new functions have been discovered. This review will introduce its pharmacological function on lifespan extension, anti-vascular aging, anti-brain aging, and anti-cancer properties, aiming to lay the ground for fully elucidating the potential mechanisms of Panax notoginseng’s anti-aging effect to promote its clinical application.


[Mechanisms of delay endothelial cell replicative senescence by extracts from Panax ginseng, Panax notoginseng and Ligusticum chuanxiong].


To explore effect of extracts from Panax ginseng, P. notoginseng and Ligusticum chuanxiong on human umbilical endothelial cells (HUVECs) replicative senescence.HUVECs were induced to aging by generation cultivating to the eighth cells in order to establish a model of endothelial cells replicative senescence. The cultured HUVECs in vitro were divided into 4 groups, the eighth generation cell-senescence untreated group, Vitamin E group, herbal treated high dose and low dose groups. Changes of HUVECs aging were observed by method of SA-beta-gal stained HUVECs and cells cycle were analyzed. Contents of ROS in cells, the levels of anti-superoxide (O2-) and nitric oxide (NO) in cell mediums were examined.
Western blot were used to analyse protein expression of NADPH oxidase p47phox, angiotensin type 1 and 2 receptor (AT1R, AT2R).Compared with Vitamin E group, the positive cell numbers of beta-gal stained HUVECs were enhanced, cell proliferation was depressed, and the fluorescence intensity of ROS was increased, at the same time, less NO and more O2- in cells were produced in the eighth generation cell-senescence untreated group. Protein expression of p47phox, AT1R and AT2R in cells increased compared with Vit E group. Chinese herbs of high dose and low dose could improve condutions of HUVECs aging. Chinese herbs of high dose and low dose could reduce the positive cell numbers of beta-gal stained HUVEC, increase cell proliferation and decrease fluorescence intensity of ROS in cells, at the same time, cells secreting more NO and less O2-.
Protein expression of p47phox, AT1R and AT2R in cells treated with Chinese herbs of high dose and low dose were decreased compared with Vit E group.The study indicated that extracts from P. ginseng, P. notoginseng and L. chuanxiong could delay endothelial cell replicative senescence. Herbal extracts downregulate the expression of NAD (P) H oxidase subunit-p47phox by means of ROS, hence decrease O2- production and ultimately delay HUVECs in vitro senescence.


To investigate the protective effect of Panax notoginseng Total Saponins (PNTS) on D-galactose-induced H9c2 cell senescence and the underlying mechanism.D-galactose was used to cause H9c2 cells senescence. Different concentrations of PNTS (5,25 and 50 μg/mL) were added into medium to protect H9c2 cells. Cell senescence was identified by senescence associated β-galactosidase. Level of reactive oxgen species (ROS) was observed according to the effect of DCFH-DA detection. The activity of superoxide dicmutase (SOD) and the content of malondialdehyde (MDA) in cells were measured by biochemical assay kits. The apoptosis of cells was tested by Hochest.Compared with the control group, the number of β-galactosidase positive cells and the fluorescence intensity of ROS in the model group were markedly increased.
Meanwhile, the activity of SOD was decreased whereas the content of MDA was increased. The apoptosis level assessed by the Hochest dyeing was significantly increased with chromatin concentration and condensation. Compared with the model group, the quantity of β-galactosidase in different PNTS treatment group was obviously decreased, the activity of SOD was increased and the content of MDA was reduced. The apoptosis rate in the cells treated with PNTS was improved. PNTS improved D-galactose-induced H9c2 cell senescence through upregulation of antioxidative ability and attenuation of cell apoptosis.

[Effect of Panax notoginseng saponins on syp and tau gene expression in brain of senescence accelerated mouse].


Objectives: To study the effect of Panax notoginseng saponins (PNS) on (synaptophysin, syp) and tau gene expression in the brain tissue in senescence accelerated mouse prone 8 (SAMP 8).
Methods: SAMP8 were randomly divided into 4 groups: PNS 23.38, 93.50 mg x kg(-1) group, huperzin A 0.038 6 mg x kg(-1) x d(-1) group and blank control group; the drug groups were treated with the designed drugs respectively per day by intragastric administration for 4 consecutive weeks, and double distilled water was given to blank control group. After treatment, the mRNA content of tau and syp were assayed by reverse transcription (RT) and real-time polymerase chain reaction (real-time PCR).
Results: Compared with blank control group, the syp mRNA contents were increased in PNS groups (P < 0.05 or P < 0.01), and the tau mRNA content were not significant difference in all groups.
Conclusions: This study suggests that PNS can up-regulate syp gene expression at transcriptional level in the brain of SAMP 8.

[Protective effect of total saponins of Panax notoginseng combined with total flavonoids of epimedium on D-galactose-incuced senescence of H9c2 cell].


To investigate the protective effect of Panax notoginseng saponins combined with total flavonoids of epimedium on D-gal-induced senescence of H9c2 cells and explore its underlying mechanisms. The 50 mol•L⁻¹ D-gal was used to induce H9c2 cells senescence. Different concentrations of TPNS, TFE, and TPNS combined with TFE were used for 4 hours for pre-treatment. D-gal was used to stimulate H9c2 cardiac muscle cells for 24 h. Then in order to determine the best combined scheme, MTT was used to detect cell viability. Cell senescence was identified by β-galactosidase staining. Levels of reactive oxygen species(ROS) was observed by DCFH-DA detection.

The changes of mitochondrial membrane potential were identified by JC-1 detection. Protein levels of silentmating type information regulation 2 Homolog-1(SIRT1), peroxisomal proliferator-activated receptor-coactivator 1α(PGC-1α) and silentmating type information regulation 2 Homolog-3(SIRT3) were detected by western blot analysis. The results showed that TPNS(5 mg•L⁻¹) combined with TFE(5 mg•L⁻¹) had significant synergistic effect on H9c2 myocardial cell proliferation(Q=1.154), so 5 mg•L-1TPNS combined with 5 mg•L⁻¹ TFE was determined as the best scheme. The quantity of β-galactosidase staining and the fluorescence intensity of ROS were apparently decreased in 5 mg•L⁻¹ TPNS combined with 5 mg•L⁻¹ TFE scheme.

