Inhabitation And Mechanism Of Huaier Aqueous In Tamoxifen-resistant Breast Cancer Cells

Background:Breast cancer is one kind of hormone-dependent diseases.At present,Tamoxifen(TAM)is the golden standard drug for estrogen receptor(ER)and/or progesterone receptor(PR)positive premenopausal breast cancer patients.TAM could inhibit the proliferation of breast cancer cells as a selective ER modulator by competitive binding ER.However,only 60-70%ER-positive breast cancers are responsive to TAM,it may be related with tamoxifen-resistant.Huaier aqueous extract is derived from Trametes robiniophila Murr.as water extactive.The main active ingredient for anticancer protein is polysaccharide,which consists 6 kinds of monosaccharide binding to 18 kinds of amino acids.Jinke Huaier granule(National Class One drug),a kind of huaier aqueous extract,is used for clinical tumor therapy.A large amount of study reports have confirmed its anticancer effects on different kinds of cancer cells.However,the mechanism of Huaier aqueous extract is not yet clear.MAPK/ERK pathway is a classic signal pathway of cell proliferation and differentiation.In the present study,different concentrations of Huaier aqueous extract were used to detect the effect on both breast cancer cell lines MCF-7/S and MCF-7/R cells,and the relevant protein expression of MAPK/ERK pathway to reveal the underlying mechanism of the inhibitory effect on tamoxifen-resistant breast cancer cells.Objective:1.Detect the inhibitory effect of Huaier aqueous extract on MCF-7/S and MCF-7/R.2.Study the change of protein expression levels of MAPK/ERK pathway,and reveal the underlying mechanism.Methods:1.MTT assay was used to detect the inhibition ratio of TAM on MCF-7/S and the inhibition ratio of Huaier aqueous extract on MCF-7/S and MCF-7/R,then calculate the IC50 of both tamoxifen and Huaier aqueous extract.2.Clonogenic assay was used to detect the effect of proliferation on MCF-7/S and MCF-7/R treated by various concentrations of Huaier aqueous extract.3.MCF-7/R cells were exposed to different treatments for 48h:negative control(T1),IC50 of Huaier aqueous extract+ IC50 of Tamoxifen(T2),IC50 of Huaier aqueous extract(T3),IC50 of Tamoxifen(T4).Cell cycle and apoptosis analyses were detected by flow cytometer.4.After 4 different kinds of treatments,the protein of MCF-7/R cells were collected.Cyclin D1,BCL-2,MEK,p-MEK,ERK,p-ERK were detected by Western Blot analysis.Results:1.MTT assay showed Huaier aqueous extract could significantly inhibited the proliferation of MCF-7/S and MCF-7/R cells in dose and time dependent manners.The IC50 of MCF-7/S at 24h,48h,72h were 5.11±0.56 mg/ml,4.51±0.35 mg/ml,3.53±0.26 mg/ml,separately.Meanwhile,the IC50 of MCF-7/R at 24h,48h,72h were 5.84±0.48 mg/ml,5.08±0.37 mg/ml,4.29±0.33 mg/ml.2.Clonogenic assay showed when incubated with 0 mg/ml,2.5 mg/ml,5 mg/ml,and 10 mg/ml Huaier aqueous extract in MCF-7/S cells,the numbers of colony were decreased.For MCF-7/S cells,when compared with 0 mg/ml group,the numbers of colony in 2.5 mg/ml,5 mg/ml,10 mg/ml groups were decreased by 32.77%(P<0.01)?67.44%(P<0.001)?87.67%(P<0.001).The difference was regarded as statistically significant.For MCF-7/R cells,when compared with 0 mg/ml group,the numbers of colony in 2.5 mg/ml,5 mg/ml,10 mg/ml groups were decreased by 37.68%(P<0.01)?63.78%(P<0.001)?89.55%(P<0.001).The difference was regarded as statistically significant.3.PI staining showed that Huaier aqueous extract inhibited the proliferation of MCF-7/R cells by changing the distribution of cell cycle and inducing apoptosis.When compared with T1 group,the percentages of G1 phase in T2 and T3 groups were increased by 73.77±7.96%(P<0.001)and 66.16%±6.19%(P<0.001).The difference was regarded as statistically significant.When compared with T1 group,the early apoptosis ratios of T2 and T3 groups were nearly 53-fold(P<0.01)and 51-fold(P<0.001)improved.The difference was regarded as statistically significant.4.Western blot showed cyclin D1?BCL-2,p-MEK and p-ERK were significantly decreased in T2 and T3 groups in MCF-7/R cells in comparison to T1 group.Conclusion:1.Huaier aqueous extract could significantly inhibit the proliferation of MCF-7/R cells in dose and time dependent manners.2.Huaier aqueous extract might change the cell cycle by down-regulating cyclin D1,induce apoptosis of MCF-7/R cells by reducing BCL-2 gene protein expression,and inhibit MCF-7/R cells by hindering the phosphorylation of MEK and ERK.