The Sensitization And Mechanisms Of Halofuginone In Epirubicin Resistance Of Basal Like Breast Cancers

Basal-like breast cancers(BLBCs)lack a target in clinical treatment due to lack of hormone receptors,are poorly sensitive to chemotherapeutic drugs and are prone to relapse.Epirubicin(EPI)is one of the most effective drugs for treating BLBCs,but the EPI resistance often affects the effect of chemotherapy.Halofuginone(HF)is derived from the traditional Chinese medicine Dichroa febrifuga and can inhibit the occurrence and development of various tumors including breast cancer.Combination of HF and other chemotherapeutic drugs can enhance the cytotoxicity of chemotherapeutic agents.HF increases the radiosensitivity of tumors by down-regulating TGF? signaling pathway.There has been no report on the role of HF in EPI resistance of BLBCs.Objective: We used EPI resistant BLBCs as the research object,to investigate the roles of Pgp-autophagy-NF-?B apoptotic pathway and TGF?-EMT signaling pathway in EPI resistance.The effect of HF in P-gp-autophagy-NF-?B apoptotic pathway,TGF?-EMT signaling pathways and EPI sensitivity of BLBCs were studied,to elucidate the mechanisms of HF regulates the EPI sensitivity in EPI resistant BLBCs.Methods:1.We established two EPI resistant cells from the parental MDA-MB-231(MDA)and BT-20(BT20)cells,named EPI-R and EPI-B cells.The drug resistance was measured from the cell growth,morphology and survival rate in chemotherapeutic drugs.2.Western blot,FACS,QPCR,and immunofluorescence assays were used to detect the expression and function of P-gp,the level of autophagy,as well as the relationship of autophagy and NF-?B signaling pathways.The association between autophagy and P-gp was investigated by sh-MDR1 and sh-BECN1.The effect of autophagy and P-gp in the survival rate of EPI resistant cells that were cultured in the medium containing EPI was examined using MTT assay.Western blot and QPCR were used to detect the levels of TGF? and EMT molecular markers.The migration ability and stem cell phenotypes of EPI resistant cells were studied by wound scratch assay,CD24/CD29 staining and tumorsphere formation assay.3.We used MTT assay,Brdu staining,mouse liver and kidney biochemical indicators and histology to reflect the effect of HF in cell survival,proliferation and the toxicity of liver and kidney in mice;MTT,colony formation assay and the volume of tumor transplantation in mice were used to observe the effect of HF in the EPI sensitivity of EPI resistant cells.4.We studied the effect of HF in P-gp-autophagy-NF-?B apoptotic pathway and TGF?-EMT signaling pathway,to investigate the mechanisms of which HF mediated EPI sensitivity in BLBCs.Western blot,QPCR,immunofluorescence,and FACS were used to detect the effect of HF in the GATA3 expression.The correlation between GATA3 and TGF?R ? was analyzed by immunohistochemistry and chromatin immunoprecipitation.We detected the changes of TGF? and EMT molecular markers by HF after EPI resistant cells transfected with sh-GATA3,and observed the survival rate of EPI resistant cells that were cultured in EPI containing medium by MTT assay.Results:1.Establishment and identification of EPI resistant cell lines: EPI resistant cells grew stably in the medium containing 8 ?g/ml EPI.The viability of the EPI resistant cells in the medium containing epirubicin,paclitaxel,etoposide and cisplatin was increased compared with the parent cells(p<0.05).2.Mechanisms of EPI resistance in basal-like breast cancer cells: Overexpression of P-gp and autophagy in EPI resistant cells compared with parental cells;Inhibition of P-gp by sh-MDR1 could downregulate the expression of autophagy protein Beclin 1 and increase the sensitivity of EPI resistant cells to EPI.sh-BECN1 or chloroquine(CQ)were used to inhibit autophagy activating NF-?B-mediated pro-apoptotic pathways and improving the sensitivity of EPI resistant cells to EPI(p<0.05).Compared with parental cells,the expression of TGF? and Vimentin were increased,the ability of cell migration was enhanced,the number of enriched luminal stem cells was decreased,the number of enriched basal stem cells(also enriched cancer stem cells)was increased,and the sphere formation of cancer stem cells ball was increased(p<0.05).3.HF enhances the EPI sensitivity of basal-like breast cancer: 200 nM HF in vitro and 0.1 mg/kg HF in vivo almost had no toxicity.Compared with DMSO group,Combination of HF with EPI significantly increased the survival and proliferation of EPI resistant cells in EPI containing medium,reduced the size of NSG mice transplanted tumors(p<0.05).4.The mechanisms of HF enhances the EPI sensitivity in basal-like breast cancer: Compared with DMSO group,HF downregulated P-gp,decreased autophagy,and increased NF-?B apoptosis signaling pathway(p<0.05).Compared with DMSO group,HF and EPI synergistically increased the expression of E-cadherin,inhibited Vimentin expression,decreased the molecular markers of TGF? and EMT,and weakened migration and stem cell phenotypes of EMT(p<0.05).Compared with DMSO group,HF and EPI synergistically increased the protein and mRNA levels of GATA3,HF increased the binding of GATA3 to TGF?RII promoter and inhibited the transcription of TGF?RII.Compared with EPI resistant cells transfected with scramble,sh-GATA3 was used in HF group,which increased the molecular markers of TGF? and EMT and survival rate of EPI resistant cells in EPI containing culture medium compared with scramble(p<0.05).Conclusion:1.In this study,we establish EPI resistant cells of BLBCs.2.P-gp-autophagy-NF-?B apoptosis signaling pathway and TGF?-EMT signaling pathway participate in the EPI resistance of BLBCs.3.HF significantly increases the sensitivity of EPI resistant cells to EPI.4.HF increases the sensitivity of EPI resistant cells to EPI by increasing GATA3 to inhibit TGF?-EMT signaling pathway.