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

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)-1H- benzo[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; Mitochondrial-dependent apoptosis; Mitotic arrest.

Fig. 1

a Chemical Structure: Chemical structure of Methyl 2-(5-fluoro-2-hydroxyphenyl)-1H- benzo[d]imidazole-5-carboxylate (MBIC). b Inhibitory effect of MBIC against HeLa cell proliferation: Cell viability graph was generated for comparison of total relative cell viability (%) after MBIC and indicated conventional drugs treatment. Experiment was done in two time points (24 and 48 h) against HeLa cells. All results were expressed as total percentage of viable cells with mean ± SD of three independent experiments. (P < 0.05)

Fig. 2

a MBIC induced apoptosis in HeLa cells: Flow cytometry analysis of HeLa cells treated with various concentration of MBIC for 24 h was carried out. Representative figures show population of viable cells in Q3 (annexin V- PI-), early apoptotic cells in Q4 (annexin V+ PI-), late apoptotic cells in Q2 (annexin V+ PI+) and necrotic cells in Q1 (annexin V- PI+). Representative figure shows apoptosis induction of MBIC (0.21, 0.42 and 1 μM) against HeLa cells 24 h after treatment. b shows early and late induced apoptosis in bar chart for HeLa cells 24 h after treatment. Data were mean ± SD of three independent experiments. All the treatment groups were compared with control. “*” indicates statistically significant at P < 0.05. c MBIC induced G₂-M arrest in HeLa cells: HeLa cells were treated with indicated concentrations of MBIC (0.21, 0.42 and 1 μM) for 24 h. Cells were permeabilized by ethanol and stained with PI. Cell cycle progression has been assessed by flow cytometry. Representative figures of cell cycle distribution (G₀-G₁, S, and G₂-M) show accumulation of MBIC-treated cells in G₂-M phase. HeLa cells were arrested in G₂-M phase 24 h after MBIC treatment

Fig. 3

a MBIC disrupts mitotic spindle: HeLa cells expressing EGFP-α-tubulin, EGFP-CENP-A and histone H2B-mCherry were treated with DMSO (upper), MBIC (10 μM, middle), or nocodazole (2 μM, lower) and imaged at 15 min intervals. Time (min) is indicated in the upper left of each panel. For middle and lower panels, arrowheads mark the cell in the center at the first frame. Scale bar = 5 μm. b Effect of MBIC on tubulin polymerization: Tubulin polymerization assay was conducted using Tubulin polymerization assay kit (Cytoskeleton, Inc.). The plate was read using Infinite® 200 PRO—Tecan regulated on 96 well plate reader spectrophotometer. The plate was read at 340 nm in kinetic mode for two hours. Figure 3 shows curves of tubulin treated by paclitaxel (10 μM), nocodazole (10 μM), colchicine (10 μM), MBIC (10 μM) and untreated tubulin. Maximal velocity (Vmax) of each drug on tubulin polymerization was calculated

Fig. 4

a MBIC induced mitotic arrest: Western blot analysis was carried out to evaluate mitotic regulators levels after MBIC treatment. HeLa cells were treated with (0.21, 0.42 and 1 μM of MBIC. Figure a shows the evaluation of Cyclin B1, CDK1, mitotic checkpoint protein (BubR1) and a chromosomal passenger complex member, Aurora B. Samples were treated with colchicine (Col) and nocodazole (Noco) (at their IC₅₀ concentration) as a positive control. β-actin served as a loading control. c, e MBIC induced mitochondria-dependent apoptosis: In order to evaluate whether MBIC caused mitochondria-dependent apoptosis, we evaluated anti-apoptotic protein Bcl-2, pro-apoptotic protein Bax, cleaved caspase-3/7/9 and cleaved PARP levels by western blot analysis (Fig. 4 c & e). HeLa cells were treated with MBIC (0.21, 0.42 and 1 μM). Samples were treated with colchicine (Col) and nocodazole (Noco) (at their IC₅₀ concentration) as a positive control. β-actin served as a loading control. b, d & f The relative intensity of each protein was normalized with β-actin. Data were mean ± SD of three independent experiments. All the treatment groups were compared with control. “*” indicates statistically significant at P < 0.05

Fig. 5

a Effect of MBIC on nuclear morphology, membrane permeabilization, MMP (Δψm) and cytochrome c release: HeLa cells were seeded in 96-well plate and treated with two different concentrations of MBIC (0.2 and 0.4 μM) for 24 h. Cells were fixed and stained according to manufacturer protocol. Images were acquired using Cellomics HCS array scan reader (objective 20 ×). Figure a displays changes in HeLa cells DNA content (blue), cell permeability (green), MMP (red) and cytochrome c (cyan). MBIC induced a considerable elevation in membrane permeability and cytochrome c release and caused an extensive reduction in mitochondrial membrane potential. b, c & d shows bar chart representing changes in membrane permeability, mitochondrial membrane potential, and cytochrome c in a dose-dependent manner. Data were mean ± SD of three independent experiments. All the treatment groups were compared with control. “*” indicates statistically significant at P < 0.05

Fig. 6

Proposed molecular mechanistic action of MBIC in HeLa cancer cell. a MBIC causes microtubule-kinetochore attachment error by interrupting microtubules polymerization. b Unattached kinetochores activate spindle assembly checkpoint (SAC). When SAC is activated, inhibits APC/C. BubR1 is a SAC member which inhibits metaphase to anaphase transition until all kinetochores are attached to microtubules correctly. Up-regulation of BubR1 is a sign of attachment error. c Cyclin B1 degradation is compulsory for exiting from mitosis. APC/C is responsible for Cyclin B1 degradation. Up-regulation of Cyclin B1 in our study shows that APC/C is not activated. d Down-regulation of Aurora B, one on chromosomal passenger complex (CPC) impairs SAC activity. e Down-regulation of Bcl-2 and up-regulation of Bax are signs of release of cytochrome c in cytosol and formation of apoptosome which activated caspase-9. Up-regulation of cleaved caspase-3/7/9 is a sign of mitochondrial-dependent apoptosis. f Up-regulation of cleaved PARP is a sign of DNA damage