Meanwhile, it markedly increased the florescence intensity of mitochondrial membrane potential and enhanced the protein expression of SIRT1, PGC-1α and SIRT3. TPNS combined with TFE could protect H9c2 cells from D-gal-induced senescence.The mechanism might be related to adjusting the signal pathways of SIRT1/PGC-1α, SIRT3, adjusting the structure and function of mitochondria and reducing oxidative stress injury.


anticancer ginsenoside 25-OCH3-PPD, a natural inhibitor of the MDM2 oncogene: Nanoparticle preparation, characterization, in vitro and in vivo anti-prostate cancer activity, and mechanisms of action


The Mouse Double Minute 2 (MDM2) oncogene plays a critical role in cancer development and progression through p53-dependent and p53-independent mechanisms. Both natural and synthetic MDM2 inhibitors have been shown anticancer activity against several human cancers. We have recently identified a novel ginsenoside, 25-OCH3-PPD (GS25), one of the most active anticancer ginsenosides discovered thus far, and have demonstrated its MDM2 inhibition and anticancer activity in various human cancer models, including prostate cancer.

However, the oral bioavailability of GS25 is limited, which hampers its further development as an oral anticancer agent. The present study was designed to develop a novel nanoparticle formulation for oral delivery of GS25. After GS25 was successfully encapsulated into PEG-PLGA nanoparticles (GS25NP) and its physicochemical properties were characterized, the efficiency of MDM2 targeting, anticancer efficacy, pharmacokinetics, and safety were evaluated in in vitro and in vivo models of human prostate cancer.

Our results indicated that, compared with the unencapsulated GS25, GS25NP demonstrated better MDM2 inhibition, improved oral bioavailability and enhanced in vitro and in vivo activities. In conclusion, the validated nano-formulation for GS25 oral delivery improves its molecular targeting, oral bioavailability and anticancer efficacy, providing a basis for further development of GS25 as a novel agent for cancer therapy and prevention.


HESPERIDIN

NF-KB INHIBITOR


Gout arthritis is a painful inflammatory disease induced by monosodium urate (MSU) crystals. We evaluate the therapeutic potential of the flavonoid hesperidin methylchalcone (HMC) in a mouse model of gout arthritis induced by intra-articular injection of MSU (100 μg/10 μL). Orally given HMC (3-30 mg/kg, 100 μL) reduced in a dose-dependent manner the MSU-induced hyperalgesia (44%, p < 0.05), edema (54%, p < 0.05), and leukocyte infiltration (70%, p < 0.05). HMC (30 mg/kg) inhibited MSU-induced infiltration of LysM-eGFP+cells (81%, p < 0.05), synovitis (76%, p < 0.05), and oxidative stress (increased GSH, FRAP, and ABTS by 62, 78, and 73%, respectively; reduced O2 and NO by 89 and 48%, p < 0.05) and modulated cytokine production (reduced IL-1β, TNF-α, IL-6, and IL-10 by 35, 72, 37, and 46%, respectively, and increased TGF-β by 90%, p < 0.05).

HMC also inhibited MSU-induced NF-κB activation (41%, p < 0.05), gp91phox (66%, p < 0.05) and NLRP3 inflammasome components mRNA expression in vivo (72, 77, 71, and 73% for NLRP3, ASC, pro-caspase-1, and pro-IL-1 β, respectively, p < 0.05), and induced Nrf2/HO-1 mRNA expression (3.9- and 5.1-fold increase, respectively, p < 0.05). HMC (30, 100, and 300 μM) did not inhibit IL-1β secretion by macrophages primed by LPS and challenged with MSU (450 μg/mL), demonstrating that the anti-inflammatory effect of HMC in gout arthritis depends on inhibiting NF-κB but not on direct inhibition of inflammasome.The pharmacological effects of HMC indicate its therapeutic potential for the treatment of gout.


UPREGULATES NRF2


KEY FINDINGS: Hesperidin treatment effectively protected aged rat heart by increasing the activity of enzymic antioxidants. Hesperidin upregulated the protein levels of nuclear factor erythroid 2-related factor 2, which is responsible for maintaining the antioxidant status of the cell.

CONCLUSIONS: Hesperidin could be useful in protecting cardiomyocytes against age-related increase in oxidative stress mediated by Nrf2 upregulation.


ANTI SENESCENCE EFFECTS


Citrus unshiu peel extract, containing hesperetin (metabolite of hesperidin), decreased expression levels of 𝛽-galactosidase, matrix metalloproteinase-1, and the number of senescent cells. Moreover, pretreatment of human fibroblasts with hesperetin glucuronides induced a 25% protection against UV-A-induced necrotic cell death [38].

In addition to upregulation of Nrf2 expression in the senescent rat heart [115], methylhesperidin, methylated derivative of hesperidin, enhanced translocation of Nrf2 from cytoplasm to nuclear, resulting in upregulation of antioxidant- related gene expression and reduction in reactive oxygen species, consequently leading to protection of epidermal keratinocytes against UVB-induced damage in keratinocyte cultures [116].


P38 MAPK INHIBITOR


AttenuationofInflammation. Developmentofinflamma- tion is a complex process involving interactions of a number of molecules in various signaling pathways, including p38 mitogen-activated protein kinase (MAPK) pathway [133]. Inhibition of p38 MAPK signaling pathway can markedly lower expression of IL-1𝛽 and IL-6, IL-8, IL-18, and TNF𝛼, in both macrophage culture and mice [134, 135]. Study showed that, prior to H2O2 stimulation, treatment of keratinocytes with hesperidin for 2 hr induced over 50% reduction in NF-𝜅B and phosphorylated p38 MAPK in comparison with those without pretreatment with hesperidin [62]. Likewise, treatment of mouse RAW 264.7 cells with hesperetin metabo- lite almost completely reversed lipopolysaccharide-induced increase in NF-𝜅B expression in addition to reductions in phosphorylated p38 MAPK and c-Jun N-terminal kinase 1/2 [61].

Thus, hesperidin-induced inhibition of p38 MAPK signaling pathway could contribute its attenuation of inflammation.


Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was in a dose-dependent manner, and IC50 levels were determined. The sensitivity of human HepG2 cells was as follows: hesperidin > naringin > neohesperidin > naringenin. Hesperidin induced HepG2 cells to undergo apoptosis in a dose-dependent manner as evidenced by the externalization of phosphatidylserine and determined by annexin V-fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Hesperidin did not induce the generation of reactive oxygen species, which was determined by using 2′,7′-dichlorohydrofluorescein diacetate and flow cytometry method.

