The investigation of the effects of resveratrol and Sirtuin1 on p53(+/+) and p53(-/-) colon carcinoma cells

Introduction: Cancer is a disease that includes heterogenic and complex molecular changes. Resveratrol is a natural polyphenol synthesized by more than 70 plant species. Nowadays, anti-carcinogenic effects of resveratrol, as well as anti-proliferative and apoptosis inducing effects have been proved in a variety of cancer cells. When recent studies on resveratrol are reviewed, activation of SIRT1, a histone deacetylase enzyme called as sirtuins has been shown. It has been known that SIRT1 has a role on deacetylation of p53. In order to in more than 50% of cancer cases have p53 mutation responses of a cancer cell to therapeutic agents or polyphenols could be very different. On the other hand, increased expression of cytosolic SIRT1 has been reported in colon cancer cells. Considering the effects of resveratrol on SIRT1 expression, the influence of the signal transduction pathways in the presence or absence of p53 of colon cancer cells is gaining importance. However we couldn't find illustrative knowledge in the literature about the effects of resveratrol on signal transduction in the presence or absence of p53 and also interaction with SIRT1. Aim: In the present study, we aim to investigate the effects of resveratrol on cell viability, apoptotic cell death levels and changes on gene expression of various signal transduction pathways under activated and inhibited SIRT1 conditions in p53(+/+) and p53(-/-) colon carcinoma cells. Material and Method: IC50 doses of resveratrol and sirtinol, which is a SIRT1 inhibitor, were determined by analysis of WST-1 in HCT116p53(+/+) and HCT116p53(-/-) colon carcinoma cells. The cell death type (apoptotic/necrotic) of resveratrol and sirtinol applications in IC50 dosages was determined by flow cytometric Annexin/PI method for 24 and 48 hours. The activity of SIRT1 enzyme was evaluated by fluorometric activity assay kit. SIRT1 and p53 protein expressions were examined by western blot analyses. Real time PCR analyses were used to evaluate the changes of 29 gene expressions of TGF-β, WNT, p53, NF-κB and JAK/STAT signal transduction pathways in all experimental groups. The effects of resveratrol on signal transduction pathways; SIRT1 activation and p53 dependency were evaluated by statistical analyses of obtained data. Results: IC50 level of resveratrol was found as 60 µM for 48 hour in HCT116p53(+/+) and HCT116p53(-/-) cell lines. Apoptotic cell death ratio was found as 34.9% for 24 hour and 45.7% for 48h and 27.4% for 24h and 40% for 48h in HCT116 p53(+/+) and HCT116p53(-/-) cell lines respectively. The inhibition dosage and timing of sirtinol application were determined by SIRT1 enzyme activity assay and found as 80 µM and 24 hour respectively. Decreases of cell viability for HCT116p53(+/+) cell line was found as 40.4% for 24h after resveratrol treatment, and % 57.3 for 24h resveratrol treatment after pre-treatment with sirtinol for 24h. Decreases of cell viability for HCT116p53(-/-) cell line was found as % 43.6 for 24h after resveratrol treatment, and 59.6% for 24h resveratrol treatment after pre-treatment with sirtinol for 24h. Apoptotic cell death ratios were determined as 17.9% for resveratrol treatment and 18.2% for consecutive treatments of sirtinol and resveratrol for 24h. Although in HCT116p53(+/+) cell line SIRT1 protein expression levels were found statistically lower (p<0.05) than the control group in sirtinol pretreated group, statistically significant results have not been found for HCT116p53(-/-) cell line (p>0.05). The results of the gene expression analysis in all experimental groups showed that resveratrol has statistically significant capacity to change on multiple gene expressions which have proliferative or apoptotic capacity, depend on the p53 and SIRT1 activity of the cell lines. Conclusion: This research has revealed that resveratrol (60µM) causes decrease in cell viability and increase in apoptotic cell death in HCT116p53(+/+) and HCT116p53(-/-) cell lines. However our research for the first time proved that the effects of resveratrol on some of gene expressions and apoptosis ratios could change by the p53 mutation and SIRT1 activity level of HCT116 colon carcinoma cells. We proposed that resveratrol might show proliferative and/or apoptotic effects according to p53 and SIRT1 activity in colon cancer cells. Consequently, we predicted that unconscious usage of resveratrol in colon cancer patient might cause adverse effects. Key Words: Colon Cancer, Resveratrol, SIRT1, p53, Apoptosis, Signal Transduction Pathways