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CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

Zhang S, Lu Z, Mao W, Ahmed AA, Yang H, Zhou J, Jennings N, Rodriguez-Aguayo C, Lopez-Berestein G, Miranda R, Qiao W, Baladandayuthapani V, Li Z, Sood AK, Liu J, Le XF, Bast RC - PLoS ONE (2015)

Bottom Line: Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase.In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells.CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America; Department of General Surgery, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi, China.

ABSTRACT
Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells.

No MeSH data available.


Related in: MedlinePlus

CDK5 knockdown induces p53-dependent growth inhibition, apoptosis and G1 arrest.(A, B) Knockdown of p53 reduced CDK5 knockdown-induced apoptosis (A) and G1 arrest (B). HEY cells were reverse transfected with control siRNA or CDK5 siRNA for 24 h and treated with 5 nM paclitaxel (Pac) or diluent (Control) for an additional 48 h, and then cell cycle analyzed by flow cytometry. Data shown are mean values from three independent experiments. (C) CDK5 knockdown increased the expression of p21Cip1, p53, and p27Kip1. HEY cells were treated with control siRNA or CDK5 siRNA for 24 h and then with paclitaxel (3 nM) or diluent for 48 h. Immunoblot analysis was performed with antibodies against p21Cip1, p53, and p27Kip. (D) Knockdown of p53 expression reduced CDK5 siRNA-induced growth inhibition and reduced the enhancement of paclitaxel sensitivity. Cells were co-transfected with CDK5 siRNA and p53 siRNA for 24 h and treated with paclitaxel (Pac) or diluent. Proliferation of cells was measured with a crystal violet cell proliferation assay. (E, F) Knockdown of p53 reduced CDK5 knockdown-induced apoptosis (E) and G1 arrest (F). HEY cells were treated as in (A) and cell cycle analyzed by flow cytometry. Data shown are mean values from three independent experiments. (G) Western analysis confirmed increasing of TP53 expression by silencing CDK5. HEY cells were treated with control siRNA or CDK5 siRNA for 24 h and then with paclitaxel (3 nM) or diluent for 48 h. Cells lysates was subjected to immunoblot analysis with p53, CDK5 and actin antibody.
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pone.0131833.g003: CDK5 knockdown induces p53-dependent growth inhibition, apoptosis and G1 arrest.(A, B) Knockdown of p53 reduced CDK5 knockdown-induced apoptosis (A) and G1 arrest (B). HEY cells were reverse transfected with control siRNA or CDK5 siRNA for 24 h and treated with 5 nM paclitaxel (Pac) or diluent (Control) for an additional 48 h, and then cell cycle analyzed by flow cytometry. Data shown are mean values from three independent experiments. (C) CDK5 knockdown increased the expression of p21Cip1, p53, and p27Kip1. HEY cells were treated with control siRNA or CDK5 siRNA for 24 h and then with paclitaxel (3 nM) or diluent for 48 h. Immunoblot analysis was performed with antibodies against p21Cip1, p53, and p27Kip. (D) Knockdown of p53 expression reduced CDK5 siRNA-induced growth inhibition and reduced the enhancement of paclitaxel sensitivity. Cells were co-transfected with CDK5 siRNA and p53 siRNA for 24 h and treated with paclitaxel (Pac) or diluent. Proliferation of cells was measured with a crystal violet cell proliferation assay. (E, F) Knockdown of p53 reduced CDK5 knockdown-induced apoptosis (E) and G1 arrest (F). HEY cells were treated as in (A) and cell cycle analyzed by flow cytometry. Data shown are mean values from three independent experiments. (G) Western analysis confirmed increasing of TP53 expression by silencing CDK5. HEY cells were treated with control siRNA or CDK5 siRNA for 24 h and then with paclitaxel (3 nM) or diluent for 48 h. Cells lysates was subjected to immunoblot analysis with p53, CDK5 and actin antibody.

Mentions: To explore the additional mechanisms by which CDK5 silencing inhibits wild type TP53 ovarian cancer cell growth and enhanced paclitaxel sensitivity, we examined the effect of CDK5 knockdown with or without paclitaxel treatment on induction of apoptosis and cell cycle arrest in Hey cells which is TP53 wild type. After treatment with CDK5 siRNA or control siRNA, flow cytometric analysis was performed. Silencing CDK5 induced G1 arrest and produced apoptotic cell death with an increased fraction of cells in sub-G1 (Fig 3A and 3B). CDK5 silencing also enhanced paclitaxel-induced apoptosis (Fig 3B).


CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

Zhang S, Lu Z, Mao W, Ahmed AA, Yang H, Zhou J, Jennings N, Rodriguez-Aguayo C, Lopez-Berestein G, Miranda R, Qiao W, Baladandayuthapani V, Li Z, Sood AK, Liu J, Le XF, Bast RC - PLoS ONE (2015)

CDK5 knockdown induces p53-dependent growth inhibition, apoptosis and G1 arrest.(A, B) Knockdown of p53 reduced CDK5 knockdown-induced apoptosis (A) and G1 arrest (B). HEY cells were reverse transfected with control siRNA or CDK5 siRNA for 24 h and treated with 5 nM paclitaxel (Pac) or diluent (Control) for an additional 48 h, and then cell cycle analyzed by flow cytometry. Data shown are mean values from three independent experiments. (C) CDK5 knockdown increased the expression of p21Cip1, p53, and p27Kip1. HEY cells were treated with control siRNA or CDK5 siRNA for 24 h and then with paclitaxel (3 nM) or diluent for 48 h. Immunoblot analysis was performed with antibodies against p21Cip1, p53, and p27Kip. (D) Knockdown of p53 expression reduced CDK5 siRNA-induced growth inhibition and reduced the enhancement of paclitaxel sensitivity. Cells were co-transfected with CDK5 siRNA and p53 siRNA for 24 h and treated with paclitaxel (Pac) or diluent. Proliferation of cells was measured with a crystal violet cell proliferation assay. (E, F) Knockdown of p53 reduced CDK5 knockdown-induced apoptosis (E) and G1 arrest (F). HEY cells were treated as in (A) and cell cycle analyzed by flow cytometry. Data shown are mean values from three independent experiments. (G) Western analysis confirmed increasing of TP53 expression by silencing CDK5. HEY cells were treated with control siRNA or CDK5 siRNA for 24 h and then with paclitaxel (3 nM) or diluent for 48 h. Cells lysates was subjected to immunoblot analysis with p53, CDK5 and actin antibody.
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pone.0131833.g003: CDK5 knockdown induces p53-dependent growth inhibition, apoptosis and G1 arrest.(A, B) Knockdown of p53 reduced CDK5 knockdown-induced apoptosis (A) and G1 arrest (B). HEY cells were reverse transfected with control siRNA or CDK5 siRNA for 24 h and treated with 5 nM paclitaxel (Pac) or diluent (Control) for an additional 48 h, and then cell cycle analyzed by flow cytometry. Data shown are mean values from three independent experiments. (C) CDK5 knockdown increased the expression of p21Cip1, p53, and p27Kip1. HEY cells were treated with control siRNA or CDK5 siRNA for 24 h and then with paclitaxel (3 nM) or diluent for 48 h. Immunoblot analysis was performed with antibodies against p21Cip1, p53, and p27Kip. (D) Knockdown of p53 expression reduced CDK5 siRNA-induced growth inhibition and reduced the enhancement of paclitaxel sensitivity. Cells were co-transfected with CDK5 siRNA and p53 siRNA for 24 h and treated with paclitaxel (Pac) or diluent. Proliferation of cells was measured with a crystal violet cell proliferation assay. (E, F) Knockdown of p53 reduced CDK5 knockdown-induced apoptosis (E) and G1 arrest (F). HEY cells were treated as in (A) and cell cycle analyzed by flow cytometry. Data shown are mean values from three independent experiments. (G) Western analysis confirmed increasing of TP53 expression by silencing CDK5. HEY cells were treated with control siRNA or CDK5 siRNA for 24 h and then with paclitaxel (3 nM) or diluent for 48 h. Cells lysates was subjected to immunoblot analysis with p53, CDK5 and actin antibody.
Mentions: To explore the additional mechanisms by which CDK5 silencing inhibits wild type TP53 ovarian cancer cell growth and enhanced paclitaxel sensitivity, we examined the effect of CDK5 knockdown with or without paclitaxel treatment on induction of apoptosis and cell cycle arrest in Hey cells which is TP53 wild type. After treatment with CDK5 siRNA or control siRNA, flow cytometric analysis was performed. Silencing CDK5 induced G1 arrest and produced apoptotic cell death with an increased fraction of cells in sub-G1 (Fig 3A and 3B). CDK5 silencing also enhanced paclitaxel-induced apoptosis (Fig 3B).

Bottom Line: Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase.In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells.CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Departments of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America; Department of General Surgery, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi, China.

ABSTRACT
Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells.

No MeSH data available.


Related in: MedlinePlus