Studies On The Anti-proliferative Effects And Molecular Mechanism Of 23-HBA Derivative B4G2 In Hepatocellular Carcinoma

Aim: To investigate anti-proliferative effect of B4G2 on hepatocellular carcinoma Hep G2 and the underlying molecular mechanisms.Methods:(1)The growth inhibitory activity of B4G2 on HCC cell lines was detected by MTT assay;(2)The long-term Hep G2 growth inhibitory activity of B4G2 was detected by cell cloning experiment;(3)The influence of B4G2 on cell cycle was detected by PI staining assay;(4)B4G2induced alteration of nuclear morphology was detected by Hoechst 33528 staining assay;(5)The apoptosis rate was measured using Annexin V-FITC/PI staining assay;(6)transmission electron microscope was applied to observe the cellular ultrastructure;(7)The expression level of apoptotic protein such as caspase,PARP and Bcl-2 were measured using western blot;(8)JC-1 staining assay was performed to detect the mitochondrial membrane potential;(9)Fluo-3-AM staining assay was used to analyse the level of Ca2+;(10)H2DCFDA probe was used to measure the amount of ROS;(11)B4G2-induced autophagosome was detected by MDC staining;(12)Mito-tracker probe was used to label mitochondria;(13)ATP Assay Kit was used to measure the level of cellular ATP.Results: B4G2 can inhibit growth of HepG2?HepG2/ADM?Hep3B?Bel-7402 and it showed obvious growth inhibitory effect on Hep G2 in a time-and dose-dependent manner,cell cycle analysis indicate that cell population in Sub G1 phase was accumulated with increased dose of B4G2.In Hoechst 33528 staining assay,Hep G2 cells showed chromatin condensation after treatment of B4G2.Moreover,many specific apoptotic characteristics were observed through transmission electron microscope.Annexin V-FITC/PI staining assay showed that B4G2 induced early and late apoptosis in a dose-dependent manner.Western blot was used to detect the key modulatory protein of apoptosis including caspase3,caspase8,caspase9 and PARP after B4G2 treatment.It was found that these apoptosis-related proteins were activated,which can not be blocked by Z-VAD-FMK,a fan-caspase inhibitor.After treated with B4G2,Hep G2 cells showed depolarization of mitochondrial membrane potential accompanied by release of cytochrome c,indicating that B4G2 kills Hep G2 cells by mitochondrial apoptotic pathway.Further research showed that B4G2 induced the generation of ROS,thus increasing the level of Ca2+,which can be blocked by antioxidants,NAC.NAC also reversed the depolarization of mitochondria and cell apoptosis.PT pore inhibitor Mg Cl2 had the same effect as NAC.Further study demonstrated that B4G2 induced mitophagy and mitophagy inhibitor increased the apoptotic rate of Hep G2.Conclusion: B4G2 has a strong anti-HCC effect and it induces ROS-dependent mitochondrial apoptosis by opening of the PT pore.B4G2 also induces mitophagy which plays a protective role against apoptosis.