Silencing of fanconi anemia complementation group f exhibits potent chemosensitization of mitomycin C activity in breast cancer cells

Abstract

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.

Keywords: Breast neoplasms; Fanconi anemia complementation group F protein; Mitomycin C; Tumor cell line.

Figure 1

Fanconi anemia complementation group F (FANCF) levels and Fanconi anemia, complementation group D2 (FANCD2) ubiquitination in cells. MCF-7 and MDA-MB-435S cells were transfected with FANCF shRNA and control shRNA (scrambled shRNA) for 24 or 48 hours. (A) and (B) is the Western blot analysis for detecting the FANCF and FANCD2 expression changes. Then protein was extracted for performing Western blotting with anti-FANCF or anti-FANCD2 antibodies (FANCD2-L=monoubiquitinated; FANCD2-S=nonubiquitinated). β-Actin was simultaneously immunodetected to verify equal loading of cell lysates. (A) FANCF and (B) FANCD2 blots representative of three independent experiments are presented. (C) The cell viability was determined by the CCK-8 kit. ^*p<0.05 versus cells with control shRNA at 48 hours; ^†p<0.05 versus cells with control shRNA at 24 hours.

Figure 2

Changes of proliferation in mitomycin C (MMC)-treated cells. Dose-response curve of FANCF silencing in cell viability assay with or without MMC. (A) MCF-7 and (B) MDA-MB-435S cells were transfected with FANCF shRNA for 48 hours, and then treated with MMC for 24 hours. The results were expressed as a percentage relative to the control (untreated with MMC). Each point on the graph represents the means±SD.

Figure 3

DNA damage measurement. (A) Comet assay showed detectable comet tails when visualized under a fluorescent microscope, indicative of DNA damage. (B) Semiquantitation analysis of the results in (A) values of tail length (means±SD) were obtain from random selected 50 cells. ^*p<0.05 versus untreated cells; ^†p<0.05 versus cells treated with control shRNA and mitomycin C. FANCF=Fanconi anemia complementation group F; MMC=mitomycin C.

Figure 4

Cell cycle analysis. (A) MCF-7 and MDA-MB-435S cells were synchronized by culturing in serem free media for 72 hours, followed by incubation in serem-containing media for 24 hours and subsequent treatment with either mitomycin C (MMC) or MMC plus Fanconi anemia complementation group F (FANCF) shRNA or control shRNA for 24 hours. Distribution of cells in different phases of cell cycle was analyzed by propidium iodide staining followed by flow cytometry. One representative experiment is shown. Enhanced accumulation of MCF-7 and MDA-MB-435S cells in the S-phase of the cell cycle was observed after cotreatment with MMC and FANCF shRNA. (B) The bar diagrams indicate the distribution of the cells in the different phases of the cell cycle. Data are means±SD of three independent experiments in triplicates.

Figure 5

The apoptosis detection in cells. (A) Apoptosis of cells were measured using FACScan after staining with FITC-annexin V and propidium iodide. Cells in the lower right-hand quadrant are early apoptotic cells with exposed phosphatidylserine (FITC-annexin V-positive) but intact membrane (PI-negative) and (B) is the quantification of apoptosis in the indicated cell lines. ^*p<0.05 versus untreated cells; ^†p<0.05 versus cells treated with control shRNA and mitomycin C. FANCF=Fanconi anemia complementation group F; MMC=mitomycin C; FITC=fluorescence isothiocyanate; PI=propidium iodide.

Figure 6

ΔΨm FACS detection in cells. (A) The cells were stained with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbe nzimidazolylcarbocyanine iodide (JC-1) fluorescence dye, and the change in ΔΨm was examined by FACS. (B) Change in ΔΨm was examined by fluorescence microscopy. (C) Densitometric analysis was done for fraction of cells. Graphs show means±SD of three independent experiments. ^*p<0.05 versus untreated cells; ^†p<0.05 versus cells treated with control shRNA and mitomycin C. FANCF=Fanconi anemia complementation group F; MMC=mitomycin C.