Autophagy In Endoplasmic Reticulum Stress-induced Hepatocellular Carcinoma Cell Death And Effects On Drug Sensitivity

Background:Hepatocellular carcinoma (HCC) is prevalent in the world, and is one of the most leading cancers in China with characteristics of high malignancy, poor prognosis and multi-drug resistance (MDR). But, the underlying mechanisms of malignant proliferation and MDR were unclear. Evidences showed that the high rate proliferation of HCC cells and other adverse microenvironment such as viral infection, hypoxia, and inflammatory factors in HCC usually result in overload of endoplasmic reticulum (ER) in cells, leading to accumulation of misfolded and/or unfolded proteins in ER, a condition referred to "ER stress". Unfolded protein response (UPR) is subsequently evoked to alleviate the stress by activating a group of signal transduction pathways and the transcription of genes. So, it is of great significance to elucidate mechanism of HCC proliferation and survival under ER stress. Autophagy is responsible for intracellular protein degradation and organelle turnover, as well as energy homeostasis under nutrition deficiency and stress. Recent studies have further extended autophagy to carcinogenesis and cancer therapy. But, whether it represents a pro-or anti-cancer mechanism is now far beyond our understanding. Usually, ER stress can cause cell death, but why HCC cells can survive in adverse microenvironment, whether ER stress itself can trigger autophagy, and the role of autophagy in HCC cells survival/death and chemotherapy sensitivity are still unsolved.Objectives:The aims of current study were to investigate autophagic responses in human HCC cell line after stimulation with ER stress inducer, tunicamycin (TM), and role of autophagy in ER stressed induced HCC cell death, as well as to explore the potential sensitization effect of autophagy inhibition in oxaliplatin treated HCC cells.Methods:Hepatocellular carcinoma cell line HepG2, HepG2.2.15and Bel-7402were cultured in DMEM, MEM, and RPMI1640respectively, with10%fetal bovine serum. Normal human liver cell L-02was cultured in DMEM with10%FBS. Cellular viability assays were performed using CCK-8. Fitting curves for dose-response were performed by OriginPro8.5.1software and IC50values were calculated. GR.P78protein expression and cleavage of caspase-3were detected by Western-Blot analysis. For comprehensive autophagy evaluation, both of morphologic, biochemical, and functional methods were used. a). Ultramicroscopic structure of autophagosome was confirmed under transmission electron microscope by using EBSS and rapamycin as positive control.b). Immunofluorescence staining for MAP1-LC3(microtubule-associated protein1light chain3, LC3) was performed, and LC3expression in cytoplasm was observed using laser confocal microscopy, c). In addition, LC3conversion from LC3-Ⅰ to LC3-Ⅱ was evaluated by Western-Blot and semi-quantity analysis of integrated optical density of the bands, d). Autophagic flux was assessed by LC3turnover assay with chloroquine (CQ) to inhibit lysosome, expression level of LC3-Ⅱ protein was compared between CQ (-) and CQ (+) group.Results:①Proliferation of three hepatocarcinoma cell lines (HepG2, HepG2.2.15and Bel-7402) and one normal hepatocyte line (L-02) were detected by CCK-8after TM stimulation. Cell viabilities of HepG2, HepG2.2.15and Bel-7402after TM incubation were higher than that in L-02while treating with same dosage of TM.②TM dose-dependently and time-dependently caused decrease of cell viability in HepG2with IC50=6.46O6μg/ml for48hours incubation. In addition, caspase-3cleavage was demonstrated to be involved in TM-induced death of HepG2cells.③Western-Blot demonstrated that expression of GRP78protein was up-regulated in TM treated HepG2cells, and the expression level correlated with concentration and duration of TM incubation.④Autophagy in ER stressed-HepG2cells, a). Transmission electron microscopy revealed that there was more autophagic compartments accumulation in TM-treated cells than that in non-treated cells. b). Immunofluorescence staining for LC3on coverslip displayed more fluorescent punctate after ER stress which reflects increase in autophagic structure, c). Western-Blot assay demonstrated LC3conversion in HepG2cells after ER stress stimulation, with decreased expression of LC3-I and increase of LC3-II. LC3ratio (LC3-II/I) elevated in2.5and5μg/ml TM-treated cells.3-MA addition could suppress LC3conversion. d). LC3turnover assay demonstrated autophagic flux enhancement in ER stressed HepG2cells. There was a remarkable increase of LC3-II accumulation after lysosomal inhibition with CQ.⑤In the presence of3-MA (with final concentrations of5mmol/L or10mmol/L),48hours of TM stimulation induced more significant decrease of cell viability than that without3-MA co-incubation, and10mmol/L3-MA exerted greater extent of cell viability suppression than5mmol/L3-MA. However, CQ did not seem to have an effect on the viability of ER stressed-HepG2cells.⑥For Oxa-treated HepG2cells, formation of autophagosome was demonstrated under TEM after Oxa treatment, as well as increased expression of LC3-Ⅱ. LC3-Ⅱ expression was markedly increased in presence of CQ, which reflected an enhancement of autophagic flux induced by Oxa.⑦CCK-8demonstrated that Oxa could dose-dependently induced HepG2cell death, and autophagy inhibition by3-MA or CQ facilitated Oxa induced cell death.Conclusions:Hepatocarcinoma cells seemed more tolerant to ER stress. ER stress in HepG2cells could elicit an enhancement of autophagic flux, the enhanced autophagic flux contributed to cell viability maintenance of HepG2and tolerance to ER stress. Oxa could also enhance autophagic flux in HepG2. Inhibition of autophagy may facilitate killing of HepG2by Oxa. The results may shed light on understanding carcinogenesis and drug resistance of HCC. Autophagy could become a potential target for HCC therapy and chemotherapy sensitization.