Raloxifene-Induced Myeloma Cell Apoptosis: A Study of Nuclear Factor- B Inhibition and Gene Expression Signature

Because multiple myeloma remains associated with a poor prognosis, novel drugs targeting specific signaling pathways are needed. The efficacy of selective estrogen receptor modulators for the treatment of multiple myeloma is not well documented. In the present report, we studied the antitumor activity of raloxifene, a selective estrogen receptor modulator, on multiple myeloma cell lines. Raloxifene effects were assessed by tetrazolium salt reduction assay, cell cycle analysis, and Western blotting. Mobility shift assay, immunoprecipitation, chromatin immunoprecipitation assay, and gene expression profiling were performed to characterize the mechanisms of raloxifeneinduced activity. Indeed, raloxifene, as well as tamoxifen, decreased JJN-3 and U266 myeloma cell viability and induced caspase-dependent apoptosis. Raloxifene and tamoxifen also increased the cytotoxic response to vincristine and arsenic trioxide. Moreover, raloxifene inhibited constitutive nuclear factorB (NFB) activity in myeloma cells by removing p65 from its binding sites through estrogen receptor interaction with p65. It is noteworthy that microarray analysis showed that raloxifene treatment decreased the expression of known NFB-regulated genes involved in myeloma cell survival and myeloma-induced bone lesions (e.g., c-myc, mip-1 , hgf, pac1,. . .) and induced the expression of a subset of genes regulating cellular cycle (e.g., p21, gadd34, cyclin G2,. . . ). In conclusion, raloxifene induces myeloma cell cycle arrest and apoptosis partly through NFB-dependent mechanisms. These findings also provide a transcriptional profile of raloxifene treatment on multiple myeloma cells, offering the framework for future studies of selective estrogen receptor modulators therapy in multiple myeloma. Multiple myeloma is a bone marrow disease characterized by uncontrolled plasma cell proliferation and by various clinical manifestations such as hyperproteinemia, renal insufficiency, anemia, and skeletal destruction. Osteolysis is a major clinical complication of multiple myeloma and is associated with bone pain and pathological fractures (Roodman, 2004). Multiple myeloma treatment remains unsatisfactory, and new drugs targeting key signaling pathways required for myeloma growth or survival are needed. Raloxifene is a selective estrogen receptor modulator (SERM) registered for the treatment of osteoporosis (Delmas et al., 2002). SERMs have both estrogen-agonistic and antagonistic properties depending on the tissue, the cell type, and even the target gene. Raloxifene thus has in vitro antiestrogen activities on breast tumor cells, and raloxifene treatment is associated with a decreased incidence of invasive breast cancer (Martino et al., 2004). However, raloxifene properties on the proliferation and survival of other cancer cells, including multiple myeloma cells, have not been explored exten-