mRNA Levels Of Sirtuin 1 Research Articles

Abstract 845: Sirtuin1, a longevity gene, is over-expressed and enhances proliferation and survival of endometrial carcinoma cells via deacetylation of p53.

Abstract Background: Sirtuin1 (SIRT1) is one of the NAD-dependent histone deacetylases, known as a longevity gene induced by caloric restriction or various stresses. SIRT1 also contributes to cell survival under stress conditions through deacetylation of apoptosis or cell cycle-related proteins. The overexpression of SIRT1 in several cancers was reported, however, the expression and function of SIRT1 in endometrial cancer remained undetermined. In this study, we examined the role of SIRT1 on the carcinogenesis and survival of endometrial carcinoma cells. Methods: The expressions of SIRT1 protein was examined by immunostaining using formalin-fixed tissue specimens of 110 endometrial carcinomas, 72 normal endometria and 24 endometrial hyperplasias. Immunoreactivity was evaluated according to the ratio of positive cells in 500 cells and described as a positivity index (PI, full score 100). All of surgical specimens were used after obtaining the consents. The mRNA and protein levels of SIRT1 in endometrial carcinoma cell lines, Ishikawa and HEC1B, were examined using real-time RT-PCR and Western blotting under stresses such as ultraviolet, hypoxia and cisplatin (CDDP) treatment. The viability of those cells under CDDP treatment was examined using WST-1 assay with/without suppression of SIRT1 by SIRT1 inhibitor (EX527) or siRNA. The effect of SIRT1 inhibition on the acetylation of p53 and the p53-related protein expression was in Ishikawa and HEC1B cells were examined using Western blotting under CDDP treatment. Results: Imunoreactivity of SIRT1 protein of carcinoma (PI=37.0±25.6) was significantly higher than that of normal endometrium (PI=25.3±26.5, P= 0.006). The expression of SIRT1 was significantly higher in grade 3 tumors (P= 0.006) and those with lympho-vascular space invasion (P= 0.044). There is a weak correlation between the expression of SIRT1 and growth potential indicated by Ki67 (P=0.051). The expression of SIRT1 protein was inversely correlated with that of p53 in carcinoma tissues and cell lines. In addition, the suppression of SIRT1 significantly reduced cell proliferation activity (P=0.01) and resistance against CDDP (P=0.004) in Ishikawa and HEC1B cells. The suppression of SIRT1 enhanced the acetylation of p53 protein and its downstream proteins. Conclusions: SIRT1 is over-expressed in endometrial carcinoma and may be involved in the carcinogenesis and development. SIRT1 may enhance the survival of endometrial carcinoma cells against various stresses, especially CDDP exposure, via inhibition of p53 by deacetylation. These findings suggest that SIRT1 can be a potent therapeutic target of endometrial carcinoma. Citation Format: Ryoichi Asaka, Tsutomu Miyamoto, Kaori Ishikawa, Yasushi Yamada, Hisanori Kobara, Akihisa Suzuki, Shun'ichiro Taniguchi, Tanri Shiozawa. Sirtuin1, a longevity gene, is over-expressed and enhances proliferation and survival of endometrial carcinoma cells via deacetylation of p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 845. doi:10.1158/1538-7445.AM2013-845

