The Mechanism Of HVJ-E Induced-apoptosis In PC3 Cell

Prostate cancer is the most commonly diagnosed malignant tumor. In USA, prostate cancer is the second leading cause of cancer-related death in men. Prostate cancer develops slowly and tumor growth is androgen dependent. Following androgen ablation or castration, prostatecancer regresses but later adopts an 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, novel therapeutic approaches are required to treat androgenrefractory prostate cancer.Studies have shown that inactivated Sendai virus (Hemaglutinating virus of Japan Envelope,HVJ-E) not only can be used as a carrier to deliver anticancer drugs,but also be able to activate multiple anti-tumor immunity.Especially,this virus has a direct anti-tumor effect. In this study we used HVJ-E to induce human Prostate cancercells (PC3) apoptosis and autophagy, to explore its anti-tumor mechanism.Firstly,the effects of HVJ-E on the proliferation and viability of PC3 cells were examined.Treatment of these cells with HVJ-E,we observed growth arrest, shrinkage and collapse.Cell inhibition and apoptosis detected by CCK-8 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, suggesting that HVJ-E could directly cause apoptosis in PC3 cells.Secondly,the Caspase-related proteins were detected by immunoblot assay;the cleavage of caspase-8,caspase-9,caspase-3,PARP can be detected with the increasing of HVJ-E concentration, indicating that HVJ-E induced apoptosis is caspase dependent.To further confirm the apoptotic induction by HVJ-E is Caspase pathway dependent,cells were pretreated with Caspase inhibitor Z-VAD-FMK,the results demonstrated that the cleavage of caspase-3 and PARP were markedly suppressed,in addition, cell apoptosis was also inhibited significantly. Theseresults demonstated that Caspase pathway is involved in HVJ-E induced PC3 cells apoptosis.To investigate whether MAPK pathways is involved in HVJ-E treated PC3 cells,the phosphorylation of p38,JNK,Erk1/2 were detetected in PC3 cells following HVJ-E treatment. The results showed that all the three proteins were activated post HVJ-E treatment.To further investigate the role of MAPK pathway in HVJ-E-induced apoptosis,specific inhibitors,SB203580,SP600125 and U0126,targeting p38,JNK and Erkl/2,respectively,were added to cells 30 minutes prior to HVJ-E treatment. Western blot results showed that the phosphorylated forms were all significantly inhibited.Meanwhile, the FACS and CCK-8 results showed these specific inhibitors could reduce HVJ-E-induced apoptosis in PC3 cells, indicating that MAPK pathway plays an important role in HVJ-E induced PC3 cells apoptosis.To investigate the potential mechanism of HVJ-E activated MAPK pathway and induced apoptosis. We found that HVJ-E could induce ROS generation in PC3 cells. Therefore, to verify MAPK activation and apoptosis in PC3 cells is depending on ROS. To evaluate the role of ROS in HVJ-E-induced apoptosis in PC3 cells, the cells were treated with an ROS scavenger, NAC, prior to HVJ-E treatment. The results revealed that NAC not only blocked ROS generation but also significantly inhibited HVJ-E-induced apoptosis. Additionally, an increase in both cell viability and the Bcl-2/Bax ratio were observed following NAC treatment compared with HVJ-E-treated cells. Furthermore, the results suggested that NAC treatment protected PC3 cells from HVJ-E-induced activation of p38, JNK, Erkl/2 and cleavage of caspase-3. These results further suggest that the MAPK pathways play a role downstream of ROS during the regulation of PC3 cellular apoptosis.Finally,we investigated the role of autophagy in HVJ-E-induced PC3 cells apoptosis.We found that HVJ-E could induce autophagy in PC3 cells by GFP-LC3 plasmid transfection. The Western blot results showed the phosphorylation of AKT,mTOR and p70S6K and the expression of becline-1 were increased with the increase of HVJ-E concentration.lt appears that autophagy is involved in the HVJ-E via positive regulatory pathway.To investigate the role of autophagy in HVJ-E-induced apoptosis in PC3 cells. Autophagy inducer and inhibitor, Rapa and CQ were prior to HVJ-E treatment.The results showed that Rapa could promote HVJ-E induced apoptosis via Western blot, FACS and CCK-8 assay.These results provide a new insightfurther investigate the application of HVJ-E on cancer therapy.