Anti-angiogenesis And Growth Inhibition Of ELB On Breast Cancer

Breast cancer is a common malignant tumor in women,and its incidence is increasing yearly,and showing a younger trend,which seriously endangers women's health.At present,drug therapy(chemotherapy)is still the main treatment for breast cancer,but chemotherapy drugs could reduce the patients' platelet content significantly during treatment.Therefore,it is imparative to find drugs that could inhibit breast cancer without affecting platelet levels.Studies have demonstrated that tumor growth and metastasis depend on angiogenesis to provide oxygen and nutrients,and inhibition of tumor angiogenesis is an effective strategy for treating tumors.At the same time,in the tumor microenvironment,a large number of macrophages can release a variety of pro-angiogenic factors,which promot tumor angiogenesis.So it is feasible to find drugs that inhibit angiogenesis by macrophages.Eltrombopag(ELB)is a thrombopoietin receptor agonist for the treatment of chronic immune thrombocytopenia.Studies have shown that ELB has inhibition effect to multiple tumor cell lines,but the mechanisms are still unclear.Therefore,this study explores whether ELB can inhibit tumor growth by inhibiting angiogenesis and maintain normal platelet levels.The details are as follows:1.Inhibition of ELB on 4T1 proliferation and angiogenesis-related factors in breast cancer cell line.Firstly,the inhibitory effect of ELB on different tumor cell lines was detected by MTT assay,and the results showed ELB had the best inhibitory effect on 4T1 cells.Secondly,the mRNAs levels of angiogenesis-related factors impacted by ELB were detected by q-PCR method to investigate whether ELB inhibited angiogenesis of 4T1 cells.The results showed that ELB can reduce mRNAs levels of angiogenesis-related factors(VEGF-A,bFGF,MMP-9)in 4T1 cells significantly,and the inhibition was most pronounced at 20 ?M.2.ELB can inhibit angiogenesis in vitro mediated by macrophages(Raw264.7).The effects of ELB on proliferation,angiogenesis-related factor mRNAs levels and VEGF protein release of Raw264.7 cells were detected by MTT assay,q-PCR method and ELISA,respectively.The results showed that ELB inhibited the proliferation of Raw264.7 cells in a dose-dependent manner,and also inhibited the mRNA levels of angiogenesis-related factors(VEGF-A,bFGF,MMP-9)and the release of VEGF protein in Raw264.7 cells.To further investigate the effect of ELB on the function of downstream vascular endothelial cells(HUVEC)through macrophages,ELB(10 ?M)was applied to macrophages for 24 h to prepare macrophage supernatants,which were then applied to HUVEC.The effects of ELB mediated by macrophages on HUVEC functions were detected by MTT assay,scratch wound assay and tube formation assay,and the direct effect of ELB on HUVEC functions was also detected.The results showed that the supernatant of ELB-treated macrophage significantly inhibited the proliferation,migration and tubule formation of HUVEC,but ELB could not impact proliferation,migration and tubule formation of HUVEC directly.This indicates that ELB can inhibit angiogenesis mediated by macrophage.3.ELB inhibits tumor growth in vivo.The 4T1 breast cancer model was constructed.The mice were divided into five groups: negative control group(normal saline),ELB low-dose group(75 mg/kg),ELB medium-dose group(150 mg/kg),ELB high-dose group(185 mg/kg)and positive control group(gemcitabine 75 mg/kg).We investigated the tumor volume,tumor weight,tumor inhibition rate,body weight and platelet changes of the mice after drug treatments.The results showed that all doses of ELB could inhibit tumor growth,and the inhibitory effect of ELB high dose group(185 mg/kg)on tumors was better than gemcitabine group(75 mg/kg).The platelets of mice were maintained at normal levels in all ELB doses,while the platelets of mice in gemcitabine group(75 mg/kg)were decreased significantly.These results suggest that ELB can inhibit the growth of tumors in vivo and produce less toxic and side effects than gemcitabine.4.ELB's effects on factors related to tumor angiogenesis in tumor-bearing mice.The formation of angiogenesis,the release of VEGF protein and the recruitment of macrophages were detected by immunofluorescence staining.And the expression of VEGF-A mRNA was detected by q-PCR method.The results showed that ELB can significantly inhibit tumor angiogenesis,recruitment of macrophages,release of VEGF protein and the VEGF-A mRNA levels in tumor tissues.This indicates that ELB can inhibit tumor growth by inhibiting angiogenesis in tumors.The results showed that ELB can inhibit the growth of breast cancer in vitro and in vivo,and maintained normal levels of platelet.The mechanism might be through inhibiting the expression of angiogenesis-related factors.