Effect And Mechanism Of Autophagy In Hep-2 Cell Treated With Resveratrol

Objectives: Autophagy is a highly regulated biological mechanism responsible for cell death and intracellular degradation. It is believed resveratrol can promote the cancer cell lead to autophagic cell death, and promote the laryngeal cancer cell lines Hep- 2 apoptosis. But whether resveratrol can cause Hep-2 cells death by induction autophagy and the promoting function between autophagy and apoptosis is not clear. This study intends to verify the promotion role of resveratrol in Heo-2 cells autophagy and the relationship between promote autophagy and apoptosis. The results of this study may help to provide a new experimental basement for the anti-tumor effect of resveratrol.Methods: The cell survival was determined by MTT. We apply Western blotting and Real Time-PCR to detect the protein and m RNA levels. The autophagic bodies and autophagic vacuoles were detected by MDC staining and transmission electron microscope respectively. Intracellular reactive oxygen species(ROS) level was assessed by DCFH-DA staining. Apoptotic cell within the Hep-2 cells was examined by Hoechest33258 staining. All data are analyzed by using SPSS 13 software. Results: Resveratrol dose- and time-dependent decreased the survival rate of Hep-2 cells, with 80μM significantly. Resveratrol dose and time dependently promote Hep-2 cells autophagy, with 40μM and 6h in the most significant. It can increase the ratio of LC3 II and LC3 I, promoting the protein and m RNA expression of beclin-1 and the number of autophagic vacuoles and autophagosomes were also increased significantly. Mechanism study found that the effect of resveratrol promoting Hep-2 cells autophagy related to ROS increased. After resveratrol treatment, the ROS in Hep-2 cells increase about 6 times. Further research found that ROS induces Hep-2 cells autophagy through the activation of AMPK-m TOR pathway. The promotion role of resveratrol on apoptosis in Hep-2 cells was weakened significantly with using 3MA and CQ inhibiting autophagy.Conclusions: Resveratrol can dose- and time-dependent promoting Hep-2 cells autophagy through AMPK-m TOR pathway, and this pathway activated by ROS. The promoting function of resveratrol on Hep-2 cells apoptosis is decreased when inhibit autophagy.