In vitro studies of the combination of imatinib mesylate (Gleevec) and arsenic trioxide (Trisenox) in chronic myelogenous leukemia

Objective. The aim of this study was the preclinical evaluation of imatinib mesylate (Gleevec, formerly STI571) in conjunction with arsenic trioxide (As ₂O ₃, Trisenox) for the treatment of chronic myelogenous leukemia (CML). Materials and Methods. Tetrazolium-based cell line proliferation assays (MTT assays) were performed to determine the cytotoxicity of As ₂O ₃ alone and in combination with imatinib. Cell lines tested in this study were Bcr-Abl-expressing cells (K562, MO7p210, 32Dp210) and parental cells (MO7e, 32D). Isobologram analysis was performed manually and using the median effect method. In vitro cytotoxicity also was determined in colony-forming assays using CML patient cells. Western blot analysis was performed to detect Bcr-Abl protein levels in K562 cells exposed to As ₂O ₃ at graded concentrations. Bcr-Abl protein level kinetics were correlated with cell viability (trypan blue count) and activated caspase-3 detected by flow cytometry. Results. We show additive to synergistic cytotoxicity in Bcr-Abl ⁺ cell lines depending on inhibitory concentrations and cell type. Results obtained by colony-forming assays confirmed the findings in cell line proliferation assays. Flow cytometric detection of activated caspase-3 revealed synergistic activity in K562 cells. Treatment of K562 cells with As ₂O ₃ alone led to down-regulation of Bcr-Abl protein within 24 hours, even at low doses. The decline of Bcr-Abl preceded activation of caspase-3 and the loss of viable cells. Conclusions. Favorable cytotoxicity and proapoptotic activity of imatinib in conjunction with As ₂O ₃ and specific down-regulation of Bcr-Abl protein levels by As ₂O ₃ in K562 cells indicate that As ₂O ₃ in combination with imatinib might be useful for circumventing resistance to imatinib monotherapy.