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Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression.

Joshi S, Braithwaite AW, Robinson PJ, Chircop M - Mol. Cancer (2011)

Bottom Line: This resulted in an accumulation of polyploid cells.Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis.Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Children's Medical Research Institute, The University of Sydney, 214 Hawkesbury Road, Westmead, NSW 2145, Australia.

ABSTRACT

Background: The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown.

Results: We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells.

Conclusion: Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.

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Over-expression of Bcl-2 protects cells from MiTMABs-induced cell death. A-D, G2/M synchronized HeLa (empty histograms) and HeLa-Bcl-2 (filled histograms) cells were synchronized at the G2/M boundary. Once released from this block, cells treated with MiTMAB, OcTMAB or indicated controls, were incubated for 20 h and their DNA contents analysed by flow cytometry. Representative flow cytometry histograms show a decrease in the <2N peak and an increase in the 4N peak in HeLa-Bcl-2 cells treated with MiTMAB (filled histograms) compared to parental HeLa cells (empty histograms; A). Graphs showing the percentage of HeLa and HeLa-Bcl-2 cells (mean ± S.D.) containing <2N (B), 4N (C) and >4N (D) DNA contents are shown. * p < 0.05, ** p < 0.01 (Student's t tests).
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Figure 4: Over-expression of Bcl-2 protects cells from MiTMABs-induced cell death. A-D, G2/M synchronized HeLa (empty histograms) and HeLa-Bcl-2 (filled histograms) cells were synchronized at the G2/M boundary. Once released from this block, cells treated with MiTMAB, OcTMAB or indicated controls, were incubated for 20 h and their DNA contents analysed by flow cytometry. Representative flow cytometry histograms show a decrease in the <2N peak and an increase in the 4N peak in HeLa-Bcl-2 cells treated with MiTMAB (filled histograms) compared to parental HeLa cells (empty histograms; A). Graphs showing the percentage of HeLa and HeLa-Bcl-2 cells (mean ± S.D.) containing <2N (B), 4N (C) and >4N (D) DNA contents are shown. * p < 0.05, ** p < 0.01 (Student's t tests).

Mentions: The activation of caspase-9 in MiTMABs-treated cells indicates that the intrinsic pathway is involved in mediating cell death. Caspase-9 is an initiator caspase activated following cytochrome c release from mitochondria [32]. Anti-apoptotic Bcl-2 family of proteins are directly responsible for maintaining mitochondrial membrane integrity, preventing cytochrome c release in the absence of apoptotic stimuli [33]. Therefore, we hypothesised that high Bcl-2 expression would inhibit MiTMAB-induced cell death. Indeed, flow cytometric quantitation of cells with <2N DNA content revealed that MiTMAB-induced apoptosis is completely blocked in HeLa cells stably expressing Bcl-2, HeLa-Bcl-2 (6.8 ± 0.3% in HeLa-Bcl-2 cells compared to 31.5 ± 0.5% in HeLa cells treated with 30 μM OcTMAB; Figure 4A and 4B). A corresponding increase in polyploid cells (4N and >4N DNA content) was observed (Figure 4C and 4D), further supporting the idea that cell death follows MiTMAB-induced cytokinesis failure. These results are analogous to those obtained in HeLa cells treated with the pan-caspase inhibitor, ZVAD (Figure 3A and 3B). We conclude that Bcl-2 over-expression renders HeLa cells resistant to MiTMAB-induced cell death, but not to MiTMAB-induced cytokinesis failure. The involvement of caspase-9 and Bcl-2 further indicate activation of the intrinsic apoptotic pathway.


Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression.

Joshi S, Braithwaite AW, Robinson PJ, Chircop M - Mol. Cancer (2011)

Over-expression of Bcl-2 protects cells from MiTMABs-induced cell death. A-D, G2/M synchronized HeLa (empty histograms) and HeLa-Bcl-2 (filled histograms) cells were synchronized at the G2/M boundary. Once released from this block, cells treated with MiTMAB, OcTMAB or indicated controls, were incubated for 20 h and their DNA contents analysed by flow cytometry. Representative flow cytometry histograms show a decrease in the <2N peak and an increase in the 4N peak in HeLa-Bcl-2 cells treated with MiTMAB (filled histograms) compared to parental HeLa cells (empty histograms; A). Graphs showing the percentage of HeLa and HeLa-Bcl-2 cells (mean ± S.D.) containing <2N (B), 4N (C) and >4N (D) DNA contents are shown. * p < 0.05, ** p < 0.01 (Student's t tests).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142233&req=5

Figure 4: Over-expression of Bcl-2 protects cells from MiTMABs-induced cell death. A-D, G2/M synchronized HeLa (empty histograms) and HeLa-Bcl-2 (filled histograms) cells were synchronized at the G2/M boundary. Once released from this block, cells treated with MiTMAB, OcTMAB or indicated controls, were incubated for 20 h and their DNA contents analysed by flow cytometry. Representative flow cytometry histograms show a decrease in the <2N peak and an increase in the 4N peak in HeLa-Bcl-2 cells treated with MiTMAB (filled histograms) compared to parental HeLa cells (empty histograms; A). Graphs showing the percentage of HeLa and HeLa-Bcl-2 cells (mean ± S.D.) containing <2N (B), 4N (C) and >4N (D) DNA contents are shown. * p < 0.05, ** p < 0.01 (Student's t tests).
Mentions: The activation of caspase-9 in MiTMABs-treated cells indicates that the intrinsic pathway is involved in mediating cell death. Caspase-9 is an initiator caspase activated following cytochrome c release from mitochondria [32]. Anti-apoptotic Bcl-2 family of proteins are directly responsible for maintaining mitochondrial membrane integrity, preventing cytochrome c release in the absence of apoptotic stimuli [33]. Therefore, we hypothesised that high Bcl-2 expression would inhibit MiTMAB-induced cell death. Indeed, flow cytometric quantitation of cells with <2N DNA content revealed that MiTMAB-induced apoptosis is completely blocked in HeLa cells stably expressing Bcl-2, HeLa-Bcl-2 (6.8 ± 0.3% in HeLa-Bcl-2 cells compared to 31.5 ± 0.5% in HeLa cells treated with 30 μM OcTMAB; Figure 4A and 4B). A corresponding increase in polyploid cells (4N and >4N DNA content) was observed (Figure 4C and 4D), further supporting the idea that cell death follows MiTMAB-induced cytokinesis failure. These results are analogous to those obtained in HeLa cells treated with the pan-caspase inhibitor, ZVAD (Figure 3A and 3B). We conclude that Bcl-2 over-expression renders HeLa cells resistant to MiTMAB-induced cell death, but not to MiTMAB-induced cytokinesis failure. The involvement of caspase-9 and Bcl-2 further indicate activation of the intrinsic apoptotic pathway.

Bottom Line: This resulted in an accumulation of polyploid cells.Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis.Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Children's Medical Research Institute, The University of Sydney, 214 Hawkesbury Road, Westmead, NSW 2145, Australia.

ABSTRACT

Background: The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown.

Results: We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells.

Conclusion: Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.

Show MeSH
Related in: MedlinePlus