Delipidating effect of resveratrol metabolites in 3T3‐L1 adipocytes

Due to the low bioavailability of resveratrol, determining whether its metabolites exert any beneficial effect is an interesting issue. 3T3-L1 maturing pre-adipocytes were treated from day 0 to day 8 of differentiation and mature adipocytes for 24 h on day 12 with 1, 10, or 25μM of resveratrol or its metabolites. Triacylglycerols were assessed by spectrophotometry and gene expression by real time RT-PCR. Resveratrol, trans-resveratrol-4'-O-glucuronide and trans-resveratrol-3-O-sulfate reduced triacylglycerol content in maturing pre-adipocytes at 25μM. In mature adipocytes, both resveratrol and its glucuronide metabolites, though not sulfate metabolite, reduced triacylglycerol content, although resveratrol was more effective than them. Resveratrol and the three metabolites reduced C/EBPβ mRNA levels. Trans-resveratrol-3-O-sulfate also reduced C/EBP-α, peroxisome proliferator-activated receptor γ (PPAR-γ), and lipoprotein lipase (LPL) expression. In mature adipocytes, resveratrol increased ATGL, CPT-1, deacetylase sirtuin 1 (SIRT-1), and PGC1-α expression. Trans-resveratrol-3-O-glucuronide reduced mRNA levels of FASN and increased those of SIRT-1. Trans-resveratrol-4'-O-glucuronide increased HSL and SIRT-1 mRNA levels. Trans-resveratrol-3-O-sulfate did not change gene expression. The present study shows for the first time the delipidating effect of (i) resveratrol metabolites in maturing pre-adipocytes and (ii) glucuronide metabolites in mature adipocytes. This suggests that both resveratrol and resveratrol metabolites may be involved in the anti-obesity effect of this polyphenol.

Metabolic programming of sirtuin 1 (SIRT1) expression by moderate energy restriction during gestation in rats may be related to obesity susceptibility in later life

In rats, 20% gestational energy restriction programmes offspring for higher food intake, which in adulthood results in higher body weight in males but not in females. Here, we aimed to assess whether the effects of moderate energy restriction during gestation and the sex-related outcomes on adult body weight may be related to the metabolic programming of sirtuin expression in different tissues. For this purpose, 25-d-old offspring of control and 20% energy-restricted (ER) rats (from days 1-12 of pregnancy) were studied. Body weight and the weight of white adipose tissue (WAT) depots and liver were recorded and mRNA expression of sirtuin 1 (SIRT1) and selected genes in the WAT, liver, muscle and hypothalamus were analysed. No differences were found in body weight or the weight of WAT and liver between the control and ER animals. A similar pattern of SIRT1 mRNA expression was found in the WAT, liver and skeletal muscle of ER animals, but in a sex-dependent manner: ER males showed lower SIRT1 mRNA levels than the controls, while no differences were found in females. A sex-different pattern was also observed in the hypothalamus. ER males, but not females, also showed lower mRNA levels of adipose TAG lipase (ATGL) and uncoupling protein 2 in WAT and of sterol response element binding protein 1c and stearoyl-CoA desaturase-1 in the liver. Both sexes of ER animals showed lower mRNA levels of 5' adenosine monophosphate-activated protein kinase and ATGL in the liver. In conclusion, moderate maternal energy restriction during gestation programmes a particular, sex-dependent gene expression profile of SIRT1 in different peripheral tissues, which may be related to obesity predisposition in adulthood; therefore SIRT1 expression emerges as a potential early biomarker of obesity susceptibility.

Resveratrol promotes expression of SIRT1 and StAR in rat ovarian granulosa cells: an implicative role of SIRT1 in the ovary

BackgroundResveratrol is a natural polyphenolic compound known for its beneficial effects on energy homeostasis, and it also has multiple properties, including anti-oxidant, anti-inflammatory, and anti-tumor activities. Recently, silent information regulator genes (Sirtuins) have been identified as targets of resveratrol. Sirtuin 1 (SIRT1), originally found as an NAD+-dependent histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. To date, the presence and physiological role of SIRT1 in the ovary are not known. Here we found that SIRT1 was localized in granulosa cells of the human ovary.MethodsThe physiological roles of resveratrol and SIRT1 in the ovary were analyzed. Immunohistochemistry was performed to localize the SIRT1 expression. SIRT1 protein expression of cultured cells and luteinized human granulosa cells was investigated by Western blot. Rat granulosa cells were obtained from diethylstilbestrol treated rats. The cells were treated with increasing doses of resveratrol, and subsequently harvested to determine mRNA levels and protein levels. Cell viability was tested by MTS assay. Cellular apoptosis was analyzed by caspase 3/7 activity test and Hoechst 33342 staining.ResultsSIRT1 protein was expressed in the human ovarian tissues and human luteinized granulosa cells. We demonstrated that resveratrol exhibited a potent concentration-dependent inhibition of rat granulosa cells viability. However, resveratrol-induced inhibition of rat granulosa cells viability is independent of apoptosis signal. Resveratrol increased mRNA levels of SIRT1, LH receptor, StAR, and P450 aromatase, while mRNA levels of FSH receptor remained unchanged. Western blot analysis was consistent with the results of quantitative real-time RT-PCR assay. In addition, progesterone secretion was induced by the treatment of resveratrol.ConclusionsThese results suggest a novel mechanism that resveratrol could enhance progesterone secretion and expression of luteinization-related genes in the ovary, and thus provide important implications to understand the mechanism of luteal phase deficiency.

