Liquiritigenin Enhances The Sensitivity Of Triple Negative Breast Cancer Cells To Doxorubicin And Its Mechanism

Background:Breast cancer is a highly heterogeneous tumor.For lack of valid therapeutic targets,the patients with triple negative breast cancer(TNBC)gain little benefit from both endocrine therapy and anti-HER2 therapy.Conventional chemotherapy has been regarded as a effective systemic therapy option for TNBC,but its therapeutic efficacy remains limited.The way to enhance the sensitivity of TNBCs to chemotherapy may be one of effective methods to improve the prognosis of triple negative breast cancer.Estrogen receptor ?(ER?)has emerged as a tumor suppressor in TNBC.TNBC patients with high ERP expression often have a better overall survival.However,the role of ER? in regulating the chemotherapeutic response in triple negative breast cancer is still unknown.Objective:The purpose of this study was to observe the effect of Liquiritigenin(Liq)and Doxorubicin(DOX)on TNBC cells in vitro and in vivo.The aim to observe the potential mechanism that ER?-specific agonist Liquiritigenin(Liq)enhanced the sensitivity of triple negative breast cancer cells to doxorubicin was also investigated.Methods:The study was divided into two parts:the effect of Liq on triple negative breast cancer cells both in vitro and in vivo,and its underlying mechanism of this effect.1.Human triple negative breast cancer cell lines MDA-MB-231,BT-549 were selected for experiments in vitro.Using CCK-8 assay,colony formation assay,wound healing assay and transwell assay to evaluate the effects of different treatments(Liq,DOX,or DOX+Liq)on the proliferation,migratory and invansive ability of MDA-MB-231 cells.CCK-8 assay was used to study the effects of different treatments(Liq,DOX,or Liq+DOX)on the proliferation ability of MDA-MB-231 and BT-549 cells,for the aim to verify the universality of Liq on different TNBC cells.2.In vivo,the MDA-MB-231 cell tumor model was established using BALB/c standard mice.Sixteen mice were randomly divided into 4 groups for different treatments:the control group,the DOX-treatment group,the Liq-treatment group,and the combination treatment group(DOX+Liq).The effect of different treatments on the growth of subcutaneous tumors was studied.3.In research of its underlying mechanism,MDA-MB-231 cells were divided into 4 groups:the control treatment group,the DOX-treatment group,the Liq-treatment group,and the combination treatment group.After 48 hours of different treatments,Real-time PCR was used to evaluate the expression levels of ER? mRNA in different groups and Western blotting was used to assess the protein expression levels of PI3K/AKT/mTOR signaling pathway-related proteins.RNA interference was used to knock down the ER? expression of MDA-MB-231 cells,for aim to clarify the mechanism of Liq effected on PI3K/AKT/mTOR signaling pathway.Result:1.In vitro,Liq inhibited cell proliferation of MDA-MB-231 cells in a dose-dependent manner.Liq inhibited the colonies formation capacity,migratory potential and invasion ability of MDA-MB-231 cells.The cell viability,colonies formation capacity,migratory potential and invasion ability were significantly inhibited by combination treatment compared with the DOX treatment(P<0.05).In BT549 cells,the cell viability was also significantly inhibited by combination treatment(P<0.05).2.In vivo,MDA-MB-231 cell tumor models were successfully constructed in nude mice.Sixteen mice were randomly assigned to 4 groups for different treatments(negative treatment,Liq-treatment,DOX-treatment,combination treatment).The volume of tumors in the combination treatment(DOX+Liq)was significantly smaller than those in other groups(P<0.05)3.Compared with the control treatment,DOX treatment,and Liq treatment,the combination treatment of DOX and Liq caused an elevated expression of ER? mRNA in vitro,and an higher ER?/?-actin ratio,a lower pAKT/AKT and p-mTOR/mTOR ratio both in vitro and in vivo(P<0.05).ER? knockdown MDA-MB-231(ER?KD MDA-MB-231)cells was successfully built;compared with NC-KD MDA-MB-231 cells,the combination treatment of DOX and Liq had a weaker inhibitory effect on ER?KD MDA-MB-231 cells.Conclusion:1.In vitro,Liq could inhibit the cell viability,colonies formation capacity,migratory potential and invasion ability of MDA-MB-231 cells.Compared with the DOX treatment,the combination treatment could suppress the cell viability,colonies formation capacity,migratory potential and invasion ability in MDA-MB-231 cells,and could enhance the sensitivity to DOX in MDA-MB-231 and BT-549 cells.2.In vivo,Liq can enhance the inhibitory effect of DOX on the tumor growth of MDA-MB-231 cells.3.In MDA-MB-231 cells,the enhanced sensitivity to doxorubicin by the combination treatment of Liq and DOX correlates with the inhibition of PI3K/AKT/mTOR signaling pathway.4.In MDA-MB-231 cells,the effect of Liq on PI3K/AKT/mTOR pathway is ER?-dependent.