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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

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
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Fig6: 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

Mentions: To investigate further the sub-cellular changes induced by MBIC, we compared the nuclear morphology, membrane permeability, mitochondrial membrane potential (MMP, Δψm) and DNA content between treated and untreated cells. Compared to untreated cells, 24 h post-MBIC treatment (0.2 and 0.42 μM), HeLa cells showed a dose-dependent loss of cells, an increase of membrane permeability, exhaustion of MMP compared to untreated cells (Fig. 5a and b). On the other hand, the nucleus of untreated samples remained rounded and uniform in size. MMP and cytochrome c were stained consistently and co-localized in the cytosol, indicating no cytochrome c release in control cells. Fig. 6 is proposing the overall mechanistic action of MBIC in HeLa cell.Fig. 5


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)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: 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
Mentions: To investigate further the sub-cellular changes induced by MBIC, we compared the nuclear morphology, membrane permeability, mitochondrial membrane potential (MMP, Δψm) and DNA content between treated and untreated cells. Compared to untreated cells, 24 h post-MBIC treatment (0.2 and 0.42 μM), HeLa cells showed a dose-dependent loss of cells, an increase of membrane permeability, exhaustion of MMP compared to untreated cells (Fig. 5a and b). On the other hand, the nucleus of untreated samples remained rounded and uniform in size. MMP and cytochrome c were stained consistently and co-localized in the cytosol, indicating no cytochrome c release in control cells. Fig. 6 is proposing the overall mechanistic action of MBIC in HeLa cell.Fig. 5

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