Promoter Hypermethylation Of FANCF Plays An Important Role In The Occurrence Of Ovarian Cancer Through Disrupting The Expression Of FANCF Gene And Fanconi Anemia-BRCA Pathway

Promoter hypermethylation of FANCF and the expression of FANCFgene in epithelial ovarian cancerObjective: Patients with Fanconi anemia (FA) are predisposed to many types of tumors. Recent reports indicated that individuals with FA are predisposed to develop ovarian cancer than those without FA, this is largely contributed to promoter methylation of the FANCF gene and subsequent disruption of the FA-BRCA pathway. In an attempt to understand more molecular genetic bases of ovarian cancer, we examined the expression of the FANCF and the status of the promoter methylation of the FANCF gene in ovarian cancer.Methods: Seven ovarian cancer cell lines and eighteen ovarian cancer specimens were selected for the purpose of this study. Both genomic DNA and total RNA were extracted from fresh tissues and cell lines. The DNA was treated with bisulfite and then analyzed with methylation-specific PCR (MSP) to detect FANCF methylation. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of FANCF mRNA. Western blot was used to detect the expression of FANCF protein.Results: The expression of FANCF mRNA decreased in most ovarian cancers as compared with those in normal ovarian tissues. Similarly, the level of FANCF protein decreased in ovarian cancers. The decrease of FANCF was due, in part, to FANCF methylation (5 of 12 specimens). The Methylation rates were 27.8% (5 of 18 specimens) in primary tumors and 14.3% (1 of 7 cell lines) in established ovarian cancer cell lines respectively. All the methylated tissues expressed down-regulated FANCF gene.Conclusions: These results indicated that FANCF methylation may have a regulatory role in the expression of FANCF at both mRNA and protein levels. These results also pointed to the possibility that methylation-induced inactivation of FANCF may play an important role in the occurrence of sporadic ovarian cancers bydisrupting the FA-BRCA pathway.Part IIThe effect of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) on the expression of FANCF in epithelial ovarian cancer cell lines and the interference with chemotherapeutic drug taxol in vitro and invivoObjective: To explore the effect of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) on expression of FANCF (Fanconi anemia complementation group F) gene and the proliferation of epithelial ovarian cancer cell lines, to observe cell's sensitivity to chemotherapeutic drug taxol in vitro and in vivo, and explore the antitumor effect and mechanism of taxol for the new treated target of epithelial ovarian cancer. Methods: OVCAR3 cell line (FANCF gene methylated) and A2780 cell line (unmethylated) were treated respectively with 5-ADC. Both genomic DNA and total RNA were extracted from cell lines. The DNA was treated with bisulfite and then analyzed with methylation-specific PCR (MSP) to detect FANCF methylation. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of FANCF mRNA. Western blot was used to detect the expression of FANCF protein. The proliferation of cell lines before and after the treatment with demethylating agent 5-ADC was examined with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in vitro. The cytotoxicity of taxol was measured by flow cytometer. We established the model of human ovarian carcinoma with nude mice to observe the weight and volume of those carcinomas in nude mice before and after treatment with 5-ADC. Immunohistochemistry was used to determine the expression of FANCF protein.Results: The treatment of ovarian cancer cells with 5-ADC contributed to the following results: the inhibition of DNA promoter methylation, the reactivation of FANCF mRNA expression and protein, the reduction in the proliferation of tumor cells both in vitro and in vivo. Before and after treated with 5-ADC, the expression of FANCF mRNA in OVCAR3 cell line was 0.05±0.01and 0.76±0.05 respectively, the expression of FANCF protein was 0.17±0.02 and 0.95±0.04 respectively. Its logarithmic growth was delayed for 24h significantly. The changes were exhibited significantly on the third to forth day (PO.01), whereas in the unmethylated ovarian cancer A2780 cell line, the proliferation and the expression of FANCF gene didn't change significantly. In the in vivo experiments, the weight of tumor in 5-ADC treated and untreated groups was 0.0351±0.0037 g versus 0.2762±0.0217 g (P<0.01), and the volume of tumor in 5-ADC treated and untreated groups was 0.24±0.02 cm³ versus 0.68±0.01 cm³, showing a statistically significant difference (P<0.01). Moreover, 2 mice died on the 45^th day and the 48^th day in the untreated groups, while none died in the treated groups until the end of the experiment. The sensitivity to taxol was a several fold decrease in treated cells compared with untreated cells. In the tissues extracted from the treated nude mice, the expression of FANCF protein increased significantly and the methylation of FANCF gene also disappeared.Conclusions: FANCF DNA methylation exists in ovarian cancer cell line OVCAR3. The treatment of 5-ADC can reactivate the expression of FANCF at both mRNA and protein levels. It also reduces the proliferation of epithelial ovarian cancer cell line in vitro and in vivo through reactivating the expression of FANCF, it may have a potentiality in the future treatment of ovarian cancer. But it can improve the resistance to chemotherapeutic drug taxol at the same time, so the use of 5-ADCmustbe cautious.