Effects Of Icariin On Macrophages Polarization In Tumor Microenvironment And Anti-metastasis In Breast Cancer

BackgroundBreast cancer is a common malignant tumor in women,which is a major threat to women’s health.Chemotherapy is an important treatment for breast cancer.However,chemotherapy drugs often have serious side effects on the body while killing cancer cells.So researchers have always to search for new cancer treatments.The interaction between tumor cells and their surrounding environment is essential for tumor development.The environment of Tumor cells is called Tumor Microenvironment(TME).In recent years,researchers have increasingly studied the tumor microenvironment in the hope of finding new targets for tumor treatment.There are a large number of tumor-associated macrophages(TAMs)in the Tumor microenvironment,which have a great influence on the progression of tumors.TAMS has two polarized phenotypes,M1 and M2.M1 cells can inhibit tumor development,while M2 cells can promote tumor.Some studies have shown that certain substances can affect the differentiation direction of TAMs,making M2 macrophages be differentiated to M1.Therefore,it is a new idea for the research and development of anti-tumor drugs to study the effect and mechanism of TAMS polarization typing.Icariin(ICA)is a flavonol glycoside with very good antitumor activity and no obvious toxicity to normal cells.Studies have reported that icariin inhibits a variety of cancer cells,including colon,thyroid,and ovarian cancer cells.However,the antitumor effect of Icariin on breast cancer is not very clear,in addition,the study of icariin on macrophage polarization has not been reported.Based on the above research background,the effect of Icariin on the polarization of macrophages and the anti-metastasis effect of breast cancer were studied in vivo and in vitro.It is mainly divided into three aspects:1.Effects of ICA on proliferation,apoptosis,invasion,metastasis,and vasculogenic mimicry formation of MDA-MB-231 cells and its mechanism.2.Effects and mechanism of ICA on the polarization of macrophages.3.Effects of polarized macrophages induced by ICA on MDA-MB-231 cells and its mechanism.Methods and results1.In vitro experiment1.1 Effects of ICA on MDA-MB-231 cellsEffects of ICA on proliferation of MDA-MB-231 cells:Breast cancer cells were treated with d ICA for 48 h.The survival rate was detected by MTT assay.The apoptosis of cells was detected by flow cytometry.The results showed that when the concentration of ICA reached 75μmol/L,it began to have a significant effect on cell survival,and with the increase of the concentration,the survival inhibition rate of MDA-MB-231 increased.Flow cytometry showed that the apoptosis rate of breast cancer cells increased with the increase of ICA concentration.Western blotting results showed that the higher the concentration of Icariin,the higher the expression of Bax and the lower the expression of Bcl-2 and DNA repair enzyme PARP in breast cancer cells.Effects of ICA on vasculogenic mimicry formation in MDA-MB-231 cells:Cells were treated with ICA to observe VM formation of cancer cells,Western blotting was used to detect the VEGF protein.The results showed that with the increase of Icariin concentration,the VM formation of MDA-MB-231 cells decreased gradually,and the expression of VEGF decreased.Effects of ICA on invasion and migration of MDA-MB-231 cells:The cells were treated with ICA at concentrations(10,20,40,80μmol/L)for 24 h,invasion and migration were measured by Transwell.The expression of E-cadherin protein was detected by Western blotting.The results showed that with the increase of Icariin concentration,the invasion and migration ability of cancer cells decreased gradually,while the expression of E-cadherin protein increased gradually.1.2 Effect of ICA on the polarization of macrophagesTHP-1 cells were induced into MO type cells by PMA and divided into two groups.One group was induced into M1 type macrophages by LPS and IFN-y,and the other group was induced into M2 type macrophages by IL-4 and IL-13.Morphological changes were observed with inverted fluorescence microscopy and M1 and M2 surface markers(CD86 and CD 163)were detected by flow cytometry to determine whether the induction was successful.Then the M2-type macrophages were treated with different concentrations of ICA,and the survival rate was detected