Crosstalk between autophagy and apoptosis induced by Rip2 and its mechanisms in human pancreatic cancer cells

AIM To investigate the crosstalk between autophagy and apoptosis caused by receptor-interacting protein 2 (Rip2) and its underling mechanisms in human pancreatic cancer cells. METHODS Plasmids (pEGFP-C2 and pEGFP-Rip2) were transfected into human pancreatic cancer Panc-1 cells by jetPRIME method. The Panc-1 cells transfected with pEGFP-Rip2 were treated with 3-methyladenine (3-MA), an autophagy inhibitor. The apoptotic rate was analyzed by flow cytometry. The levels of apoptosis-associated proteins were measured by Western blot. The activity of caspase-8, -9 and -3 was examined by colorimetric method. Moreover, the Panc-1 cells transfected with pEGFP-Rip2 were treated with Z-VAD-FMK, a broad inhibitor of caspases. Subsequently, the levels of autophagy- and PI3K/Akt/mTOR signaling pathway-related proteins were assessed by Western blot. The autophagosomes were observed under transmission electron microscope. RESULTS (1) The apoptotic rate in pEGFP-Rip2 group markedly increased as compared with control group and pEGFP-C2 group, while the apoptotic rate in pEGFP-Rip2+3-MA group was further elevated compared with pEGFP-Rip2 group (P<0.05). Meanwhile, the protein levels of Fas, Bax and cytoplasmic cytochrome c (Cyt-c) were significantly increased, and the protein expression of Bcl-2 was markedly reduced in pEGFP-Rip2+3-MA group as compared with pEGFP-Rip2 group (P<0.05). The activity of caspase-8, -9 and -3 in pEGFP-Rip2+3-MA group was higher than that in pEGFP-Rip2 group. (2) The protein expression of beclin-1 and LC3-Ⅱ was significantly increased and more accumulated autophagosomes were observed under transmission electron microscope in pEGFP-Rip2+Z-VAD-FMK group as compared with pEGFP-Rip2 group. Furthermore, the protein levels of p-mTOR and p-Akt in pEGFP-Rip2+Z-VAD-FMK group were markedly reduced compared with pEGFP-Rip2 group, while no significant difference of mTOR and Akt protein expression was found between the 2 groups. CONCLUSION Inhibition of autophagy promotes apoptosis induced by Rip2 in the pancreatic cancer cells. Its mechanism may be associated with the further activation of the intrinsic and extrinsic apoptotic pathways. Suppression of apoptosis accelerates autophagy induced by Rip2 in the pancreatic cancer cells, and the mechanism may be related to the further down-regulation of PI3K/Akt/mTOR signaling pathways. There is a mutual antagonistic effect between autophagy and apoptosis caused by Rip2 in pancreatic cancer cells.