Sorafenib induces autophagic cell death and apoptosis in hepatoma HepG2 cells via PERK/EIF2α signaling pathway

Abstract

Backgound & AimsPrevious studies have documented cellular apoptosis, autophagy and endoplasmic reticulum (ER) stress participated in the hepatocellular carcinoma (HCC). Sorafenib has been reported to regulate autophagy and apoptosis in hepatoma cells associated with different unfolded protein response (UPR). However, resistance problem dramatically limited the usage of sorafenib in HCC with uncertain mechanism. Therefore, this research is focused on whether and how sorafenib induces autophagy and apoptosis via specific PERK/EIF2a signaling pathway of ER stress in HepG2 cells.MethodsFirstly, stable infected HepG2 cell lines of PERK knockdown and vehicle control cells were established with lentivirus‐mediated RNA interference, RT‐PCR and western blot were employed to identify the efficiency of PERK silencing. Trypen blue assay was then applied to investigate the effect of PERK knockdown, and sorafenib (10 μM) induced‐lethality in these established HepG2 cell lines in presence or absence of thapsigargin (Tg, 500 nM). Western blot was further performed to analyze the protein expression of PERK, p‐PERK, EIF2α, p‐EIF2α, CHOP, caspase3 and P62. Finally, the numbers of apoptotic cells in these cells were measured by flow cytometry.ResultsAfter PERK silencing, the expression of PERK mRNA and protein were significantly lower (P<0.05) in the established HepG2 cell lines v.s. control. In parallel, the expression of p‐PERK, EIF2α, p‐EIF2α, CHOP and cleaved caspase3 protein were significantly decreased (P<0.05) and apoptotic numbers were dramatically inhibited in PERK silencing cells (P<0.05). In basal condition, sorafenib was not only found to induce autophagic flux but also drive up‐regulation of PERK, p‐PERK, EIF2α, p‐EIF2α, CHOP in vehicle HepG2 cells. Interestingly, PERK overexpression further induced autophagy with decrement in P62 in HepG2 cells in response to sorafenib or sorafenib plus Tg (P<0.05). Meanwhile, trypan blue assay found that the sorafenib alone and sorafenib combined with Tg induced‐cell death was substantially decreased with about 10% and 11% in PERK silencing cells but increased with 30% in PERK overexpression cells, respectively (P<0.05).ConclusionPERK, one of the hallmarks of ER stress signaling, might cause procelldeath autophagy and thereby contribute to enhance sorafenib derived apoptosis in hepatoma HepG2 cells, which may provide a potential critical target for overcoming the resistance of sorafenib in HCC in the future.Support or Funding InformationSupported by NSFC Grants 81360077, 81860654‐30630145, 81172260 and GXNSFC Grants 2015GXNSFDA139022 to Guo ZhangThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.