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Loss of p21Cip1/CDKN1A renders cancer cells susceptible to Polo-like kinase 1 inhibition.

Kreis NN, Louwen F, Zimmer B, Yuan J - Oncotarget (2015)

Bottom Line: While the preclinical results are encouraging, the clinical outcomes are rather less inspiring by showing limited anticancer activity.Intriguingly, upon treatment with Polo-like kinase 1 inhibitors, p21 is increased in the cytoplasm, associated with anti-apoptosis, DNA repair and cell survival.We suggest that the p21 status may be a useful biomarker for predicting the efficacy of Plk1 inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology and Obstetrics, J. W. Goethe-University, 60590 Frankfurt, Germany.

ABSTRACT
The deregulation of Polo-like kinase 1 is inversely linked to the prognosis of patients with diverse human tumors. Targeting Polo-like kinase 1 has been widely considered as one of the most promising strategies for molecular anticancer therapy. While the preclinical results are encouraging, the clinical outcomes are rather less inspiring by showing limited anticancer activity. It is thus of importance to identify molecules and mechanisms responsible for the sensitivity of Polo-like kinase 1 inhibition. We have recently shown that p21Cip1/CDKN1A is involved in the regulation of mitosis and its loss prolongs the mitotic duration accompanied by defects in chromosome segregation and cytokinesis in various tumor cells. In the present study, we demonstrate that p21 affects the efficacy of Polo-like kinase 1 inhibitors, especially Poloxin, a specific inhibitor of the unique Polo-box domain. Intriguingly, upon treatment with Polo-like kinase 1 inhibitors, p21 is increased in the cytoplasm, associated with anti-apoptosis, DNA repair and cell survival. By contrast, deficiency of p21 renders tumor cells more susceptible to Polo-like kinase 1 inhibition by showing a pronounced mitotic arrest, DNA damage and apoptosis. Furthermore, long-term treatment with Plk1 inhibitors induced fiercely the senescent state of tumor cells with functional p21. We suggest that the p21 status may be a useful biomarker for predicting the efficacy of Plk1 inhibition.

