The Mechanism Of Dimethyl-celecoxib In Inhibiting The Proliferation Of Tamoxifen-sensitive And Drug-resistant Cells

Objective:Acquired resistance to tamoxifen is a major challenge in endocrine therapy of breast cancer,and research on its resistance mechanism has always been a focus of attention.The purpose of this study was to investigate the effects of 2,5-dimethyl-celecoxib（DMC）on the proliferation of tamoxifen（TAM）sensitivity and resistant breast cancer cells,and to explore its possible mechanism.Methods:1.MTT assay was used to detect the proliferation inhibitory effect of tamoxifen on breast cancer cell line（MCF-7）and Tamoxifen-resistant cell line（MCF-7/Tam R）.SPSS software was used to calculate each cell line IC₅₀ value at 72 hours and the resistance index（RF）of the tamoxifen-resistant cell line for breast cancer（MCF-7/Tam R）was also determined.Western blotting was used to detect the basic protein expression in the breast cancer cell line（MCF-7）and Tamoxifen-resistant cell line（MCF-7/Tam R）and to evaluate the resistance characteristics of breast cancer tamoxifen-resistant cell lines（MCF-7/Tam R）.2.We employed MTT assay and cell clone formation test to detect the proliferation inhibitory effect of dimethyl-celecoxib on breast cancer cell line（MCF-7）and Tamoxifen resistant cell lines（MCF-7/Tam R）,and screen the drug concentration and action time for subsequent experiments.3.Four groups were set up in follow-up experiments:control group（control）,tamoxifen intervention group（TAM）,dimethyl-celecoxib intervention group（DMC）,tamoxifen combined with dimethyl-celecoxib intervention group（TAM+DMC）.MTT method was used to detect the effects of different interventions on cell proliferation,and western blotting was evaluate to detect the effects of interventions on proliferation-related proteins.4.The effects of different interventions on apoptosis and apoptosis-related proteins were detected by flow cytometry and western blotting respectively.5.Western blotting,cellular immunofluorescence and comet assay were performed to explore the possible inhibition mechanism of dimethyl-celecoxib on breast cancer cell line（MCF-7）and tamoxifen-resistant cell line（MCF-7/Tam R）.6.Conducting tumor formation experiments in mice and immunohistochemical staining,we test the tumor suppressive effects of different interventions in animals,to further verify the results of in vitro cell experiments.result:1.The results of the MTT assay indicated that the half maximal inhibitory concentration(IC₅₀)values of TAM in the MCF7 and MCF-7/Tam R cell lines were 4.392μM and 9.800μM,respectively.also,the TAM resistance factor（RF）of MCF-7/Tam R cell line was 2.231.Furthermore,western blotting assays revealed that the protein expression levels of p-Rb and Rb in the MCF-7/Tam R cells were lower than those in the parental cells.2.IC₅₀ of dimethyl-celecoxib on breast cancer cell line（MCF-7）and tamoxifen resistant cell line（MCF-7/Tam R）were 35.930μM and 34.538μM respectively.30μM dimethyl-celecoxib had a significant long-term proliferation inhibition effect on MCF-7,MCF-7/Tam R cells.Therefore,subsequent experiments used 30μM dimethyl-celecoxib to act on cell lines.3.Dimethyl-celecoxib combined with tamoxifen significantly inhibited the proliferation of breast cancer cell line（MCF-7）and tamoxifen resistant cell line（MCF-7/Tam R）,and the proliferation-related protein P-Rb,Rb,MCM7,and PCNA expression was reduced.4.Dimethyl-celecoxib combined with tamoxifen significantly induced apoptosis in breast cancer cell line（MCF-7）and tamoxifen resistant cell line（MCF-7/Tam R）,and apoptosis-related protein Bcl-2,survivin expression was decreased;Cleaved PARP,Caspase-3 expression was increased.5.Dimethyl-celecoxib treatment significantly reduced the expression of MCM7/Rb protein in cells,increased the expression ofγ-H2AX protein,and further increased the expression ofγ-H2AX protein in combination with tamoxifen.DNA damage was induced by dimethyl-celecoxib treatment,and DNA damage was further aggravated after combined treatment with tamoxifen.After dimethyl-celecoxib treatment,DNA damage repair-related proteins were activated,and the activation was more pronounced after combined treatment with tamoxifen.6.Tumor volume and tumor species in animals were significantly reduced after dimethyl-celecoxib treatment;Rb/MCM7 protein expression in tumors in animals was significantly reduced.conclusion:1.Dimethyl-celecoxib alone or in combination with tamoxifen inhibits tamoxifen sensitive and resistant breast cancer cell proliferation in vitro and in vivo.2.Dimethyl-celecoxib alone or in combination with tamoxifen inhibits the proliferation of tamoxifen sensitive and resistant breast cancer cells may be related to DNA damage.