A Novel Nitrobenzoate Microtubule Inhibitor that Overcomes Multidrug Resistance Exhibits Antitumor Activity

www.nature.com/scientificreports OPEN received: 10 May 2016 accepted: 20 July 2016 Published: 11 August 2016 A Novel Nitrobenzoate Microtubule Inhibitor that Overcomes Multidrug Resistance Exhibits Antitumor Activity Yan-Bo Zheng, Jian-Hua Gong, Xiu-Jun Liu, Shu-Ying Wu, Yi Li, Xian-Dong Xu, Bo-Yang Shang, Jin-Ming Zhou, Zhi-Ling Zhu, Shu-Yi Si & Yong-Su Zhen Multidrug resistance is a major limitation for microtubule-binding agents in cancer treatment. Here we report a novel microtubule inhibitor (2-morpholin-4-yl-5-nitro-benzoic acid 4-methylsulfanylbenzyl ester, IMB5046), its cytotoxicity against multidrug-resistant cell lines and its antitumor efficacy in animal models. IMB5046 disrupted microtubule structures in cells and inhibited purified tubulin polymerization in vitro. It bound to the colchicine pocket of tubulin. IMB5046 displayed potent cytotoxicity against multiple tumor cell lines with an IC50 range of 0.037–0.426 μM. Notably, several multidrug-resistant cell lines which were resistant to colchicine, vincristine and paclitaxel remained sensitive to IMB5046. IMB5046 was not a P-glycoprotein substrate. IMB5046 blocked cell cycle at G2/M phase and induced cell apoptosis. Microarray assay indicated that the differentially expressed genes after IMB5046 treatment were highly related to immune system, cell death and cancer. In a mouse xenograft model IMB5046 inhibited the growth of human lung tumor xenograft by 83% at a well-tolerated dose. It is concluded that IMB5046 is a tubulin polymerization inhibitor with novel chemical structure and can overcome multidrug resistance. It is a promising lead compound for cancer chemotherapy, especially for treatment of multidrug-resistant tumors. Microtubules are highly dynamic cytoskeletal fibers composed of α- and β-tubulin heterodimers, and are involved in a variety of fundamental cellular processes, such as the maintenance of cell shape, intracellular trafficking, cell movement, and most recognized mitosis1,2. The fast dynamics of spindle microtubules make the rapidly dividing cells exquisitely sensitive to microtubule-binding agents3,4. These agents are broadly classified into two main groups: the microtubule-destabilizing agents (such as colchicine and vincristine), and the microtubule-stabilizing agents (such as paclitaxel and laulimalide). Given the success of taxanes and vinca alkaloids in the clinic, microtubule-binding agents represent one of the most active classes of drugs in the treatment of cancer3,4. However, resistance to these drugs is common5,6. The expression of P-glycoprotein (P-gp) efflux pump and different β-tubulin isotypes play important roles in the development of drug resistance3,5,7. As reported, both taxanes and vinca alkaloids are substrates of P-gp8. P-gp executes efflux of these drugs, and consequently reduces their intracellular concentrations and their cytotoxic activity3. Other limitations of microtubule-binding agents include neurological toxicity, poor solubility, cumbersome synthesis/purification, etc4,6,9. Searching for new microtubule inhibitors with novel chemical structure will help to overcome these limitations. During the screening of anticancer drugs using the EMT-mimetic (epithelial-mesenchymal transition) assay, we found a highly active sample designated IMB5046. This was a nitrobenzoate compound, verified as 2-morpholin-4-yl-5-nitro-benzoic acid 4-methylsulfanyl-benzyl ester (Fig. 1a). Herein, we report its effect on microtubule assembly and its cytotoxicity against multidrug-resistant cell lines. Moreover, its antitumor efficacy against human tumor xenografts is also presented. To our knowledge, this is the first report that a nitrobenzoate compound can inhibit tubulin polymerization and overcome multidrug resistance. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R.China. Correspondence and requests for materials should be addressed to Y.-S.Z. (email: ) Scientific Reports | 6:31472 | DOI: 10.1038/srep31472 1 www.nature.com/scientificreports/ Figure 1. Chemical structure of IMB5046 and its effect on microtubules. (a) Chemical structure of IMB5046. (b) Effect of IMB5046 on the cytoskeleton of NIH/3T3 cells. NIH/3T3 cells were incubated with or without 100 nM IMB5046 for 6 h. Microtubule was stained with anti-tubulin antibody and F-actin was stained with phalloidin-FITC. IMB5046 at 100 nM partially disrupted the microtubule structures characterized by short microtubule fragments, but had no effect on F-actin networks. Scale bar, 10 μm. (c) IMB5046 disrupted the microtubule structures in A431 cells. A431 cells were treated with 100 nM IMB5046, 500 nM paclitaxel or 100 ng/mL nocodazole for 6 h. Insets are mitotic spindles from the same preparation. Scale bar, 10 μm. (d) Microtubule assembly assay in A431 cells. IMB5046 increased the free tubulin content in a concentrationdependent manner. Representative result of three independent experiments is shown. The histogram shows the relative density of tubulin. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 versus Ctrl. Ctrl, control; Noc, nocodazole; Col, colchcine; Ptx, paclitaxel. Scientific Reports | 6:31472 | DOI: 10.1038/srep31472 2 www.nature.com/scientificreports/ Figure 2. Inhibitory effects of IMB5046 on tubulin polymerization and its binding kinetics in vitro. (a) Tubulin polymerization assay in vitro. IMB5046 inhibited tubulin polymerization, evaluated by turbidity changes at a wavelength of 340 nm. The experiment was repeated twice. Data from representative experiment are shown. (b) Electron micrographs of microtubules assembled in the presence or absence of IMB5046. Negatively stained samples. Scale bar, 500 nm. (c) Binding kinetics of IMB5046 (1.25, 2.5, 5, 10, 20, 40 μM) and colchicine (1.25, 2.5, 5, 10, 20, 40 μM) to tubulin (2,000 RU) determined by SPR technology. The experiment was repeated twice. Data from representative experiment are shown. RU, Resonance Units. Results IMB5046 induces morphological changes and disrupts microtubule networks in cells. In the primary EMT-mimetic screening, we found that IMB5046 induced morphological changes of NIH/3T3 cells in a time and concentration-dependent manner similar to that induced by nocodazole, a microtubule-destabilizing agent, with rounding up of the initially spindle-shaped cells (data not presented). Because of the pivotal role of the cytoskeleton in the maintenance of cell shape, the effects of IMB5046 on the cytoskeleton were investigated. As shown in Fig. 1b, 100 nM IMB5046 disrupted the microtubule structures in NIH/3T3 cells but had no effect on F-actin networks. Human epidermoid carcinoma A431 cells were also treated with 100 nM IMB5046 for 6 h, and stained with anti-α-tubulin antibody for the detection of microtubules. As shown in Fig. 1c, untreated cells contained well-organized microtubule networks and mitotic spindles, whereas IMB5046 treated cells sho (...truncated)