Beclin1is A Key Molecule Of Resveratrol-induced Autophagy Under P53Regulation Of Colon Cancer Cells

Objective：Resveratrol （RSV）, an extracted phytoalexin from plants, hasmany biological effects. In vitro assays showed that resveratrol is capable ofinhibiting tumor growth and activating autophagy and apoptosis in cancercells. However, to date, the mechanism for resveratrol to inhibit cancer isunclear. p53, a tumor suppressor gene, could induce cell autophagy andpromote apoptosis. But the effects of p53varied in different conditions. Thepurpose of the present study is to elucidate the mechanisms of the resveratrolinhibits colon cell growth, test cell autophagy, and the interaction of p53withthe autophagy related protein. Analyzes the relations between autophagy andapoptosis.Methods:1. HCT-116^p53+/+and HCT-116p53-/-cells grown in the medium containing50,100μM RSV for24h were used to observe the response of colon cells toRSV.2. MTT assay was performed to confirm the cell viability of the treatedHCT-116^p53+/+and HCT-116p53-/-cells with50,100μM RSV.3. western-blot assays were used to detect expression of LC3, p53, Beclin1,p-Raptor in HCT-116^p53+/+and HCT-116p53-/-cells.4. The expression of LC3was detected by immunofluorescence.5. HCT-116^p53+/+and HCT-116p53-/-cells was treated with the combination ofRSV with p53activator （Nutlin3）. The relationship between p53and LC3was determined.6. SPSS13.0was used to analyze all the data. The data from threeindependent experiments performed in triplicate presentedare mean±S.D.Used t-test to analyze the data. P<0.05was regarded as statisticalsignificant. Results:1. The expression of p53mRNA in HCT-116^p53+/+and HCT-116p53-/-cells.There was a significant difference in transcriptional level of p53mRNAbetween HCT-116^p53+/+and HCT-116p53-/-cells （1v.s.0.002±0.002, P<0.05）.After the treatment with0,50,100μM of RSV for24h, the level of p53mRNA in HCT-116^p53+/+cells was1,2.013,2.171, respectively. There was asignificant increase of p53in the groups treated with100μM RSV comparedwith the control group （P <0.05）.2. The morphological changes of HCT-116^p53+/+and HCT-116p53-/-cells aftertreatment with0μM,50μM,100μM RSV for24h.After treatment at0,50,100μM of RSV for24h, HCT-116^p53+/+andHCT-116p53-/-cells exhibited marked morphological changes. Compared withthe control, cells surface refraction were weakened and cells were shrinkedwith uneven surface. There were more individual cells floated with theincreased RSV concentration.3. The viability of HCT-116^p53+/+and HCT-116p53-/-cells treated with0μM,50μM,100μM RSV for24h.There was significant difference between the viability of HCT-116^p53+/+cells and that of HCT-116p53-/-cell after treatment with50μM RSV （0.913v.s.0.687, P<0.05）. When the concentration of RSV was increased to100μM,there still was significant difference between the viability of HCT-116^p53+/+and the viability of HCT-116p53-/-（0.753v.s.0.570, P<0.05）.4. The expression of LC3in HCT-116^p53+/+and HCT-116p53-/-cells wasdetected by immunofluorescence.In the control group of HCT-116^p53+/+cell，there was only nucleus of blue,and in the experimental group of100μM RSV treatment, red LC3could beobserved. While in HCT-116p53-/-cells, red LC3could be observed, and LC3was increased in the100μM RSV treatment group.5. The expression of autophagy-related protein p-Raptor, Beclin1and LC3.The expression of autophagy-related protein p-Raptor was enhanced inHCT-116^p53+/+and HCT-116p53-/-cells. After treatment with different concentration of RSV （0,50,100μM） for24h, the level of LC3inHCT-116^p53+/+cells was increased. Expression of Beclin1was increased inHCT-116^p53+/+cell and reduced in HCT-116p53-/-cell treated with differentconcentration of RSV （0,50,100μM） for24h.6. The relation of p53and autophagy treated with Nutlin3combined with ornot RSV in HCT-116^p53+/+cells.Expression of p53protein was increased in HCT-116^p53+/+cell treatedwith Nutlin3. The HCT-116^p53+/+cell’s LC3II expression with Nutlin3treatment was reduced compared with control group. Samely, compared withRSV treatment group, LC3II expression was reduced while HCT-116^p53+/+cellwas treated with RSV and Nutlin3.Conclusions:1. The growth was inhibited by resveratrol on HCT-116^p53+/+thanHCT-116p53-/-cells.2. Autophagy of HCT-116^p53+/+cells was inhibited by the p53in thecytoplasm.3. Resveratrol could induced HCT-116^p53+/+cells autophagy.4. Beclin1was a key molecule of p53regulating human colon cancer cellsfrom autophagy to apoptosis.