Cytoplasmic M-CSF Induces The Resistance To5-FU And ADR By Downregulating The Activities Of DNA Methyltransferase3b In Breast Cancer MCF-7Cells

OBJECTIVE: To investigate the mechanisms of the resistance to5-FU and ADRinduced by cytoplasmic macrophage colony-stimulating factor (M-CSF) in breastcancer MCF-7cells.METHODS: The parental MCF-7cells (referred as MCF-7), pCMV/cyto/myc-transfected MCF-7cells (referred as MCF-7-C)and pCMV/cyto/myc-M-CSF-transfected MCF-7cells (referred as MCF-7-M) were established in our lab.The geneexpression, protein expression and cell proliferation was measured by QuantitativePCR,Western Blotting and MTT, respectively. Epiquik DNA methyltransferase3BActivity/Inhibitor Screening Assay Core Kit was used to analyze the activity ofDNMT3b. Promoter methylation of MDR-1gene was identified by MSP.RESULTS: The results from Quantitative PCR showed that higher expressionof MDR-1mRNA and lower expression of DNMT3b mRNA was significantlyfound in MCF-7-M cells(p<0.05), compared to MCF-7cells and MCF-7-C cells. Theexpression level of MDR-1mRNA and DNMT3b mRNA in MCF-7-C cells wassimilar to that in MCF-7cells (p>0.05).The results from Epiquik DNAmethyltransferase3B Activity/Inhibitor Screening Assay Core Kit revealed that theactivities of DNMT3b was decreased in MCF-7-M cells (p<0.05),compared to MCF-7cells and MCF-7-C cells.Furthermore, lower activities of DNMT3b induced lowermethylation of MDR-1promoter in MCF-7-M cells (p<0.05).The results fromWestern blotting indicated that MCF-7-M cells had a higher expression of P-gp and alower expression of DNMT3b compared to MCF-7cells and MCF-7-C cells (p<0.05).MTT assay illustrated that the proliferation of MCF-7-M cells was stronger inMCF-7-M cells than that MCF-7cells and MCF-7-C cells after treatments with5-FU and ADR. The IC50of MCF-7cells, MCF-7-C cells and MCF-7-M cells to5-FU was641.741±119.337,668.295±78.188and3359.326±343.345μmol/L(p<0.05), respecti-vely, and three cells mentioned above to ADR was3.073±0.059,2.853±0.086and7.234±0.047μmol/L (p<0.05), respectively.CONCLUSIONS: Cytoplasmic M-CSF decreased the activities of DNAmethyltransferase3b and the methylation of MDR-1gene promoter and up-regulatedP-gp expression in MCF-7cells. Cytoplasmic M-CSF induced the resistance to5-FUand ADR in MCF-7cells by decreasing activities of DNA methytransferase3b andthe methylation of MDR-1gene promoter and by up-regulating P-gp expression inMCF-7cells.