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PLK1 is a critical determinant of tumor cell sensitivity to CPT11 and its inhibition enhances the drug antitumor efficacy in squamous cell carcinoma models sensitive and resistant to camptothecins.

Zuco V, De Cesare M, Zaffaroni N, Lanzi C, Cassinelli G - Oncotarget (2015)

Bottom Line: Downregulation of the mitotic kinase PLK1 was found associated with apoptosis induced by SN38 (CPT11 active metabolite).The ability to activate an efficient G2/M cell cycle checkpoint allowing PLK1 ubiquitination and degradation was found associated with SN38-induced apoptosis in SCC cells.A well-tolerated CPT11/BI2536 cotreatment resulted in improved antitumor effect against SCC xenografts in mice compared to single agent treatments.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

ABSTRACT
Intrinsic and acquired tumor drug resistance limits the therapeutic efficacy of camptothecins (CPTs). Downregulation of the mitotic kinase PLK1 was found associated with apoptosis induced by SN38 (CPT11 active metabolite). We investigated the role of PLK1 in the cell response to CPTs in squamous cell carcinoma (SCC) and pediatric sarcoma cell lines and explored the therapeutic potential of the combination of CPT11 and the PLK1 inhibitor BI2536 in CPT-sensitive and -resistant tumor models. Gain- and loss-of-function experiments established a direct role for PLK1 in counteracting SN38 antiproliferative and pro-apoptotic effects. The ability to activate an efficient G2/M cell cycle checkpoint allowing PLK1 ubiquitination and degradation was found associated with SN38-induced apoptosis in SCC cells. However, the synergistic interaction between SN38 and BI2536 enhanced apoptosis in cell lines both sensitive and resistant to SN38-induced apoptotic cell death. A well-tolerated CPT11/BI2536 cotreatment resulted in improved antitumor effect against SCC xenografts in mice compared to single agent treatments. The increased apoptosis induction was reflected in a high rate of complete responses and cures in mice harboring SCC, including tumors with intrinsic or acquired resistance to CPTs. PLK1 inhibition represents a promising strategy to improve the antitumor efficacy of CPT11-based regimens.

No MeSH data available.


