The Inhibitory Effect Of Calycosin Derivative On Endothelial Cell Angiogenesis Indirectly Through Breast Cancer Cells

Objective Breast cancer is an estrogen-dependent tumor,and Calycosin derivative CA028 is derived from calycosin,an isoflavone phytoestrogen.Our group has been devoted to studying the anti-breast cancer effect and related mechanism of calycosin in recent years.ER-α36 is a novel splice variant of the classical estrogen receptor ER-α66 with a molecular weight of 36 Kda.It can cooperate with a variety of signaling factors and rapidly activate the nongenomic estrogen signaling pathway to regulate the biological behavior of tumors.Considering the correlation between lactate level in the tumor microenvironment and endothelial cell proliferation,metastasis and tumor angiogenesis,we investigated the role of signaling molecule lactate in the regulation of "Warburg effect" and lactate synthesis and secretion in breast cancer cells and endothelial cells by co-culture.Indirect effects on the possibility of endothelial cell metastasis and angiogenesis.To explore the effect of reduced lactate secretion by breast cancer cells on migration,invasion and angiogenesis of co-cultured human umbilical vein endothelial cells(HUVEC),which is mediated by inhibition of ER-α36-mediated Warburg effect in breast cancer cells with treatment of calycosin derivative CA028.It may provide a new target and strategy for anti-angiogenic treatment of breast tumors by phytoestrogen isoflavones and their derivatives.Methods 1.Estrogen receptor(ER)negative breast cancer cell line MDAMB-231 and ER positive breast cancer cell line MCF-7 were treated with different concentrations of calycosin derivative CA028(0,16,32 μM)for 48 h.Then the drug containing medium was removed,and these cells pre-treated with CA028 were co-cultured with HUVEC.The content of lactic acid in the comedium was measured.2.The migration,invasion and angiogenic abilities of co-cultured HUVEC were detected by wound healing assay,transwell assay and matrigel angiogenesis assay,respectively.3.Western blot was performed to determine the ERα36 expression level and the activities of key glycolytic enzymes in co-cultured breast cancer cells.4.Western blot was also performed to determine the expression of angiogenesis-related factors VEGF,MMP-9 and β-catenin in co-cultured HUVEC.5.MCF-7 cell line was transfected with GV358-ERα36 or empty vector(GV358-ERα36-NC),followed by pre-treatment with 32 μM CA028 for 48 h.Next,these transfected cells were co-cultured with HUVEC cells.6.The experimental groups were as follows: control group,vector group,ERα36 group,vector+32 μM group,ERα36+32 μM group.After another continuous culture of 48 h,the lactic acid content in the co-culture medium was detected.7.Meanwhile,all the above experiments were repeated in the co-cultured HUVEC and breast cancer cells.Results 1.Calycosin derivative CA028 reduced the production and secretion of lactate by breast cancer cells MDA-MB-231 and MCF-7,resulting in the decrease in lactate content of the co-incubation medium.2.Accompanied with this decrease,the migration,invasion and angiogenic abilities of co-cultured HUVEC was significantly inhibited(p<0.05 or 0.01).3.Compared with control group,the ER-α36 expression and the activities of PKM2 and PDK1 were down-regulated in breast cancer cells with the pretreatment of CA028,while the expression levels of MMP9,VEGF and β-catenin were significantly decreased in co-cultured HUVEC cells(p<0.05 or 0.01).4.Next,in the co-culture system of HUVEC and transfected MCF-7,ERα36 overexpression in breast cancer cells enhanced the activities of key enzymes of glycolysis PDK1 and PKM2 in cancer cells,increased the content of lactic acid in the co-culture medium,indirectly induced the expression of angiogenic factors VEGF,MMP-9 and β-catenin in co-cultured HUVEC.5.Meanwhile,ER-α36 overexpression partially reversed the inhibitory effect of CA028 on "Warburg effect" in breast cancer cells,and impaired the inhibitory effect of CA028 on lactate secretion and activities of enzymes responsible for "Warburg effect".Correspondingly,the CA028-induced inhibition of migration,invasion and angiogenesis of co-cultured HUVEC was blocked by ERα36 overexpression,the same as the decrease of angiogenic factors in HUVEC cells(p<0.05 or 0.01).Conclusion ER-α36 in breast cancer cells promotes the "Warburg effect" of breast cancer cells,thereby indirectly promoting the migration,invasion and angiogenesis of endothelial cells co-cultured with breast cancer cells.Moreover,Calycoisoflavone derivative CA028 can indirectly inhibit the migration,invasion and angiogenesis of endothelial cells co-cultured with breast cancer cells,which may be mediated by reversing ERα36-mediated Warburg effect in breast cancer cells and reducing lactate secretion in breast cancer cells.