Targeting of tubulin polymerization and induction of mitotic blockage by Methyl 2-(5-fluoro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylate (MBIC) in human cervical cancer HeLa cell

Hasanpourghadi et al. Journal of Experimental & Clinical Cancer Research (2016) 35:58 DOI 10.1186/s13046-016-0332-0 RESEARCH Open Access Targeting of tubulin polymerization and induction of mitotic blockage by Methyl 2-(5-fluoro-2-hydroxyphenyl)-1Hbenzo[d]imidazole-5-carboxylate (MBIC) in human cervical cancer HeLa cell Mohadeseh Hasanpourghadi1, Chandrabose Karthikeyan2, Ashok Kumar Pandurangan1, Chung Yeng Looi1, Piyush Trivedi2, Kinue Kobayashi3, Kozo Tanaka3, Won Fen Wong4 and Mohd Rais Mustafa1* Abstract Background: Microtubule Targeting Agents (MTAs) including paclitaxel, colchicine and vinca alkaloids are widely used in the treatment of various cancers. As with most chemotherapeutic agents, adverse effects and drug resistance are commonly associated with the clinical use of these agents. Methyl 2-(5-fluoro-2-hydroxyphenyl)-1Hbenzo[d]imidazole-5-carboxylate (MBIC), a benzimidazole derivative displays greater toxicity against various cancer compared to normal human cell lines. The present study, focused on the cytotoxic effects of MBIC against HeLa cervical cancer cells and possible actions on the microtubule assembly. Methods: Apoptosis detection and cell-cycle assays were performed to determine the type of cell death and the phase of cell cycle arrest in HeLa cells. Tubulin polymerization assay and live-cell imaging were performed to visualize effects on the microtubule assembly in the presence of MBIC. Mitotic kinases and mitochondrial-dependent apoptotic proteins were evaluated by Western blot analysis. In addition, the synergistic effect of MBIC with low doses of selected chemotherapeutic actions were examined against the cancer cells. Results: Results from the present study showed that following treatment with MBIC, the HeLa cells went into mitotic arrest comprising of multi-nucleation and unsegregated chromosomes with a prolonged G2-M phase. In addition, the HeLa cells showed signs of mitochondrial-dependant apoptotic features such as the release of cytochrome c and activation of caspases. MBIC markedly interferes with tubulin polymerization. Western blotting results indicated that MBIC affects mitotic regulatory machinery by up-regulating BubR1, Cyclin B1, CDK1 and down-regulation of Aurora B. In addition, MBIC displayed synergistic effect when given in combination with colchicine, nocodazole, paclitaxel and doxorubicin. Conclusion: Taken together, our study demonstrated the distinctive microtubule destabilizing effects of MBIC against cervical cancer cells in vitro. Besides that, MBIC exhibited synergistic effects with low doses of selected anticancer drugs and thus, may potentially reduce the toxicity and drug resistance to these agents. Keywords: Cervical cancer, Microtubule dynamics, Mitotic arrest, Mitochondrial-dependent apoptosis * Correspondence: 1 Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia Full list of author information is available at the end of the article © 2016 Hasanpourghadi et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hasanpourghadi et al. Journal of Experimental & Clinical Cancer Research (2016) 35:58 Background Cervical cancer is the third most common cancer and the fourth cause of death in women worldwide. More than 85 % of deaths due to cervical cancer occur mostly in developing countries. Globally, South-central Asia is reported to have among the highest incidences of cervical cancer [1]. In Malaysia, cervical cancer is rated as second most common cancer among women [2]. Human papillomavirus (HPV) infection is the main cause of cervical cancer [3]. By now it is well documented that cervical cancer cannot develop in the absence of the persistent HPV DNA [4]. Chemotherapy is a category of cancer treatment using one or more chemotherapeutic antitumor drugs. A more efficacious chemotherapy is expected when it is given to patients concurrently with radiation because chemotherapy and radiotherapy synergistically suppress DNA repair after the DNA damage caused by radiation [5]. In this regard, Cisplatin is a chemotherapeutic drug used to treat cervical cancer. Cisplatin-based chemotherapy with radiotherapy significantly improves survival for high-risk, early-stage cervical cancer patients. However, cisplatin displays acute and late toxicity which limits its therapeutic effectiveness. At the same time, many patients develop drug resistance to cisplatin after prolonged treatment [6]. Therefore, there is a need for a new anticancer agent with better efficacy and less toxicity. Microtubule-targeting agents (MTAs) or microtubule inhibitors such as paclitaxel, docetaxel, vinblastine, estramustine, epothilones, colchicine, and nocodazole are commonly used in cancer chemotherapy [7]. Microtubules are the fundamental element of mitotic spindles and they are vital for numerous functions such as intracellular trafficking, maintenance of cellular architecture and cell movement and migration [8]. MTAs arrest cell cycle progression in mitosis by perturbing the microtubule dynamics and function [9]. MTAs interfere with microtubule dynamics and consequently, the formation of the spindle is disrupted and mitotic kinases function is restricted [10]. As a result, chromosomes are not able to attach to microtubule fibers precisely. In pursuance of protecting genome integrity and to reduce the occurrence of aneuploidy, the spindle assembly checkpoint delays metaphase-anaphase transition and causes mitotic arrest [11]. Following prolonged mitotic arrest, cancer cells either die in suspended mitosis through mitotic catastrophe [12], or exit mitosis without dividing (a process is known as mitotic slippage) to form multi-nucleated cells [13]. The effectiveness of MTAs has been hampered by the appearance of severe adverse effects particularly hematological and neurological toxicities and development of drug resistance. Development of new MTAs with decreased side effects and overcoming drug resistance may provide more effective therapeutic options for cancer patients [14]. Page 2 of 13 Benzimidazoles are nitrogen heterocycles that contained a phenyl ring fused to an imidazole ring [15]. Benzimidazole derivatives are well documented as anticancer agents [16]. It was known that benzimidazole derivatives are potent inhibitors of tubulin polymerization [17]. Karthikeyan et al. [18] synthesized a series of 2phenyl benzimidazole derivatives and showed that methyl 2-(5-f (...truncated)