Related in: MedlinePlus

Effects of loss- and gain-of-function on SCC cell linesA) SiHa cells were treated with transfection reagent (vehicle), aspecific RNA oligonucleotide (control siRNA) or PLK1-directed siRNA (PLK1 siRNA). Left panel, the effect of PLK1 knockdown on cell growth (cell counting), induction of apoptosis (TUNEL assay) and mitotic cell number (MPM-2 detection by immunofluorescence) was assayed 72h after transfection. Values of cell growth are given in percentage ± SD referred to the negative control siRNA-transfected cells (100%). Central panel, cells were lysed 48h after transfection to assess levels of PLK1 and apoptotic or G2/M cell cycle phase specific markers by Western blot analysis. Tubulin is shown as a loading control. Right panel shows FACS analysis of DNA content and cell cycle distribution of cells stained with propidium-iodide 72h after transfection B) SN38 antiproliferative activity and apoptosis induction were examined in SiHa cells transiently transfected with control or PLK1-directed siRNA. Twenty four hours after transfection, cells were exposed to solvent (−) or to the indicated concentrations of SN38 for 1h. Three days after the end of treatment, the drug antiproliferative activity was evaluated by cell counting (left panel). Values are expressed as percentage ± SD of untreated cells (100%) from three independent experiments. Apoptosis was assessed by TUNEL assay (central panel) and Western blot analysis of PLK1 and cleavage of caspase-3 and PARP was performed in cells exposed to 3 μM SN38 (right panel). Protein loading is shown by vinculin. C) CaSki cells were transiently transfected with control or PLK1-directed siRNAs (Loss-of-function) or, alternatively, with empty or PLK1-expressing vector (Gain-of-function). Left, 24h after siRNA transfection, cells were exposed to SN38 for 72h to assess drug antiproliferative activity by cell counting. Apoptosis induction by SN38 was evaluated in siRNA-transfected cells by TUNEL assay 72h after treatment. Western blots show, on the left, levels of PLK1 after 72h of PLK1 siRNA transfection. Right, 24h after transfection with the PLK1 expression vector, cells were exposed to SN38 and IC50s were calculated after 72h. Western blots in the upper panel show PLK1 levels after 72h of PLK1 vector transfection. PLK1 bands were quantified using ImageJ software and normalized to vinculin. Values are expressed as arbitrary units referred to v-Empty-transfected cells (two independent experiments). In the lower panel, caspase-3 and PARP cleavage after 72h of SN38 treatment is shown (96h after transfection). Vinculin is shown as a control of protein loading. Columns and bars: mean percentage ± SD from three independent experiments. *P < 0.05; **P < 0.01, ***P<0.001 by Student's t test
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Figure 3: Effects of loss- and gain-of-function on SCC cell linesA) SiHa cells were treated with transfection reagent (vehicle), aspecific RNA oligonucleotide (control siRNA) or PLK1-directed siRNA (PLK1 siRNA). Left panel, the effect of PLK1 knockdown on cell growth (cell counting), induction of apoptosis (TUNEL assay) and mitotic cell number (MPM-2 detection by immunofluorescence) was assayed 72h after transfection. Values of cell growth are given in percentage ± SD referred to the negative control siRNA-transfected cells (100%). Central panel, cells were lysed 48h after transfection to assess levels of PLK1 and apoptotic or G2/M cell cycle phase specific markers by Western blot analysis. Tubulin is shown as a loading control. Right panel shows FACS analysis of DNA content and cell cycle distribution of cells stained with propidium-iodide 72h after transfection B) SN38 antiproliferative activity and apoptosis induction were examined in SiHa cells transiently transfected with control or PLK1-directed siRNA. Twenty four hours after transfection, cells were exposed to solvent (−) or to the indicated concentrations of SN38 for 1h. Three days after the end of treatment, the drug antiproliferative activity was evaluated by cell counting (left panel). Values are expressed as percentage ± SD of untreated cells (100%) from three independent experiments. Apoptosis was assessed by TUNEL assay (central panel) and Western blot analysis of PLK1 and cleavage of caspase-3 and PARP was performed in cells exposed to 3 μM SN38 (right panel). Protein loading is shown by vinculin. C) CaSki cells were transiently transfected with control or PLK1-directed siRNAs (Loss-of-function) or, alternatively, with empty or PLK1-expressing vector (Gain-of-function). Left, 24h after siRNA transfection, cells were exposed to SN38 for 72h to assess drug antiproliferative activity by cell counting. Apoptosis induction by SN38 was evaluated in siRNA-transfected cells by TUNEL assay 72h after treatment. Western blots show, on the left, levels of PLK1 after 72h of PLK1 siRNA transfection. Right, 24h after transfection with the PLK1 expression vector, cells were exposed to SN38 and IC50s were calculated after 72h. Western blots in the upper panel show PLK1 levels after 72h of PLK1 vector transfection. PLK1 bands were quantified using ImageJ software and normalized to vinculin. Values are expressed as arbitrary units referred to v-Empty-transfected cells (two independent experiments). In the lower panel, caspase-3 and PARP cleavage after 72h of SN38 treatment is shown (96h after transfection). Vinculin is shown as a control of protein loading. Columns and bars: mean percentage ± SD from three independent experiments. *P < 0.05; **P < 0.01, ***P<0.001 by Student's t test

Mentions: To assess whether PLK1 directly contributes to the cellular outcome in response to SN38, we modulated PLK1 expression in SCC cell lines. Fig. 3A shows that, in SiHa cells, PLK1 knockdown by siRNA resulted in a marked inhibition of cell growth (about 60%) and in the accumulation of mitotic and apoptotic cells. The occurrence of a mitotic arrest [33] was also supported by the enhancement of M phase markers (i.e. cyclin B1, phospho-Ser10 histone H3 and MPM-2) and by the accumulation of cells with 4N DNA content. The induction of apoptotic cell death by PLK1 silencing was confirmed by increased number of TUNEL positive cells and processing of caspase-3. Coherently, Hoechst nuclei staining showed the coexistence of aberrant mitoses and nuclei with apoptotic features in the silenced cell population (not shown). These data indicated that also in the CPT-resistant SiHa cells, PLK1 plays a prosurvival role and that reluctance of these cells to SN38 cytotoxicity was not related to defects in the apoptotic machinery.


