Mechanism Of The Arsenic Compounds Induced-apoptosis In HepG2 Cell

Objective To investigate the proliferation inhibition and apoptosis induced by As2O3 and organoarsenium compound in HepG2 cell line and clarify the mechnism and provide a theoretical basis for new drug development of HCC.Methods HepG2 cells and PAO were used in present study.Firstly,MTT assay is used to detect the proliferation inhibition of HepG2 cells,which were treated with arsenic trioxide(iAsⅢ),heat shock(43 ℃),or the combination of iAsⅢ and heat shock(cells were pretreated in 43 ℃ for 1h and then treated with iAsⅢ).Then we investigated the proliferation inhibition of HepG2 cells treated with the combination of HSP90 inhibitor(BIIB021)and heat shock or the combination of iAsⅢ and heat shock(43℃).At last,we treated HepG2 cells with organoarsenium compound(Phenylarsine Oxide;PAO)and detected the viability of cells.We investigated the apoptosis of HepG2 cells induced by PAO and its potientially molecular mechanism(such as mitochondrial membrane potential changes,reactive oxygen species generation,and ER stress)by Flow cytometry,Western-blot and confocal microscopy.Results First,the results of MTT assay showed that the proliferation inhibition of HepG2 cells treated by iAsⅢ was not satisfied(IC50 is more than 100μM),demonstrated that liver cancer cells have great tolerance to iAsⅢ.What’s more,the combination of heat shock(pretreat in 43℃ for 1h)or HSP90 inhibitor with iAsⅢfailed to improve the proliferation inhibition effect of iAsⅢ.Secondly,we found that Phenylarsine Oxide(PAO)could induce the proliferation and induce the apoptosis of HepG2 cells.And it was time and dose dependence.Moreover western-blot results showed that after treated with 0.5μM PAO for 12h,cytochrome C was released from mitochondria membrane to cytoplasm,and apoptosis related protein PARP、Caspase 3、Caspase 9 were also activated and their cleavage fragments were increased.Interestingly we also found that PAO could induce the generation of ROS in cells.And confocal microscopy showed that ROS in cells was generated at mitochondria and endoplasmic reticulum.After pretreated with ROS scavenger N-actyl-L-cystine(NAC),the PAO induced apoptosis were obviously decreased.Furthermore we found that endoplasmic reticulum protein kinase PERK could be phosphorylated at early phase induced by PAO,and led to its downstream protein such as phosphorylated eIF2α(p-eIF2α),ATF4 and CHOP distinctly upregulating.It suggested that PAO could induce cell apoptosis through mitochondria pathway and ER stress pathway.After pretreated with NAC,PARP、caspase-9 and ER stress related protein which were induced by PAO were obviously inhibited,it suggested that PAO-induced cell apoptosis was ROS-dependent.When pretreated with Z-VAD-FMK(Caspase enzyme inhibitor),cell apoptosis was also inhibited,it indicated that PAO-induced cell apoptosis was depended on caspase pathway.Conclusion In conclusion,arsenic trioxide(iAsⅢ),or combination wit HSP90 inhibitor or heat shock failed to induce the apoptosis of Hep G2.However Phenylarsine Oxide effeciently induced cell apoptosis at low concentration,and PAO-induced cell apoptosis is mainly mediated by ROS and induced through mitochondria and ER stress pathway.