Effects of resveratrol on H2O2‐induced apoptosis and expression of SIRTs in H9c2 cells
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Abstract Resveratrol, a polyphenol found in fruits, has been demonstrated to activate Sir2. Though many studies have demonstrated that resveratrol can activate SIRT1, whether it has effect on other sirtuins (SIRT2–7) are unknown. The present study shows that exposure of H9c2 cells to 50 µM H 2 O 2 for 6 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry (FCM), but pretreatment of resveratrol (20 µM) eliminated H 2 O 2 ‐induced apoptosis. Resveratrol also prevented H 2 O 2 ‐induced caspase‐3 activation. Exposure of cells to resveratrol caused rapid activation of SIRT1,3,4,7. Sirtuin inhibitor, nicotinamide (20 mM) attenuated resveratrol's inhibitory effect on cell apoptosis and caspase‐3 activity. These results suggest that resveratrol protects cardiomyocytes from H 2 O 2 ‐induced apoptosis by activating SIRT1,3,4,7. J. Cell. Biochem. 107: 741–747, 2009. © 2009 Wiley‐Liss, Inc.Keywords:
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Resveratrol (3,5,4-trihydroxystilbene) is a polyphenol present in black grapes and its derivatives. Several reports have shown that resveratrol can prevent or slow the progression of a wide variety of illnesses, including cancer, cardiovascular disease and ischemic injuries as well as enhances stress resistance and extends the lifespan of various organisms from yeast to mammals. The mechanism by which resveratrol exerts such a range of beneficial effects across species and disease models is not yet clear, although at the beginning it was proposed that the antioxidant properties of this drug may explain the majority of its beneficial effects. Another mechanism by which resveratrol could combat tumour formation is induction of cell cycle arrest and apoptosis. The last protective mechanism related with resveratrol is its role as activator of sirtuin 1. Resveratrol increases the affinity of sirtuin 1 for its acetylated substrates, possibly inducing a conformational change of the enzyme. The axis sirtuin 1/PGC-1 activated by the resveratrol is a signalling pathway involved in several cellular contexts and tissues, each of the actors involved may promote a separate slow down in the neurodegenerative process. This neuroprotective action is very likely due the fact that the central factor of this signal, PGC-1, promotes mitochondrial activity while neurodegenerative diseases are linked to mitochondrial failures. It is strongly suggested that the activation of the axis sirtuin 1 /PGC-1 by resveratrol could be a key feature of the mechanisms of neuroprotection by this polyphenol and give birth to new therapeutic prospects. Keywords: Oxidative stress, cell cycle, sirtuin 1
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Sirtuins (SirT1-7) are the human homologues of the NAD+-dependent histone deacetylase Sir2 from Saccharomyces cerevisiae (ySir2). Since the overexpression of Sir2 in yeast increases lifespan, the study of mammalian sirtuins has gained widespread interest. Each sirtuin is characterized by a conserved 275 amino-acid catalytic core domain as well as by unique additional N-terminal and/or C-terminal sequences of variable length. Sirtuins hold promise as potential drug targets for the treatment of a variety of conditions, including cancer, metabolic diseases, diabetes and aging. Although, a number of sirtuin inhibitors as well as activators are known, the inhibitory mechanism of sirtuins is still unknown. In this study, we sought to identify novel inhibitors of SirT1, and we assessed their in vivo potential as chemotherapeutic agents. For this purpose, we established a fluorescence-based deacetylation assay using methyl-aminocoumarin-acetyllysine (MAL) as a substrate that is suitable for high-throughput screening. Two compound libraries (500 and 18.000 compounds, respectively) were screened for SirT1-modulating activities, and we identified 14 potential inhibitors and 12 potential activators of SirT1. Of the inhibitors, 9 showed inhibition of SirT1-dependent deacetylation of an acetylated p53 peptide. Interestingly, two of them also inhibited SirT2. Both SirT2 inhibitors were also able to inhibit the p53 deacetylation with IC50 values comparable to those determined in the MAL deacetylation assay. Moreover, we observed that the first 220 amino acids of the N-terminal region of SirT1 had an influence on the inhibitory effect of one inhibitor identified here. The potential activators failed to enhance the activity of SirT1 to deacetylate the p53 peptide. Surprisingly, one of them showed strong inhibition of SirT1 in this assay (Hill 2012) as well as inhibition of MAL deacetylation by ySir2 and SirT2. Subsequently, we determined the anticancer potential of the inhibitors identified in this study by different in vivo experiments with the lung cancer cell lines A549 and H1299. Three compounds that inhibited cell viability and proliferation of these cancer cells in a dose-dependent manner were pursued in more detail. These three inhibitors induced an additional increase of apoptosis after combined treatment with the chemotherapeutic agent etoposide. We observed that the additional increase of apoptosis mediated by one of theses inhibitors was p53-dependent. In summary, this study has led us to identify one SirT1 inhibitor as well as two SirT2 inhibitors that showed antiproliferation potential and can be developed further for cancer therapy.
