The Role Of Prion Protein In Breast Cancer MDA-MB-435Cell Apoptosis

Objective:To investigate the role of cellular prion protein (PrPc) in breast cancer cells apoptosis.Methods:Using Western-bloting method for protein detection; immune fluorescence method for target protein localization and semi quantitative analysis; retroviral vector RNAi for establishing PrPc knockdown stable cell lines; MTT method for the determination of cell viability and normal cell growth curve; trypan blue staining method for determining live/dead cell ratio.Results:The MDA-MB-435cell line is a estrogen receptor negative and PrPc high expression human breast cancer cell line. PrPc knockdown stable cell lines (abbreviated as MDA-MB-435-siPrP:siPrP) and control cell lines (abbreviated as MDA-MB-435-siLuc:siLuc) were successfully constructed using retroviral vectors expressing RNAi targeting PrP or luciferase.Our results revealed that PrPc plays dramatically different roles in cell death caused by various factors.(1) In Doxorubicin treatment or glucose deprivation, siPrP cell line exhibited significantly more tolerance than siLuc cell lines, indicating a pro-apoptotic function of PrPc;(2) however, in rotenone-induced cell death, a higher cell death was observed in siPrP cell line compared to the control siLuc cells, suggesting an anti-apoptotic function of PrPc;(3) in hydrogen peroxide (H2O2), paclitaxel and staurosporine treatments, siPrP cells and siLuc cell lines did not show clear differences, suggesting that PrPc does not play a significant role in these cell death processes. Our results showed that, in the same cell line, PrPc may play different roles in cell deaths induced by various factors. In order to remove the additional effect caused by the stable cell line establishment process due to antibiotic screening of cell lines, we in transiently transfected siPrP and controlled siLuc plasmid to MDA-MB-435cell lines;48hours after transfection, repeat the doxorubicin experiment,results showed that PrPc downregulation of cell lines are still on adriamycin tolerance phenomenon; that of doxorubicin on siLuc and siPrP two cell lines of different effect was not due to stable cell lines construction process, but because of PrPc expression downregulated.Moreover,research on the mechanism of siPrP cell tolerance by doxorubicin treatment, we found that siPrP cell line p-p53expression was higher in siLuc cell line after doxorubicin treatment,; due to MDA-MB-435cell line p53mutants, we transfect widetype p53in stable cell lines, the experimental results show that after up-regulation of p53,siPrP-p53were still more tolerance on doxorubicin treatment,we also showed that the pro-apoptotic function of PrPc in Doxorubicin-treated siPrP cells is p53pathway independent.