PLK1 is a critical determinant of tumor cell sensitivity to CPT11 and its inhibition enhances the drug antitumor efficacy in squamous cell carcinoma models sensitive and resistant to camptothecins.

Zuco V, De Cesare M, Zaffaroni N, Lanzi C, Cassinelli G - Oncotarget (2015)

Effects of loss- and gain-of-function on SCC cell linesA) SiHa cells were treated with transfection reagent (vehicle), aspecific RNA oligonucleotide (control siRNA) or PLK1-directed siRNA (PLK1 siRNA). Left panel, the effect of PLK1 knockdown on cell growth (cell counting), induction of apoptosis (TUNEL assay) and mitotic cell number (MPM-2 detection by immunofluorescence) was assayed 72h after transfection. Values of cell growth are given in percentage ± SD referred to the negative control siRNA-transfected cells (100%). Central panel, cells were lysed 48h after transfection to assess levels of PLK1 and apoptotic or G2/M cell cycle phase specific markers by Western blot analysis. Tubulin is shown as a loading control. Right panel shows FACS analysis of DNA content and cell cycle distribution of cells stained with propidium-iodide 72h after transfection B) SN38 antiproliferative activity and apoptosis induction were examined in SiHa cells transiently transfected with control or PLK1-directed siRNA. Twenty four hours after transfection, cells were exposed to solvent (−) or to the indicated concentrations of SN38 for 1h. Three days after the end of treatment, the drug antiproliferative activity was evaluated by cell counting (left panel). Values are expressed as percentage ± SD of untreated cells (100%) from three independent experiments. Apoptosis was assessed by TUNEL assay (central panel) and Western blot analysis of PLK1 and cleavage of caspase-3 and PARP was performed in cells exposed to 3 μM SN38 (right panel). Protein loading is shown by vinculin. C) CaSki cells were transiently transfected with control or PLK1-directed siRNAs (Loss-of-function) or, alternatively, with empty or PLK1-expressing vector (Gain-of-function). Left, 24h after siRNA transfection, cells were exposed to SN38 for 72h to assess drug antiproliferative activity by cell counting. Apoptosis induction by SN38 was evaluated in siRNA-transfected cells by TUNEL assay 72h after treatment. Western blots show, on the left, levels of PLK1 after 72h of PLK1 siRNA transfection. Right, 24h after transfection with the PLK1 expression vector, cells were exposed to SN38 and IC50s were calculated after 72h. Western blots in the upper panel show PLK1 levels after 72h of PLK1 vector transfection. PLK1 bands were quantified using ImageJ software and normalized to vinculin. Values are expressed as arbitrary units referred to v-Empty-transfected cells (two independent experiments). In the lower panel, caspase-3 and PARP cleavage after 72h of SN38 treatment is shown (96h after transfection). Vinculin is shown as a control of protein loading. Columns and bars: mean percentage ± SD from three independent experiments. *P < 0.05; **P < 0.01, ***P<0.001 by Student's t test
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Figure 3: Effects of loss- and gain-of-function on SCC cell linesA) SiHa cells were treated with transfection reagent (vehicle), aspecific RNA oligonucleotide (control siRNA) or PLK1-directed siRNA (PLK1 siRNA). Left panel, the effect of PLK1 knockdown on cell growth (cell counting), induction of apoptosis (TUNEL assay) and mitotic cell number (MPM-2 detection by immunofluorescence) was assayed 72h after transfection. Values of cell growth are given in percentage ± SD referred to the negative control siRNA-transfected cells (100%). Central panel, cells were lysed 48h after transfection to assess levels of PLK1 and apoptotic or G2/M cell cycle phase specific markers by Western blot analysis. Tubulin is shown as a loading control. Right panel shows FACS analysis of DNA content and cell cycle distribution of cells stained with propidium-iodide 72h after transfection B) SN38 antiproliferative activity and apoptosis induction were examined in SiHa cells transiently transfected with control or PLK1-directed siRNA. Twenty four hours after transfection, cells were exposed to solvent (−) or to the indicated concentrations of SN38 for 1h. Three days after the end of treatment, the drug antiproliferative activity was evaluated by cell counting (left panel). Values are expressed as percentage ± SD of untreated cells (100%) from three independent experiments. Apoptosis was assessed by TUNEL assay (central panel) and Western blot analysis of PLK1 and cleavage of caspase-3 and PARP was performed in cells exposed to 3 μM SN38 (right panel). Protein loading is shown by vinculin. C) CaSki cells were transiently transfected with control or PLK1-directed siRNAs (Loss-of-function) or, alternatively, with empty or PLK1-expressing vector (Gain-of-function). Left, 24h after siRNA transfection, cells were exposed to SN38 for 72h to assess drug antiproliferative activity by cell counting. Apoptosis induction by SN38 was evaluated in siRNA-transfected cells by TUNEL assay 72h after treatment. Western blots show, on the left, levels of PLK1 after 72h of PLK1 siRNA transfection. Right, 24h after transfection with the PLK1 expression vector, cells were exposed to SN38 and IC50s were calculated after 72h. Western blots in the upper panel show PLK1 levels after 72h of PLK1 vector transfection. PLK1 bands were quantified using ImageJ software and normalized to vinculin. Values are expressed as arbitrary units referred to v-Empty-transfected cells (two independent experiments). In the lower panel, caspase-3 and PARP cleavage after 72h of SN38 treatment is shown (96h after transfection). Vinculin is shown as a control of protein loading. Columns and bars: mean percentage ± SD from three independent experiments. *P < 0.05; **P < 0.01, ***P<0.001 by Student's t test
Mentions: To assess whether PLK1 directly contributes to the cellular outcome in response to SN38, we modulated PLK1 expression in SCC cell lines. Fig. 3A shows that, in SiHa cells, PLK1 knockdown by siRNA resulted in a marked inhibition of cell growth (about 60%) and in the accumulation of mitotic and apoptotic cells. The occurrence of a mitotic arrest [33] was also supported by the enhancement of M phase markers (i.e. cyclin B1, phospho-Ser10 histone H3 and MPM-2) and by the accumulation of cells with 4N DNA content. The induction of apoptotic cell death by PLK1 silencing was confirmed by increased number of TUNEL positive cells and processing of caspase-3. Coherently, Hoechst nuclei staining showed the coexistence of aberrant mitoses and nuclei with apoptotic features in the silenced cell population (not shown). These data indicated that also in the CPT-resistant SiHa cells, PLK1 plays a prosurvival role and that reluctance of these cells to SN38 cytotoxicity was not related to defects in the apoptotic machinery.

