The Role Of Reactive Oxygen Species (ROS) In Induction Of Apoptosis By Inactivated Sendai Virus In Murine Melanoma Cells

Melanoma is one of the common malignant tumor. It has some features, such as high degree of malignancy, strongly metastatic, rapid development, poor prognosis and high mortality. In recent years, the incidence and mortality have been at an upward trend, but the conventional treatment methods can not achieve the desired results. Studies have shown that inactivated Sendai virus (Hemaglutinating virus of Japan Envelope, HVJ-E) can induce apoptosis in tumor cells, but the mechanism has not been fully elucidated. Based on the murine melanoma cells (B16F10), this paper is to study the role of reactive oxygen species (ROS) in induction of apoptosis by inactivated Sendai virus both in vitro and vivo. And thus, we can provide new references for revealing the anti-tumor mechanism of Sendai virus.Firstly, B16F10 cells were treated with different concentrations of HVJ-E. And then, we were through the method of FACS (DCFH-DA) to detect the generation of ROS in cells; the cell morphology was observed by inverted microscope; through the method of CCK-8 to detect the cell viability; through the method of FACS (Annexin V/PI double staining) to detect the cell apoptosis rate; Hoechst 33258 staining was used to observe nuclear morphological changes. The results show that on the one hand, HVJ-E can cause the increase generation of ROS in B16F10 cells, on the other hand, HVJ-E can cause the changes of cell morphology and nuclear morphology and can decrease the cell viability and increase the apoptosis rate. These can indicate that ROS may be involved in the induction of apoptosis by HVJ-E in B16F10 cells.Secondly, B16F10 cells were treated with different concentrations of HVJ-E. And then, we were through the method of Western blotting to detect the expression of p53 and Caspase, MAPK signaling pathways proteins. The results show that with the increase of the virus concentration, the expression of p53, Bax, Cleaved Caspase-3, PARP protein gradually increased, Bcl-2 protein expression decreased gradually; the expression of p-JNK, p-Erkl/2, p-p38 was also gradually increased. These can explain that p53, Bax, Bcl-2 and Caspase, MAPK signaling pathways may be involved in the induction of apoptosis by HVJ-E in B16F10 cells.Thirdly, we were through the scavenger of ROS (NAC) to analysis. The B16F10 cells were treated with HVJ-E for 24 h after 30 min pretreatment with NAC. And then, we were through the method of CCK-8 to detect the cell viability; FACS to detect the generation of ROS and the cell apoptosis rate; Western blotting to detect the expression of apoptosis-related proteins. The results show that, NAC pretreatment can suppress the decrease of the cell viability; can inhibit the increase of the cell apoptosis rate; can suppress the increase generation of ROS; meanwhile, NAC pretreatment can suppress the increase expression of p53, Bax, Cleaved Caspase-3, PARP and the decrease expression of Bcl-2; p-JNK, p-Erkl/2, p-p38 proteins expression through the NAC pretreatment is also declined. These can explain that, ROS is indeed involved in the HVJ-E-induced apoptosis in B16F10 cells, and should play a regulatory role at the upstream of the signaling pathway.Finally, we come to study in vivo by establishing the murine melanoma model. Once every two days to measure tumor volume from the tumor-bearing mice, and then we were based on the measured data to draw the tumor growth curve. The results show that, compared with the PBS group, the tumor volume of HVJ-E group grew slowly, while the NAC+HVJ-E group had rapid growth compared to the HVJ-E group, indicating that NAC can inhibit the decrease growth of tumor volume by HVJ-E. Through the method of Western blotting to detect the proteins from tumor tissue, the results show that, NAC can inhibit the increase expression of p53, Bax, Cleaved Caspase-3 and the decrease expression of Bcl-2; meanwhile, the expression of p-JNK, p-Erkl/2, p-p38 is also declined. These can explain that the results from the vivo study are consistent with the vitro study, and thus we can further validate the role of ROS in HVJ-E-induced apoptosis in B16F10 cells.