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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 caspase-mediated apoptosis and downregulates Bcl-2.(A) CDK5 knockdown induced caspase-3 activation in the presence and absence of paclitaxel. HEY cells were transfected with control siRNA or CDK5 siRNA for 24 h, treated with paclitaxel (3 nM) or diluent for 48 h, lysed and total protein analyzed by immunoblot using antibodies against activated caspase-3 and GAPDH. (B) A pan-caspase inhibitor blocked CDK5 knockdown–induced apoptosis. HEY cells were pretreated for 2 h with 20 μM of a pan-caspase inhibitor (Z-VAD-FMK), or negative control (Z-FA-FMK), followed by treatment for 24 h with or without paclitaxel (3 nM). The cells were then fixed in 70% ethanol, stained with propidium iodide, and subjected to cell cycle analysis by flow cytometry. The sub-G1 cell population was considered apoptotic and expressed as a percentage of the whole-cell population. (C) CDK5 knockdown decreased the expression of Bcl-2 mRNA. HEY cells were treated as in (A) and total RNA was extracted for real-time PCR analysis of Bcl-2 mRNA expression. Data shown were from three independent experiments.
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pone.0131833.g005: CDK5 knockdown induces caspase-mediated apoptosis and downregulates Bcl-2.(A) CDK5 knockdown induced caspase-3 activation in the presence and absence of paclitaxel. HEY cells were transfected with control siRNA or CDK5 siRNA for 24 h, treated with paclitaxel (3 nM) or diluent for 48 h, lysed and total protein analyzed by immunoblot using antibodies against activated caspase-3 and GAPDH. (B) A pan-caspase inhibitor blocked CDK5 knockdown–induced apoptosis. HEY cells were pretreated for 2 h with 20 μM of a pan-caspase inhibitor (Z-VAD-FMK), or negative control (Z-FA-FMK), followed by treatment for 24 h with or without paclitaxel (3 nM). The cells were then fixed in 70% ethanol, stained with propidium iodide, and subjected to cell cycle analysis by flow cytometry. The sub-G1 cell population was considered apoptotic and expressed as a percentage of the whole-cell population. (C) CDK5 knockdown decreased the expression of Bcl-2 mRNA. HEY cells were treated as in (A) and total RNA was extracted for real-time PCR analysis of Bcl-2 mRNA expression. Data shown were from three independent experiments.

Mentions: To elucidate the mechanism by which CDK5 knockdown induced apoptosis, we asked whether CDK5 knockdown and paclitaxel treatment activated caspase-3. Western analysis showed that CDK5 knockdown could induce the activation of caspase-3 and this was further increased by treatment with paclitaxel (Fig 5A). Z-VAD-FMK, a pan-caspase inhibitor, blocked apoptosis in cells transfected CDK5 siRNA (Fig 5B). Consequently, the apoptotic cell death induced by CDK5 silencing was caspase dependent. Bcl-2 is an important inhibitor of apoptosis. CDK5 silencing was found to decrease the expression of Bcl-2 mRNA level by qRT-PCR (Fig 5C), which can be accounted for the upregulation of p27Kip1, as we previously reported [1]. The combination of CDK5 siRNA and paclitaxel showed greater inhibition of Bcl-2 mRNA than did CDK5 siRNA alone.


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 caspase-mediated apoptosis and downregulates Bcl-2.(A) CDK5 knockdown induced caspase-3 activation in the presence and absence of paclitaxel. HEY cells were transfected with control siRNA or CDK5 siRNA for 24 h, treated with paclitaxel (3 nM) or diluent for 48 h, lysed and total protein analyzed by immunoblot using antibodies against activated caspase-3 and GAPDH. (B) A pan-caspase inhibitor blocked CDK5 knockdown–induced apoptosis. HEY cells were pretreated for 2 h with 20 μM of a pan-caspase inhibitor (Z-VAD-FMK), or negative control (Z-FA-FMK), followed by treatment for 24 h with or without paclitaxel (3 nM). The cells were then fixed in 70% ethanol, stained with propidium iodide, and subjected to cell cycle analysis by flow cytometry. The sub-G1 cell population was considered apoptotic and expressed as a percentage of the whole-cell population. (C) CDK5 knockdown decreased the expression of Bcl-2 mRNA. HEY cells were treated as in (A) and total RNA was extracted for real-time PCR analysis of Bcl-2 mRNA expression. Data shown were from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492679&req=5

pone.0131833.g005: CDK5 knockdown induces caspase-mediated apoptosis and downregulates Bcl-2.(A) CDK5 knockdown induced caspase-3 activation in the presence and absence of paclitaxel. HEY cells were transfected with control siRNA or CDK5 siRNA for 24 h, treated with paclitaxel (3 nM) or diluent for 48 h, lysed and total protein analyzed by immunoblot using antibodies against activated caspase-3 and GAPDH. (B) A pan-caspase inhibitor blocked CDK5 knockdown–induced apoptosis. HEY cells were pretreated for 2 h with 20 μM of a pan-caspase inhibitor (Z-VAD-FMK), or negative control (Z-FA-FMK), followed by treatment for 24 h with or without paclitaxel (3 nM). The cells were then fixed in 70% ethanol, stained with propidium iodide, and subjected to cell cycle analysis by flow cytometry. The sub-G1 cell population was considered apoptotic and expressed as a percentage of the whole-cell population. (C) CDK5 knockdown decreased the expression of Bcl-2 mRNA. HEY cells were treated as in (A) and total RNA was extracted for real-time PCR analysis of Bcl-2 mRNA expression. Data shown were from three independent experiments.
Mentions: To elucidate the mechanism by which CDK5 knockdown induced apoptosis, we asked whether CDK5 knockdown and paclitaxel treatment activated caspase-3. Western analysis showed that CDK5 knockdown could induce the activation of caspase-3 and this was further increased by treatment with paclitaxel (Fig 5A). Z-VAD-FMK, a pan-caspase inhibitor, blocked apoptosis in cells transfected CDK5 siRNA (Fig 5B). Consequently, the apoptotic cell death induced by CDK5 silencing was caspase dependent. Bcl-2 is an important inhibitor of apoptosis. CDK5 silencing was found to decrease the expression of Bcl-2 mRNA level by qRT-PCR (Fig 5C), which can be accounted for the upregulation of p27Kip1, as we previously reported [1]. The combination of CDK5 siRNA and paclitaxel showed greater inhibition of Bcl-2 mRNA than did CDK5 siRNA alone.

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