The number of hesperidin-treated HepG2 cells with the loss of mitochondrial transmembrane potential increased concentration dependently, using 3,3′-dihexyloxacarbocyanine iodide employing flow cytometry. Caspase-9, -8, and -3 activities were activated and increased in hesperidin-treated HepG2 cells. Bcl-xL protein was downregulated whereas Bax, Bak, and tBid protein levels were upregulated after treatment with hesperidin in a dose-dependent manner. In conclusion, the bioflavanone from Citrus seeds, hesperidin, induced human HepG2 cell apoptosis via mitochondrial pathway and death receptor pathway. Citrus seed flavonoids are beneficial and can be developed as anticancer drug or food supplement, which still needs further in vivo investigation in animals and human beings.



BLOCKS TLR4

HES decreased the expression of TLR4 protein and increased the expression of GLUT2 protein. Meanwhile, the expression of GLUT2 protein would increased when TLR4 was blocked. Based on the above knowledge, we speculated that HES ameliorated IR by directly affecting the expression of TLR4 and NF‐κB proteins and indirectly affecting the expression of GLUT2 protein by regulating TLR4.

However, HES could not affect GLUT2 by TLR4 when TLR4 was blocked. Therefore, the effect of HES on the expression of GLUT2 protein in cells was attenuated compared with the cells that were not treated with HTA125, which reveals that TLR4 was an underlying key target for HES in ameliorating IR.


Honokiol

CDK1⊥, Bcl-2↓, Bax↑, cyclin D1↓, pAKT↓, γ-secretase activity↓, γ-secretase complex proteins↓, PPAR-γ⊥, COX-2⊥, NF-κB⊥, EGFR/P13K/AKt↓; JunB ↓ and JunD↓ caspase-8↑, caspase-9↑, caspase-3↑, PARP↑, p53↑, CD31 staining↓, LH↑, p38⊥, NF-κB⊥, Bcl-XL↓, Bad↑, cyclin E↓, (Cdk2 and Cdk4)↓, Cdk↑, p21 and p27↑, NF-κB↓, Bcl-2↓, Mcl-1↓, surcivin↓, VEGF↓, STAT3⊥, HG-induced IL1β⊥, IL18⊥, TNFα⊥, -PGE2⊥, NO⊥, and TGFβ1⊥, MCP-1⊥, MIP-1α⊥, EGFR targeting TKI⊥, Akt⊥ erlotinib⊥, EGFR signaling⊥, MAPK⊥, cyclin D1⊥

Honokiol: a novel natural agent for cancer prevention and therapy


Honokiol ((3’,5-di-(2-propenyl)-1,1’-biphenyl-2,2’-diol) is a bioactive natural product derived from Magnolia spp. Recent studies have demonstrated anti-inflammatory, anti-angiogenic, anti-oxidative and anti-cancer properties of honokiol in vitro and in preclinical models. Honokiol targets multiple signaling pathways including nuclear factor kappa B (NF-κB), signal transducers and activator of transcription 3 (STAT3), epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (m-TOR), which have great relevance during cancer initiation and progression.

Furthermore, pharmacokinetic profile of honokiol has revealed a desirable spectrum of bioavailability after intravenous administration in animal models, thus making it a suitable agent for clinical trials. In this review, we discuss recent data describing the molecular targets of honokiol and its anti-cancer activities against various malignancies in pre-clinical models. Evaluation of honokiol in clinical trials will be the next step towards its possible human applications.


INHIBITS STAT3

Honokiol suppressesSTAT3 activity induced by IL-6, one of the many growth factors that activate STAT3(25). Furthermore, STAT3 inhibition by honokiol has also been correlated with the repression of upstream protein tyrosine kinases c-Src, JAK1 and JAK2 (26). Although not yet demonstrated, suppression of STAT3 and NF-κB activation by honokiol could also be inter-linked. The p65 subunit of NF-κB has been shown to interact with STAT3 (27) and it is reported that STAT3 prolongs NF-κB nuclear retention through acetylation (28). Activation of both STAT3 and NF-κB in tumor cells by factors in tumor microenvironment (interleukins and chemokines) and release of cytokines and chemokines by tumor cells as a response (21, 28) also suggest their role in mediating the cross-talk between tumor and its microenvironment.

All these observations clearly imply that NF-κB and STAT3 inhibition by honokiol can have a multifaceted impact on the growth and spread of the tumor cells.


PI3k AKT MTOR Inhibitor / Autophagy activator

Polyphenolic compounds such as can honokiol act as anticar cinogenic agents. Honokiol (3-,5-di-(2-propenyl)-1,1′ -biphenyl- 2,2′-diol), a naturally occurring dietary product isolated from an extract of seed cones from Magnolia grandiflora, can induce cellular autophagy by modulating the PI3K/Akt/mTOR signaling pathway in neuroblastoma cells (Yeh et al., 2016). Treatment with honokiol has been shown to reduce PI3K content, and in turn, inhibit Akt, which down regulates phosphorylation of mTOR. mTOR inhibition induces autophagyby upregulating the downstream protein kinases, such as ULK1and ATG13 (Yeh et al., 2016).


DOWNREGULATES BCL-XL


Honokiol is a phenolic compound purified from Magnolia officinalis, which induced the apoptotic cell death in several types of human cancer cells. In the present study, the molecular mechanism of honokiol-mediated apoptotic process was examined in human squamous lung cancer CH27 cells. Here, we found that honokiol-induced apoptotic cell death was accompanied by upregulation of Bad and downregulation of Bcl-XL, while honokiol had no effect on the levels of Bcl-2, Bcl-XS, Bag-1, Bax and Bak proteins. Moreover, honokiol treatment caused the release of mitochondrial cytochrome c to cytosol and sequential activation of caspases. Proteolytic activation of caspase-3 and cleavage of PARP, an in vivo substrate for caspase-3, were observed in honokiol-treated CH27 cells. Furthermore, treatment with caspase inhibitors z-DEVD-fmk and z-VAD-fmk markedly blocked honokiol-induced apoptosis. These results demonstrated that modulation of Bcl-XL and Bad proteins, release of mitochondrial cytochrome c and activation of caspase-3, participated in honokiol-triggered apoptotic process in human squamous lung cancer CH27 cells.

