Mitotic Kinesin Inhibitors Induce Mitotic Arrest and Cell Death in Taxol-resistant and -sensitive Cancer Cells

Adam I Marcus,Ulf Peters,Shala L Thomas,Sarah Garrett,Amelia Zelnak,Tarun M Kapoor,Paraskevi Giannakakou

doi:10.1074/jbc.m413471200

Adam I Marcus, Ulf Peters + Show 5 more

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https://doi.org/10.1074/jbc.m413471200

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Abstract

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.

Highlights

Taxanes represent one of the most successful classes of anticancer drugs and have validated microtubules as excellent chemotherapeutic targets [1]
Using in vitro assays it was previously shown that HR22C16 inhibits the mitotic kinesin, Eg5 with an IC50 of 800 Ϯ 10 nM; it is not known if this compound binds and inhibits Eg5 or if it binds other microtubule motor proteins
We sought to determine if small molecule inhibitors of the mitotic kinesin, Eg5, can overcome drug resistance in two model cancer cell lines of taxane resistance

Summary

Introduction

Taxanes represent one of the most successful classes of anticancer drugs and have validated microtubules as excellent chemotherapeutic targets [1]. Taxanes bind microtubules directly leading to a potent suppression of microtubule dynamics, increased microtubule stabilization, and interphase microtubule bundling; cells undergo robust mitotic arrest and subsequent apoptotic cell death [2, 3] Despite their clinical success against several solid tumors including ovarian, breast, prostate, and non-small cell. Second generation Eg5 inhibitors have been discovered in drug screens One such compound, CK0106023, is a specific allosteric inhibitor of Eg5 and possesses antitumor activity in an ovarian cancer xenograft [17]. CK0106023, is a specific allosteric inhibitor of Eg5 and possesses antitumor activity in an ovarian cancer xenograft [17] Another Eg5 inhibitor, HR22C16 was discovered in a microscopy-based forward chemical genetics screen of ϳ16,000 compounds [18]. Since HR22C16 and its analogs target Eg5 but not interphase microtubules, we wanted to assess their efficacy in Taxol-resistant and -sensitive human ovarian carcinoma cells

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