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Sirtuins are NAD+ dependent lysine deacylases involved in many regulatory processes like control of metabolic pathways, DNA repair, and stress response. Modulators of sirtuin activity are needed as tools for uncovering the biological function of these enzymes and as potential therapeutics. Systematic discovery of such modulators is hampered by the lack of efficient and simple continuous activity assays running at low sirtuin concentrations in microtiter plates. Here we describe an improved continuous sirtuin 5 assay based on the coupling of the sirtuin reaction to a proteolytic cleavage using internally fluorescence-quenched substrates. Systematic optimization of a carbamoyl phosphate synthetase 1 derived, glutarylated peptide yielded a Sirt5 substrate with kcat/KM value of 337 000 M–1 s–1, which represents the best sirtuin substrate described so far. These extraordinary substrate properties allowed reliable determination of Ki values for different inhibitors in the presence of only 10 nM sirtuin in microtiter plate format. Assay conditions could be transferred effectively to other lysine deacetylases, like sirtuin 2 and sirtuin 3, which now enables more efficient development of sirtuin targeting drugs.
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Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology.Protein levels of SIRT1 and SIRT2 were determined in non-small cell lung cancer (NSCLC) cell lines and primary tumors from 105 patients. Changes in proliferation were assessed after SIRT1 and SIRT2 downregulation in lung cancer cell lines using siRNA-mediated technology or tenovin-1, a SIRT1 and SIRT2 inhibitor.High SIRT1 and SIRT2 protein levels were found in NSCLC cell lines compared with non-tumor lung epithelial cells. The expression of SIRT1 and SIRT2 proteins was also significantly higher in lung primary tumors than in normal tissue (P<0.001 for both sirtuins). Stronger nuclear SIRT1 staining was observed in adenocarcinomas than in squamous cell carcinomas (P=0.033). Interestingly, in NSCLC patients, high SIRT1 and SIRT2 expression levels were associated with shorter recurrence-free survival (P=0.04 and P=0.007, respectively). Moreover, the combination of high SIRT1 and SIRT2 expression was an independent prognostic factor for shorter recurrence-free survival (P=0.002) and overall survival (P=0.022). In vitro studies showed that SIRT1 and/or SIRT2 downregulation significantly decreased proliferation of NSCLC.Our results support the hypothesis that SIRT1 and SIRT2 have a protumorigenic role in lung cancer, promoting cell proliferation. Moreover, the expression of these proteins is associated with poor prognosis in NSCLC patients and may help to identify those NSCLC patients with high risk of recurrence that could benefit from adjuvant therapy after resection.