HSP90 INHIBITOR


Here, LHK increased HSP90 acetylation levels , which resulted in inhibition of HSP90 and HCP binding (Fig. 3E). LHK was reported to inhibit class I histone deacetylase [40,41]. The increased HSP90 acetylation levels re- ported here may be due to class I histone deacetylase-mediated inhibition by LHK. Additionally, HK destabilized EGFR proteins in cancer cell lines [40,42]; these findings were recapitulated in the current study. The Akt and Erk1/2 pathways are activated to sustain cell survival in NSCLC cells that have EGFR-activating and genfitinib-resistant mutations. In H1975 and HCC827 cell lines, LHK promoted degradation of EGFR, C-Raf and Akt, and the Akt and Erk1/2 pathways were both inhibited . Taken together, these data indicated that LHK inhibited the Akt and Erk1/2 path- ways by promoting the degradation of HCP in NSCLC cell lines harboring EGFR-activating and resistant mutations.


INHIBITS c-FLIP


Honokiol (HNK) is a natural compound isolated from the magnolia plant with numerous pharmacological activities, including inhibiting epithelial-mesenchymal transition (EMT), which has been proposed as an attractive target for anti-tumor drugs to prevent tumor migration. In this study we investigated the effects of HNK on EMT in human NSCLC cells in vitro and the related signaling mechanisms. TNF-α (25 ng/mL) in combination with TGF-β1 (5 ng/mL) was used to stimulate EMT of human NSCLC A549 and H460 cells. Cell proliferation was analyzed using a sulforhodamine B assay. A wound-healing assay and a transwell assay were performed to examine cell motility. Western blotting was used to detect the expression levels of relevant proteins. siRNAs were used to knock down the gene expression of c-FLIP and N-cadherin. Stable overexpression of c-FLIP L (H157-FLIP L) or Lac Z (H157-Lac Z) was also performed. Treatment with TNF-α+TGF-β1 significantly enhanced the migration of A549 and H460 cells, increased c-FLIP, N-cadherin (a mesenchymal marker), snail (a transcriptional modulator) and p-Smad2/3 expression, and decreased IκB levels in the cells; these changes were abrogated by co-treatment with HNK (30 μmol/L).

Further studies demonstrated that expression level of c-FLIP was highly correlated with the movement and migration of NSCLC cells, and the downstream effectors of c-FLIP signaling were NF-κB signaling and N-cadherin/snail signaling, while Smad signaling might lie upstream of c-FLIP. HNK inhibits EMT-mediated motility and migration of human NSCLC cells in vitro by targeting c-FLIP, which can be utilized as a promising target for cancer therapy, while HNK may become a potential anti-metastasis drug or lead compound.


Honokiol exerts dual effects on browning and apoptosis of adipocytes


Honokiol exhibits a dual modulatory role in adipocytes.

•Honokiol regulates lipid catabolism and activates AMPK.

•Honokiol induces brown adipocyte-like phenotype through activation of ERK.

Induction of brown adipocyte-like phenotype (browning) in white adipocytes and promotion of apoptosis by dietary and pharmacological compounds is considered a novel strategy against obesity. Here, we show that honokiol exerts dual modulatory effects on adipocytes via induction of browning in 3T3-L1 white adipocytes and apoptosis as well as activation of HIB1B brown adipocytes combined with inhibition of apoptosis.


Honokiol-induced browning and apoptosis were investigated by determining expression levels of brown adipocyte-specific genes and proteins by RT-PCR and immunoblot analysis, respectively. Apoptotic data were validated by immunofluorescence and ROS levels were measured by FACS analysis.


Honokiol treatment induced browning by elevating expression levels of brown adipocyte-specific genes such as Cidea, Cox8, Fgf21, Pgc-1α, and Ucp1. Honokiol promoted apoptosis of 3T3-L1 white adipocytes and inhibited apoptosis of HIB1B brown adipocytes viaopposite regulation of the pro-apoptotic protein BAX and anti-apoptotic protein Bcl-2. Honokiol also significantly increased protein expression levels of ACOX1, CPT1, p-HSL, and p-PLIN and reduced ROS levels, suggesting its possible role in fat oxidation and lipid catabolism. Honokiol-induced browning could be mediated by activation of ERK, as inhibition of ERK by FR180204 abolished expression of PGC-1α and UCP1.


Our findings suggest that honokiol exhibits a modulatory role in adipocytes via induction of browning and apoptosis in white adipocytes, promotion of catabolic lipid metabolism, as well as activation and inhibition of apoptosis in HIB1B brown adipocytes, thereby exhibiting therapeutic potential against obesity.



We previously reported that hyperforin, a phloro- glucinol purified from Hypericum perforatum, induces the mitochondrial pathway of caspase-dependent apoptosis in chronic lymphocytic leukemia (CLL) cells ex vivo, and that this effect is associated with upregulation of Noxa, a BH3-only protein of the Bcl-2 family. Here, we investigated the role of this upregulation in the pro-apoptotic activity of hyperforin in the cells of CLL patients and MEC-1 cell line. We found that the increase in Noxa expression is a time- and concentration- dependent effect of hyperforin occurring without change in Noxa mRNA levels. A post-translational regulation is suggested by the capacity of hyperforin to inhibit proteasome activity in CLL cells. Noxa silencing by siRNA reduces partially hyperforin-elicited apoptosis.

Furthermore, treatment with hyperforin, which has no effect on the expression of the prosurvival protein Mcl-1, induces the interaction of Noxa with Mcl-1 and the dissociation of Mcl-1/ Bak complex, revealing that upregulated Noxa displaces the proapoptotic protein Bak from Mcl-1. This effect is accompanied with Bak activation, known to allow the release of apoptogenic factors from mitochondria. Our data indicate that Noxa upregulation is one of the mechanisms by which hyperforin triggers CLL cell apoptosis.They also favor that new agents capable of mimicking specifically the BH3-only protein Noxa should be developed for apoptosis-based therapeutic strategy in CLL.


