SENOLYTIC: “ZOMBIE CELL KILLER” 200:1

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 p21^Waf1/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.

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.

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 [38]. 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 [50]. On the contrary, NF-kB activation was involved in resistance to oxidative stress and p53-mediated programmed cell death [51–53]. 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

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

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

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 [130]. The compounds were shown to activate CHEK2, and degradation of CDC25A, and the subsequent inactivation of CDK1, as indicated in [130]. Berberine also downregulated the cyclin D1 expression, and induced the acetylation of α-tubulin [130].

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.

Campesterol

Carnosic acid

down-regulation of c-FLIP and Bcl-2

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.

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

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.

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.

Cycloastragenol

STAT3 INHIBITOR

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

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.

Diosmin

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.

(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 (1–13) 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 (1–13) 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 $T_H$ and $T_C/T_S$ 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

HOELEN (Poria Cocos)

downregulateS AKT & C-FLIP

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 (39–42). 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

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.

EGCG-induced JNK activation

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

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.

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.

Fisetin

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

New agents that target senescent cells: the flavone, fisetin, and the BCL-X_L 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-X_L 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

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 ³².

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.

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.

FUCOIDAN

Caspase 3 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[6–8,18,19].

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.

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.

Ganoderma lucidum (Reishi)

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

Genistein

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

inhibits tyrosine kinase

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

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.

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.