Expression Patterns of ERα, ERß, AR, SIRT1, SIRT2, and SIRT3 in Prostate Cancer Tissue and Normal Prostate Tissue.

BACKGROUND/AIM: The purpose of this study was to examine the expression of estrogen receptor α (ERα) and ß (ERß), androgen receptor (AR), SIRT1, SIRT2 and SIRT3 in prostate cancer (PCa). MATERIALS AND METHODS: From October 2010 to January 2015, 70 patients who had undergone radical prostatectomy following a PCa diagnosis were enrolled in our study. Normal prostate tissue (NPT) and prostate cancer tissues (PCAT) were separated, and the expression of each receptor in each tissue was analyzed with immunochemical staining. Univariate and multivariate analyses were performed to identify factors affecting the development of PCa. RESULTS: ERß and AR were highly expressed in PCAT compared with NPT (p<0.05). SIRT2 was highly expressed in NPT and PCAT (p<0.05). Univariate and multivariate analyses showed that AR and SIRT2 affect PCa development. CONCLUSION: AR is a risk factor for PC, and SIRT2 is associated with a lower incidence of PCa.

Altered levels of sirtuin genes (SIRT1, SIRT2, SIRT3 and SIRT6) and their target genes in adipose tissue from individual with obesity.

INTRODUCTION: Sirtuins regulate energy metabolism and insulin sensitivity through their ability to act as energy sensors and regulators in several metabolic tissues. AIM: To evaluate the expression levels of sirtuin genes SIRT1, SIRT2, SIRT3 and SIRT6 and their target genes (PPAR-α, PGC1-α, NRF1, DGAT1, PPAR-γ and FOXO3a) in subcutaneous adipose tissue collected from individuals with normoweight, overweight and obesity. METHODS: Adipose tissue samples, obtained by lipoaspiration during liposuction surgery, were processed to obtain RNA, which was reverse-transcribed to cDNA. Then, we measured the expression levels of each gene by qPCR. RESULTS: We found differences in the mRNA expression of SIRT1, SIRT2, SIRT3 and SIRT6 and their target genes (PPAR-α, PGC1-α, NRF1, DGAT1, PPAR-γ and FOXO3a) in adipose tissue from overweight or obese subjects when compared to normoweight subjects. All genes analyzed, except SIRT2, showed correlation with BMI. CONCLUSIONS: Our findings in human subcutaneous adipose tissue show that increased body mass index modifies the expression of genes encoding sirtuins and their target genes, which are metabolic regulators of adipose tissue. Therefore, these could be used as biomarkers to predict the ability of adipose tissue to gain mass of adipose tissue.

Sirtuin 2 (Sirt2) Expression Predicts Lymph Node Metastasis and Poor Overall Survival of Patients with Esophageal Squamous Cell Carcinoma.

BACKGROUND: Altered expression of Sirtuin 2 (Sirt2) was associated with cancer development and progression. This study further assessed the association of Sirt2 expression with clinicopathological data and prognosis of patients with esophageal squamous cell carcinoma (ESCC) after postoperative concurrent chemoradiotherapy. METHODS: Tissue specimens from 95 ESCC patients were collected for immunohistochemical analysis of Sirt2 expression, which was used to determine association with patient clinicopathological and survival data. RESULTS: Sirt2 protein was expressed in 53.7% of ESCC tissue specimens but only in 25.3% of normal squamous epithelium (p = 0.000). Sirt2 expression was associated with tumor invasion (p = 0.005), lymph node metastasis (p = 0.003), and advanced clinical stage (p = 0.000), but not with tumor size (p = 0.199), or differentiation (p = 0.177). Sirt2 expression was associated with poor overall and progression-free survival (p = 0.034). The multivariate analysis showed that Sirt2 expression was an independent predictor for overall survival of patients with resected ESCC followed by concurrent chemoradiotherapy (p = 0.048). CONCLUSIONS: Sirt2 protein expression in ESCC tissue specimens was associated with ESCC invasion, lymph node metastasis, and advanced tumor clinical stage, as well as poor overall and poor progression-free survival. Sirt2 expression is an independent prognostic predictor for ESCC patients.

Helicobacter pylori infection increases sirt2 gene expression in gastric epithelial cells of gastritis patients.

