title: The role of p27(Kip1) in dasatinib-enhanced paclitaxel cytotoxicity in human ovarian cancer cells.
abstract: 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 p27(Kip1) decreased dasatinib- and paclitaxel-induced apoptosis compared with a negative control siRNA (sub-G1 fraction, control siRNA vs. p27(Kip1) 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 p27(Kip1) enhanced paclitaxel-induced apoptosis by negatively regulating Bcl-2 and Cdk1 expression.
  
  
  CONCLUSIONS
  Inhibition of Src family and Abl kinases with either siRNAs or dasatinib enhances paclitaxel sensitivity of ovarian cancer cells through p27(Kip1)-mediated suppression of Bcl-2 and Cdk1 expression.
authors.affiliation: Department of Experimental Therapeutics, the University of Texas M. D. Anderson Cancer Center, Unit 354, Rm Y6.5343, 1515 Holcombe Blvd, Houston, TX, USA. xfle@mdanderson.org
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Le
authors.forename: Weiqun
authors.initials: W
authors.lastname: Mao
authors.forename: Guangan
authors.initials: G
authors.lastname: He
authors.forename: Francois-Xavier
authors.initials: FX
authors.lastname: Claret
authors.forename: Weiya
authors.initials: W
authors.lastname: Xia
authors.forename: Ahmed Ashour
authors.initials: AA
authors.lastname: Ahmed
authors.forename: Mien-Chie
authors.initials: MC
authors.lastname: Hung
authors.forename: Zahid H
authors.initials: ZH
authors.lastname: Siddik
authors.forename: Robert C
authors.initials: RC
authors.lastname: Bast
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors; D053148:Caspase 3 / Q000032:analysis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015986:Confounding Factors, Epidemiologic; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D000069439:Dasatinib; D004357:Drug Synergism; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D019940:Genes, bcl-1; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D051379:Mice; D008819:Mice, Nude; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000378:metabolism; D012107:Research Design; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology*; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
chemical_list: D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013844:Thiazoles; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D016315:Proto-Oncogene Proteins c-abl; D019061:src-Family Kinases; D016203:CDC2 Protein Kinase; D053148:Caspase 3; D017239:Paclitaxel; D000069439:Dasatinib