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Co-inhibition of polo-like kinase 1 and Aurora kinases promotes mitotic catastrophe.

Li J, Hong MJ, Chow JP, Man WY, Mak JP, Ma HT, Poon RY - Oncotarget (2015)

Bottom Line: Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536).We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells.Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, Center for Cancer Research, and State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

ABSTRACT
Mitosis is choreographed by a number of protein kinases including polo-like kinases and Aurora kinases. As these kinases are frequently dysregulated in cancers, small-molecule inhibitors have been developed for targeted anticancer therapies. Given that PLK1 and Aurora kinases possess both unique functions as well as co-regulate multiple mitotic events, whether pharmacological inhibition of these kinases together can enhance mitotic catastrophe remains an outstanding issue to be determined. Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536). We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells. Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells. These observations underscore the mechanism and potential benefits of targeting PLK1 and Aurora kinases to induce mitotic catastrophe in cancer cells.

No MeSH data available.


Related in: MedlinePlus

Inhibition of PLK1 with BI 2536 induces metaphase defects and mitotic catastrophe(A) Inhibition of PLK1 promotes G2/M delay and apoptosis in a dose-dependent manner. HeLa cells were incubated with buffer or the indicated concentrations of BI 2536 (PLK1i). After 24 h, the cells were harvested and the DNA content was analyzed with flow cytometry. The positions of 2N and 4N DNA content are indicated. (B) Inhibition of PLK1 induces mitotic catastrophe. HeLa cells were incubated with buffer or the indicated concentrations of PLK1i for 24 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. Actin analysis was included to assess protein loading and transfer. (C) Treatment with the PLK1 inhibitor GW843682X induces mitotic catastrophe. HeLa cells were incubated with buffer or the indicated concentrations of GW843682X for 24 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. (D) Inhibition of PLK1 affects AURKA activity. Mitotic HeLa cells were isolated by treating cells with nocodazole for 16 h followed by mechanical shake off. The cells were either untreated or incubated with PLK1i, AURKAi, or AURKBi. The proteasome inhibitor MG132 was added to prevent the cells from exiting mitosis. The cells were harvested after 2 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. (E) Low concentrations of PLK1i stimulate cell proliferation. HeLa cells expressing iRFP (~200 cells) were seeded onto 6-well culture plates and cultured in the presence of buffer or different concentrations of PLK1i. On different days, the plate was scanned with an Odyssey infrared imaging system and the iRFP signal was quantified (average± SD of three independent experiments). Note that the PLK1i was left in the medium continuously throughout the experiment. At 0.078 nM, PLK1i significantly increased cell proliferation (P < 0.001; Student's t-test). (F) Inhibition of PLK1 induces a delay in mitosis. HeLa cells expressing histone H2B-GFP were exposed to buffer or the indicated concentrations of PLK1i. Individual cells were then tracked for 24 h with time-lapse microscopy. Each horizontal bar represents one cell (n = 50). Light grey: interphase; black: mitosis (from DNA condensation to anaphase or mitotic slippage); truncated bars: cell death. (G) PLK1i inhibits metaphase–anaphase transition. Cells were treated and imaged as described in panel (F). The duration from prometaphase to metaphase and from metaphase to the end of mitosis (anaphase, apoptosis, or the end of imaging period) was quantified (average ±90% CI). PLK1i treatment significantly extended mitosis after the metaphase was formed (****: P < 0.0001; **: P < 0.01; Student's t-test).
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Figure 2: Inhibition of PLK1 with BI 2536 induces metaphase defects and mitotic catastrophe(A) Inhibition of PLK1 promotes G2/M delay and apoptosis in a dose-dependent manner. HeLa cells were incubated with buffer or the indicated concentrations of BI 2536 (PLK1i). After 24 h, the cells were harvested and the DNA content was analyzed with flow cytometry. The positions of 2N and 4N DNA content are indicated. (B) Inhibition of PLK1 induces mitotic catastrophe. HeLa cells were incubated with buffer or the indicated concentrations of PLK1i for 24 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. Actin analysis was included to assess protein loading and transfer. (C) Treatment with the PLK1 inhibitor GW843682X induces mitotic catastrophe. HeLa cells were incubated with buffer or the indicated concentrations of GW843682X for 24 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. (D) Inhibition of PLK1 affects AURKA activity. Mitotic HeLa cells were isolated by treating cells with nocodazole for 16 h followed by mechanical shake off. The cells were either untreated or incubated with PLK1i, AURKAi, or AURKBi. The proteasome inhibitor MG132 was added to prevent the cells from exiting mitosis. The cells were harvested after 2 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. (E) Low concentrations of PLK1i stimulate cell proliferation. HeLa cells expressing iRFP (~200 cells) were seeded onto 6-well culture plates and cultured in the presence of buffer or different concentrations of PLK1i. On different days, the plate was scanned with an Odyssey infrared imaging system and the iRFP signal was quantified (average± SD of three independent experiments). Note that the PLK1i was left in the medium continuously throughout the experiment. At 0.078 nM, PLK1i significantly increased cell proliferation (P < 0.001; Student's t-test). (F) Inhibition of PLK1 induces a delay in mitosis. HeLa cells expressing histone H2B-GFP were exposed to buffer or the indicated concentrations of PLK1i. Individual cells were then tracked for 24 h with time-lapse microscopy. Each horizontal bar represents one cell (n = 50). Light grey: interphase; black: mitosis (from DNA condensation to anaphase or mitotic slippage); truncated bars: cell death. (G) PLK1i inhibits metaphase–anaphase transition. Cells were treated and imaged as described in panel (F). The duration from prometaphase to metaphase and from metaphase to the end of mitosis (anaphase, apoptosis, or the end of imaging period) was quantified (average ±90% CI). PLK1i treatment significantly extended mitosis after the metaphase was formed (****: P < 0.0001; **: P < 0.01; Student's t-test).

