Role And Mechanism Of CHOP In Apoptosis Of Human Hepatocellular Carcinoma Cell Lines Induced By Sorafenib Combined With Suberoylanilide Hydroxamic Acid

Objective: To investigate the role and mechanism of C/EBP homologous protein(CHOP)in apoptosis of human hepatocellular carcinoma cell lines MHCC97 L and Hep G2 induced by sorafenib(SOR)combined with suberoylanilide hydroxamic acid(SAHA).Methods: Different concentrations of SOR(0.5,1,3,6,12,25 and 50 ?mol/L),SAHA(0.5,1,3,6,12,25 and 50 ?mol/L)and SOR combined with SAHA(the same dose as before)were administered to MHCC97 L and Hep G2 cells for 48 h,the inhibitory effect of SOR combined with SAHA on proliferation of MHCC97 L and Hep G2 cells was detected by CCK-8 assay,and then cells were treated with 12 ?mol/L SOR,6 ?mol/L SAHA and 12 ?mol/L SOR combined with 6 ?mol/L SAHA according to the results of CCK-8 assay.Subsequently,the control group,the 12 ?mol/L SOR group,the 6 ?mol/L SAHA group and the two-drug combination group(12 ?mol/L SOR + 6 ?mol/L SAHA)were treated for 48 h,flow cytometry was used to detect the effect of sorafenib combined with SAHA on cell cycle and apoptosis.Western blot was used to detect the expression of glucose-regulated protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),p-PERK and activated transcription factor 4(ATF4),as well as CHOP.To further clarify the role of CHOP in apoptosis of human hepatocellular carcinoma cells induced by SOR combined with SAHA,the apoptosis of MHCC97 L and Hep G2 cells was observed by flow cytometry after CHOP was silenced by CHOP si RNA.Results: Compared with the control group,the cell proliferation of MHCC97 L and Hep G2 cells were treated with different concentrations of SOR or SAHA significantly decreased in a dose-dependentmanner,the combination with SOR and SAHA could further inhibit cell proliferation(P<0.05),and the cell cycle was arrested in G1 phase.The results of flow cytometry showed that 12 ?mol/L SOR combined with 6 ?mol/L SAHA resulted in significant apoptosis,the apoptotic rate was(37.88 ± 7.68)% in MHCC97 L cells and(23.20 ±1.06)% in Hep G2 cells,which were also significantly higher than control group(0.80± 0.87)% ?(5.23 ± 0.27)% and that of cells treated with SOR(13.18 ± 1.08)% ?(13.57 ± 1.12)% or SAHA(12.18 ± 1.84)% ?(10.85 ± 0.86)%(P<0.01).Simultaneously,the results of Western blot showed that SOR combined with SAHA up-regulated the expression of GRP78,p-PERK,ATF4 and CHOP.Specific knockdown of CHOP by CHOP si RNA significantly attenuated sorafenib combined with SAHA-induced apoptosis.Conclusion: Sorafenib combined with SAHA significantly inhibits the viability and promotes the apoptosis of MHCC97 L and Hep G2 cells,which may be related to the up-regulation of CHOP expression in the apoptotic pathway of endoplasmic reticulum stress.