Résumé
Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.
Sujets)
Humains , Antinéoplasiques/pharmacologie , Mouvement cellulaire/génétique , Prolifération cellulaire/génétique , /génétique , Protéine du groupe de complémentation F de l'anémie de Fanconi/génétique , Lignée cellulaire tumorale , Mouvement cellulaire/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Résistance aux substances , Tests de criblage d'agents antitumoraux , Régulation de l'expression des gènes tumoraux , Techniques de knock-down de gènes , Interférence par ARN , Petit ARN interférentRésumé
PURPOSE: Fanconi anemia complementation group F (FANCF) is a key factor to maintaining the function of Fanconi anaemia/BRCA (FA/BRCA) pathway, a DNA-damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. In the present study, we evaluated the chemosensitization effect of FANCF in breast cancer cells. METHODS: We performed specific knockdown of the endogenous FANCF in breast cancer cells by transfecting the cells with an FANCF short hairpin RNA (shRNA) vector. Cell viability was measured with a Cell Counting Kit-8, and DNA damage was assessed with the alkaline comet assay. The apoptosis, cell cycle, and drug accumulation were measured by flow cytometric analysis. Protein expression levels were determined by Western blot analysis, using specific antibodies. RESULTS: The analyses of two breast cancer cell lines (MCF-7 and MDA-MB-435S) demonstrated that the FANCF shRNA could effectively block the FA/BRCA pathway through the inhibition of Fanconi anemia complementation group D2 ubiquitination. Moreover, FANCF silencing potentiated the sensitivity of cells to mitomycin C (MMC), where combined FANCF shRNA/MMC treatment inhibited cell proliferation, induced S-phase arrest, apoptosis, and DNA fragmentation, and reduced the mitochondrial membrane potential, compared with MMC treatment alone. CONCLUSION: Taken together, this study demonstrates that the inhibition of FANCF by its shRNA leads to a synergistic enhancement of MMC cytotoxicity in breast cancer cells. These results suggest that the inhibition of the FA/BRCA pathway is a useful adjunct to cytotoxic chemotherapy for the treatment of breast cancer.
Sujets)
Apoptose , Technique de Western , Région mammaire , Tumeurs du sein , Numération cellulaire , Cycle cellulaire , Lignée cellulaire , Lignée cellulaire tumorale , Prolifération cellulaire , Survie cellulaire , Test des comètes , Protéines du système du complément , Altération de l'ADN , Fragmentation de l'ADN , Anémie de Fanconi , Protéine du groupe de complémentation F de l'anémie de Fanconi , Potentiel de membrane mitochondriale , Mitomycine , Petit ARN interférent , Ubiquitine , UbiquitinationRésumé
Fanconi anemia (FA) is an rare autosomal recessive inherited disease which manifests progressive marrow failure, congenital bone malformation, high risk to cancers and so on. Chromatin of FA cells display auto-instability and high hypersensitivity to interstrand DNA cross-links such as mitomycin C. As normally FA develop into acute myeloid leukemia easily, it has been regarded as pre-leukemia state. Till now 11 FA genes have been found and play a role in sustaining stability of gene groups through the same mechanism. As an active connecting protein, FANCF protein play an important part in correct FA complex formation. Which makes FANCD2 single ubiquitin. Ubiquitin FANCD2 induces chromatin and BRCA1 interact, and repair injured DNA. FA gene defect makes gene group instable and increases the risk of chromatin collapse, which finally leads to acute myeloid leukemia and myelodysplastic syndrome.