Mentions: To study possible synergism between the antimitotic effects of inhibitors of PLK1 and Aurora kinases, newer generation of small-molecule inhibitors that show relatively high specificity were used in this study. We first verified the effects of an inhibitor of PLK1 called BI 2536 [22,23] (designated PLK1i herein) as a single agent on the cell cycle. Flow cytometry analysis revealed that PLK1i induced a G2/M cell cycle delay in a dose-dependent manner (Figure 2A). Mitotic delay was confirmed by the increase in histone H3Ser10 phosphorylation (Figure 2B). Moreover, the stimulation of apoptosis (as indicated by PARP1 cleavage, caspase 3 cleavage, and increase of sub-G1 population) suggested that mitotic catastrophe was induced by PLK1i in these cells (Figure 2B). PLK1 itself accumulated upon incubation with PLK1i, possibly reflecting the increase of mitotic population (see below). Similar induction of accumulation of PLK1, histone H3Ser10 phosphorylation, and PARP1 cleavage were obtained by targeting PLK1 with another inhibitor called GW843682X [24], indicating that the effects were not specific for BI 2536 only (Figure 2C). Another PLK1 inhibitor called BI 6727 (also called Volasertib) [25] also induced mitotic block and apoptosis in a dose-dependent manner (Supplementary Figure S1).


Co-inhibition of polo-like kinase 1 and Aurora kinases promotes mitotic catastrophe.

Li J, Hong MJ, Chow JP, Man WY, Mak JP, Ma HT, Poon RY - Oncotarget (2015)