CASPASE 3 & 9 ACTIVATOR : Induces Apoptosis through Mitochondrial Pathway

We examined the antiproliferation effect of Jaceosidin (4′, 5, 7-trihydroxy-3′, 6-dimethoxyflavone) isolated from the herb of Artemisia vestita Wall on several human cancer cell lines. Jaceosidin significantly reduced the proliferation of CAOV-3, SKOV- 3, HeLa, and PC3 cells in a concentration-dependent manner. A time-dependent inhibition was also observed in CAOV-3 cells by Jaceosidin. By flow cytometric analysis, we found that Jaceosidin treatment resulted in an increased apoptosis in CAOV-3 cells. The cells treated with Jaceosidin exhibited a decreased mitochondrial membrane potential. Jaceosidin also increased the level of cleaved caspase-9 and induced the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP), while caspase-3 inhibitor Z-DEVD-FMK significantly reversed the proapoptotic effect of Jaceosidin in CAOV-3 cells.

Moreover, Jaceosidin elevated the level of cytochrome c in cytosol. These findings suggest that the anticancer effect of Jaceosidin may be contributed by an induction of apoptosis involving cytochrome c release from mitochondria to cytosol.


DOWNREGULATES CYCLIN B1, CDK1; UPREGULATES P53 AND P21

Flavonoid compounds have been shown to trigger cell cycle arrest at G0/G1, S and G2/M checkpoints, allowing cells to repair DNA damage before entry into mitosis. Jaceosidin, a flavonoid compound, has been reported to induce apoptosis in various cancer cell lines. In our previous study, we established that jaceosidin induces apoptosis in U87 glioblastoma cells through G2/M phase arrest. However the molecular mechanisms oremained unclear. In the present study, mRNA and protein expression levels of major cell cycle regulatory genes were analyzed by semi-quantitative RT-PCR and Western blot studies respectively.

The results demonstrated that jaceosidin-induced G2/M phase arrest in U87 cells is associated with DNA fragmentation, up-regulation of p53 and p21and subsequent down-regulation of cyclin B1 and CDK1 expression at mRNA as well as at protein level. These findings provide insights into jaceosidin-induced G2/M phase arrest in U87 glioblastoma cells.


Juglanin


inhibits BCL-2, BCL-XL; enhances BAX, BAD

Juglanin (Jug) is obtained from the crude extract of Polygonum aviculare, exerting suppressive activity against cancer cell progression in vitro and in vivo. Juglanin administration causes apoptosis and reactive oxygen species (ROS) in different types of cells through regulating various signaling pathways. In our study, the effects of juglanin on non-small cell lung cancer were investigated. A significant role of juglanin in suppressing lung cancer growth was observed. Juglanin promoted apoptosis in lung cancer cells through increasing Caspase-3 and poly ADP-ribose polymerase (PARP) cleavage, which is regulated by TNF-related apoptosis-inducing ligand/Death receptors (TRAIL/DRs) relied on p53 activation. Anti-apoptotic members Bcl-2 and Bcl-xl were reduced, and pro-apoptotic members Bax and Bad were enhanced in cells and animals receiving juglanin. Additionally, nuclear factor-κB (NF-κB), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinases (MAPKs) activation were inhibited by juglanin. Further, juglanin improved ROS and induced autophagy. ROS inhibitor N-acetyl-l-cysteine (NAC) reversed apoptosis induced by juglanin in cancer cells. The formation of autophagic vacoules and LC3/autophagy gene7 (ATG7)/Beclin1 (ATG6) over-expression were observed in juglanin-treated cells. Also, juglanin administration to mouse xenograft models inhibited lung cancer progression. Our study demonstrated that juglanin could be a promising candidate against human lung cancer progression.



Icariin

Icariin (from Epimedium aka “Horny Goat Weed”)

strongly inhibits STAT3 activation


Signal transducer and activator of transcription-3 (STAT3) is critical for cancer progression by regulating tumor cell survival, proliferation, and angiogenesis. Herein, we investigated the regulation of STAT3 activation and the therapeutic effects of Icaritin, a prenyl flavonoid derivative from Epimedium Genus, in renal cell carcinoma (RCC). Icaritin showed significant anti-tumor activity in the human and mouse RCC cell lines, 786-O and Renca, respectively. Icaritin inhibited both constitutive and IL-6-induced phospho-STAT3 (STAT3(Y705)) and reduced the level of STAT3-regulated proteins Bcl-xL, Mcl-1, Survivin, and CyclinD1 in a dose-dependent manner. Icaritin also inhibited activation of Janus-activated kinase-2 (JAK2), while it showed minimal effects on the activation of other key signaling pathways, including AKT and MAPK. Expression of the constitutively active form of STAT3 blocked Icaritin-induced apoptosis, while siRNA directed against STAT3 potentiated apoptosis.

Finally, Icaritin significantly blunted RCC tumor growth in vivo, reduced STAT3 activation, and inhibited Bcl-xL and Cyclin E, as well as VEGF expression in tumors, which was associated with reduced tumor angiogenesis. Overall, these results suggest thatIcaritin strongly inhibits STAT3 activation and is a potentially effective therapeutic option for the treatment of renal cell carcinoma.


Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade


Because of the essential role of signal transducer and activator of transcription 3 (STAT3) in proliferation, anti-apoptosis, and chemoresistance of multiple myeloma (MM), we investigated whether icariin, a prenylated flavonol glycoside, inhibits both constitutive and inducible STAT3 activation in human myeloma cell lines. We noted that icariin could block constitutive STAT3 phosphorylation as well as its nuclear translocation and DNA binding ability in U266 cells.

Icariin also suppressed IL-6-induced STAT3 activation through the inhibition of upstream kinases (Janus activated kinase-1 and -2, and c-Src). We found that icariin downregulated the protein expression of STAT3 downstream target gene products such as Bcl-2, Bcl-xl, survivin, IAP-1/2, COX-2, VEGF, and matrix metallopeptidase 9 (MMP-9) in a concentration-dependent manner. Moreover, this flavonoid also exhibited the capacity to significantly induce apoptosis and suppress proliferation of MM cells. Interestingly, this agent also significantly potentiated the apoptotic effects of bortezomib through the suppression of STAT3 activation in MM cells. Altogether, our data indicates that the potential application of icariin as a STAT3 blocker in myeloma therapy.


icariin and icaritin ameliorated hippocampus neuroinflammation via inhibiting HMGB1-related pro-inflammatory signals in lipopolysaccharide-induced inflammation …


Highlights

HMGB1-RAGE signaling and TLR4-XBP1s-NF-κB signaling were involved in neuroinflammation.

