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. 2013 May 1;12(9):1340-51.
doi: 10.4161/cc.24573. Epub 2013 Apr 10.

Polo-like kinase 1 inhibitors, mitotic stress and the tumor suppressor p53

Mourad Sanhaji  1 , Frank Louwen, Brigitte Zimmer, Nina-Naomi Kreis, Susanne Roth, Juping Yuan
Affiliations

Polo-like kinase 1 inhibitors, mitotic stress and the tumor suppressor p53

Mourad Sanhaji et al. Cell Cycle. .

Abstract

Polo-like kinase 1 has been established as one of the most attractive targets for molecular cancer therapy. In fact, multiple small-molecule inhibitors targeting this kinase have been developed and intensively investigated. Recently, it has been reported that the cytotoxicity induced by Plk1 inhibition is elevated in cancer cells with inactive p53, leading to the hypothesis that inactive p53 is a predictive marker for the response of Plk1 inhibition. In our previous study based on different cancer cell lines, we showed that cancer cells with wild type p53 were more sensitive to Plk1 inhibition by inducing more apoptosis, compared with cancer cells depleted of p53. In the present work, we further demonstrate that in the presence of mitotic stress induced by different agents, Plk1 inhibitors strongly induced apoptosis in HCT116 p53(+/+) cells, whereas HCT116 p53(-/-) cells arrested in mitosis with less apoptosis. Depletion of p53 in HCT116 p53(+/+) or U2OS cells reduced the induction of apoptosis. Moreover, the surviving HCT116 p53(-/-) cells showed DNA damage and a strong capability of colony formation. Plk1 inhibition in combination with other anti-mitotic agents inhibited proliferation of tumor cells more strongly than Plk1 inhibition alone. Taken together, the data underscore that functional p53 strengthens the efficacy of Plk1 inhibition alone or in combination by strongly activating cell death signaling pathways. Further studies are required to investigate if the long-term outcomes of losing p53, such as low differential grade of tumor cells or defective DNA damage checkpoint, are responsible for the cytotoxicity of Plk1 inhibition.

Keywords: BI 2536; BI 6727; Poloxin; monastrol; p53.

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Figures

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Figure 1. Plk1 inhibitors trigger more apoptosis in HCT116 p53+/+ cells than in HCT116 p53−/− cells in the presence of mitotic stress. (A) Illustration of experimental schedule. Cells were pretreated with a low dose of mitotic stress inducers (15 µM monastrol, 7 nM paclitaxel, 1.2 nM vincristine or 50 ng/ml nocodazole) for 10 h, then Plk1 inhibitor (25 µM Poloxin, 25 nM BI 2536 or BI 6727) was added for further 28 h. (B) Western blot analysis for cells treated with monastrol and Plk1 inhibitors. HCT116 p53+/+ or HCT116 p53−/− cells were treated as described in (A) and harvested for western blot analysis using indicated antibodies. β-actin served as loading control. (C) Western blot analysis for cells treated with paclitaxel and Plk1 inhibitors. (D) Western blot analysis for cells treated with vincristine and Plk1 inhibitors. (E) Western blot analysis for cells treated with nocodazole and Plk1 inhibitors. (F) Quantification of relative activity of caspase-3/7 for cells treated as in (B). (G) Quantification of relative activity of caspase-3/7 for cells treated as in (D). The results are presented as mean ± SD; mona, monastrol; vincris, vincristine.
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Figure 2. Depletion of p53 reduces apoptosis in HCT116 p53+/+ and U2OS cells. (A) Experimental schedule. (B) HCT116 p53+/+ cells were treated with 20 nM control siRNA (siRNA con) or siRNA against p53 (siRNA p53) for 24 h and then further incubated as described in (A). Cellular lysates were prepared for western blot analysis with antibodies as indicated. β-actin served as loading control. (C) Cellular lysates were also used for measuring the activity of caspase-3/7. The results are presented as mean ± SD (D) U2OS cells were treated as described in (A). Cellular lysates were prepared for western blot analysis with antibodies as indicated. β-actin served as loading control. (E) Cellular lysates were also used for measuring the activity of caspase-3/7. The results are presented as mean ± SD.
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Figure 3. Activation of the spindle assembly checkpoint in HCT116 p53−/− cells. (A) Experimental schedule. (B) HCT116 p53+/+ and HCT116 p53−/− cells were treated as described in (A) and harvested at 0, 6, 12, 18, 24 and 36 h for western blot analysis with indicated antibodies. (C) The same lysates were also used for measurement of the caspase-3/7 activity. The results are presented as mean ± SD (D) Treated cells with monastrol and BI 6727 were fixed and stained for BubR1, pericentrin and DNA. The representatives of confocal microscopy are shown. Scale: 5 µm
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Figure 4. Phenotype of treated HCT116 cells with or without p53. Cells were treated with monastrol and Plk1 inhibitors as shown in Figure 1A and stained for Plk1, pericentrin, tubulin and DNA. (A and B) The representatives from CLSM are shown for HCT116 p53+/+ cells (A) and for HCT116 p53−/− cells (B). Scale: 5 µm. (C) Quantification of monopolar spindle. About 300 to 400 mitotic cells were evaluated from each cell line. The results are presented as mean ± SD; con, control; mona,: monastrol. (D) Quantification of multipolar spindle. About 300 to 400 mitotic cells were evaluated from each cell line. The results are presented as mean ± SD; con, control; mona,: monastrol.
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Figure 5. Survived HCT116 p53−/− cells show more mitotic DNA damage than the cells with p53. (A and B) HCT116 p53+/+ cells (A) and HCT116 p53−/− cells (B) were pre-treated with paxlitaxel for 10 h and then treated with BI 6727 for 28 h. Cells were fixed and stained for DNA damage marker γH2AX, for centromere (ACA, anti-centromere antibody), for tubulin and DNA. The representatives are shown. Scale: 5 µm. (C) Quantification of γH2AX foci in about 100–150 mitotic nuclei. The results are presented as mean ± SD. PTX, paclitaxel.
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Figure 6. Long-time kinetics. (A) Cells were treated with monastrol for 10 h and further incubated together with Plk1 inhibitor BI 6727 for 1 d, 2 d and 3 d. Cells were harvested and cellular lysates were prepared for western blot analysis with indicated antibodies. β-actin served as loading control. (B and C) Treated HCT116 p53+/+ cells (B) and HCT116 p53−/− cells (C) were also collected for phospho-histone H3 (S10) staining and measured by FACS. The results are presented as mean ± SD. (D and E) HCT116 p53+/+ cells (D) and HCT116 p53−/− cells (E) were treated with paclitaxel for 10 h and further incubated with Plk1 inhibitor BI 6727 for 1 d, 2 d and 3 d. Cells were then collected for active caspase-3 staining and measured by FACS. The results are presented as mean ± SD and statistically analyzed. * p < 0.05. ***p < 0.001.
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Figure 7. More HCT116 without p53 survived upon combined treatment. (A) Cells were seeded in 96-well plates, pre-treated with paclitaxel for 10 h then incubated with Plk1 inhibitors for indicated time periods and cell viability was measured. The results are presented as mean ± SD (B) Colony-formation assay. Treated cells were seeded in 6-well plates and cultured for 12–14 d. Cells were then stained, and the colony numbers were counted. The results are presented as mean ± SD.
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