Targeting class II and class IVb β-tubulins hypersensitizes non-small cell lung cancer cells to vinca alkaloids Free

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The effectiveness of microtubule disrupting agents such as vincristine and paclitaxel in the treatment of cancer is often hampered by the development of drug resistance. These agents disrupt microtubules by binding to the β-tubulin subunit of α/β-tubulin, interfering with microtubule dynamics, inducing mitotic arrest and cell death. The existence of multiple tubulin isotypes together with their tissue-restricted expression suggests that different isotypes are functionally diverse. Aberrant expression of specific β tubulin isotypes is frequently described in tumor tissues and cell lines that are selected for resistance to antimicrotubule agents. It is not clear how different β-tubulin isotypes influence response to antimicrotubule agents. We hypothesized that a cells response to antimicrotubule agents could be influenced by the β-tubulin isotype composition. We have previously shown that decreased expression of βIII by antisense or small interfering RNA (siRNA) hypersensitized non-small cell lung cancer (NSCLC) cells to both paclitaxel and vincristine. To gain insights into the role of other β-tubulin isotypes in response to various antimicrotubule agents, we employed siRNA mediated specific knockdowns of class II and class IVb β-tubulins in two NSCLC cell lines, NCI-H460 (H460) and Calu-6. Drug-treated clonogenic assays showed that specific downregulation of both βII and βIVb tubulins hypersensitized the NSCLC cells to vinca alkaloids but not taxanes. Increased drug sensitivity was not attributable to changes in drug accumulation since intracellular vincristine accumulation was unchanged in βIVb transfectants. Immunofluorescent staining demonstrated that microtubule organization and structure were not affected by either βII or βIV tubulins. However upon treatment with 10 nM of vincristine, the βII and βIVb siRNA treated cells showed extensive disruption of the microtubules as compared to the controls. In addition, downregulation of βIVb tubulin, but not βII tubulin, abrogated vincristine-induced G2/M arrest. This was accompanied by an increase in the sub G1 population which is indicative of cell fragmentation. This study provides the first evidence that βII and βIVb tubulin directly affect a cancer cells response to antimicrotubule agents. These findings may have important implications for improving the targeting and consequently, at designing better therapies for the treatment of NSCLC.