Bortezomib Is Synergistic with Rituximab and Cyclophosphamide in Inducing Apoptosis of Mantle Cell Lymphoma Cells In Vitro and In Vivo.

Introduction: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor clinical outcome. Although frontline therapy induces a high rate of complete remission, relapse is inevitable and new regimens are needed for relapsed MCL. The proteasome inhibitor bortezomib (BTZ) induces apoptosis and sensitizes MCL cells to chemotherapy in relapsed MCL, but as a single agent, response rate is low, duration of response is short and side effects are severe. Here we evaluated whether BTZ is additive or synergistic with cyclophosphamide (CTX) and rituximab (RTX). Material and Methods: Four human MCL cell lines SP53, MINO, Grant 519, and Jeko-1 and freshly isolated primary tumor cells from three MCL patients were treated with BTZ, CTX, RTX individually or in combination of RTX and CTX (RC), or BTZ plus RTX and CTX (BRC regimen). Cell proliferation and apoptosis were evaluated to determine if there was additive or synergistic effect of the BRC regimen. Western blot analysis was used to elucidate the molecular mechanism by which BTZ, RTX, CTX, RC and BRC induces apoptosis in MCL cells. In addition, in vivo experiments using severe combined immunodeficiency mice with human mantle cell lymphoma xenografts were performed to examine the in vivo efficacy of the regimen to control the growth of and eradicate MCL cells. Results: BTZ and CTX as single agents inhibited the growth of MCL cell lines in a dose-dependent manner (P < 0.01). The IC50 (inhibitory concentration at 50%) for BTZ and for CTX were between 10 and 20 nM and between 5 and 20 mM, respectively. Increasing doses of BTZ with a fixed dose of RTX (10 μg/mL) and CTX (10 mM) resulted in markedly synergistic growth inhibition of MCL cells (P < 0.01). The BRC regimen induced apoptosis in about 69.7% of MCL cell lines and 92.6% of primary tumor cells (P < 0.05 and P < 0.01, compared with those induced by BTZ, RTX, CTX or RC). Furthermore, western blotting analysis showed that BRC induced apoptosis earlier via activation and cleavage of caspases-8, -9, and -3, and PARP as compared with BTZ, RTX, CTX or RC. The pan-caspase inhibitor z-VAD-FMK completely blocked apoptosis induced by BRC. In vivo studies demonstrated that BRC regimen eradicated subcutaneous tumors in MCL-bearing SCID mice and significantly prolonged the long-term event-free survival up to 10 weeks in 70% of the mice, whereas all tumor-bearing mice receiving BTZ, RTX, CTX or RC or PBS (control) died of aggressive MCL within 6 weeks. Conclusion: Cytoreductive chemotherapy with both BTZ and anti-CD20 antibody effectively inhibited the growth and induced apoptosis of MCL cells in vitro and in vivo. Bortezomib-rituximab-cyclophosphamide (BRC) regimen may offer a better therapeutic modality for MCL patients. Thus, our data lay the basis for a clinical trial in relapsed MCL using the BRC combination treatment.