Abstract
Epothilones are a new class of microtubule stabilizing agents with promising preclinical and clinical activity. Their cellular target is β-tubulin and factors influencing intrinsic sensitivity to epothilones are not well understood. In this study, the functional significance of specific β-tubulin isotypes in intrinsic sensitivity to epothilone B was investigated using siRNA gene knockdown against βII-, βIII- or βIVb-tubulins in two independent non-small cell lung cancer (NSCLC) cell lines, NCI-H460 and Calu-6. Drug-treated clonogenic assays showed that sensitivity to epothilone B was not altered following knockdown of βII-tubulin in both NSCLC cell lines. In contrast, knockdown of βIII-tubulin significantly increased sensitivity to epothilone B. Interestingly, βIVb-tubulin knockdowns were significantly less sensitive to epothilone B, compared to mock- and control siRNA cells. Cell cycle analysis of βIII-tubulin knockdown cells showed a higher percentage of cell death with epothilone B concentrations as low as 0.5 nM. In contrast, βIVb-tubulin knockdown cells displayed a decrease in epothilone B-induced G2-M cell cycle accumulation compared to control siRNA cells. Importantly, βIII-tubulin knockdowns displayed a significant dose-dependent increase in the percentage of apoptotic cells upon treatment with epothilone B, as detected using caspase 3/7 activity and Annexin-V staining. Higher concentrations of epothilone B were required to induce apoptosis in the βIVb-tubulin knockdowns compared to control siRNA, highlighting a potential mechanism underlying decreased sensitivity to this agent. This study demonstrates that specific β-tubulin isotypes can influence sensitivity to epothilone B and may influence differential sensitivity to this promising new agent.
Highlights
The taxanes are established drugs widely used in the treatment of several types of solid tumours, including ovarian, breast, lung and head and neck cancer, either singly or in combination with other chemotherapeutic agents
The naturally occurring epothilone B, has shown promising activity in various preclinical models that are resistant to taxane-based chemotherapy and is currently under phase II/III clinical trials [2,3,4,5]
Knockdown of bIVbtubulin significantly reduced sensitivity to epothilone B in both cell lines, compared to mock- and control siRNA-transfected cells (Fig. 1C), suggesting that tumors expressing high levels of this isotype may be more sensitive to epothilone B than tumors with low levels of this isotype
Summary
The taxanes (including paclitaxel and docetaxel) are established drugs widely used in the treatment of several types of solid tumours, including ovarian, breast, lung and head and neck cancer, either singly or in combination with other chemotherapeutic agents. Epothilones are a novel class of nontaxane microtubule-stabilizing agents that have shown promising anticancer activity. The epothilone B analogue, Ixabepilone (BMS-247550, aza-EpoB) was approved in 2007 by the Food and Drug Administration for the treatment of metastatic or locally advanced breast cancer resistant to anthracyclines, taxanes and capecitabine, either singly or in combination with these agents [1]. The naturally occurring epothilone B (patupilone, EPO906), has shown promising activity in various preclinical models that are resistant to taxane-based chemotherapy and is currently under phase II/III clinical trials [2,3,4,5]. Epothilones and taxanes stabilize microtubules against depolymerization, they exhibit distinct differences in activity and efficacy (reviewed in [9,10])
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