ACT001 Can Reverse Tamoxifen Resistance In Human Breast Cancer Cell Lines By Inhibiting NFκB Activation

Objective: To investigate the role of NFκB signaling pathway in the resistance of breast cancer to tamoxifen and its mechanism.Methods: MCF7,T47 D and ZR7530 cell lines were selected as research cells.MTT assay was used to detect the inhibitory effect of tamoxifen and ACT001 on the proliferation of breast cancer cells.Effect of different drugs on cell clone formation ability were detected by colony formation assay.The lentivirus was produced,and the fluorescent apoptosis protein VC3 AI was transfected into the cells to observe the apoptosis by observing the green fluorescence with a fluorescence microscope.Annexin V-FITC/PI double staining was used to detect apoptosis rate by flow cytometer.The NFκB signaling pathway key protein mutant plasmids(p CDH-CMV-IKBα(S32A,S262A)、p CDH-CMV-IKKβ(S177E,S181E))and p CDH-CMV-VECTOR were transfected into cells.Puro was used to screen NFκB signaling pathway continued activation cell lines and NFκB signaling pathway continued inhibition cell lines.The inhibitory effect of tamoxifen on cell proliferation was tested by MTT.Western blot was used to detect the expression of IκBα,a negative correlation protein of NFκB signaling,in transfected cells and cells after treated with tamoxifen for 48 h.Immunofluorescence was used to detect the nuclear translocation of p65 after treatment.Real-time fluorescent quantitative PCR was used to detect the m RNA level of NFκB and NFκB-related apoptosis protein and anti-apoptotic protein in tamoxifen-resistant MCF7R/LCC9 cells.Wound healing assay and Transwell migration assay were used to detect the effects of drugs on cell migration.The effect of drugs on cell invasion was detected by Transwell assay.Statistical analysis of differences between groups were made by SPSS19.0 software,the results were considered statistically significant where p<0.05.Results:1.Combination of ACT001 and tamoxifen inhibited cell proliferation and cell clone formation ability.2.Combination of ACT001 and tamoxifen treatment promoted the cell apoptosis3.Tamoxifen activated NFκB cell signaling.Tamoxifen promoted the nuclear translocation of p65,which was inhibited by ACT001.Tamoxifen decreased the protein expression of IKBα,an inhibitor of NFκB translocation.Tamoxifen also increased NF-κB m RNA levels,which could be decreased by ACT001.4.We constructed cell lines expressing an NFκB signaling pathway-activating plasmid(m IKKβ)and an NFκB inactivated plasmid(m IKBα).In MTT assays,we found that the cells with the activated NFκB construct developed tamoxifen resistance,whereas those with the inactivated NFκB construct were more responsive to tamoxifen5.Combination of ACT001 and tamoxifen inhibited the proliferation of MCF7R/LCC9 cells.The m RNA level of NFκB was higher in MCF7 R cells than in MCF7 wild type cells,which was decreased upon treatment with ACT001.The m RNA levels of pro-apoptotic and anti-apoptotic genes were higher in MCF7 R cells than in MCF7 cells,which could be decreased by treatment with ACT001.The most dramatic change was in the expression of TRAF1,an anti-apoptotic gene.6.Tamoxifen could promote cell migration.Combined ACT001 and tamoxifen could inhibit cell invasion,migration,and wound healing ability.Conclusion:1.The activation of NFκB signaling pathway is involved in the development of tamoxifen resistance in hormone-receptor-positive breast cancer cells.ACT001 increased the inhibitory effect of tamoxifen on the proliferation and apoptosis induction of hormone receptor-positive breast cancer cells by inhibiting NFκB signaling pathway,which reversed the sensitivity of tamoxifen-resistant breast cancer cells to tamoxifen.2.Low-level tamoxifen can promote the migration of breast cancer cells,ACT001 can inhibit the promotion of migration induced by tamoxifen.