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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 M, Karthikeyan C, Pandurangan AK, Looi CY, Trivedi P, Kobayashi K, Tanaka K, Wong WF, Mustafa MR - J. Exp. Clin. Cancer Res. (2016)

Bottom Line: As with most chemotherapeutic agents, adverse effects and drug resistance are commonly associated with the clinical use of these agents.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia.

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.

No MeSH data available.


Related in: MedlinePlus

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. 4c & 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
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Fig4: 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. 4c & 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

Mentions: Since cell cycle is governed by a group of proteins called cyclin-dependent kinases (CDKs) and mitotic kinases, we performed Western blot analysis to examine whether MBIC affects these targets. Cells were also treated with colchicine or nocodazole as positive controls. First, we decided to evaluate Cyclin B1 and CDK1 levels. Cyclin B1-CDK1 complex is known as a mitosis-promoting factor (MPF). Also, this complex is inactive in G2 phase and its activation begins exactly before nuclear envelope breakdown which leads on to set up the events in prophase [23]. As shown in Fig. 4a, we observed up-regulation of Cyclin B1 and CDK1, indicating the occurrence of mitotic arrest.Fig. 4


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 M, Karthikeyan C, Pandurangan AK, Looi CY, Trivedi P, Kobayashi K, Tanaka K, Wong WF, Mustafa MR - J. Exp. Clin. Cancer Res. (2016)

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. 4c & 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
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Fig4: 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. 4c & 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
Mentions: Since cell cycle is governed by a group of proteins called cyclin-dependent kinases (CDKs) and mitotic kinases, we performed Western blot analysis to examine whether MBIC affects these targets. Cells were also treated with colchicine or nocodazole as positive controls. First, we decided to evaluate Cyclin B1 and CDK1 levels. Cyclin B1-CDK1 complex is known as a mitosis-promoting factor (MPF). Also, this complex is inactive in G2 phase and its activation begins exactly before nuclear envelope breakdown which leads on to set up the events in prophase [23]. As shown in Fig. 4a, we observed up-regulation of Cyclin B1 and CDK1, indicating the occurrence of mitotic arrest.Fig. 4

Bottom Line: As with most chemotherapeutic agents, adverse effects and drug resistance are commonly associated with the clinical use of these agents.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia.

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.

No MeSH data available.


Related in: MedlinePlus