Silencing Of Pokemon Enhances Caspase-Dependent Apoptosis Via Fas-and Mitochondria-mediated Pathways In Hepatocellular Carcinoma Cells

The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, highly expressed in a wide variety of tumors,but in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT-and ERK-dependent manner. Classical apoptosis can be initiated via two major pathways:the intrinsic or mitochondria-mediated pathway and the extrinsic or death receptor-mediated pathway. Activation of both pathways results in the activation of caspases. Oxaliplatin, a third-generation platinum-based chemotherapeutic agent, displays a broader spectrum of antitumor activity than cisplatin and carboplatin.We hypothesized that Pokemon can through the regulation of the two apoptotic pathway to in hepatocellular carcinoma (HCC) occurrence and development control under the action of oxaliplatin.In the present study, We employed stable transfection with short hairpin RNA to stably silence the expression of Pokemon in the HepG2, SMMC-7721hepatocellular carcinoma cell lines.We used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway,expression of pro-apoptotic Bcl-2family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10and caspase-8were activated, causing increased release of caspase-8active fragments p18and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9and caspase-3was observed in HepG2si-Pokemon cells as compared to control. Silencing Pokemon induces cell cycle arrest and up-regulation of cell cycle checkpoints in HepG2cells as shown by flow cytometry and RT-PCR analyses. Therefore, silencing of Pokemon enhances caspase-dependent apoptosis via Fas-and mitochondria-mediated pathways in hepatocellular carcinoma cells,and Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, Pokemon may through the regulation of cell cycle checkpoints to induces cell cycle arrest. So our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.