Bottom Line: Downregulation of the mitotic kinase PLK1 was found associated with apoptosis induced by SN38 (CPT11 active metabolite).The ability to activate an efficient G2/M cell cycle checkpoint allowing PLK1 ubiquitination and degradation was found associated with SN38-induced apoptosis in SCC cells.A well-tolerated CPT11/BI2536 cotreatment resulted in improved antitumor effect against SCC xenografts in mice compared to single agent treatments.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

ABSTRACT
Intrinsic and acquired tumor drug resistance limits the therapeutic efficacy of camptothecins (CPTs). Downregulation of the mitotic kinase PLK1 was found associated with apoptosis induced by SN38 (CPT11 active metabolite). We investigated the role of PLK1 in the cell response to CPTs in squamous cell carcinoma (SCC) and pediatric sarcoma cell lines and explored the therapeutic potential of the combination of CPT11 and the PLK1 inhibitor BI2536 in CPT-sensitive and -resistant tumor models. Gain- and loss-of-function experiments established a direct role for PLK1 in counteracting SN38 antiproliferative and pro-apoptotic effects. The ability to activate an efficient G2/M cell cycle checkpoint allowing PLK1 ubiquitination and degradation was found associated with SN38-induced apoptosis in SCC cells. However, the synergistic interaction between SN38 and BI2536 enhanced apoptosis in cell lines both sensitive and resistant to SN38-induced apoptotic cell death. A well-tolerated CPT11/BI2536 cotreatment resulted in improved antitumor effect against SCC xenografts in mice compared to single agent treatments. The increased apoptosis induction was reflected in a high rate of complete responses and cures in mice harboring SCC, including tumors with intrinsic or acquired resistance to CPTs. PLK1 inhibition represents a promising strategy to improve the antitumor efficacy of CPT11-based regimens.

No MeSH data available.


Related in: MedlinePlus