The Signaling Pathways And Mechanisms Of HVJ-E-induced Autophagy In Hela Cells

Autophagy is a highly conserved eukaryotic cellular recycling process.Through the degradation of cytoplasmic organelles,proteins,and macromolecules as well as the recycling of the breakdown products,autophagy plays important roles in cell survival and maintenance.Accordingly,dysfunction of this process contributes to the pathologies of many human diseases.Early studies suggested that cellular autophagy accounts for the mechanism of cell survival,however,growing evidences have confirmed that autophagy also controls cell death.At present,the interplay between autophagy and cell death modes is very complicated,and regulation of autophagy has become a hot topic in the ongoing study of cell death-related diseases.In recent years,the inactivated Sendai virus(Hemagglutinating virus of Japan evelope,HVJ-E)has attracted wide attention due to its high potential for cancer treatment.According to our previous studies,in this study,cervical cancer Hela cells were used to investigate the roles of autophagy played in HVJ-E induced cell death.When HeLa cells were treated with HVJ-E,the changed cell morphology and reduced adherent cells were observed.Subsequently,CCK-8 detection revealed that the survival rate of Hela cells decreased in a dose-dependent manner after HVJ-E treatment,suggesting that HVJ-E could induce cell death in Hela cells.Then,transmission electron microscopy revealed that autophagosomes and autophagosomes were present in HVJ-E treated Hela cells,and when HeLa cells transfected with GFP-LC3 plasmid following HVJ-E treatment,the number of green punta increased significantly,indicating that HVJ-E could induce autophagy in Hela cells.Furthermore,the results of western-blot showed that HVJ-E treatment increased the conversion of LC3-II,an autophagy marker protein,and decreased the expression of autophagy-degrading substrate protein SQSTML/p62 after HVJ-E treatment,further confirming that HVJ-E could induce autophagy in HeLa cells.The class ? phosphoinositide 3-kinase(PI3K)/Akt/mTOR/p70S6K pathway negatively regulates the induction of autophagy,whereas the class ? PI3K/Beclin-1 positively regulates autophagy.Both of these signaling pathways are involved in regulating apoptosis and autophagy.In the current study,western-blot showed that HVJ-E treatment inhibited the phosphorylation of Akt and mTOR,indicating that the PI3K/Akt/mTOR/p70S6K involves in HVJ-E-induced autophagy.To further verify the above findings,Hela cells were pretreated with 740Y-P(PI3K activator)and SC79(Akt agonist),respectively,and then treated with HVJ-E for 6 h.The results showed that in the presence of 740Y-P or SC79,down-regulated Akt or mTOR activity was significantly restored.These results confirmed the findings that the PI3K/Akt/mTOR pathway negatively regulates HVJ-E-induced autophagy in HeLa cells.Our previous studies have shown that HVJ-E induces apoptosis in Hela cells mainly via the activation of the Erkl/2 signaling pathway,and Erkl/2 signaling also plays an important role in autophagy regulation.To further elucidate the role of Erkl/2 played in HVJ-E-induced autophagy in Hela cells,the Erkl/2 inhibitor was added to Hela cells prior to HVJ-E treatment.As a result,it was found that the Erkl/2 inhibition could both inhibit autophagy and apoptosis.To further investigate the relationship between apoptosis and autophagy induced by HVJ-E in Hela cells,the rapamycin(autophagy inducer)and CQ(autophagy inhibitor)were used to explain the relationship between autophagy and apoptosis,and the outcome revealed that rapamycin could augment HVJ-E-induced apoptosis,in contrast,inhibition of autophagy reduced HVJ-E induced apoptosis.