Study On The Mechanism Of Inactivated Sendai Virus Strain Tianjin-induced Apoptosis In Human Breast Cancer MDA-MB-231 Cells And Cervical Cancer HeLa Cells

Purpose: It has made great progress in using virus for tumor therapy in these years. Sendai virus strain Tianjin is a new genetype of Sendai virus. The purpose of this article is to evaluate the anticancer effect of ultraviolet-inactivied Sendai virus strain Tianjin(UV-Tianjin) by detecting the apoptosis and the possible mechanism in human breast cancer MDA-MB-231 cells or human cervical cancer He La cells. We hope that our studies can provide a reference for studying the anti-tumor mechanism of virus and developing novel biological agent for breast cancer and cervical cancer therapies.Methods: Human breast cancer MDA-MB-231 cells or human cervical cancer He La cells were treated by different doses of UV-Tianjin, 24 h later,(1) MTT assay was used to detect the proliferation of MDA-MB-231 cells, He La cells or MCF 10 A cells treated with UV-Tianjin(MOI:10, 20, 40, 80, 160, 320) or not.(2) The morphological changes of apoptosis were observed under fluorescence microscope after Hoechst stain in UV-Tianjin-treated(MOI: 40 or 80) MDA-MB-231 or He La cells.(3) The percent of apoptosis cells was determined by flow cytometry after Annexin V / PI double staining in MDA-MB-231 cells or He La cells treated with UV-Tianjin(MOI:20, 40 or 80).(4) The activities of caspase-3,-8 and-9 were assayed by spectrophotometry in MDA-MB-231 cells or He La cells treated by UV-Tianjin(MOI:20, 40 or 80).(5) The mitochondrial membrane potential(MMP) was detected by flow cytometry after JC-10 staining in MDA-MB-231 cells treated with UV-Tianjin(MOI:5, 20 or 80) or He La cells treated with UV-Tianjin(MOI:20, 40 or 80).(6) Western blot was used to determine the expression levels of Bcl-2, Bax, Cyt c, caspase-9, Fas, Fas L, caspase-8 and caspase-3 proteins in UV-Tianjin-treated MDA-MB-231 cells(MOI: 20 or 80) or FADD, APAF-1, Cyt c, caspase-9, Fas, Fas L, caspase-8 and caspase-3 proteins in UV-Tianjin-treated He La cells(MOI: 20, 40 or 80).Results:(1) MTT assay showed that UV-Tianjin inhibited the proliferation of MDA-MB-231 cells or He La cells in a dose-dependent manner, but not normal breastepithelium cell MCF 10 A.(2) Hoechst 33342 staining revealed that the number of cells with characteristic bright blue fluorescence increased in a dose-dependent manner in UV-Tianjin-treated MDA-MB-231 cells or He La cells, which indicated condensed or fragmented chromatin(3) The flow cytometric analysis after Annexin V / PI staining showed that UV-Tianjin induced apoptosis in MDA-MB-231 cells or He La cells in a dose-dependent manner(P<0.01).(4) The activities of caspase-3,-8 and-9 increased in a dose-dependent manner in MDA-MB-231 cells or He La cells after UV-Tianjin treated(P<0.05).(5) JC-10 staining showed UV-Tianjin induced MMP changes in both MDA-MB-231 cells and He La cells in a dose-dependent manner by flow cytometry(P<0.01).(6) Western blot analysis showed that there was a significant difference between high-dose UV-Tianjin group and control group in the expression levels of cytochrome c, Bax, Bcl-2, caspase-9, caspase-3, Fas, Fas L and caspase-8 in MDA-MB-231 cells, as well as FADD, APAF-1, Cyt c, caspase-9, Fas, Fas L, caspase-8 and caspase-3 proteins in He La cells(P<0.05).Conclusion: Our study suggests that UV-Tianjin exhibits the anticancer activity in both human breast cancer MDA-MB-231 cells and human cervical cancer He La cells through inducing apoptosis, which may involve in both the endogenous mitochondrial pathway and the exogenous death receptor pathway.