Overcoming Chemoresistance in Pancreatic Cancer: Targeting the Apoptotic Protein Machinery using Raloxifene

Abstract

Pancreatic cancer is chemoresistant, with the lowest 5-year survival rate of all cancers (5%). We hypothesized that differential regulation of apoptotic proteins contributed to chemoresistance in pancreatic cancer. Preliminary data showed that raloxifene, a Selective Estrogen Receptor Modulator, induced apoptosis in the human pancreatic cancer cell line MIA PaCa-2. We investigated whether raloxifene treatment altered the 1) interaction between anti- and pro-apoptotic proteins, and 2) their mitochondrial recruitment. Briefly, following a 72 hour treatment with 0, 0.05, 0.5, 5, or 20 µM raloxifene, we immunoprecipitated Bax from MIA PaCa-2 cells, and probed the immunoprecipiate with anti-Mcl1L monoclonal antibody. Our results show a dose-dependent reduction in interaction between Bax and Mcl-1L, indicating that raloxifene specifically reduced the recruitment of Mcl-1L to this protein complex. In similar experiments, but with the immunoprecipitating event reversed, immunoprecipitated anti-apoptotic Bcl-2, evinced increased co-precipitated Bim. Further, raloxifene induced dose-dependent mitochondrial translocation of Bax. Taken together, these data suggest a selective pro-apoptotic protein effect of raloxifene. Raloxifene also resulted in cytochrome c release into the cytoplasm, and PARP, and caspase-9 cleavage confirming activation of the mitochondrial apoptotic pathway. In conclusion, raloxifene altered mitochondrial recruitment and molecular interaction between pro- and anti-apoptotic proteins.