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Related in: MedlinePlus

Poloxin induces strong apoptosis and more DNA damage in HCT116 cells without p21(A) Western blot analysis of Poloxin concentration kinetics. HCT116 p21+/+ and HCT116 p21−/− cells were treated with 10, 15, 20, 25, 35 and 50 μM Poloxin for 24 h. Cellular lysates were prepared for Western blot analyses with indicated antibodies. DMSO treated cells served as vehicle and β-actin as loading control. (B) Quantification of the cleaved PARP signals from three independent Western blot analyses, relative to the corresponding β-actin signal, cells treated as in (A). The value of each DMSO control is defined as 100%. The quantification was performed with ImageJ (National Institutes of Health). The results are shown as mean ± SEM and statistically analyzed compared to its DMSO control (*p < 0.05). (C) Quantification of early apoptosis. Cells were treated as in (A). Annexin positive cells (Ann+) were evaluated by FACS using a Vybrant apoptosis kit. The results from four independent experiments are shown as mean ± SEM and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01, ***p < 0.001) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05, ##p < 0.01). (D) Evaluation of late apoptosis. Cells were treated as in (A). Annexin and PI (propidium iodide) positive cells (Ann+PI+) were identified by FACS measurements (Vybrant). The results from four independent experiments are shown as mean ± SEM and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05). (E) HCT116 p21+/+ and HCT116 p21−/− cells were treated with DMSO, 10, 15, 20 and 25 μM Poloxin for 24 h. The relative activities of caspase-3/7 were measured. The results are based on three independent experiments, presented as mean ± SEM, and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05). (F) Western blot analysis of Poloxin time kinetics. HCT116 p21+/+ and HCT116 p21−/− cells were treated with 25 μM Poloxin for 0, 4, 8, 12, 24, 36 and 48 h. Western blot analyses were carried out with indicated antibodies. β-actin served as loading control. (G) HCT116 cells were treated without or with the pan-caspase inhibitor Z-VAD (10 μM) 1 h prior to Poloxin treatment (25 μM) for 24 h and Western blot analyses were performed with indicated antibodies and β-actin as loading control. (H) Cells were treated with control siRNA or a mixture of two siRNAs against Plk1 (each 10 nM) for 24 h and Western blot analyses were performed with indicated antibodies. β-actin served as loading control. (I) HCT116 p21+/+ and HCT116 p21−/− cells were treated as in (H). The relative activities of caspase-3/7 were measured in triplicate and presented as mean ± SD (***p < 0.001).
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Figure 4: Poloxin induces strong apoptosis and more DNA damage in HCT116 cells without p21(A) Western blot analysis of Poloxin concentration kinetics. HCT116 p21+/+ and HCT116 p21−/− cells were treated with 10, 15, 20, 25, 35 and 50 μM Poloxin for 24 h. Cellular lysates were prepared for Western blot analyses with indicated antibodies. DMSO treated cells served as vehicle and β-actin as loading control. (B) Quantification of the cleaved PARP signals from three independent Western blot analyses, relative to the corresponding β-actin signal, cells treated as in (A). The value of each DMSO control is defined as 100%. The quantification was performed with ImageJ (National Institutes of Health). The results are shown as mean ± SEM and statistically analyzed compared to its DMSO control (*p < 0.05). (C) Quantification of early apoptosis. Cells were treated as in (A). Annexin positive cells (Ann+) were evaluated by FACS using a Vybrant apoptosis kit. The results from four independent experiments are shown as mean ± SEM and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01, ***p < 0.001) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05, ##p < 0.01). (D) Evaluation of late apoptosis. Cells were treated as in (A). Annexin and PI (propidium iodide) positive cells (Ann+PI+) were identified by FACS measurements (Vybrant). The results from four independent experiments are shown as mean ± SEM and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05). (E) HCT116 p21+/+ and HCT116 p21−/− cells were treated with DMSO, 10, 15, 20 and 25 μM Poloxin for 24 h. The relative activities of caspase-3/7 were measured. The results are based on three independent experiments, presented as mean ± SEM, and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05). (F) Western blot analysis of Poloxin time kinetics. HCT116 p21+/+ and HCT116 p21−/− cells were treated with 25 μM Poloxin for 0, 4, 8, 12, 24, 36 and 48 h. Western blot analyses were carried out with indicated antibodies. β-actin served as loading control. (G) HCT116 cells were treated without or with the pan-caspase inhibitor Z-VAD (10 μM) 1 h prior to Poloxin treatment (25 μM) for 24 h and Western blot analyses were performed with indicated antibodies and β-actin as loading control. (H) Cells were treated with control siRNA or a mixture of two siRNAs against Plk1 (each 10 nM) for 24 h and Western blot analyses were performed with indicated antibodies. β-actin served as loading control. (I) HCT116 p21+/+ and HCT116 p21−/− cells were treated as in (H). The relative activities of caspase-3/7 were measured in triplicate and presented as mean ± SD (***p < 0.001).

Mentions: (A) Cells were treated with indicated Plk1 inhibitors (25 μM Poloxin, 25 nM BI 2536 or 25 nM BI 6727) for 24 h and cell cycle analyses were performed for HCT116 p21+/+ (left panel) and HCT116 p21−/− cells (right panel). The results are presented as mean ± SEM from three independent experiments and statistically analyzed compared to DMSO treated cells. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Examples of FACS profiles are shown. Upper panel: HCT116 p21+/+ cells; lower panel: HCT116 p21−/− cells. (C) Cellular extracts were prepared for Western blot analyses with indicated antibodies. β-actin served as loading control. DMSO treated cells served as vehicle control. (D) HCT116 cells were treated with control siRNA or a mixture of two siRNAs against Plk1 (each 10 nM) for 24 h and cell cycle analyses were performed in triplicate. Examples of FACS profiles were shown. Upper panel: HCT116 p21+/+; lower panel: HCT116 p21−/−. (E) Results of (D) are presented as mean ± SD. ***p < 0.001. Control Western blot is shown in Fig. 4H.


Loss of p21Cip1/CDKN1A renders cancer cells susceptible to Polo-like kinase 1 inhibition.

