Sirtuin 1 promotes autophagy and proliferation of endometrial cancer cells by reducing acetylation level of LC3

Endometrial cancer (EC) constitutes a common female genital tract tumor with a rising incidence rate. Sirtuin 1 (SIRT1) is a member of histone deacetylase, which extensively participates in the progression of aging, cell death, and tumorigenesis. This study explored the effect of SIRT1-mediated LC3 acetylation on autophagy and proliferation of EC cells. SIRT1 expression in EC tissues and adjacent tissues, EC cell lines and normal human epithelial cells was detected. SIRT1 expression was elevated in EC cell lines and tissues. Knockdown of SIRT1 inhibited proliferation, migration, and invasion of EC cells. Then, EC cells were starved in serum-free medium, and levels of autophagy-related proteins were detected. Starvation induced autophagy of EC cells. The starvation-treated EC cells showed an increased SIRT1 expression, a decreased LC3 acetylation level and an increased autophagy level. The proliferation and autophagy of EC cells under different treatments were evaluated. In EC cells transfected with overexpressing SIRT1, LC3 acetylation was inhibited and cell proliferation was promoted. Moreover, overexpressing SIRT1 facilitated growth and autophagy of transplanted tumors in nude mice. In conclusion, SIRT1 promoted autophagy and proliferation of EC cells by reducing acetylation level of LC3.     

Extension of human cell lifespan by nicotinamide phosphoribosyltransferase

Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD+ salvage from nicotinamide. Replicative senescence of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated beta-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD+-dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation.     

SIRT3 deregulation is linked to mitochondrial dysfunction in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Despite decades of study, effective treatments for AD are lacking. Mitochondrial dysfunction has been closely linked to the pathogenesis of AD, but the relationship between mitochondrial pathology and neuronal damage is poorly understood. Sirtuins (SIRT, silent mating type information regulation 2 homolog in yeast) are NAD‐dependent histone deacetylases involved in aging and longevity. The objective of this study was to investigate the relationship between SIRT3 and mitochondrial function and neuronal activity in AD. SIRT3 mRNA and protein levels were significantly decreased in AD cerebral cortex, and Ac‐p53 K320 was significantly increased in AD mitochondria. SIRT3 prevented p53‐induced mitochondrial dysfunction and neuronal damage in a deacetylase activity‐dependent manner. Notably, mitochondrially targeted p53 (mito‐p53) directly reduced mitochondria DNA‐encoded ND2 and ND4 gene expression resulting in increased reactive oxygen species (ROS) and reduced mitochondrial oxygen consumption. ND2 and ND4 gene expressions were significantly decreased in patients with AD. p53‐ChIP analysis verified the presence of p53‐binding elements in the human mitochondrial genome and increased p53 occupancy of mitochondrial DNA in AD. SIRT3 overexpression restored the expression of ND2 and ND4 and improved mitochondrial oxygen consumption by repressing mito‐p53 activity. Our results indicate that SIRT3 dysfunction leads to p53‐mediated mitochondrial and neuronal damage in AD. Therapeutic modulation of SIRT3 activity may ameliorate mitochondrial pathology and neurodegeneration in AD.     

Progressive loss of SIRT1 with cell cycle withdrawal

Sir2 is an NAD+-dependent deacetylase that regulates lifespan in yeast, worms and flies. The mammalian orthologs of Sir2 include SIRT1 in humans and mice. In this study, we analyzed the level of SIRT1 in human lung fibroblasts (IMR90) and mouse embryonic fibroblasts (MEFs) from mice with normal, accelerated, and delayed aging. SIRT1 protein, but not mRNA, decreased significantly with serial cell passage in both human and murine cells. Mouse SIRT1 decreased rapidly in prematurely senescent (p44 Tg) MEFs, remained high in MEFs with delayed senescence (Igf-1r-/-), and was inversely correlated with senescence-activated beta-galactosidase (SA-betaGal) activity. Reacquisition of mitotic capability following spontaneous immortalization of serially passaged wild-type MEFs restored the level of SIRT1 to that of early passage, highly proliferative MEFs. In mouse and human fibroblasts, we found a significant positive correlation between the levels of SIRT1 and proliferating cell nuclear antigen (PCNA), a DNA processing factor expressed during S-phase. In the animal, we found that SIRT1 decreased with age in tissues in which mitotic activity also declines, such as the thymus and testis, but not in tissues such as the brain in which there is little change in mitotic activity throughout life. Again, the decreases in SIRT1 were highly correlated with decreases in PCNA. Finally, loss of SIRT1 with age was accelerated in mice with accelerated aging but was not observed in long-lived growth hormone-receptor knockout mice. Thus, as mitotic activity ceases in mouse and human cells in the normal environment of the animal or in the culture dish, there is a concomitant decline in the level of SIRT1.     

