TY - JOUR
T1 - The role of p27Kip1 in dasatinib-enhanced paclitaxel cytotoxicity in human ovarian cancer cells
AU - Le, Xiao Feng
AU - Mao, Weiqun
AU - He, Guangan
AU - Claret, Francois Xavier
AU - Xia, Weiya
AU - Ahmed, Ahmed Ashour
AU - Hung, Mien Chie
AU - Siddik, Zahid H.
AU - Bast, Robert C.
PY - 2011/9/21
Y1 - 2011/9/21
N2 - Background:Less than 50% of ovarian cancers respond to paclitaxel. Effective strategies are needed to enhance paclitaxel sensitivity. Methods:A library of silencing RNAs (siRNAs) was used to identify kinases that regulate paclitaxel sensitivity in human ovarian cancer SKOv3 cells. The effect of dasatinib, an inhibitor of Src and Abl kinases, on paclitaxel sensitivity was measured in ovarian cancer cells and HEY xenografts. The roles of p27 Kip1, Bcl-2, and Cdk1 in apoptosis induced by dasatinib and paclitaxel were assessed using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, siRNA knockdown of gene expression, transfection with Bcl-2 and Cdk1 expression vectors, and flow cytometry. All statistical tests were two-sided. Results:Src family and Abl kinases were identified as modulators of paclitaxel sensitivity in SKOv3 cells. The siRNA knockdown of Src, Fyn, or Abl1 enhanced paclitaxel-mediated growth inhibition in ovarian cancer cells compared with a control siRNA. HEY cells treated with dasatinib plus paclitaxel formed fewer colonies than did cells treated with either agent alone. Treatment of HEY xenograft-bearing mice with dasatinib plus paclitaxel inhibited tumor growth more than treatment with either agent alone (average tumor volume per mouse, dasatinib + paclitaxel vs paclitaxel:0.28 vs 0.81 cm3, difference = 0.53 cm3, 95% confidence interval [CI] = 0.44 to 0.62 cm3, P =.014); dasatinib + paclitaxel vs dasatinib:0.28 vs 0.55 cm3, difference = 0.27 cm3, 95% CI = 0.21 to 0.33 cm3, P =.035). Combined treatment induced more TUNEL-positive apoptotic cells than did either agent alone. The siRNA knockdown of p27Kip1 decreased dasatinib-and paclitaxel-induced apoptosis compared with a negative control siRNA (sub-G1 fraction, control siRNA vs p27Kip1 siRNA:42.5% vs 20.1%, difference = 22.4%, 95% CI = 20.1% to 24.7%, P =.017). Studies with forced expression and siRNA knockdown of Bcl-2 and Cdk1 suggest that dasatinib-mediated induction of p27Kip1 enhanced paclitaxel-induced apoptosis by negatively regulating Bcl-2 and Cdk1 expression. Conclusion:Inhibition of Src family and Abl kinases with either siRNAs or dasatinib enhances paclitaxel sensitivity of ovarian cancer cells through p27Kip1-mediated suppression of Bcl-2 and Cdk1 expression.
AB - Background:Less than 50% of ovarian cancers respond to paclitaxel. Effective strategies are needed to enhance paclitaxel sensitivity. Methods:A library of silencing RNAs (siRNAs) was used to identify kinases that regulate paclitaxel sensitivity in human ovarian cancer SKOv3 cells. The effect of dasatinib, an inhibitor of Src and Abl kinases, on paclitaxel sensitivity was measured in ovarian cancer cells and HEY xenografts. The roles of p27 Kip1, Bcl-2, and Cdk1 in apoptosis induced by dasatinib and paclitaxel were assessed using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, siRNA knockdown of gene expression, transfection with Bcl-2 and Cdk1 expression vectors, and flow cytometry. All statistical tests were two-sided. Results:Src family and Abl kinases were identified as modulators of paclitaxel sensitivity in SKOv3 cells. The siRNA knockdown of Src, Fyn, or Abl1 enhanced paclitaxel-mediated growth inhibition in ovarian cancer cells compared with a control siRNA. HEY cells treated with dasatinib plus paclitaxel formed fewer colonies than did cells treated with either agent alone. Treatment of HEY xenograft-bearing mice with dasatinib plus paclitaxel inhibited tumor growth more than treatment with either agent alone (average tumor volume per mouse, dasatinib + paclitaxel vs paclitaxel:0.28 vs 0.81 cm3, difference = 0.53 cm3, 95% confidence interval [CI] = 0.44 to 0.62 cm3, P =.014); dasatinib + paclitaxel vs dasatinib:0.28 vs 0.55 cm3, difference = 0.27 cm3, 95% CI = 0.21 to 0.33 cm3, P =.035). Combined treatment induced more TUNEL-positive apoptotic cells than did either agent alone. The siRNA knockdown of p27Kip1 decreased dasatinib-and paclitaxel-induced apoptosis compared with a negative control siRNA (sub-G1 fraction, control siRNA vs p27Kip1 siRNA:42.5% vs 20.1%, difference = 22.4%, 95% CI = 20.1% to 24.7%, P =.017). Studies with forced expression and siRNA knockdown of Bcl-2 and Cdk1 suggest that dasatinib-mediated induction of p27Kip1 enhanced paclitaxel-induced apoptosis by negatively regulating Bcl-2 and Cdk1 expression. Conclusion:Inhibition of Src family and Abl kinases with either siRNAs or dasatinib enhances paclitaxel sensitivity of ovarian cancer cells through p27Kip1-mediated suppression of Bcl-2 and Cdk1 expression.
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U2 - 10.1093/jnci/djr280
DO - 10.1093/jnci/djr280
M3 - Article
C2 - 21813412
AN - SCOPUS:80053291558
SN - 0027-8874
VL - 103
SP - 1403
EP - 1422
JO - Journal of the National Cancer Institute
JF - Journal of the National Cancer Institute
IS - 18
ER -