Abstract

The proteasome inhibitor bortezomib (BZ) has significant antineoplastic activity in multiple myeloma, lymphoma and acute leukemia. Recently, it has been shown that BZ can potentiate the activity of other antitumor agents in vitro. Topoisomerase IIα (topo IIα) is known to be regulated by proteasomal degradation, suggesting that proteasomal inhibition might increase intracellular topo IIα levels and thereby enhance sensitivity to topo IIα-targeted agents, including etoposide, daunorubicin and mitoxantrone. Incubation with sublethal concentrations of BZ led to rapid upregulation of topo IIα protein levels in the human TF-1 AML cell line, the human 8226 myeloma cell line and the 8226-derived, topo IIα-deficient, anthracycline-resistant Dox1V line (kindly provided by William S. Dalton), as measured by Western blot assay. Enhanced topo IIα expression was evident at 3 hrs, peaked at 6 hrs and fell off markedly 24 hrs after BZ addition. Maximal upregulation was 25–90 fold as measured by optical densitometry. BZ induced a shift in the apparent molecular weight of topo IIα, suggesting that upregulation resulted, at least in part, from the stabilization of the ubiquitinated form(s) of the enzyme following proteasomal inhibition. The kinetics of topo IIα upregulation inversely correlated with measured 20S proteasomal activity, further supporting a post-translational mechanism. Nuclear extracts prepared from BZ-treated cells showed a corresponding 36-fold increase in topo IIα enzymatic activity. The cytotoxicity of BZ in combination with other antineoplastic agents was evaluated by MTT assay. Synergistic cytotoxicity was observed for BZ plus mitoxantrone, etoposide or daunorubicin, but no significant augmentation was observed in this system for BZ plus cytarabine, melphalan, camptothecin, dexamethasone, vincristine or fludarabine. In TF-1 AML cells, sublethal doses of BZ reduced the IC50 for daunorubicin, mitoxantrone and etoposide 16-, 52- and 22-fold, respectively. In 8226 myeloma cells, BZ enhanced sensitivity to daunorubicin and etoposide 11- and 4-fold, respectively. Importantly, BZ treatment overcame chemoresistance in topo IIα-deficient Dox1V myeloma cells, enhancing the sensitivity to daunorubicin and etoposide 110- and 7- fold, respectively. The observed synergy was sequence-dependent and correlated with BZ-induced upregulation of topo IIα. Maximal synergy occurred within 3–6 hrs of BZ pretreatment and diminished after 24 hrs. Minimal synergy occurred when topo IIα-targeted agents preceded BZ by ≥ 6 hrs. These findings demonstrate that BZ has synergistic cytotoxic effects with topo IIα-targeted agents in AML and myeloma cell lines, which correlate with BZ-induced upregulation of topo IIα protein and nuclear enzymatic activity. Furthermore, these data provide a rationale for timed-sequential cytotoxic therapy in patients with hematologic malignancies.

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