Inhibition of PLK1 with BI 2536 induces metaphase defects and mitotic catastrophe(A) Inhibition of PLK1 promotes G2/M delay and apoptosis in a dose-dependent manner. HeLa cells were incubated with buffer or the indicated concentrations of BI 2536 (PLK1i). After 24 h, the cells were harvested and the DNA content was analyzed with flow cytometry. The positions of 2N and 4N DNA content are indicated. (B) Inhibition of PLK1 induces mitotic catastrophe. HeLa cells were incubated with buffer or the indicated concentrations of PLK1i for 24 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. Actin analysis was included to assess protein loading and transfer. (C) Treatment with the PLK1 inhibitor GW843682X induces mitotic catastrophe. HeLa cells were incubated with buffer or the indicated concentrations of GW843682X for 24 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. (D) Inhibition of PLK1 affects AURKA activity. Mitotic HeLa cells were isolated by treating cells with nocodazole for 16 h followed by mechanical shake off. The cells were either untreated or incubated with PLK1i, AURKAi, or AURKBi. The proteasome inhibitor MG132 was added to prevent the cells from exiting mitosis. The cells were harvested after 2 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. (E) Low concentrations of PLK1i stimulate cell proliferation. HeLa cells expressing iRFP (~200 cells) were seeded onto 6-well culture plates and cultured in the presence of buffer or different concentrations of PLK1i. On different days, the plate was scanned with an Odyssey infrared imaging system and the iRFP signal was quantified (average± SD of three independent experiments). Note that the PLK1i was left in the medium continuously throughout the experiment. At 0.078 nM, PLK1i significantly increased cell proliferation (P < 0.001; Student's t-test). (F) Inhibition of PLK1 induces a delay in mitosis. HeLa cells expressing histone H2B-GFP were exposed to buffer or the indicated concentrations of PLK1i. Individual cells were then tracked for 24 h with time-lapse microscopy. Each horizontal bar represents one cell (n = 50). Light grey: interphase; black: mitosis (from DNA condensation to anaphase or mitotic slippage); truncated bars: cell death. (G) PLK1i inhibits metaphase–anaphase transition. Cells were treated and imaged as described in panel (F). The duration from prometaphase to metaphase and from metaphase to the end of mitosis (anaphase, apoptosis, or the end of imaging period) was quantified (average ±90% CI). PLK1i treatment significantly extended mitosis after the metaphase was formed (****: P < 0.0001; **: P < 0.01; Student's t-test).
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Figure 2: Inhibition of PLK1 with BI 2536 induces metaphase defects and mitotic catastrophe(A) Inhibition of PLK1 promotes G2/M delay and apoptosis in a dose-dependent manner. HeLa cells were incubated with buffer or the indicated concentrations of BI 2536 (PLK1i). After 24 h, the cells were harvested and the DNA content was analyzed with flow cytometry. The positions of 2N and 4N DNA content are indicated. (B) Inhibition of PLK1 induces mitotic catastrophe. HeLa cells were incubated with buffer or the indicated concentrations of PLK1i for 24 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. Actin analysis was included to assess protein loading and transfer. (C) Treatment with the PLK1 inhibitor GW843682X induces mitotic catastrophe. HeLa cells were incubated with buffer or the indicated concentrations of GW843682X for 24 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. (D) Inhibition of PLK1 affects AURKA activity. Mitotic HeLa cells were isolated by treating cells with nocodazole for 16 h followed by mechanical shake off. The cells were either untreated or incubated with PLK1i, AURKAi, or AURKBi. The proteasome inhibitor MG132 was added to prevent the cells from exiting mitosis. The cells were harvested after 2 h. Lysates were prepared and the indicated proteins were detected with immunoblotting. (E) Low concentrations of PLK1i stimulate cell proliferation. HeLa cells expressing iRFP (~200 cells) were seeded onto 6-well culture plates and cultured in the presence of buffer or different concentrations of PLK1i. On different days, the plate was scanned with an Odyssey infrared imaging system and the iRFP signal was quantified (average± SD of three independent experiments). Note that the PLK1i was left in the medium continuously throughout the experiment. At 0.078 nM, PLK1i significantly increased cell proliferation (P < 0.001; Student's t-test). (F) Inhibition of PLK1 induces a delay in mitosis. HeLa cells expressing histone H2B-GFP were exposed to buffer or the indicated concentrations of PLK1i. Individual cells were then tracked for 24 h with time-lapse microscopy. Each horizontal bar represents one cell (n = 50). Light grey: interphase; black: mitosis (from DNA condensation to anaphase or mitotic slippage); truncated bars: cell death. (G) PLK1i inhibits metaphase–anaphase transition. Cells were treated and imaged as described in panel (F). The duration from prometaphase to metaphase and from metaphase to the end of mitosis (anaphase, apoptosis, or the end of imaging period) was quantified (average ±90% CI). PLK1i treatment significantly extended mitosis after the metaphase was formed (****: P < 0.0001; **: P < 0.01; Student's t-test).
Mentions: To study possible synergism between the antimitotic effects of inhibitors of PLK1 and Aurora kinases, newer generation of small-molecule inhibitors that show relatively high specificity were used in this study. We first verified the effects of an inhibitor of PLK1 called BI 2536 [22,23] (designated PLK1i herein) as a single agent on the cell cycle. Flow cytometry analysis revealed that PLK1i induced a G2/M cell cycle delay in a dose-dependent manner (Figure 2A). Mitotic delay was confirmed by the increase in histone H3Ser10 phosphorylation (Figure 2B). Moreover, the stimulation of apoptosis (as indicated by PARP1 cleavage, caspase 3 cleavage, and increase of sub-G1 population) suggested that mitotic catastrophe was induced by PLK1i in these cells (Figure 2B). PLK1 itself accumulated upon incubation with PLK1i, possibly reflecting the increase of mitotic population (see below). Similar induction of accumulation of PLK1, histone H3Ser10 phosphorylation, and PARP1 cleavage were obtained by targeting PLK1 with another inhibitor called GW843682X [24], indicating that the effects were not specific for BI 2536 only (Figure 2C). Another PLK1 inhibitor called BI 6727 (also called Volasertib) [25] also induced mitotic block and apoptosis in a dose-dependent manner (Supplementary Figure S1).

Bottom Line: Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536).We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells.Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, Center for Cancer Research, and State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

ABSTRACT
Mitosis is choreographed by a number of protein kinases including polo-like kinases and Aurora kinases. As these kinases are frequently dysregulated in cancers, small-molecule inhibitors have been developed for targeted anticancer therapies. Given that PLK1 and Aurora kinases possess both unique functions as well as co-regulate multiple mitotic events, whether pharmacological inhibition of these kinases together can enhance mitotic catastrophe remains an outstanding issue to be determined. Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536). We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells. Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells. These observations underscore the mechanism and potential benefits of targeting PLK1 and Aurora kinases to induce mitotic catastrophe in cancer cells.

No MeSH data available.


Related in: MedlinePlus