“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.”
“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.”
“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 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.”
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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 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.
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.”
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.
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 (35). We recently provided a large-scale characterization of the SASP, using antibody arrays to quantitatively measure factors secreted by human fibroblasts and epithelial cells (18), 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 (36–46). Among the cells that have been shown to senesce and secrete biologically active molecules are liver stellate cells (47), endothelial cells (36, 48–51), and epithelial cells of the retinal pigment, mammary gland, colon, lung, pancreas, and prostate (8, 18, 36, 41, 52–56).
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 (147–150). 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.
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]
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 ZOMBIESENESCENT CELLS AND PREMATURELY AGES YOU. THE SASP MUST GO!
THE KEY TO ELIMINATING ZOMBIE SENESCENT CELLS CONSISTS OF TWO PARTS:
DOWN-REGULATING ANT-APOPTOTIC PATHWAYS THAT PROTECT THEM
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.
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.
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.
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].
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.
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 p53–p21CIP1 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 familyproteins 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.
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.
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).
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.
Apigenin has been shown to induce apoptosis in different types of cells [46, 70, 84, 85]. 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 [55]. In human prostate cancer cells, apigenin treatment has been shown to alter the Bax/Bcl-2 ratio in favor of apoptosis [46]. 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 [70]. 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 [51].
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 (51, 52). 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 (53, 54). In addition, apigenin has been shown to induce apoptosis in a wide range of malignant cells (55–57). 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 (54).
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 (2)/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 (86).
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, 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 p