by MTT assay.According to the results,the concentration(50μmol/L)that had little effect on the survival rate of cells was selected for the following experiment.Effect of polarized macrophages on breast cancer cells:THP-1 cells were induced into M0,M1 and M2 macrophages.M2 macrophages was divided into two groups,one group treated with ICA,another group as control group,and discard culture medium containing ICA,then cultured with fresh medium for 24 hours,respectively take M0,M1 M2,and M2+ICA supernatant as conditioned medium to treat MDA-MB-231 cells for migration and vasculogenic mimicry formation experiment.Results showed that the ability of migration,invasion,and vasculogenic mimicry formation of MDA-MB-231 cell treated with M1 macrophages conditioned medium reduced.The ability of migration,invasion and vasculogenic mimicry formation of MDA-MB-231 cell treated with M2 macrophages conditioned medium enhanced,and after processing M2 macrophage with ICA,the conditioned medium of M2 macrophages will reduce the abilities of migration,invasion,and angiogenesis of MDA-MB-231 cells.Effects of ICA on polarized macrophages:THP-1 cells were induced into M0,Ml and M2 macrophages.M2 macrophages was divided into two groups,one group treated with ICA,another group as control group,and then cells of four groups were used to detect the expression of macrophage marker CD163 by Flow cytometry.for RT-PCR to detect the expression of marker genes IL-6,IL-12,IL-10,Arg-1 and for Western blotting to detect the expression of PI3K,P-PI3K,Akt,P-Akt,mTOR,P-mTOR protein.The results showed that after ICA treatment,the expression of marker CD 163 in M2 macrophages decreased,the mRNA expression of marker genes IL-10 and Arg-1 in M2 macrophages decreased,while the mRNA expression of IL-6 and IL-12 in M1 macrophages increased.Western blotting results showed that the expressions of P-PI3K,P-Akt and P-mTOR were significantly increased in M2 macrophages,and the expression of these proteins in M2+ICA group was decreased and like that in control group of M1 macrophages.2.In vivo experiments15-20g(5-6weeks)female BALB/c-nu nude mice were selected and subcutaneously inoculated MDA-MB-231 cells.Then the tumor was removed when it grew,cut up as much as possible into tumor inoculation to the left armpit of other nude mice in vivo,nude mice were randomly divided into control group,low dose group(40 mg/kg),middle dose group(80 mg/kg),high dose(160 mg/kg).Four groups of five nude mice in each group,according to the dose of ICA,lavage,once every two days,record tumor growth and the weight changes within 20 days,and then kill the mice.Tumor-associated macrophages were extracted from the tumor,and the expression changes of macrophage markers CD86,CD 163 and F4/80 were detected by flow cytometry.The results showed that compared with the control group,the tumor growth of nude mice in low-dose,medium-dose and high-dose groups decreased in turn,while the expression of M1-type macrophage marker CD86 increased in turn,and the M2-type macrophage marker CD 163 decreased in turn.Conclusions1.Icariin could promote the apoptosis of breast cancer cells by increasing the expression of pro-apoptotic protein Bax and inhibiting the expression of anti-apoptotic proteins Bcl-2 and PARP,and Icariin could inhibit vasculogenic mimicry formation,invasion,and metastasis of breast cancer cells by inhibiting the expression of VEGF and up-regulating the expression of E-cadherin.2.Icariin could inhibit vasculogenic mimicry formation,invasion,and metastasis of breast cancer by promoting the polarization of M2 macrophages to M1 type.3.Icariin could promote the polarization of M2 tumor-associated macrophages to M1 type,which may be achieved by inhibiting the phosphorylation of P13K,Akt and mTOR in M2 macrophages.SignificanceThis study discussed the effects of ICA on proliferation,apoptosis,invasion and migration in MDA-MB-231 cells and its mechanisms and explored the effect and mechanism of ICA on the polarization of macrophages in tumor microenvironment and its influence on breast cancer cells.The results showed that ICA could inhibit the invasion and metastasis of breast cancer by promoting the polarization of M2 macrophages to M1 type through inhibiting the phosphorylation of PI3K,Akt and mTOR in M2 macrophages.It provides the basis for the research and development of anti-breast cancer drugs.