BACKGROUND: Helicobacter pylori Infection causes some clinical features of the human stomach such as gastritis, duodenal ulcer, and gastric cancer. It has been shown that Helicobacter pylori infection increases proinflammatory cytokine gene expressions in Gastric Epithelial Cells by activation of NF-kB signaling. Sirt1 and sirt2 as deacetylases play a certain role in the progress of inflammation in arthritis and lung infection by impacting the NF-kB. AIMS: Sirt1 and sirt2 gene expressions in Gastric Epithelial cells of gastritis patients were surveyed with and without Helicobacter pylori infection and rate of prevalence of cagA and hopQ genes in Helicobacter pylori strains were investigated. METHODS: 25 biopsy samples of gastritis patients with Helicobacter pylori infection and 25 biopsy samples of gastritis patients without Helicobacter pylori infection were collected from Tohid Hospital in the city of Sanandaj throughout the year 2016. CDNA was made from total RNA extracted from biopsy samples (Qiagen® Kit). Sirt1 and sirt2 gene expressions were determined using the Corbett machine (Rotor-Gene 6000 Software). CagA and hopQ genes of Helicobacter pylori strains were determined by PCR using specific primers. RESULTS: The sirt2 gene expression was increased in Gastric Epithelial Cells of gastritis patients with Helicobacter pylori infection. No significant relationship was found between sirt1 and sirt2 gene expressions as well as cagA and hopQ as Helicobacter pylori virulence genes. CONCLUSIONS: This study shows the Helicobacter pylori infection duo to sirt2 gene up-expression. There is not a statistically significance relationship between cagA and hopQ Helicobacter pylori genotypes and sirt2 gene up-expression in Gastric Epithelial Cells of gastritis patients.

Reduced gene expression of sirtuins and active AMPK levels in children and adolescents with obesity and insulin resistance.

BACKGROUND: Sirtuins, including SIRT1 and SIRT2, are longevity-associated deacetylase enzymes that modulate metabolic homeostasis in response to the cellular energy state. Adenosine monophosphate activated protein kinase (AMPK) and SIRT1 are interrelated and share several common target pathways. This study aimed to evaluate the SIRT1 and SIRT2 gene expression in peripheral blood mononuclear cells (PBMCs) as well as plasma levels of AMPK, in obese children and adolescents. MATERIALS AND METHODS: Participants included 60 children and adolescents (30 obese and 30 age- and gender-matched control subjects). Real-time polymerase chain reaction (PCR) was used to assess the SIRT1 and SIRT2 gene expression in PBMCs. Serum phospho-AMPK and insulin were measured using enzyme-linked immunosorbent assay (ELISA), and insulin resistance (IR) was calculated by the Homeostasis Model of Assessment of Insulin Resistance (HOMA-IR). Glucose and lipid profile were also measured. RESULTS: SIRT1 gene expression and phospho-AMPK plasma levels were significantly diminished in obese subjects compared to the control group, and both SIRT1 and SIRT2 were significantly lower in obese children with IR compared to those without IR. SIRT1 expression revealed significant negative correlations with body mass index and waist circumference as well as insulin and HOMA-IR and a positive correlation with AMPK. SIRT2 negatively correlated with SIRT1 and positively correlated with high density lipoprotein-cholesterol (HDL-C). CONCLUSION: SIRT1 and SIRT2 expression and AMPK levels decrease in children with obesity and IR. Targeting SIRT1 can be valuable in preventing obesity-associated IR in childhood and adolescence.

Mutant A53T α-Synuclein Improves Rotarod Performance Before Motor Deficits and Affects Metabolic Pathways.

