title: Effect of the WWOX gene on the regulation of the cell cycle and apoptosis in human ovarian cancer stem cells.
abstract: In order to examine new ideas for gene therapy in ovarian cancer, the specific mechanism underlying the effects of the WW domain containing oxidoreductase (WWOX) gene on cell cycle regulation and apoptosis in human ovarian cancer stem cells was investigated. Ovarian cancer stem cells were transfected with a eukaryotic expression vector carrying the WWOX gene in vitro (recombinant plasmid) and cells transfected with the empty plasmid (empty plasmid) or untransfected cells were used as controls. Stably transfected cells were screened and amplified in culture and the WWOX protein was detected by western blot analysis in the three groups of cells. Western blot analysis was performed to detect the expression of cell cycle regulatory proteins cyclin E, cyclin-dependent kinase (CDK) 2, cyclin D1, CDK4 and apoptosis-related protein Wnt-5α and c-Jun N-terminal kinase (JNK), while polymerase chain reaction (PCR) was used to detect alterations in the mRNA expression levels of caspase-3. The results demonstrated that the WWOX protein was stably expressed in cells of the recombinant plasmid group, but was not detected in cells of the empty plasmid group and the control group. Cell proliferation at each time point decreased significantly in the recombinant plasmid group compared with the empty plasmid group and the control group. Flow cytometric analysis demonstrated that the proportion of cells in the G0/G1 phase in the recombinant plasmid group was significantly higher than that of cells in the empty plasmid group and the control group. The rate of apoptosis in the recombinant plasmid group was significantly higher than that of cells in the empty plasmid group and the control group. Western blot analysis demonstrated that the expression levels of cyclin E, CDK2, cyclin D1 and CDK4 in the recombinant plasmid group were significantly lower than those in the empty plasmid group and the control group; however, the expression levels of Wnt-5α and JNK were significantly higher than those in the empty plasmid group and the control group. PCR results demonstrated that the mRNA expression level of caspase-3 in the recombinant plasmid group was significantly higher than that in the empty plasmid group and the control group. In conclusion, the present study demonstrated that the WWOX gene can be stably expressed in ovarian cancer stem cells and that it inhibits the proliferation of ovarian cancer stem cells. The WWOX gene can downregulate the expression levels of cell cycle proteins cyclin E-CDK2 and cyclin D1-CDK4, which affects the cell cycle of ovarian cancer stem cells. Furthermore, the WWOX gene can upregulate the mRNA expression levels of Wnt-5α, JNK and caspase-3, thus contributing to apoptosis of ovarian cancer stem cells. The present study demonstrated that the WWOX gene may be an important molecular target for the treatment of ovarian cancer in the future.
authors.affiliation: Department of Obstetrics and Gynecology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, P.R. China.
authors.forename: Hongchao
authors.initials: H
authors.lastname: Yan
authors.affiliation: Department of Obstetrics and Gynecology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, P.R. China.
authors.forename: Jianye
authors.initials: J
authors.lastname: Tong
authors.affiliation: Department of Obstetrics and Gynecology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, P.R. China.
authors.forename: Xiaoman
authors.initials: X
authors.lastname: Lin
authors.affiliation: Department of Obstetrics and Gynecology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, P.R. China.
authors.forename: Qiuyu
authors.initials: Q
authors.lastname: Han
authors.affiliation: Department of Obstetrics and Gynecology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, P.R. China.
authors.forename: Hongxiang
authors.initials: H
authors.lastname: Huang
mesh_terms: D017209:Apoptosis; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D015536:Down-Regulation; D005260:Female; D059585:G1 Phase Cell Cycle Checkpoints; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000166:cytology / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D000074584:WW Domain-Containing Oxidoreductase; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism; D000071818:Wnt-5a Protein
chemical_list: C530104:CCND1 protein, human; D019927:Cyclin E; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D019938:Cyclin D1; D010088:Oxidoreductases; D000074584:WW Domain-Containing Oxidoreductase; C486071:WWOX protein, human; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D048031:JNK Mitogen-Activated Protein Kinases; D053148:Caspase 3