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21^CIP1 and p16^INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21^CIP1 and p16^INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.     

p16(INK4a) suppression by glucose restriction contributes to human cellular lifespan extension through SIRT1-mediated epigenetic and genetic mechanisms

Although caloric restriction (CR) has been shown to increase lifespan in various animal models, the mechanisms underlying this phenomenon have not yet been revealed. We developed an in vitro system to mimic CR by reducing glucose concentration in cell growth medium which excludes metabolic factors and allows assessment of the effects of CR at the cellular and molecular level. We monitored cellular proliferation of normal WI-38, IMR-90 and MRC-5 human lung fibroblasts and found that glucose restriction (GR) can inhibit cellular senescence and significantly extend cellular lifespan compared with cells receiving normal glucose (NG) in the culture medium. Moreover, GR decreased expression of p16(INK4a) (p16), a well-known senescence-related gene, in all of the tested cell lines. Over-expressed p16 resulted in early replicative senescence in glucose-restricted cells suggesting a crucial role of p16 regulation in GR-induced cellular lifespan extension. The decreased expression of p16 was partly due to GR-induced chromatin remodeling through effects on histone acetylation and methylation of the p16 promoter. GR resulted in an increased expression of SIRT1, a NAD-dependent histone deacetylase, which has positive correlation with CR-induced longevity. The elevated SIRT1 was accompanied by enhanced activation of the Akt/p70S6K1 signaling pathway in response to GR. Furthermore, knockdown of SIRT1 abolished GR-induced p16 repression as well as Akt/p70S6K1 activation implying that SIRT1 may affect p16 repression through direct deacetylation effects and indirect regulation of Akt/p70S6K1 signaling. Collectively, these results provide new insights into interactions between epigenetic and genetic mechanisms on CR-induced longevity that may contribute to anti-aging approaches and also provide a general molecular model for studying CR in vitro in mammalian systems.     

Mutant telomerase RNAs induce DNA damage and apoptosis via the TRF2-ATM pathway in telomerase-overexpressing primary fibroblasts

Mutant template human telomerase RNAs (MT-hTers) have been shown to induce apoptosis in various cancer cells with high telomerase activity. However, the mechanism by which MT-hTers inhibit the growth of cancer cells and their effects on normal cells remain unknown. To determine the effects of MT-hTers on normal cells, MT-hTer-47A and -AU5 were introduced into IMR90 lung fibroblasts, which have low telomerase levels. Growth of IMR90 cells after MT-hTers infection was not significantly impaired; however, similar treatments in telomerase-overexpressing IMR90 [IMR90 wild-type (WT)hTERT] cells inhibited cell proliferation and induced apoptosis. Confocal microscopy showed that MT-hTers induced DNA damage foci (i.e. 53BP1 and γ-H2AX) in IMR90 WThTERT cells. Microarray analysis revealed that GADD45γ was significantly elevated in MT-hTer-treated IMR90 WThTERT cells. MT-hTers also induced ATM phosphorylation at Ser1981 in IMR90 WThTERT cells, and western blot analysis revealed high levels of phosphorylated p53 after the down-regulation of cellular TRF2 expression in MT-hTer-treated IMR90 WThTERT cells. Taken together, we have shown that MT-hTers induce double-stranded DNA break-like damages in telomerase positive IMR90 WThTERT cells after phosphorylation of ATM and p53 via suppression of TRF2, which may eventually lead to apoptosis via elevation of GADD45γ.     

Human SIRT1 associates with mitotic chromatin and contributes to chromosomal condensation

SIRT1 is a NAD-dependent deacetylase that participates in cellular controls of gene expression, metabolism, genomic stability and anti-aging. Here we report that SIRT1 levels rise in prometaphase leading to SIRT1 global association with mitotic chromatin until telophase. Moreover, SIRT1 contributes to chromosomal condensation by mediating chromosomal loading of histone H1 and the condensin I complex. Consistently, SIRT1 knockdown led to improper condensation and overall aberrant mitosis. Our data highlight new role for SIRT1 in maintenance of chromosome stability in mitosis and suggests how diminished SIRT1 activity during aging and tumorigenesis may lead to aneuploidy and genomic instability.     

