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@# Objective: To investigate the expression profile and function of FANCF gene (a key gene in FA/BRCA pathway) in both cisplatin (DDP)-resistant and DDP-sensitive human triple-negative breast cancer cell lines and to analyze its correlation with DDP-resistance in breast cancer. Methods: The DDP-resistant breast cancer MDA-MB-231 cell line (MDA-MB-231/DDP) was established by induction of gradient DDP. The expression of FANCF gene in both sensitive and resistant cell lines was knocked-down by RNAi interference technology and the knockdown efficiency was validated at both RNA and protein level. The cell viability of MDA-MB-231 cells and MDA-MB-231/DDP cells was determined by the CCK8 assay; Flow cytometry was used to examine the cell cycle distribution and apoptosis; the mRNAand protein expressions of FANCF gene were examined by using qRT-PCR and western blotting, respectively. Results: The resistance index of MDA-MB-231/DDP cells was 13.5 after 3-month induction. The mRNA and protein expressions of FANCF were significantly increased in MDA-MB-231/DDP cells (all P<0.01). Cell cycle analysis indicated that the DDP treatment significantly induced G0/G1 arrest and decreased the cell proportion in phase S and G2/M. siRNA-mediated knockdown of FANCF could not only be able to increase sensitivity of MDA-MB-231 to DDP but also promote the cell apoptosis (all P<0.01). Conclusion: FANCF attributes to the occurrence of DDP-resistance through anti-apoptosis effect, which might be served as a potential treatment target for drug-resistant human breast cancer.
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@#[Abstract] Objective: To establish paclitaxel(PTX)-resistant human triple negative breast cancer cell line and to examine the expression profile of FA-related genes and FANCF, the correlation between the expression of FA-related genes, FANCF and PTX-resistance in breast cancer were further analyzed. Methods: PTX-resistant MDA-MB-231 cell line was established by means of long-term PTX-exposed culture. The sensitivity of the cells to paclitaxel was determined by the CCK8 assay. The cell cycle distribution was examined by flow cytometry after exposure to the paclitaxel. The expression of FA-related gene mRNA and FANCF protein were examined by using real time quantitative PCR and Western blotting. The expression of FANCF in the cells was reduced by RNAi interference technology and the effect of the RNAi was verified. Results: MDA-MB-231/PTX cell showed a 9.9-fold resistance to paclitaxel, indicating that the cell had acquired resistance to PTX. PTX treatment significantly induced G0/G1 arrest and the number of cells in phase S markedly decreased after exposure to PTX. The mRNA and protein expression of FANCF was significantly higher in PTX-resistant cell than that in PTX-sensitve parental cell，Knockdown of FANCF induced apoptosis in MDA-MB-231/PTX cell as well as in parental cell. FANCF knockdown increased the sensitivity of paclitaxel to both MDA-MB-231 and MDA-MB-231/PTX cells (P<0.05 or P<0.01). Conclusion: FANCF played an important role in PTX resistance of the breast cancer cells and FANCF might be a target for therapy aimed at reversing chemoresistance.
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Objective: To investigate the effect of methyltransferase inhibitor 5-Aza-dC (5-aza-2'- deoxycytidine) on the expression of FANCF (Fanconi anemia complementation group F) gene in ovarian cancer OVCAR3 cells, and examine its effect on the sensitivity to chemotherapeutic drug paclitaxel in ovarian cancer in vitro , and to explore the antitumor effect of 5-Aza-dC as a new target for treatment of ovarian cancer. Methods: The ovarian cancer OVCAR3 cells (methylated FANCF gene) and A2780 cells (unmethylated FANCF gene) were treated with 5-Aza-dC. The methylation status of FANCF gene and the expression levels of FANCF mRNA and protein in OVCAR3 and A2780 cells before and after the treatment with 5-Aza-dC were detected by MSP (methylation-specific PCR), RT-PCR and Western blotting, respectively. The proliferation rate of these ovarian cancer cells was detected by MTT method, and the apoptosis rate of these cells treated with paclitaxel was measured by FCM (flow cytometry). The xenografts were induced in nude mice transplanted with OVCAR3 cells or A2780 cells. The effect of 5-Aza-dC on the growth of the xenografts in nude mice was observed. The expression of FANCF protein in the xenografts was detected by immunohistochemistry. Results: DNA promoter methylation disappears in OVCAR3 cells after treatment with 5-Aza-dC, and the expression levels of FANCF mRNA and protein were increased in vitro . The growth of OVCAR3 cells was also slowed down. The volume and the weight of OVCAR3 ovarian cancer xenografts treated with 5-Aza-dC were both significantly decreased as compared with those of the untreated xenografts in nude mice; the expression of FANCF protein in the xenografts was increased after treatment with 5-Aza-dC. These changes were not observed in A2780 ovarian cancer xenografts in nude mice. Conclusion: The methyltransferase inhibitor 5-Aza-dC may become a potential therapeutic drug in the treatment of ovarian cancer through inhibiting the proliferation of ovarian cancer cells, but it can also increase the risk of resistance to paclitaxel. © 2012 by Tumor.
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[Objective]To evaluate FANCF gene methylation status and its roles in the pathogenesis acute myeloid leukemia (AML).[Methods]Genomic DNA from primary 30 AML patients and 21 AML cell lines were subjected to FANCF methylation analysis by PCR based restriction enzyme digestion assay.FANCF protein expression was detected by Western blot.In addition,FANCF gene methylation status was further analyzed using bisulfate sequencing.[Results] No FANCF methylation was found in primary AML patients.One (4.76 ％) AML cell line contained FANCF methylation in the promoter region.The AML cell line was hypersensitive to MMC with absence of FANCF protein expression.[Conclusion] FANCF methylation is a rare event in AML,and does not contribute to the initiation of AML, but may contribute to the clonal transformation and cellular phenotype maintenance in some AML cell lines.
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Objective:To evaluate the disruption of FANCF gene by methylation in cervical cancer and analyze the relation between cervical cancer and the methylation of FANCF DNA so as to find a method to inhibit the development of cervical cancer.Methods:The level of FANCF mRNA was detected with reverse transcription polymerase chain reaction(RT-PCR) before and after treatment with 5-aza-2'-deoxycytidine(5-Aza-CdR).Protein was tested by Western Blot.Methylation specific PCR(MSP) was used to check whether it was methylated in exon of FANCF.Normal cervical cancer tissues were used as controls.Results:The expression of FANCF mRNA in cervical cancer cell line SiHa was absent,and so was the expression of protein.The expression of FANCF mRNA and protein was decreased in Hela cell line.After treated with(5-Aza-CdR),SiHa expressed FANCF mRNA and protein,and time for SiHa cells to proliferate double was prolonged.Compared with the controls,the expression of FANCF mRNA in cervical cancer tissues decreased significantly or absolutely defaulted,and expression of FANCF protein also decreased(P