The role of sirtuin 1 in osteoblastic differentiation in human periodontal ligament cells

Activation of sirtuin 1 (SIRT1) promotes the differentiation of keratinocytes and mesenchymal stem cells, but inhibits the differentiation of muscle and fat cells. However, the involvement of SIRT1 in the differentiation of human periodontal ligament cells into osteoblast-like cells remains unclear. To identify the role of SIRT1 in human periodontal ligament cells, we measured SIRT1 mRNA and SIRT1 protein levels during the osteoblastic differentiation of human periodontal ligament cells. Additionally, we investigated the effects of overexpressing and underexpressing SIRT1 on the differentiation of human periodontal ligament cells, and the signaling mechanisms involved. Expression of SIRT1 and osteoblastic differentiation markers was assessed by RT-PCR, real-time PCR, Alizarin red staining and western blotting. Marked upregulation of SIRT1 mRNA and SIRT1 protein was observed in cells grown for 3 d in osteogenic induction medium (OM). Activation of SIRT1 using resveratrol and isonicotinamide stimulated osteoblastic differentiation in a dose-dependent manner, as assessed by the expression of mRNAs encoding alkaline phosphatase, osteopontin, osteocalcin, osterix and Runx2, and induced calcium deposition. In contrast, inhibition of SIRT1 using sirtinol, nicotinamide and gene silencing by RNA interference suppressed mineralization and the expression of osteoblast marker mRNAs. Further mechanistic studies revealed that resveratrol treatment increased the phosphorylation of Akt, adenosine monophosphate kinase (AMPK), Smad 1/5/8 and c-Jun N-terminal kinase, but reduced OM-induced activation of nuclear factor-κB. Conversely, application of sirtinol suppressed the phosphorylation of Akt, AMPK, Smad 1/5/8, p38, ERK and c-Jun N-terminal kinase, and enhanced nuclear factor-κB activity, in OM-stimulated cells. These data suggest that SIRT1 is a potent regulator of differentiation of human periodontal ligament cells and may have clinical implications for periodontal bone regeneration.

The effects of resveratrol on cyclooxygenase-1 and -2, nuclear factor kappa beta, matrix metalloproteinase-9, and sirtuin 1 mRNA expression in hearts of streptozotocin-induced diabetic rats

Resveratrol (RSV) has a beneficial role in the prevention of diabetes and alleviates some diabetic complications, such as cardiomyopathy. We investigated cyclooxygenase-1 (COX-1), COX-2, nuclear factor κB (NF-κB), matrix metalloproteinase-9 (MMP-9), and sirtuin 1 (SIRT1) mRNA expression levels in heart tissue after RSV treatment in streptozotocin (STZ)-induced diabetic rats. After induction of chronic diabetes with STZ, 10 mg RSV/kg per day was administered to DM and DM+RSV groups for four weeks. At the end of the experiment, all rats were sacrificed and heart tissues were stored at -80°C; mRNA expression levels of COX-1, COX-2, NF-κB, MMP-9, and SIRT1 genes were analyzed with quantitative real-time PCR. We did not find any significant effect of RSV on MMP-9, COX-1, COX-2, or NF-κB mRNA levels among the groups. However, SIRT1 mRNA levels decreased in the DM group compared to controls and increased in the DM+RSV group when compared to the DM group. SIRT1 is activated by RSV treatment in diabetic heart tissue. Activation of SIRT1 by RSV may lead to a new therapeutic approach for diabetic heart tissue. We conclude that RSV treatment can alleviate heart dysfunction by inhibiton of inflammatory gene expression such as SIRT1.