Kreis NN, Louwen F, Zimmer B, Yuan J - Oncotarget (2015)

Poloxin induces strong apoptosis and more DNA damage in HCT116 cells without p21(A) Western blot analysis of Poloxin concentration kinetics. HCT116 p21+/+ and HCT116 p21−/− cells were treated with 10, 15, 20, 25, 35 and 50 μM Poloxin for 24 h. Cellular lysates were prepared for Western blot analyses with indicated antibodies. DMSO treated cells served as vehicle and β-actin as loading control. (B) Quantification of the cleaved PARP signals from three independent Western blot analyses, relative to the corresponding β-actin signal, cells treated as in (A). The value of each DMSO control is defined as 100%. The quantification was performed with ImageJ (National Institutes of Health). The results are shown as mean ± SEM and statistically analyzed compared to its DMSO control (*p < 0.05). (C) Quantification of early apoptosis. Cells were treated as in (A). Annexin positive cells (Ann+) were evaluated by FACS using a Vybrant apoptosis kit. The results from four independent experiments are shown as mean ± SEM and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01, ***p < 0.001) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05, ##p < 0.01). (D) Evaluation of late apoptosis. Cells were treated as in (A). Annexin and PI (propidium iodide) positive cells (Ann+PI+) were identified by FACS measurements (Vybrant). The results from four independent experiments are shown as mean ± SEM and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05). (E) HCT116 p21+/+ and HCT116 p21−/− cells were treated with DMSO, 10, 15, 20 and 25 μM Poloxin for 24 h. The relative activities of caspase-3/7 were measured. The results are based on three independent experiments, presented as mean ± SEM, and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05). (F) Western blot analysis of Poloxin time kinetics. HCT116 p21+/+ and HCT116 p21−/− cells were treated with 25 μM Poloxin for 0, 4, 8, 12, 24, 36 and 48 h. Western blot analyses were carried out with indicated antibodies. β-actin served as loading control. (G) HCT116 cells were treated without or with the pan-caspase inhibitor Z-VAD (10 μM) 1 h prior to Poloxin treatment (25 μM) for 24 h and Western blot analyses were performed with indicated antibodies and β-actin as loading control. (H) Cells were treated with control siRNA or a mixture of two siRNAs against Plk1 (each 10 nM) for 24 h and Western blot analyses were performed with indicated antibodies. β-actin served as loading control. (I) HCT116 p21+/+ and HCT116 p21−/− cells were treated as in (H). The relative activities of caspase-3/7 were measured in triplicate and presented as mean ± SD (***p < 0.001).
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Figure 4: Poloxin induces strong apoptosis and more DNA damage in HCT116 cells without p21(A) Western blot analysis of Poloxin concentration kinetics. HCT116 p21+/+ and HCT116 p21−/− cells were treated with 10, 15, 20, 25, 35 and 50 μM Poloxin for 24 h. Cellular lysates were prepared for Western blot analyses with indicated antibodies. DMSO treated cells served as vehicle and β-actin as loading control. (B) Quantification of the cleaved PARP signals from three independent Western blot analyses, relative to the corresponding β-actin signal, cells treated as in (A). The value of each DMSO control is defined as 100%. The quantification was performed with ImageJ (National Institutes of Health). The results are shown as mean ± SEM and statistically analyzed compared to its DMSO control (*p < 0.05). (C) Quantification of early apoptosis. Cells were treated as in (A). Annexin positive cells (Ann+) were evaluated by FACS using a Vybrant apoptosis kit. The results from four independent experiments are shown as mean ± SEM and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01, ***p < 0.001) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05, ##p < 0.01). (D) Evaluation of late apoptosis. Cells were treated as in (A). Annexin and PI (propidium iodide) positive cells (Ann+PI+) were identified by FACS measurements (Vybrant). The results from four independent experiments are shown as mean ± SEM and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05). (E) HCT116 p21+/+ and HCT116 p21−/− cells were treated with DMSO, 10, 15, 20 and 25 μM Poloxin for 24 h. The relative activities of caspase-3/7 were measured. The results are based on three independent experiments, presented as mean ± SEM, and statistically analyzed compared to DMSO control (*p < 0.05, **p < 0.01) or between HCT116 p21+/+ and HCT116 p21−/− cells (#p < 0.05). (F) Western blot analysis of Poloxin time kinetics. HCT116 p21+/+ and HCT116 p21−/− cells were treated with 25 μM Poloxin for 0, 4, 8, 12, 24, 36 and 48 h. Western blot analyses were carried out with indicated antibodies. β-actin served as loading control. (G) HCT116 cells were treated without or with the pan-caspase inhibitor Z-VAD (10 μM) 1 h prior to Poloxin treatment (25 μM) for 24 h and Western blot analyses were performed with indicated antibodies and β-actin as loading control. (H) Cells were treated with control siRNA or a mixture of two siRNAs against Plk1 (each 10 nM) for 24 h and Western blot analyses were performed with indicated antibodies. β-actin served as loading control. (I) HCT116 p21+/+ and HCT116 p21−/− cells were treated as in (H). The relative activities of caspase-3/7 were measured in triplicate and presented as mean ± SD (***p < 0.001).
Mentions: (A) Cells were treated with indicated Plk1 inhibitors (25 μM Poloxin, 25 nM BI 2536 or 25 nM BI 6727) for 24 h and cell cycle analyses were performed for HCT116 p21+/+ (left panel) and HCT116 p21−/− cells (right panel). The results are presented as mean ± SEM from three independent experiments and statistically analyzed compared to DMSO treated cells. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Examples of FACS profiles are shown. Upper panel: HCT116 p21+/+ cells; lower panel: HCT116 p21−/− cells. (C) Cellular extracts were prepared for Western blot analyses with indicated antibodies. β-actin served as loading control. DMSO treated cells served as vehicle control. (D) HCT116 cells were treated with control siRNA or a mixture of two siRNAs against Plk1 (each 10 nM) for 24 h and cell cycle analyses were performed in triplicate. Examples of FACS profiles were shown. Upper panel: HCT116 p21+/+; lower panel: HCT116 p21−/−. (E) Results of (D) are presented as mean ± SD. ***p < 0.001. Control Western blot is shown in Fig. 4H.