•Icariin and icaritin could ameliorate neuroinflammation via suppressing HMGB1-RAGE signaling.

•Icariin and icaritin might promote neuroregeneration via activating TLR4NF-κB signaling.

Inflammation is a defensive response of the body and is at the center of many diseases’ process like depression. High mobility group protein box 1 (HMGB1), has been proved to function as a pro-inflammatory cytokine. We aim to explore the role of HMGB1 played in the neuroinflammation here. In this study, we used LPS to induce an acute inflammatory response, and to measure the anti-neuroinflammation effect of icariin (ICA) and icaritin (ICT). We found that LPS could increase the expression of HMGB1 in serum and hippocampus, along with a high expression of HMGB1 in the cytoplasm and a high expression of RAGE, which could be rescued by ICA and ICT, and ethyl pyruvate (EP) pretreatment showed similar effects here. We speculated that the translocation of HMGB1 from the nucleus to the cytoplasm played an important role in neuroinflammatory process, and HMGB1-RAGE signal was involved in this process. Furthermore, we found that ICA and ICT treatment activated TLR4-XBP1s related NF-κB signal, which we thought was relevant with the neuroprotective effect of ICA and ICT.

However, EP pretreatment suppressed TLR4-XBP1s- endoplasmic reticulum stress related NF-κB signal to anti-inflammatory response, which was almost absolutely opposite with ICA and ICT treatment. We speculated that it might be caused by the duration of inflammation. We supposed that ICA and ICT could ameliorate neuroinflammation in hippocampus via suppressing HMGB1-RAGE signaling and might show a neuroprotective effect via activating TLR4-XBP1s related NF-κB signal at the same time, making it possible to act as an anti-neuroinflammatory drugs.

icariin and icaritin ameliorated hippocampus neuroinflammation via mediating HMGB1 expression in social defeat model in mice


Depression is a chronic, severe, and often life-threatening disease accompanied with impaired neurogenesis. Evidence showed that neuroinflammation played a key role in the process of depression. High mobility group protein box 1 (HMGB1) has been proved to function as a pro-inflammatory cytokine. In this study, we used a social defeat (SD) stress to induce inflammatory response, aiming to explore the relationship between HMGB1 and neuroinflammation.

We found that the expression of HMGB1 decreased in mice exposure to SD stress, but showed a high expression of cytoplasmic HMGB1 and a high expression of RAGE, which could be rescued by ICA and ICT. So, we speculated that the translocation of HMGB1 from the nucleus to the cytoplasm might play an important role in neuroinflammatory process, and HMGB1-RAGE signaling was involved in this process. Furthermore, we also found that TLR4-XBP1s-ER stress related NF-κB signaling activation was also involved in HMGB1-related neuroinflammation.

However, ICA and ICT treatment activated NF-κB signaling, and we also observed the translocation of HMGB1 into the nucleus and the increased number of neurons in mice hippocampus, indicating that the activation of NF-κB signaling might be related to neuroregeneration. Moreover, recombinant human HMGB1 protein (rHMGB1) pretreatment could suppress HMGB1-RAGE signaling and TLR4-XBP1s-ER stress related NF-κB signaling, resulted in a suppressed microglia activation in mice hippocampus. We supposed that ICA and ICT could ameliorate neuroinflammation in hippocampus via suppressing HMGB1-RAGE signaling and show neuroprotective effects via activating TLR4– NF-κB signaling at the same time, resulting in improving depressive behaviors in mice.

HMGB1 is a major pro-inflammatory SASP factor


Epigenetic manipulation through long non-coding RNAs, miRNA modulators, and other oligonucleotides has yielded promising outcomes in experimental settings.162–165 Some of these molecules are currently clinically evaluated in CVD.166 Epigenetic modification through histone modification via bromodomain inhibitors could suppress atherosclerosis,167 senescence,168 and the SASP,169 indicating high potency for such interventions. The non-histone DNA-binding protein high mobility group protein B1 (HMGB1) is a major pro-inflammatory SASP factor, when externalized from the nucleus into the extracellular environment.39 Soluble HMGB1 promoted atherosclerosis40,41 and CVD risk.170

It remains to be seen if HMGB1 nuclear retention ameliorates the epigenetic landscape of senescent cells and provides benefits in atherosclerosis. Another promising strategy is the epigenetic blockade of p66Shc, since pharmacological inhibitors are not currently available. p66Shc is a promoter of oxidative stress70 and is highly expressed in senescent cells68 and atherosclerosis.171Inhibiting p66Shc its key regulator MiR-34a is promising,172 as p66Shc knock-out led to senescence prevention,68 30% animal lifespan extension,173 and strong inhibition of atherogenesis.


INDIRUBIN (INDIGO BLUE EXTRACT)

Inhibits CDK1

The bisindole indirubin has been described, more than 30 years ago, as being clinically active in the treatment of human chronic myelocytic leukaemia. However, the underlying mechanism of action has remained unclear. We have reported previously that indirubin and its analogues are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, we investigated the influence of indirubin and derivatives on CDK1/cyclin B kinase in human tumour cells at concentrations known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G(1)/G(0)phase of the cell cycle in the presence of 2 microM indirubin-3′-monoxime. At higher drug concentrations (> or = 5 microM) an increase of the cell population in the G(2)/M phase is additionally observed.

Cells synchronized in G(2)/M phase by nocodazole remain arrested in the G(2)/M phase after release, in the presence of indirubin-3′-monoxime (> or =5 microM). After 24 h treatment with 10 microM indirubin-3′-monoxime a sub-G(2)peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concentrations of indirubin-3′-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a reduction of cyclin B in complex with CDK1 is observed. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumour cells is a major mechanism by which indirubin derivatives exert their potent antitumour efficacy.