The protein α-synuclein (α-Syn) interferes with glucose and lipid uptake and also activates innate immune cells. However, it remains unclear whether α-Syn or its familial mutant forms contribute to metabolic alterations and inflammation in synucleinopathies, such as Parkinson's disease (PD). Here, we address this issue in transgenic mice for the mutant A53T human α-Syn (α-SynA53T), a mouse model of synucleinopathies. At 9.5 months of age, mice overexpressing α-SynA53T (homozygous) had a significant reduction in weight, exhibited improved locomotion and did not show major motor deficits compared with control transgenic mice (heterozygous). At 17 months of age, α-SynA53T overexpression promoted general reduction in grip strength and deficient hindlimb reflex and resulted in severe disease and mortality in 50 % of the mice. Analysis of serum metabolites further revealed decreased levels of cholesterol, triglycerides and non-esterified fatty acids (NEFA) in α-SynA53T-overexpressing mice. In fed conditions, these mice also showed a significant decrease in serum insulin without alterations in blood glucose. In addition, assessment of inflammatory gene expression in the brain showed a significant increase in TNF-α mRNA but not of IL-1ß induced by α-SynA53T overexpression. Interestingly, the brain mRNA levels of Sirtuin 2 (Sirt2), a deacetylase involved in both metabolic and inflammatory pathways, were significantly reduced. Our findings highlight the relevance of the mechanisms underlying initial weight loss and hyperactivity as early markers of synucleinopathies. Moreover, we found that changes in blood metabolites and decreased brain Sirt2 gene expression are associated with motor deficits.

High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses sirtuin deacetylase 2 (SIRT2) and 6 (SIRT6) expression leading to histone acetylation and gene expression. SIRT down-regulation mediates the teratogenicity of diabetes leading to (NTD) formation. The study provides a mechanistic basis for the development of natural antioxidants and SIRT activators as therapeutics for diabetic embryopathy.

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 ß-galactosidase (SA-ß-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-ß-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.

Expression/localization patterns of sirtuins (SIRT1, SIRT2, and SIRT7) during progression of cervical cancer and effects of sirtuin inhibitors on growth of cervical cancer cells.

Sirtuins belong to the family of class III histone deacetylases; its role in neoplasia is controversial as both tumor-suppressive and promoting functions have been reported. There are very few reports available, where expressions of sirtuin isoforms are comprehensively analyzed during neoplasia. Therefore, in the present study, the expression of SIRT1, SIRT2, and SIRT7 during different stages of cervical cancer progression was analyzed. The normal cervical epithelium showed feeble expression of sirtuin isoforms, SIRT1, SIRT2, and SIRT7. A significant increase in SIRT1 expression was noted in the cytoplasm as well as in the nucleus of proliferative layers of cervical epithelium in squamous intraepithelial lesions (SIL); however, in the squamous cell carcinomas (SCC), a heterogeneous pattern of SIRT1 expression varying from low to high was noted. A progressive increase in the expression of both SIRT2 and SIRT7 was noted during cancer progression in the following order: normal < preneoplasia < cancer. Cervical cancer cell lines, HeLa and SiHa, showed higher levels of SIRT1 and SIRT2 in comparison to the immortalized cell counterpart, HaCaT. Specific inhibitors of SIRT1 (Ex527) and SIRT2 (AGK2) impaired the growth of the cervical cancer cells, SiHa, but not of the HaCaT cells. SIRT1 inhibition caused cell death, while SIRT2 inhibition resulted in cell cycle arrest. In conclusion, we report the overexpression of SIRT2 and SIRT7 proteins in cervical cancer and suggest probable application of sirtuin inhibitors as therapeutic targets. Further, a specific increase in the levels of SIRT1 in intraepithelial lesion makes it a promising candidate for identification of preneoplastic changes.

Regulation of the mitogen-activated protein kinase kinase (MEK)-1 by NAD(+)-dependent deacetylases.

Sirtuins are class III deacetylases that regulate many essential processes, including cellular stress, genome stability and metabolism. Although these NAD(+)-dependent deacetylases control adaptive cellular responses, identification of sirtuin-regulated signaling targets remain under-studied. Here, we demonstrate that acetylation of the mitogen-activated protein kinase kinase-1 (MEK1) stimulates its kinase activity, and that acetylated MEK1 is under the regulatory control of the sirtuin family members SIRT1 and SIRT2. Treatment of cells with sirtuin inhibitors, or siRNA knockdown of SIRT1 or SIRT2 proteins, increases MEK1 acetylation and subsequent phosphorylation of the extracellular signal-regulated kinase. Generation of an acetyl-specific MEK1 antibody demonstrates that endogenous acetylated MEK1 is extensively enriched in the nucleus following epidermal growth factor  stimulation. An acetyl-mimic of MEK1 increases inappropriate growth properties, suggesting that acetylation of MEK1 has oncogenic potential.

Changes in microtubule-related proteins and autophagy in long-term vitamin E-deficient mice.