Bottom Line: While the preclinical results are encouraging, the clinical outcomes are rather less inspiring by showing limited anticancer activity.Intriguingly, upon treatment with Polo-like kinase 1 inhibitors, p21 is increased in the cytoplasm, associated with anti-apoptosis, DNA repair and cell survival.We suggest that the p21 status may be a useful biomarker for predicting the efficacy of Plk1 inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology and Obstetrics, J. W. Goethe-University, 60590 Frankfurt, Germany.

ABSTRACT
The deregulation of Polo-like kinase 1 is inversely linked to the prognosis of patients with diverse human tumors. Targeting Polo-like kinase 1 has been widely considered as one of the most promising strategies for molecular anticancer therapy. While the preclinical results are encouraging, the clinical outcomes are rather less inspiring by showing limited anticancer activity. It is thus of importance to identify molecules and mechanisms responsible for the sensitivity of Polo-like kinase 1 inhibition. We have recently shown that p21Cip1/CDKN1A is involved in the regulation of mitosis and its loss prolongs the mitotic duration accompanied by defects in chromosome segregation and cytokinesis in various tumor cells. In the present study, we demonstrate that p21 affects the efficacy of Polo-like kinase 1 inhibitors, especially Poloxin, a specific inhibitor of the unique Polo-box domain. Intriguingly, upon treatment with Polo-like kinase 1 inhibitors, p21 is increased in the cytoplasm, associated with anti-apoptosis, DNA repair and cell survival. By contrast, deficiency of p21 renders tumor cells more susceptible to Polo-like kinase 1 inhibition by showing a pronounced mitotic arrest, DNA damage and apoptosis. Furthermore, long-term treatment with Plk1 inhibitors induced fiercely the senescent state of tumor cells with functional p21. We suggest that the p21 status may be a useful biomarker for predicting the efficacy of Plk1 inhibition.

Show MeSH
Related in: MedlinePlus