Study On The Inhibition Of MCF-7/TAM~R Breast Cancer Cells Proliferation And Down Regulation Of FUT4 And LeY By Cetuximab

Objective: Molecular targeted therapy has achieved outstanding results in clinical cancer treatment practice in recent years. Monoclonal antibodies against membrane receptors in the inhibition of tumor cell proliferation showed good results. The expression level of Lewis Y oligosaccharides (LeY) has associated with the process of tumor growth, metastasis, and prognosis of malignancy. FucosyltransferasesⅣ(FUT4) is the key enzyme of LeY synthesis. FUT4 and LeY have been confirmed abnormal overexpression in many kinds of epithelial tumors. In our experiment, we used epidermal growth factor receptor (EGFR) monoclonal antibody, cetuximab, to treat on tamoxifen (TAM) resistant MCF-7 breast cancer cell strain (MCF-7/TAM~R) in vitro to observe the inhibition of MCF-7/TAM~R cell proliferation. We were aiming to explore the mechanism of cetuximab treated inhibited the MCF-7/TAM~R cell proliferation by detecting the expression of FUT4 and LeY before and after treatment.Methods: MCF-7/TAM~R cells were the object of our study, which were established from epithelial breast cancer MCF-7 cell lines by the methods previously reported. The experiments were as follows: (1) We detected if there were some differences in EGFR and p-EGFR expression before and after the induction of resistance. (2) Adding different concentrations of cetuximab in the cell culture medium, the different inhibition efficiency of drug concentrations to MCF-7/TAM~R cells proliferation was measured by MTT colorimetric assay. We assessed the best effective drug concentration by calculating its IC50. (3) The best experimentally measured drug concentration has been selected and added in the culture medium of MCF-7/TAM~R cells for continuous culture. By comparing the differences of cell morphology in control group and dosing group under the optical microscope, we observed if there were some reduction in cell numbers or morphological change. (4) RT-PCR and Western blot were used to detect the differences in the amount of the PCNA mRNA and protein between control group and dosing group. (5) We used Western blot and indirect immunofluorescence staining to detect the effect of cetuximab on the activation EGFR and the ERK1/2 signal transduction pathway in MCF-7/TAM~R cells. (6) RT-PCR, Western blot and indirect immunofluorescence were used to detect the reduction of FUT4 and LeY expression in cetuximab or PD98059 treated MCF-7/TAM~R cells.Results: (1) The EGFR and the phosphorylated EGFR (p-EGFR) were higher in MCF-7/TAM~R cells than that in wild type MCF-7 cells. (2) MTT colorimetric assay results showed that cetuximab inhibited the proliferation of MCF-7/TAM~R cells in a time and dose all dependent. (3) After continuous cell culture for 7 days, the morphological changes occured in cetuximab group and the cell number was decreased significantly compared with control group. (4) PCNA synthesis of cetuximab treated MCF-7/TAM~R cells was decreased. (5) Cetuximab inhibited phosphorylation of EGFR and blocked the ERK1/2 signaling pathway. (6) Cetuximab and PD98059 treatments both reduced FUT4 and LeY synthesis in MCF-7/TAM~R cells.Conclusions: (1) The expression level of EGFR and p-EGFR are increased in tamoxifen-resistant breast cancer cell strain MCF-7/TAM~R. (2) Cetuximab inhibits MCF-7/TAM~R cell proliferation. (3) Cetuximab blocks ERK1/2 signaling pathway. (4) FUT4 and LeY synthesis both are reduced in MCF-7/TAM~R cells by cetuximab via blocking the ERK1/2 signaling pathway.