Vitamin E deficiency induces neuronal dysfunction and while oxidative stress is likely to be involved in mediating this process, the detailed mechanisms remain to be elucidated. Previously, we found axonal degeneration in the hippocampal CA1 region in vitamin E-deficient mice of 6 months of age (long-term). However, 3 month-old (short-term) vitamin E-deficient mice did not exhibit axonal degeneration in same region. In order to characterize the mechanisms involved in axonal degeneration in long-term vitamin E-deficient mice, we examined changes in microtubule-related proteins. Long-term vitamin E-deficiency led to significantly increased expression of the phosphorylated form of collapsin response mediator protein (CRMP)-2 compared to short-term deficiency. It is well known that CRMP-2 plays a crucial role in the maintenance of neurite function. Similarly, long-term vitamin E-deficiency significantly decreased the expression of silent mating type information regulation (SIRT)-2 mRNA compared to short-term deficiency. SIRT-2 belongs to a family of class III histone deacetylases (HDACs) and functions in the deacetylation of tubulins. Furthermore, the expression of microtubule-associated protein light chain (MAP-LC)3-2, which is a key autophagy protein was significantly higher in the short-term vitamin E-deficiency than the long-term deficiency. These results indicate that the mechanisms of axonal injury in long-term vitamin E-deficient mice are related to dysfunction in microtubules assembly via alterations in microtubule-related proteins and autophagy.

Aging: filtering out bad mitochondria.

During yeast cytokinesis an aged mother cell gives rise to an immaculate daughter cell. A new study now demonstrates that this rejuvenation encompasses a novel Sir2- and actin-cable-dependent filtering process that prevents feeble mitochondria from entering the daughter cell.

Discovery of a potent small molecule SIRT1/2 inhibitor with anticancer effects.

SIRT1 and SIRT2 are deacetylase enzymes that belong to the sirtuin family and are involved in tumorigenesis. In our screen for small molecules inhibiting SIRT1/2 toxoflavin was identified. Toxoflavin potently inhibited SIRT1 activity in in vitro deacetylase assay using purified SIRT1 protein. SIRT2 activity was also inhibited by toxoflavin less potently than SIRT1 in deacetylase assay in vitro. Toxoflavin exhibited growth inhibition of various cancer cell lines including A549 lung cancer cells with a GI(50) of 48 nM. Toxoflavin treatment in A549 cells increased the acetylated form of p53, which is a substrate of SIRT1. The acetylation levels of α-tubulin, a SIRT2 substrate, were also increased by toxoflavin treatment dose-dependently. Several toxoflavin derivatives were synthesized to determine the preliminary structure-activity relationship of toxoflavin. Some of the toxoflavin derivatives showed highly selective inhibition against SIRT1. In conclusion, this study presented toxoflavin as a potent SIRT1/2 inhibitor with anticancer activity.

A molecular study of pathways involved in the inhibition of cell proliferation in neuroblastoma B65 cells by the GSK-3 inhibitors lithium and SB-415286.

Pharmacological GSK-3 inhibitors are potential drugs for the treatment of neurodegenerative diseases, cancer and diabetes. We examined the antiproliferative effects of two GSK-3 inhibitors, lithium and SB-415286, on B65 neuroblastoma cell line. Treatment of B65 cells with either drug administered separately caused a decrease in cell proliferation that was associated with G(2)/M cell cycle arrest. Cell-cycle proteins such as cyclins D, E, A, cdk4 and cdk2 were up-regulated. Since lithium and SB-415286-induced G(2)/M arrest we studied changes in the expression of proteins involved in this phase, specifically cyclin B, cdc2 and the phosphorylated form of this protein (tyr15-cdc2). Both drugs increased the expression of tyr15-cdc2, thus inhibiting mitosis. On the other hand, SB-415286 increased the expression of SIRT2, involved in the regulation of proliferation. Moreover, cell-cycle arrest mediated by SB-415286 was accompanied by apoptosis that was not prevented by 100 microM of zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), a pan-caspase inhibitor. Likewise, GSK-3 inhibitors did not affect the mitochondrial release of apoptosis inducing factor (AIF). We conclude that inhibitors of GSK-3 induced cell-cycle arrest, mediated by the phosphorylation of cdc2 and, in the case of SB-415286, SIRT2 expression, which induced apoptosis in a caspase-independent manner.