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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 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
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Fig3: 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

Mentions: As cells were arrested in G2-M phase, we decided to examine MBIC’s action against microtubule dynamics and spindle formation in live-cell imaging. We observed HeLa cells stably expressing EGFP-α-tubulin, EGFP-CENP-A and histone H2B-mCherry (Fig. 3a). Control cells treated with DMSO formed bipolar spindle with aligned chromosomes (Fig. 3a, upper, 45 min) and segregated chromosomes properly without delay (Fig. 3a, upper, 90 min). In contrast, cells treated with MBIC did not form the spindle and stayed in mitosis for a long time before dying with pyknosis and cell shrinkage, i.e., characteristics of apoptotic cell death (Fig. 3a, middle), similar to cells treated with nocodazole (Fig. 3a, lower). The result indicated that MBIC disrupts spindle formation, consistent with its role as a MTA.Fig. 3


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 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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4815073&req=5

Fig3: 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
Mentions: As cells were arrested in G2-M phase, we decided to examine MBIC’s action against microtubule dynamics and spindle formation in live-cell imaging. We observed HeLa cells stably expressing EGFP-α-tubulin, EGFP-CENP-A and histone H2B-mCherry (Fig. 3a). Control cells treated with DMSO formed bipolar spindle with aligned chromosomes (Fig. 3a, upper, 45 min) and segregated chromosomes properly without delay (Fig. 3a, upper, 90 min). In contrast, cells treated with MBIC did not form the spindle and stayed in mitosis for a long time before dying with pyknosis and cell shrinkage, i.e., characteristics of apoptotic cell death (Fig. 3a, middle), similar to cells treated with nocodazole (Fig. 3a, lower). The result indicated that MBIC disrupts spindle formation, consistent with its role as a MTA.Fig. 3

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