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Sirtuins are a class of histone deacetylases which have been shown to regulate a range of physiopathological processes such as cellular aging, inflammation, metabolism and cell proliferation. Although there are seven mammalian sirtuins (SIRT1-7), more detail is known about the function, distribution and substrate of the SIRT1 compared to the remaining sirtuins. Numerous studies have reported changes in SIRT1 in neurodegenerative diseases such as Alzheimer's disease (AD). Since aging is the biggest risk factor in the development of AD, we investigated changes in sirtuin levels in these conditions. We tested mRNA and protein expression levels levels in post-mortem human control and AD brain tissue (Hippocampus, Frontal, Occipital and Temporal lobes) using western blotting and real time PCR. To confirm whether these changes are due to Alzheimer's disease pathology or a response to aging, we characterised the changes in sirtuin expression in normal aged wistar rats. We found a significant increase in SIRT2 mRNA and protein expression in the occipital lobe. Our data also shows that SIRT5 is downregulated in the temporal lobe. From our results it seems that SIRT2 is the most abundant sirtuin in the human brain. In the aged female wistar rat brain, we found a significant upregulation in SIRT1, SIRT3, SIRT4, mRNA and protein expression in the frontal lobe, while SIRT2 is upregulated in the occipital lobe consistent with AD. SIRT5 and SIRT6 levels are reduced in the aging hippocampus, and temporal lobe, while SIRT7 was significantly increased in the temporal lobe. This study suggests that aberrant expression levels of sirtuins is present in AD and aging, and may represent metabolic differences between humans and rodents.
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Abstract Sirtuins 1‐7 (SIRT1‐7) belong to the third class of deacetylase enzymes, which are dependent on NAD + for activity. Sirtuins activity is linked to gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection, and healthy aging. Because sirtuins modulation could have beneficial effects on human diseases there is a growing interest in the discovery of small molecules modifying their activities. We review here those compounds known to activate or inhibit sirtuins, discussing the data that support the use of sirtuin‐based therapies. Almost all sirtuin activators have been described only for SIRT1. Resveratrol is a natural compound which activates SIRT1, and may help in the treatment or prevention of obesity, and in preventing tumorigenesis and the aging‐related decline in heart function and neuronal loss. Due to its poor bioavailability, reformulated versions of resveratrol with improved bioavailability have been developed (resVida, Longevinex ® , SRT501). Molecules that are structurally unrelated to resveratrol (SRT1720, SRT2104, SRT2379, among others) have been also developed to stimulate sirtuin activities more potently than resveratrol. Sirtuin inhibitors with a wide range of core structures have been identified for SIRT1, SIRT2, SIRT3 and SIRT5 (splitomicin, sirtinol, AGK2, cambinol, suramin, tenovin, salermide, among others). SIRT1 inhibition has been proposed in the treatment of cancer, immunodeficiency virus infections, Fragile X mental retardation syndrome and for preventing or treating parasitic diseases, whereas SIRT2 inhibitors might be useful for the treatment of cancer and neurodegenerative diseases.
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Abstract The NAD + ‐dependent deacylase family of sirtuin enzymes have been implicated in biological ageing, late‐life health and overall lifespan, though of these members, a role for sirtuin‐2 (SIRT2) is less clear. Transgenic overexpression of SIRT2 in the BubR1 hypomorph model of progeria can rescue many aspects of health and increase overall lifespan, due to a specific interaction between SIRT2 and BubR1 that improves the stability of this protein. It is less clear whether SIRT2 is relevant to biological ageing outside of a model where BubR1 is under‐expressed. Here, we sought to test whether SIRT2 over‐expression would impact the overall health and lifespan of mice on a nonprogeroid, wild‐type background. While we previously found that SIRT2 transgenic overexpression prolonged female fertility, here, we did not observe any additional impact on health or lifespan, which was measured in both male and female mice on standard chow diets, and in males challenged with a high‐fat diet. At the biochemical level, NMR studies revealed an increase in total levels of a number of metabolites in the brain of SIRT2‐Tg animals, pointing to a potential impact in cell composition; however, this did not translate into functional differences. Overall, we conclude that strategies to enhance SIRT2 protein levels may not lead to increased longevity.
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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, p21CIP1 and p16INK4A 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, p21CIP1 and p16INK4A 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.
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