Limits...
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 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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: 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

Mentions: Since MBIC demonstrated higher cytotoxicity and selectivity in HeLa, subsequent assays were performed using this cancer cell-line. During early apoptosis, membrane phosphatidylserine (PS) translocate from the inner face of the cell membrane to the cell surface. Annexin V can bind to exposed PS with high affinity, whereas PI molecules intercalate inside the DNA double helix in cells with a compromised plasma membrane. Therefore, cells stained strongly with Annexin V signifies early apoptosis and PI-stained cells indicate late apoptosis or necrosis [21]. To examine whether MBIC-treated HeLa cells undergo apoptosis or necrosis, MBIC treated cells were stained with annexin V and PI. As shown in Fig. 2a, MBIC exposure at different concentrations (0.21, 0.42 and 1 μM) resulted in a higher population of late apoptotic cells (44.8 ± 2.3 % to 74.8 ± 4.2 %) compared to control (0.0 ± 0.0 %). Our results indicated that MBIC-induced dose-dependent apoptosis in HeLa cells as shown in the bar graphs (Fig. 2b).Fig. 2


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 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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: 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
Mentions: Since MBIC demonstrated higher cytotoxicity and selectivity in HeLa, subsequent assays were performed using this cancer cell-line. During early apoptosis, membrane phosphatidylserine (PS) translocate from the inner face of the cell membrane to the cell surface. Annexin V can bind to exposed PS with high affinity, whereas PI molecules intercalate inside the DNA double helix in cells with a compromised plasma membrane. Therefore, cells stained strongly with Annexin V signifies early apoptosis and PI-stained cells indicate late apoptosis or necrosis [21]. To examine whether MBIC-treated HeLa cells undergo apoptosis or necrosis, MBIC treated cells were stained with annexin V and PI. As shown in Fig. 2a, MBIC exposure at different concentrations (0.21, 0.42 and 1 μM) resulted in a higher population of late apoptotic cells (44.8 ± 2.3 % to 74.8 ± 4.2 %) compared to control (0.0 ± 0.0 %). Our results indicated that MBIC-induced dose-dependent apoptosis in HeLa cells as shown in the bar graphs (Fig. 2b).Fig. 2

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