Effects And Mechanisms Of Huaier Extract On Doxorubicin Resistance In Breast Cancer

Background and AimsDoxorubicin is the first-line drug used in chemotherapy for breast cancer,but the development of chemoresistance leads to treatment failure.Huaier extract,a traditional Chinese medicine,has been increasingly applied to treat cancers.We investigated whether Huaier extract would increase doxorubicin toxicity as a chemosensitizer in a doxorubicin-resistant 231(231/Dox)breast cancer cell line.As compared to parental 231 cells,the 231/Dox cells were 3.32 fold more resistant to doxorubicin at 48h post-exposure(Dox IC50=0.261 luM vs.0.8680uM);while Huaier extract(2mg/ml or 4mg/ml)decreased the Dox-IC50 by 1.38-fold(0.6295uM)and 1.75-fold(0.4940uM)respectively(p<0.05).That Huaier extract increased toxicity of doxorubicin in 231/DOX cells was also verified in colony formation,and invasion and migration assays.Huaier extract downregulated P-gP expression and enhanced apoptosis by suppressing the AKT signaling pathway in 231/Dox cells,as measured by drug-efflux assays,fluorescence microscopy,flow cytometry,TUNEL,and western blot analysis.In xenografts mice model,Dox combined with Huaier extract decreased tumor growth more significantly(p<0.01)than either agent alone.In conclusion,Huaier extract,as a chemosensitizer,sensitizes 231/Dox breast cancer cells to doxorubicin by downregulating AKT signaling pathway.Methods and ResultsIn this study,we intend to explore the mechanism of Huaier extract in doxorubicin-resistance in Breast Cancer;specific research includes the following four parts:Part ? Huaier extract reduces doxorubicin-resistance inhibition in 231/dox breast cancer cellsMethods:The doxorubicin resistant MDA-MB-231 breast cancer cell line was isolated by stepwise selection of MDA-MB-231 cells via exposing to increasing concentrations of doxorubicin for 8 months.The doxorubicin resistant phenotype was maintained by DMEM supplemented with 50 ng/mL doxorubicin.We compared susceptibility to doxorubicin in both 231 and 231/doxorubicin breast cancer cells by MTT.In addition,we measured the cytotoxicity of Huaier extract exposure in 231/doxorubicin cells.To confirm Huaier extract could reverse doxorubicin-resistance,doxorubicin-induced cytotoxicity in Huaier-treated 231/doxorubicin cells extract was measured by MTT.Results:As measured by MTT,231/Dox breast cancer cells were less sensitive to doxorubicin than that of parental MDA-MB-231 breast cancer cells.Meanwhile,MTT assay revealed that Huaier extract had inhibitory effect on 231/Dox breast cancer cells and increased the susceptibility to doxorubicin,reversing the doxorubicin-resistance of 231/Dox breast cancer cells significantly.This demonstrated that Huaier extract enhanced doxorubicin-induced cytotoxicity and promoted sensitivity to doxorubicin of 231/doxorubicin cells.Part ? Huaier extract promotes cell apoptosis in 231/dox breast cancer cellsMethods:In order to dig out the specific mechanism of the inhibitory effect and reversing the doxorubicin-resistance in 231/Dox breast cancer cells of Huaier extract,we conducted further research such as detection of apoptosis by PE-Annexin-V staining via FACScan flow cytometry and TUNEL assay,and detection the expressions of apoptotic proteins revolved in apoptosis pathway with Huaier extract exposures.Results:As measured by Flow cytometry and TUNEL assay,Huaier extract showed the stimulative effect of cell apoptosis in 231/Dox breast cancer cells;Western blot assays revealed that the expression of apoptotic proteins were modulated by Huaier extract,leading to apoptosis of 231/Dox breast cancer cells.In a word,Huaier extract could reverse the doxorubicin-resistance of 231/Dox breast cancer cells by promoting cell apoptosis.Part ? Effects of exposure to Huaier extract on drug-efflux functionMethods:Drug-efflux function was quantified in 231/doxorubicin cells by using Calcein-AM as the fluorescent dye and verapamil as the P-gp inhibitor.Intracellular Calcein retention was determined by a fluorescence microplate reader(BioTek)after Huaier extract treatment.Intracellular doxorubicin accumulation assay was carried out in the similar way to determine the effect of Huaier effect.On the meanwhile,we further investigate the differences of P-gP expression in 231/Dox breast cancer cells and parental MDA-MB-231 breast cancer cells,and the P-gP expression regulated by Huaier extract in 231/Dox breast cancer cells.Results:As measured in drug-efflux assays and intracellular doxorubicin accumulation assays,Huaier extract played the inhibitory effects in cell drug-efflux function and increased intracellular doxorubicin accumulation;In immunoblotting analysis,P-gp was confirmed to be overexpressed in 231/doxorubicin cells,comparing to parental cells;Huaier extract suppressed P-gp expression in 231/doxorubicin cells in a dose-and time-dependent manner.Part IV Huaier extract inhibits doxorubicin resistance of breast cancer cells by targeting AKT signaling pathwayMethods:Construction of AKT overexpression plasmid,transfections of AKT-pcDNA3.1 plasmid,establishment of AKT overexpression cell line was used to test the effect of AKT on doxorubicin-resistant breast cancer cells.MTT assay was carried out to show whether AKT activation and overexpression promote the doxorubicin-resistance in breast cancer.Wetsern blotting was used to confirm the influences of Huaier extract on the statement of AKT/mTOR signaling pathway,cell apoptosis and the expression of P-gP.Results:It was proved that Huaier extract could inhibit AKT expression and activation,suppress AKT signaling pathway by western blots carried out in 231/doxorubicin breast cancer cells.While overexpressed AKT enhanced the doxorubicin resistance in breast cancer cells via MTT assay.Further western blot analysis revealed that AKT overexpression inhibited cell apoptosis related proteins and increased P-gP expression;while the changes caused by AKT were reversed by Huaier extract.In turn,the transfection of AKT-pcDNA3.1 reversed the inhibitory effects of Huaier extract in MTT assay and immunoblot analysis.It was profoundly confirmed that Huaier extract could reverse doxorubicin-resistance by promoting apoptosis and inhibiting P-gp expression via AKT signaling pathway in breast cancer.Conclusions:1.Huaier extract induces cell apoptosis in doxorubicin-resistant breast cancer cells;2.Huaier extract inhibits P-gP protein-mediated drug efflux function of doxorubicin-resistant breast cancer cells by downregulating the expression of P-gP;3.Huaier extract inhibits breast cancer doxorubicin resistance through the regulation of AKT signaling pathway.Significances:1.Elucidated the mechanisms of the anti-drug resistance of doxorubicin in breast cancer;2.Confirmed the anti-tumor effect of Huaier extract in breast cancer and provided a theoretical basis for Huaier extract as a clinical adjuvant therapy for breast cancer.