INHIBITS STAT3


Stat3 protein has an important role in oncogenesis and is a promising anticancer target. Indirubin, the active component of a traditional Chinese herbal medicine, has been shown previously to inhibit cyclin-dependent kinases, resulting in cell cycle arrest. Here, we show that the indirubin derivatives E564, E728, and E804 potently block constitutive Stat3 signaling in human breast and prostate cancer cells. In addition, E804 directly inhibits Src kinase activity (IC50 = 0.43 μM) in an in vitro kinase assay. Levels of tyrosyl phosphorylation of c-Src are also reduced in cultured cells 30 min after E804 treatment. Tyrosyl phosphorylation of Stat3, which is known to be phosphorylated by c-Src, was decreased, and constitutive Stat3 DNA binding-activity was suppressed in cells 30 min after E804 treatment. The antiapoptotic proteins Mcl-1 and Survivin, which are encoded in target genes of Stat3, were down-regulated by indirubin derivatives, followed by induction of apoptosis.

These results demonstrate that E804 directly blocks the Src-Stat3 signaling pathway, suggesting that the antitumor activity of indirubin compounds is at least partially due to inhibition of this pathway.


indole-3-carbinol (I3C)

downregulation of Bcl-2, Bcl-XL


I3C, also abundant in cruciferous vegetables, induces apoptosis by significant downregulation of Bcl-2, Bcl-XL, IAP, X chromosome-linked IAP, and cellular FLICE inhibitory protein (c-FLIP)in human prostate cancer cells.


MDM2 INHIBITOR


Coimmunoprecipitation analysis revealed that I3C reduced by 4‐fold the level of MDM2protein that associated with p53. The p53–MDM2 interaction and absence of p21 production were restored in cells treated with I3C and the ATM inhibitor wortmannin. Significantly, I3C does not increase the number of 53BP1 foci or H2AX phosphorylation, indicating that ATM is activated independent of DNA double‐strand breaks.

Taken together, our results demonstrate that I3C activates ATM signaling through a novel pathway to stimulate p53 phosphorylation and disruption of the p53–MDM2 interaction, which releases p53 to induce the p21 CDK inhibitor and a G1 cell cycle arrest.


Isoalantolactone (Tumuxiang)

induces apoptosis in SGC-7901 cells via mitochondrial and PI3K/Akt signaling pathways.

Isoalantolactone, a sesquiterpene lactone, possesses anti-fungal as well as cytotoxic properties. In this study, the effects of Isoalantolactone on cell viability, cell cycle, and apoptosis were investigated in human gastric adenocarcinoma SGC-7901 cells. The results demonstrated that Isoalantolactone induced morphological changes and decreased cell viability. Subsequently, we found that Isoalantolactone induced G2/M and S phase arrest, which was associated with a decrease in the expression level of cyclin B1. Apoptosis triggered by Isoalantolactone was visualized using propidium iodide (PI) and Annexin V-FITC/PI staining. Isoalantolactone-induced apoptosis of SGC-7901 cells was associated with the dissipation of mitochondrial membrane potential (ΔΨ m) that was due to the down-regulation of Bcl-2 and up-regulation of Baxthat led to the cleavage of caspase-3.

Additionally, it was found that Isoalantolactone was involved in the inhibition of phosphorylation of PI3K/Akt. Isoalantolactone-induced cytotoxicity and apoptosis of SGC-7901 cells involve mitochondria-caspase and PI3K/Akt dependent pathways, which gives the rationale for in vivo studies on the utilization of Isoalantolactone as a potential cancer therapeutic compound.


Isoorientin

induces apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cancer cells


Isoorientin (ISO) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum; however, its biological activity remains poorly understood. The present study investigated the effects and putative mechanism of apoptosis induced by ISO in human hepatoblastoma cancer (HepG2) cells. The results showed that ISO induced cell death in a dose-dependent manner in HepG2 cells, but no toxicity in human liver cells (HL-7702) and buffalo rat liver cells (BRL-3A) treated with ISO at the indicated concentrations. ISO-induced cell death included apoptosis which characterized by the appearance of nuclear shrinkage, the cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation.

ISO significantly (p < 0.01) increased the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), increased the release of cytochrome c, activated caspase-3, and enhanced intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO). In addition, ISO effectively inhibited the phosphorylation of Akt and increased FoxO4 expression. The PI3K/Akt inhibitor LY294002 enhanced the apoptosis-inducing effect of ISO. However, LY294002 markedly quenched ROS and NO generation and diminished the protein expression of heme peroxidase enzyme (HO-1) and inducible nitric oxide synthase (iNOS). Furthermore, the addition of a ROS inhibitor (N-acetyl cysteine, NAC) or iNOS inhibitor (N-[3-(aminomethyl) benzyl] acetamidine, dihydrochloride, 1400W) significantly diminished the apoptosis induced by ISO and also blocked the phosphorylation of Akt. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells and indicate that this apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway, and has no toxicity in normal liver cells, suggesting that ISO may have good potential as a therapeutic and chemopreventive agent for liver cancer.

MAPK signaling pathways regulate mitochondrial-mediated apoptosis induced by isoorientin
Isoorientin (ISO)is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum. ISO is able to induce apoptosis through mitochondrial dysfunction and inhibition of PI3K/Aktsignaling pathway in HepG2 cells, however, the effects of ISO on MAPK signaling pathways remain unknown. The present study investigated the effects of ISO on this pathway, and the roles of MAPK kinases on mitochondrial-mediated apoptosis in HepG2 cells. The results showed that ISO induced cell death in a dose- and time-dependent manner, and induction apoptosis is main cause for ISO-induced cytotoxicity in HepG2 cells. ISO significantly inhibited the levels of ERK1/2 kinase and increased the expression of JNK and p38 kinases.

Furthermore, U0126 (an ERK1/2 inhibitor) significantly enhanced the ISO-induced the Bax/Bcl-2 ratio, the release of cytochrome c to the cytosol fraction, and the levels of cleaved caspase-3. While SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) markedly prevented the expression of these proteins induced by ISO. Furthermore, the ROS inhibitor (NAC) notably promoted the inhibited effect of ISO on the ERK1/2 kinase. NAC also suppressed the p-JNK and p-p38, but failed to reverse the effects of ISO. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells through inactivating ERK1/2 kinase and activating JNK and p38 kinases, and ROS stimulated by ISO is able to activate the MAPK singaling pathway as the upstream signaling molecules. Initiating event of the mitochondrial-mediated apoptosis induced by ISO is MAPK signals.

