| The mortality rate of HER2+ and triple-negative breast cancer(TNBC)still remains high due to few drugs for these two kinds of estrogen receptor negative(ER-)breast cancers.Tamoxifen(TAM)is the most widely used drug targeting estrogen receptor,which is often used for ER+ breast cancer therapy and prevention for women at high risk of breast cancer.However,it's mainly used in the treatment of ER+ breast cancer patients and often develops resistance after treating,resulting in limited application.Lots of studies have found that 1,25-dihydroxyvitamin D3(1,25D)inhibits various kinds of breast cancer cells including the ER-breast cancer.Therefore,in order to testify whether 1,25 D combined with TAM inhibit the growth of ER-breast cancer cells,SK-BR-3(HER-2+ breast cancer cell)and HCC1806(triple negative breast cancer cell)were used in this study.We analyzed the effects of 1,25 D combined with TAM on cell proliferation,cell cycle,cell apoptosis,signaling pathways and tumor growth in HCC1806 breast xenografts.The following results are drawn:(1)Cell counting,CCK-8 assay and cell morphology were used to assess the effects of 1,25 D combined with TAM on cell proliferation of SK-BR-3 and HCC1806 breast cancer cells.Results showed that 5 ?M of 1,25 D or 5 ?M TAM alone had no significant effects on the cell proliferation of both subtypes of cells.However,compared with 5 ?M TAM alone,the combination of 1,25 D and TAM could significantly reduce the number of HCC1806 cells,but increase the cell number of SK-BR-3 cells.These results indicate different effects of 1,25 D combined with TAM on the cell proliferation in different subtypes of ER-breast cancer cells.(2)To investigate whether these different effects were induced by the influences on cell cycle and apoptosis;flow cytometry and western blotting were used to examine cell cycle and apoptosis in this study.Results showed that the combination of 1,25 D and TAM did not lead to cell cycle arrest and alter the expression levels of p21 and p27 both in SK-BR-3 and HCC1806 than the individual regent.The combination had no significant effect on the cell apoptosis of SK-BR-3,but it led to significant cell apoptosis of HCC1806,with decreased expression levels of Caspase3,Caspase6,Bcl-2,p53 and increased expression levels of cleaved-PARP.These results indicated that different effects of 1,25 D combined with TAM on the cell proliferation might be associated with cell apoptosis,but not cell cycle.(3)To further investigate the different mechanisms of 1,25 D combined with TAM on the apoptosis of SK-BR-3 and HCC1806 cells compared to single TAM treatment,we examined the cell number after inhibiting the degradation of p53 protein.However,the cell number was similar by the intervention with protein degradation inhibitor CQ and MG132 in HCC1806 and SK-BR-3,suggesting the different effects in cell apoptosis of HCC1806 and SK-BR-3 cells induced by the combination of 1,25 D and TAM were independent of the degradation of p53 protein.(4)To further testify the cause that 1,25 D combined with TAM significantly increased the number of SK-BR-3 compare to single TAM treatment,the expression of p-AKT was analyzed.Results showed that 1,25 D combined with TAM led to an significant increase on the expression of p-AKT in SK-BR-3.Increased apoptotic cell number,cleaved-PARP and decreased Bcl-2 levels were observed after inhibiting the phosphorylation of AKT by MK2206 compound,suggesting AKT-related signals might lead to drug resistance in SK-BR-3 cells.(5)The effect of 1,25 D combined with TAM on inhibiting the ER-breast cancer cell proliferation in vivo was examined by immunohistochemistry and Western blotting.The results showed that 1,25 D combined with TAM could significantly inhibit the growth of tumor in xenograft model,decreased the expression levels of Ki67,p53 and Bcl-2 proteins in tissues and increased the expression of cleaved-PARP protein.These results indicated that 1,25 D combined with TAM could inhibit the growth of tumor in vivo via inducing the cell apoptosis of tumors.Taken together,we find that there is a difference in the effects of 1,25 D combined with TAM on different subtypes of ER-breast cancer cells.1,25 D combined with TAM lead to an increased drug-resistance to SK-BR-3 cells,which is dependent on the phosphorylation of AKT.However,the combination of 1,25 D and TAM inhibit the growth of HCC1806 cells both in vivo and in vitro and lead to cell apoptosis.These results provide an evidence of functional food for assistance for chemotherapy drugs in breast cancer patients. |
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