Serum withdrawal up‐regulates human SIRT1 gene expression in a p53‐dependent manner

SIRT1, a nicotinamide adenine dinucleotide (NAD⁺)‐dependent histone/protein deacetylase, has been extensively studied recently for its critical role in the regulation of physiology, calorie restriction and aging. Studies on laboratory mice showed that expression of SIRT1 can be induced by starvation in a p53‐dependent manner and requires the p53‐binding sites present in the Sirt1 promoter. However, it remains to be determined whether these findings based on rodents apply to human beings. In this paper, we characterized a putative p53‐binding element in the human SIRT1 promoter that might be required for the up‐regulation of SIRT1 in response to nutritional stress. The p53‐binding site in the promoter of human SIRT1 is more deviant from the consensus sequence than the corresponding sequence in the mouse Sirt1. There is a C to A change at the second half site in human SIRT1, thus disrupting the core‐binding element CWWG in the canonical RRRCWWGYYY. To test whether such sequence change would affect its binding with p53 and the SIRT1 expression under stress, we studied various human cell lines with different p53 status and cells with ectopic expression of functionally distinct p53. We found that serum withdrawal also up‐regulates human SIRT1 gene expression in a p53‐dependent manner and that the p53‐binding element in SIRT1 is required for the up‐regulation. Thus, the mechanism responsible for the regulation of SIRT1 expression by p53 is conserved between mice and human beings.     

Novel role of silent information regulator 1 in acute endothelial cell oxidative stress injury

Silent information regulator 1 (SIRT1), a class III histone deacetylase, retards aging and plays roles in cellular oxidative stress injury (OSI). However, the biological context in which SIRT1 promotes oxidative injury is not fully understood. Here, we show that SIRT1 essentially mediates hydrogen peroxide (H₂O₂)-induced cytotoxicity in human umbilical vein endothelial cell (HUVEC). In HUVECs, SIRT1 protein expression was significantly increased in a dose-dependent manner after H₂O₂ treatment, whereas the acetylation levels of the NF-κB p65 subunit and p53 were decreased. EX527 (a specific SIRT1 inhibitor) conferred protection to the HUVECs against H₂O₂, as indicated by an improved cell viability, adhesion, an enhanced migratory ability, a decreased apoptotic index, decreased reactive oxygen species (ROS) production and reductions in several biochemical parameters. Immunofluorescence and Western blot analyses demonstrated that H₂O₂ treatment up-regulated SIRT1, phosphorylated-JNK (p-JNK), p-p38MAPK, and p-ERK expression. EX527 pretreatment reversed these effects on SIRT1, p-JNK, and p-p38MAPK but further increased the p-ERK levels. Similar results were confirmed in SIRT1 siRNA experiments. In summary, SIRT1 signaling pathway inhibition imparts protection against acute endothelial OSI, and modulation of MAPKs (JNK, p38MAPK, and ERK) may be involved in the protective effect of SIRT1 inhibition.     

Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins

Sir2 is a NAD+-dependent protein deacetylase that extends lifespan in yeast and worms. This study examines seven human proteins homologous to Sir2 (SIRT1 through SIRT7) for cellular localization, expression profiles, protein deacetylation activity, and effects on human cell lifespan. We found that: 1) three nuclear SIRT proteins (SIRT1, SIRT6, and SIRT7) show different subnuclear localizations: SIRT6 and SIRT7 are associated with heterochromatic regions and nucleoli, respectively, where yeast Sir2 functions; 2) SIRT3, SIRT4, and SIRT5 are localized in mitochondria, an organelle that links aging and energy metabolism; 3) cellular p53 is a major in vivo substrate of SIRT1 deacetylase, but not the other six SIRT proteins; 4) SIRT1, but not the other two nuclear SIRT proteins, shows an in vitro deacetylase activity on histone H4 and p53 peptides; and 5) overexpression of any one of the seven SIRT proteins does not extend cellular replicative lifespan in normal human fibroblasts or prostate epithelial cells. This study supports the notion that multiple human SIRT proteins have evolutionarily conserved and nonconserved functions at different cellular locations and reveals that the lifespan of normal human cells, in contrast to that of lower eukaryotes, cannot be manipulated by increased expression of a single SIRT protein.     

Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage

Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.     

SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53

Nephrotoxicity is one of the important dose-limiting factors during cisplatin treatment. There is a growing body of evidence that activation of p53 has a critical role in cisplatin-induced renal apoptotic injury. The nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 decreases apoptosis through deacetylating of p53, and resveratrol is known as an activator of SIRT1. To study the role of SIRT1 in cisplatin-induced renal injury through interaction with p53, mouse proximal tubular cells (MPT) were treated with cisplatin and examined the expression level of SIRT1, acetylation of p53, PUMA-α, Bax, the cytosolic/mitochondrial cytochrome c ratio, and active caspase-3. The expression of SIRT1 was decreased by cisplatin. Resveratrol, a SIRT1 activator, ameliorated cisplatin-induced acetylation of p53, apoptosis, and cytotoxicity in MPT cells. In addition, resveratrol remarkably blocked cisplatin-induced decrease of Bcl-xL in MPT cells. Further specific SIRT1 inhibition with EX 527 or small interference RNA specific to SIRT1 reversed the effect of resveratrol on cisplatin-induced toxicity. Inhibition of p53 by pifithrin-α reversed the effect of EX527 in protein expression of PUMA-α, Bcl-xL, and caspase-3 and cytotoxicity in MPT cells. SIRT1 protein expression after cisplatin treatment was significantly decreased in the kidney. SIRT1 activation by resveratrol decreased cisplatin-induced apoptosis while improving the glomerular filtration rate. Taken together, our findings suggest that the modulation of p53 by SIRT1 could be a possible target to attenuate cisplatin-induced kidney injury.     

Natural products with anti-aging potential: Affected targets and molecular mechanisms

In recent years, there has been a great deal of attention toward the molecular machinery relevant to age-related progression controlled through the external intervention of polyphenols- an epigenetic-modulating diet. Natural products modulate cellular longevity through histone post-translational modification and can also induce the upregulation of autophagy, thus reducing the level of acetyl coenzyme A (AcCoA). In addition, the effect of caloric restriction (CR) on cancer-related chronic inflammation is of great significance in aging. In line with this, SIRT1 protein levels are expanded in response to calorie restriction mimetics (CRM), in this way acting as autophagy inducers relevant to cancer prevention.     

Metformin and tenovin‐6 synergistically induces apoptosis through LKB1‐independent SIRT1 down‐regulation in non‐small cell lung cancer cells

Sirtuin 1 (SIRT1) is known to play a role in a variety of tumorigenesis processes by deacetylating histone and non‐histone proteins; however, antitumour effects by suppressing SIRT1 activity in non‐small cell lung cancer (NSCLC) remain unclear. This study was designed to scrutinize clinicopathological significance of SIRT1 in NSCLC and investigate effects of metformin on SIRT1 inhibition. This study also evaluated new possibilities of drug combination using a SIRT1 inhibitor, tenovin‐6, in NSCLC cell lines. It was found that SIRT1 was overexpressed in 300 (62%) of 485 formalin‐fixed paraffin‐embedded NSCLC tissues. Its overexpression was significantly associated with reduced overall survival and poor recurrence‐free survival after adjusted for histology and pathologic stage. Thus, suppression of SIRT1 expression may be a reasonable therapeutic strategy for NSCLC. Metformin in combination with tenovin‐6 was found to be more effective in inhibiting cell growth than either agent alone in NSCLC cell lines with different liver kinase B1 (LKB1) status. In addition, metformin and tenovin‐6 synergistically suppressed SIRT1 expression in NSCLC cells regardless of LKB1 status. The marked reduction in SIRT1 expression by combination of metformin and tenovin‐6 increased acetylation of p53 at lysine 382 and enhanced p53 stability in LKB1‐deficient A549 cells. The combination suppressed SIRT1 promoter activity more effectively than either agent alone by up‐regulating hypermethylation in cancer 1 (HIC1) binding at SIRT1 promoter. Also, suppressed SIRT1 expression by the combination synergistically induced caspase‐3‐dependent apoptosis. The study concluded that metformin with tenovin‐6 may enhance antitumour effects through LKB1‐independent SIRT1 down‐regulation in NSCLC cells.