The present study showed that ISO induced autophagy, which was correlated with the formation of autophagic vacuoles and the overexpression of Beclin-1 and LC3-II. The autophagy inhibitor 3-methyladenine (3-MA) markedly inhibited apoptosis, and the apoptosis inhibitor ZVAD-fmk also decreased ISO-induced autophagy. In addition, the PI3K/Akt inhibitor LY294002 enhanced Beclin-1, LC3-II, and poly(ADP-ribose) polymerase (PARP) cleavage levels. Also, the reactive oxygen species (ROS) inhibitor N-acetyl-l-cysteine (NAC), the JNK inhibitor SP600125, and the p38 inhibitor SB203580 efficiently downregulated the levels of these proteins. Moreover, the p53 inhibitor pifithrin-α and the nuclear factor (NF)-κB inhibitor pyrrolidinedithiocarbamic acid (PDTC) clearly suppressed Beclin-1 and LC3-II and increased cytochrome c release, caspase-3 activation, and PARP cleavage.

These results demonstrated for the first time that ISO simultaneously induced apoptosis and autophagy by ROS-related p53, PI3K/Akt, JNK, and p38 signaling pathways. Furthermore, ISO-induced apoptosis by activating the Fas receptor-mediated apoptotic pathway and suppressing the p53 and PI3K/Akt-dependent NF-κB signaling pathway, with the subsequent increase in the release of cytochrome c, caspase-3 activation, and PARP cleavage.


BCL-W, BCL-2, BCL-XL MCL-1 INHIBITOR


Isorhamnetin

blocks Hsp70 expression to promote apoptosis

The roots of Codonopis bulleynana Forest ex diels (cbFed), locally known as Tsoong, have been used as a tonic food. Tsoong has wide range of pharmacological effects, including anticancer efficacy. In the present study, the anticancer activity of Tsoong and its potential molecular mechanisms were investigated. Isorhamnetin, a flavonol aglycone, is important compound and metabolite in Tsoong. It can promote apoptosis of colon cancer cells through up-regulating apoptosis-related genes (Apaf1, Casp3 and Casp9) because it blocks Hsp70 genes (Hspa1a, Hspa1b and Hspa8). These were verified by in vitro and in vivo experiments. In vitro, cell counting kit-8 (CCK-8) assays and flow cytometry in HCT116 and SW480 colon cancer cell were used to assess the anti-proliferation and apoptosis-promoting activities of Tsoong. In vivo, the antitumor effect of Tsoong was assessed in colon cancer-bearing nude mice as a xenograft model. These results show that Isorhamnetin is very critical in Tsoong because Tsoong can down-regulate Hsp70 genes and promote apoptosis of colon cancer cells by inhibiting Hsp70 largely due to the efficacy of Isorhamnetin. Our results may ultimately help in the development of diagnostic and therapeutic strategies to control this devastating disease.


Isorhamnetin is an O-methylated flavonol that is predominantly found in the fruits and leaves of various plants, which have been used for traditional herbal remedies. Although several previous studies have reported that this flavonol has diverse health-promoting effects, evidence is still lacking for the underlying molecular mechanism of its anti-cancer efficacy. In this study, we examined the anti-proliferative effect of isorhamnetin on human bladder cancer cells and found that isorhamnetin triggered the gap 2/ mitosis (G2/M) phase cell arrest and apoptosis.
Our data showed that isorhamnetin decreased the expression of Wee1 and cyclin B1, but increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, and increased p21 was bound to Cdk1. In addition, isorhamnetin-induced apoptosis was associated with the increased expression of the Fas/Fas ligand, reduced ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein (Bax) expression, cytosolic release of cytochrome c, and activation of caspases.Moreover, isorhamnetin inactivated the adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway by diminishing the adenosine triphosphate (ATP) production due to impaired mitochondrial function. Furthermore, isorhamnetin stimulated production of intracellular reactive oxygen species (ROS); however, the interruption of ROS generation using a ROS scavenger led to an escape from isorhamnetin-mediated G2/M arrest and apoptosis.
Collectively, this is the first report to show that isorhamnetin inhibited the proliferation of human bladder cancer cells by ROS-dependent arrest of the cell cycle at the G2/M phase and induction of apoptosis. Therefore, our results provide an important basis for the interpretation of the anti-cancer mechanism of isorhamnetin in bladder cancer cells and support the rationale for the need to evaluate more precise molecular mechanisms and in vivo anti-cancer properties.

Isovitexin

induces apoptosis and autophagy

•Isovitexin inhibits the proliferation and induces apoptosis in human liver cancer.
•Isovitexin triggers the interplay between apoptosis and autophagy in liver cancer cells.
•ER stress participates in isovitexin-induced apoptosis and autophagy.
•Isovitexin displays anti-tumor effect in HepG2 tumor-bearing mice.

Liver cancer is a leading cause of cancer death worldwide, and novel chemotherapeutic drugs to suppress liver cancer are urgently required. Isovitexin(IV), a glycosylflavonoid, is extracted from rice hulls of Oryza sativa, and has various biological activities. However, the anti-tumor effect of IV against liver cancer has not yet been demonstrated in vitro or in vivo. In the present study, we showed that IV significantly suppressed the growth of liver cancer cells. Mechanistic studies indicated that IV induced apoptosis by the mitochondrial apoptotic pathway, as evidenced by the increase of Bax, cleaved Caspase-3, poly (ADP-ribose) polymerase(PARP), and cytoplasm Cyto-c released from mitochondria. In addition, IV resulted in autophagy in liver cancer cells, supported by the enhancement of LC3II, autophagy-related protein (Atg) 3, Atg5 and Beclin1. Suppressing autophagy using bafilomycin A1 (BFA) or siRNA Atg-5 reduced apoptotic cells in IV-treated cells, demonstrating that autophagy induction regulated apoptosis.

Moreover, IV was found to cause endoplasmic reticulum (ER) stress in liver cancer cells, along with the promotion of ER stress-related molecules, including inositol-requiring enzyme 1α (IRE1α), X-box-binding protein-1s (XBP-1s), C/EBP homologous protein (CHOP) and glucose-regulated protein (GRP)-78. Of note, inhibition of ER stress by use of its inhibitor, tauroursodeoxycholate (TUDCA), significantly reversed IV-induced apoptosis and autophagy. In vivo, IV treatment showed significant tumor growth inhibition compared to the non-treated group. IV could therefore be a strong candidate for liver cancer prevention.


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