Targeting IP3-dependent Calcium Signaling Enhances Sorafenib Lethality For Hepatoma Via Endoplasmic Reticulum Stress Related Apoptosis

Objectives:IRE1,hallmark of endoplasmic reticular stress,has been previously shown as a prosurvival molecule in cancer therapy by multikinase inhibitor sorafenib.However,given that the detail mechanism by which trigger IRE1 to impair the sorafenib lethality remains unclear,the present study was designed to clarify whether calcium signaling is involved in this pharmacologic processin hepatocellular carcinoma(HCC).Methods:In HepG2 cells,confocal microscopy was performed to monitor dramatic intracellular calcium mobilizations with sorafenib or combination with calcium channel-related drugs existed.The expression of IRE 1 was examined by Western-blotting and the expression level of spliced XBP1 mRNA(XBP1S)was determined by qRT-PCR.Flow cytometry and light microscope were used to analyze the apoptosis of cells.Results:1.By cofocal microscopy,in certain concentrations range,sorafenib(20～200μM)was found to drive intracelluer free calcium into a rapid-rise-fall mobilization in HepG2 cells,and the elevated level of calcium was in a sorafenib dose-dependent manner.2.IRE1 and the expression level of XBP1S mRNA were significantly increased in cells either in the presence of sorafenib(20μM)or directly addition of low dose of CaCl2(50μM)for 24h by western blot and qRT-PCR.3.While using 2-Aminoethyl diphenylborinate(2-APB)(75μM),the inhibitor of inositol-1,4,5-trisphosphate(IP3)receptor,to blunt the sorafenib induced-calcium release in HepG2,the increment of IRE1 and its activity evoked by sorafenib was substantially attenuated in these cells.4.Meanwhile,flow cytometry and light microscope shew that 2-APB and sorafenib(20μM)amazingly caused a synergistic effect versus sorafenib alone with about 62.8%augmentation of apoptosis,P<0.05.Conclusions:1.Sorafenib-induced intracellular calcium release was in a sorafenib dose-dependent manner.2.The inhibitor of IP3 receptor weakened both the sorafenib induced-calcium release and the increment of IRE1.3.The combination of the inhibitor of IP3 receptor with sorafenib was more effective than alone with sorafeinib in the treatment of hepatoma.Therefore,IP3-elicited Ca2+ release likely plays a critical role in the sorafenib up-regulating IRE1 activity and that is emerging as a novel potential target for anti-HCC therapeutic strategy in the future.