RESUMO
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.
Assuntos
Apoptose , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/genética , Oxirredutases/genética , Proteínas Supressoras de Tumor/genética , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Regulação para Baixo , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células-Tronco Neoplásicas/citologia , Neoplasias Ovarianas/patologia , Oxirredutases/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMO
The aim of this study was to explore the effects of 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methylation inhibitor, on the methylation state and function of the WWOX gene in the HO-8910 ovarian cancer cell line. The HO-8910 cells were divided into two groups, a control group and a 5-Aza-CdR-treated group. The methylation state of the WWOX gene was evaluated using a methylation-specific PCR assay. The effect of 5-Aza-CdR on the HO-8910 cells was analyzed using MTT and cell invasion assays, as well as flow cytometry. The animal models were established by intraperitoneal transplantation of the cells into nude mice. Following treatment with 5-Aza-CdR, a demethylation state was detected in the HO-8910 cells. WWOX protein expression was significantly higher in the 5-Aza-CdR-treated group compared with that in the control group. The cell growth rate at each tested time point and the number of invasive cells were lower in the 5-Aza-CdR-treated group compared with that in the control group. Flow cytometry revealed that 67.13% of the cells were arrested at the G0/G1 stage in the 5-Aza-CdR-treated group. The tumorigenic ability of the 5-Aza-CdR-treated group was lower compared with that of the control group. In conclusion, the methylation state of the WWOX gene in HO-8910 cells may be reversed using 5-Aza-CdR, which may also inhibit the growth of these cells.
