The Role Of ROS In The Process Of Autophagy Induced By Resveratrol In HCT116 Cells

Objective: This research aimed to discover the role of ROS and its regulation involved in the p53 interaction with NQO1 in the process of RSV induced autophagy. Our results will explain the specific regulatory mechanism of RSV induced autophagy in colon caner, as may give a sufficient and reliable theoretical support for the treatment of the colon cancer.Methods:1 HCT116 cells were cultured in DMEM/F12 medium with 10% fetal bovine serum. When the cells took up 80% of the culture dish, we first pretreated the cells with the ROS scavenger(NAC) for 1h, then with different doses of RSV for 24 h. Finally, The changes of morphology on cells were observed under inverted microscope.2 The cell survival rate was detected by MTT on HCT116 cells processed with different doses of RSV, and NBB was used to compare the cell survival rate between the NAC pretreated group and RSV group.3 The change of the acidic vesicular organelles was observed by MDC on HCT116 cells processed with different doses of RSV.4 The change of the cell nucleus and the chromosome was detected by DAPI staining on HCT116 cells.5 The expression of p53, NQO1,Nrf2 protein were explored by Western Blot on HCT116 cells after they were processed with diverse doses of drugs(NAC,Nutlin-3,PFT-α,dicoumarol) and RSV together for 24 h and transfected with sip53.6 Flow cytometry was used to measure the trend of ROS on HCT116 cells after they were processed with diverse doses of RSV.7 SPSS13.0 was used to deal with the repeat and control results with t-test. The final result were in the form of mean±SD. If the data P was less than 0.05, the results was significant.Results:1 MTT assay showed that the survival rate of HCT116 cells was in a dose dependent manner after treatment with different doses RSV(0-200μM) for 24 h. The inverted microscope was used for watching the changes of morphology in HCT116 cells after treatment with various doses of RSV. If the density of RSV was increased, the morphology would be also altered. The results showed that swell and quantity of cells reduced, the space between the cells get bigger and the quantity of the cells get smaller. After treatment with RSV of 100μM on HCT116 cells for different time(0h, 12 h, 24 h, 48h), the change of the morphology and the quantity of HCT116 cells had the same feature with the concentration group.2 DAPI staining and Western Blot methods showed that when the cells were treated with the different doses of RSV and in the different time of RSV, there was no notable phenomenon of apoptosis. After treatment with diverse doses of RSV for 24 h, we found that the quantity of autophagosome and the phenomenon of autophagy became increased and obvious with the increased doses of RSV. After treatment with 100μM RSV, the variation of HCT116 cells in time group(0h, 12 h, 24 h, 48h)had the same feature with the concentration group. These results were checked by MDC method. The datas of Western Blot made it clear that the expression of some autophagy related protein became added with the increased concentrations of RSV, and as with the anti-apoptotic protein bcl-2 and the pro-apoptotic protein Bax. The proportion of bcl-2/Bax was also increased.All these results showed that there was the autophagy in HCT116 cells.3 Flow cytometry was used to measure the content of ROS in the HCT116 cells after they were processed with diverse doses of RSV. The results indicated that the amount of ROS would be added with the increased doses of RSV.4 The results of NBB indicated that the survival rate of HCT116 cells in ROS scavenger(NAC) group and RSV group was obviously decreased. But the cell survival rate in NAC group was higher than that in RSV group. The inverted microscope showed that the number of survival cells in NAC group was more than that in RSV group.5 MDC method demonstrated that the quantity of the autophagic vacuole in NAC group was less than that in RSV group. Western Blot method showed that the expression of Beclin-1, LC3 protein in NAC group was less than that in RSV group.6 The HCT116 cells were pretreated with ROS scavenger(NAC), p53 activator(Nutlin-3), p53 inhibitor(PFT-α), NQO1 activity inhibitor(dicoumarol) and RSV together respectively for 24 h.Western Blot analysis demonstrated that the expression of related protein in NAC group, PFT-α group and dicoumarol group was lower than that in RSV group, but it had the opposite trend in Nutlin-3 group.Conclusions:1 RSV inhibits the development of HCT116 cells in time and dose dependent manner.2 RSV induces the autophagy of HCT116 cells in a range of dose and time, and the autophagy was related with ROS. But there was no apoptosis in the cells.3 ROS, p53, NQO1 and Nrf2 played a role in the process of autophagy, and there is a link among them.