p8 (candidate of metasiasis 1) drives ER-stress/autophagy/apoptosis axis induced by the synthetic cannabinoid WIN in HCC cells.

Abstract

Background: Today, evidence is emerging for the role of autophagy in the regulation of life and death of tumour cells and its relationship with ER-stress signaling. Our previous results demonstrated that hepatoma HepG2 cells are sensitive to apoptotic effects induced by WIN, a synthetic cannabinoid, which acts through a mechanism involving the reduction in the levels of some survival factors and the activation of pro-apoptotic ones. Since WIN effects were observed after 36−48 hours of treatment, we investigated the possible activation of ER-stress and autophagic process in the first hours of WIN treatment focusing our attention on p8, a factor whose expression is upregulated in response to cannabinoid-mediated stress. Material and Methods: ER-stress- and autophagy-related proteins were studied by RT-PCR and western blotting analysis. The autophagic morphology was estimated by MDC staining and immunofluorescence. Gene silencing was performed using small interfering RNA against p8. Results: WIN induced ER-stress activating a pathway involving p8-CHOP-TRB3 proteins and increased the expression of the ER chaperone GRP78 which could mediate the transfer of the proapoptotic protein PAR-4 on plasma membrane. Our results indicate that WIN induced the increase in phospho-PAR-4(Thr163) level and the decrease of the pro-survival protein phospho-AKT which is responsible for an inactivating phosphorylation of PAR-4 in Ser249. Moreover, after 16 h of treatment, WIN induced the appearance of autophagic vacuoles and the increase in the lipidated form of LC3 (LC3-II) which is associated with the autophagosomal membrane. The study of beclin-1 revealed a non-canonical beclin-1 independent autophagy. To evaluate the role of p8 as an activator of death pathway we carried out experiments using specific siRNA (sip8). After p8 silencing, either the markers of ER-stress (CHOP, TRB3 and GRP78) as well as those of autophagic process (LC3-II and vacuoles formation) were significantly reduced with respect to the levels observed in WIN-treated non transfected cells. Conclusions: These findings demonstrate that ER-stress and autophagic activation are early events in WIN-induced apoptosis of HCC cells. In particular, ER-stress-related protein p8 seems to have a key role in triggering the WINdependent ER-stress/autophagy/apoptosis cascade in HCC cells. Moreover, the modulation of pAKT/pPAR4 balance contributes to these events.