Arsenic trioxide induces depolymerization of microtubules in an acute promyelocytic leukemia cell line

Abstract

Background

Arsenic trioxide (As ₂O ₃) is a well-known and effective treatment that can result in clinical remission for patients diagnosed with acute promyelocytic leukemia (APL). The biologic efficacy of As ₂O ₃ in APL and solid tumor cells has been explained through its actions on anti-proliferation, anti-angiogenesis, and apoptotic signaling pathways. We theorize that As ₂O ₃ activates a pathway that disrupts microtubule dynamics forming abnormal, nonfunctioning mitotic spindles, thus preventing cellular division. In this study, we investigated how As ₂O ₃ induces apoptosis by causing microtubule dysfunction.

Methods

Cultured NB4 cells were treated with As ₂O ₃, paclitaxel, and vincristine. Flow cytometric analysis was then performed. An MTT assay was used to determine drug-mediated cytotoxicity. For tubulin polymerization assay, each polymerized or soluble tubulin was measured. Microtubule assembly-disassembly was measured using a tubulin polymerization kit. Cellular microtubules were also observed with fluorescence microscopy.

Results

As ₂O ₃ treatment disrupted tubulin assembly resulting in dysfunctional microtubules that cause death in APL cells. As ₂O ₃ markedly enhanced the amount of depolymerized microtubules. The number of microtubule posttranslational modifications on an individual tubulin decreased with As ₂O ₃ concentration. Immunocytochemistry revealed changes in the cellular microtubule network and formation of polymerized microtubules in As ₂O ₃-treated cells.

Conclusion

The microtubules alterations found with As ₂O ₃ treatment suggest that As ₂O ₃ increases the depolymerized forms of tubulin in cells and that this is potentially due to arsenite's negative effects on spindle dynamics.