Induction Of Apoptosis In PC3Cells By Inactivated Sendai Virus

Prostate cancer is one of the most common solid tumors in men and one of the major leading causes of cancer-related deaths worldwide. Prostate cancer cells in patients initially respond to androgen withdrawal therapy by undergoing apoptosis, but then the apoptosis-resistant cells survive and develop into androgen-refractory phenotype. Androgen-refractory prostate cancer is more resistant to a number of therapeutic modalities, including chemotherapy and radiotherapy, and is ultimately lethal. Therefore, there is an urgent need for novel treatment strategies for androgen-refractory prostate cancers. Inactivated Sendai virus particle [hemagglutinating virus of Japan envelope (HVJ-E)] has a potential oncolytic effect due to its ability to induce apoptosis in tumor cells. However, the molecular mechanism of apoptosis induction in cancer cells mediated by HVJ-E has not been fully elucidated. Here, to explore the anti-tumor mechanism, we use HVJ-E to induce apoptosis in PC3hormone-resistant human prostate cancer cells.PC3cells were treated with HVJ-E and RIG-I, IFN-β were observed by ELISA and western blot. The results showed that RIG-I was activated, HVJ-E can induce IFN-β production and was dose-dependent. The results indicated that HVJ-E fused effectively with PC3cells and the broken viral genome were recognized by RIG-I, which resulted in an increasing amount of IFN-P production in PC3cells with the increment of HVJ-E.The effects of HVJ-E on the proliferation and viability of PC3cells were examined. HVJ-E treated cells not only represented the reduction of cell number but also represented shrinkage and cell disintegration by microscopy. Cell inhibition and apoptosis detected by MTT and FACS assay indicated that the proliferation of treated cells was inhibited, and the apoptosis of the cells was dose-dependent by HVJ-E administration. Apoptotic chromatin condensation and nuclear fragmentation were readily observed in HVJ-E-treated cells by Hoechst33258staining. The activation of statl and downstream caspase-8, caspase-3and PARP was assayed. The results showed that phosphorylation of statl was detected with HVJ-E at all indicated MOI, meanwhile, the cleavage of caspase-8, caspase-3and PARP was also detected from100to1000MOI by western blot. To further confirm the apoptotic induction by HVJ-E in PC3cells is caspase pathway dependent, cells were pretreated with Jak inhibitor, and then the cleavage of caspase-3and PARP in HVJ-E-treated cells were determined, meanwhile, the treated cells were submitted to apoptotic analysis and microscopic observation. The results showed that pretreatment with Jak inhibitor for2hours markedly suppressed the phosphorylation and the cleavage of caspase-3and PARP, cell apoptosis was also inhibited significantly.To investigate the regulation of the MAPK and Akt pathways in HVJ-E treated PC3cells, phosphorylation of Erkl/2, Jnk, p38MAPK and Akt were detected by IB assay using appropriate antibodies. The results showed that phosphorylation of p38MAPK and Jnk were activated while the phosphorylation of Erk was undetectable. In addition, the phosphorylation level of Akt in PC3cells remains unchanged after HVJ-E treatment. To further investigate the role that MAPK pathways play in HVJ-E induced apoptosis in PC3cells, specific inhibitors, SB203580and SP600125, targeting p38MAPK and Jnk, respectively, were added to PC3cells30min prior to HVJ-E treatment and cell viability was determined by the MTT-based assay. The results showed that PC3cell proliferation was not affected by treatment with the inhibitors at their specific concentration, but the two inhibitors showed significant inhibition of cytotoxicity at24,48and72hours post treatment, meanwhile, the microscopic observation also confirmed that the inhibitors could reduce the cytotoxic effect induced by HVJ-E on PC3cells. In addition, inactivation of the target pathway by specific inhibitors was confirmed by IB assay.PC3cells were incoculated into intradermal space in the back of BALB/C naked mice. Then the tuomor-bearing mice were devided into two groups randomly. Particles of HVJ-E or PBS were injected into tumors thrice when the diameter of tumor reached to4-6mm. The tumor volume of the tumor bearing mice was observed every day post injection. The results showed that the toumors treated by HVJ-E were strongly inhibited compared with the controls.