Mfn2 Induces Human Cervix Carcinoma HeLa Cells Apoptosis Via Mitochondrial Apoptosis Pathway

Part ? Mfn2 Induces He La Cells Apoptosis through Mitochondrial Pathway in vitroPurpose To investigate the cell apoptotic effects of Mfn2 on cervix carcinoma He La cells in vitro and explore the underlying mechanisms.Methods Adenovirus-Mfn2(Adv-Mfn2)was used to delivery Mfn2 into He La cells.The CCK-8 and western blot were performed to detect the effects of Mfn2 on He La cells.The effect of Mfn2 on cell apoptosis was investigated by flow cytometry.Flow cytometry was used to assess the change of mitochondrial membrane potential of cells treated by JC-1 assay.Mfn2,Bax,Bcl-2,cytochome c,cleaved caspase-3,cleaved caspase-9,p53 protein levels were analyzed by western blot.Results Data from CCK-8 and flow cytometry showed that Mfn2 could inhibit proliferation and induce apoptosis in a dose-and time-dependent manner in He La cells.JC-1 Kit assay results revealed that mitochondrial membrane potential decreased in a dose-dependent manner in He La cells after Adv-Mfn2 treatment.The data from western blot confirmed higher cytosolic amounts of cytochrome c with increasing doses of Adv-Mfn2 signified onset of intrinsic apoptotic pathway.To further validate the apoptotic control by Mfn2,expression levels of important apoptotic proteins were analyzed by western blot.Levels of cleaved caspase-3 and cleaved caspase-9 increased in He La cells with Adv-Mfn2 treatment.Meanwhile,significant increment in Bax levels and decreased Bcl-2 levels with Adv-Mfn2 treatment were found.Results of western blot showed that the expression of p53 protein was upregulated obviously compared with the Adv-control groupConclusion Mfn2 overexpression could inhibit He La cells proliferation in a concentration-and timedependent manner.Mfn2 could induce cervix carcinoma He La cells apoptosis via mitochondrial pathway in vitro.Part ? Anti-tumor Effect of Mfn2 on He La Cells Xenografts Nude MicePurpose This study intends to investigate the anticancer potential of Mfn2 in cervix carcinoma mouse model.Methods Adenovirus-Mfn2(Adv-Mfn2)was used to delivery mfn2 into tumor tissues in nude mouse model.BALB/c nude mice(4-5 weeks old)were obtained and He La cells were collected and injected subcutaneously at the right armpit of nude mice.When the tumors were visible,the mice were divided into 2 groupsfor the 9-day treatment with Adv-Mfn2 and Adv-control,respectively.The TUNEL assay,western blot and immunohistochemical staining were performed to detect the effects of Mfn2.Mfn2,Bax,Bid,p-Bad,Bcl-2,cleaved caspase-3 and p53 protein levels of cervix tumor tissues were analyzed by western blot.Results The human cervical carcinoma He La cells were injected subcutaneously at the right armpit of female BALB/c nude mouse.After 5 days,the tumor tissues were found at the right armpit of the mice,and the mice were divided into 2 groups for treatment with Adv-Mfn2 and Adv-control.After the treatment,the volume of xenografted cervix carcinomas treated with Adv-mfn2 declined gradually,whereas the volume of the tumors treated with Adv-control increased.TUNEL assay of tumor sections showed that Mfn2 induced a more prominent He La cells apoptosis than control group.Results of western blot and immunohistochemical staining proved that caspase-3 was activated in xenograftedtumor of Adv-Mfn2 group,compared with the Adv-control group aftertreatment.Western blot demonstrated that overexpression of Mfn2 could not only increase the levels of Bax and Bid in cervix tumor cells,but also decrease the phosphorylation of Bad and the expression of Bcl-2.Expression of p53 protein was upregulated obviously compared with the Adv-control group analyzed by western blot.Conclusion These studie ssuggested overexpression of Mfn2 could trigger the cervix tumor apoptosis in vivo,which was related to mitochondrial pathway,and may provide a new treatment target of cervical carcinoma.