Kinetic Stabilization of Microtubule Dynamics by Estramustine Is Associated with Tubulin Acetylation, Spindle Abnormalities, and Mitotic Arrest Renu Mohan and Dulal Panda School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Mumbai, India Abstract Estramustine (EM) alone or in combination with other anticancer agents is clinically used for the treatment of hormone refractory prostate cancer. Furthermore, EM has been shown to potently inhibit the proliferation of different types of cancer cells in culture apparently by targeting micro- tubules; however, the antiproliferative mechanism of action of EM is not clear. In this work, we have shown that EM strongly suppressed the dynamic instability of individual microtubules in MCF-7 cells by reducing the rates of growing and shorten- ing excursions and increasing the time microtubule spent in the pause state. At its half maximal proliferation inhibitory concentration (IC 50 ), EM exerted strong suppressive effects on the dynamics of microtubules in MCF-7 cells without detect- ably affecting either the organization or the polymerized mass of microtubules. At relatively high concentrations (5  IC 50 ), EM significantly depolymerized microtubules in the cells. Furthermore, the microtubules were found highly acetylated, supporting the conclusion that they were stabilized by the drug. EM treatment induced spindle abnormalities in MCF-7 cells, and a major population of the arrested mitotic cells was multipolar. EM also perturbed the microtubule-kinetochore interaction, thereby activating the spindle assembly check- point and leading to apoptotic cell death. [Cancer Res 2008; 68(15):6181–9] Introduction Estramustine (EM), a conjugate of nor-nitrogen mustard and estradiol phosphate, has become one of the most valuable drugs for the treatment of hormone refractory prostate cancer (HRPC). When used alone to treat HRPC, it has shown response rates ranging from 19% to 69% (1). EM has also shown promising activity against HRPC in combination with other drugs and is currently undergoing clinical trials in combination with docetaxel, etoposide, carboplatin, and vinblastine (2, 3). Fizazi and colleagues (2007) have reported that the overall survival rate for metastatic HRPC is significantly increased when EM is combined with docetaxel, paclitaxel, vinblastine, or ixabepilone rather than used by itself (4). The promising response of the combined application of EM with docetaxel led to the Southwest Oncology Group trial, which was a phase 3–randomized study for evaluating the combination of EM and docetaxel in 770 HRPC patients. Patients treated with this combination showed a significant increase in the overall survival and also a significant reduction in the risk of death (5). In addition, EM when used singly was found effective in advanced breast cancer (6) and in combination with docetaxel was found to increase the overall survival and improve the quality of life of patients having refractory metastatic breast carcinoma (7). EM is given via the oral route in the form of EM phosphate (EMP) with 70% to 75% of the oral dose absorbed. EMP is more soluble than the parent compound but is not active in cells because it does not penetrate the plasma membrane. However, it is rapidly dephosphorylated in the gastrointestinal tract and the dephosphorylated form predom- inates f4 h after ingestion (8). The most important adverse effects of EM are cardiovascular and gastrointestinal toxicities, which can be avoided by careful treatment measures (9). Chemically, EM consists of an estradiol moiety linked to nor- nitrogen mustard by a carbamate bridge. Originally, it was designed to treat breast cancer based on the notion that the estradiol moiety may specifically direct the nor-nitrogen mustard to the breast cancer cells, wherein the alkylating activity of the nor-nitrogen mustard can kill the breast cancer cells (10). However, contrary to this idea, EM was found to be highly effective in treating prostate cancer patients. It inhibits cell proliferation and induces mitotic arrest in many types of cancer cells but is especially active in prostate cancer cells (11). The high efficacy of EM against prostate cancer cells is thought to be due to the presence of an EM binding protein in these cells (12). The antitumor activity of EM is thought to be due to its action on microtubules. EM has been found to bind weakly to microtubule-associated proteins (MAP) and to inhibit microtubule assembly in vitro (13, 14). EM has been shown to bind to tubulin dimers (15, 16) and weakly inhibits the polymerization of MAPs- free tubulin into microtubules (16). Furthermore, EM has been shown to suppress the dynamic instability of individual MAP-free microtubules in vitro (16). The EM binding site on tubulin has been suggested to be distinct from the colchicine and vinblastine sites (16) and may partially overlap with the Taxol-binding site in tubulin (15). Interestingly, EMP has been shown to bind to brain MAPs and to depolymerize MAP-rich microtubules in vitro (17). Whereas the antimitotic activity of EM seems to be due to its actions on microtubules, the mechanism by which it inhibits cell cycle progression and mitosis is poorly understood. In this study, we have shown that EM suppresses the dynamic instability of individual microtubules in living MCF-7 cells. The kinetic stabilization of microtubule dynamics occurred in the absence of a significant depolymerization of the microtubules. EM also increased the acetylation levels of the interphase microtubules in the MCF-7 cells, further supporting the idea that EM kinetically stabilizes the microtubules. EM also interfered with the microtu- bule-kinetochore interaction, thereby activating the spindle checkpoint leading to apoptosis. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Dulal Panda, School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Mumbai 400076, India. Phone: 91-22-2576- 7838; Fax: 91-22-2572-3480; E-mail: panda@iitb.ac.in. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-0584 www.aacrjournals.org 6181 Cancer Res 2008; 68: (15). August 1, 2008 Research Article Research. on January 8, 2016. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from