In-vitro Intervention And Mechanism Of A Calycosin Derivative On Endothelial Angiogenesis Induced By Breast Cancer Cells

Objective: This study is to investigate the effects of decreased lactate secretion from breast cancer cells by isoflavone derivative CA028 on proliferation,apoptosis,migration,invasion and angiogenesis of endothelial cells in co-culture model,along with its mechanism of action.This may provide new insights into isoflavone phytoestrogens action in preventing breast cancer metastasis,as well as new molecular targets for cancer therapeutics.Methods: The estrogen receptor(ER)-positive breast cancer cells MCF-7and ER-negative breast cancer cells MDA-MB-231 were incubated with different concentrations of isoflavone derivative CA028(0,8 and 16 μM)for48 h.Then,these breast cancer cells and human umbilical vein endothelial cells(HUVECs)were grown in the transwell co-culture system,Next,the concentrations of lactate in medium were measured so as to determine the reliability of the co-culture model.The migration and invasion ability of HUVECs in co-culture model was respectively measured by Wound-healing assay,matrigel-coated and-uncoated transwell assays.The cell proliferation was measured by MTT,while the cell apoptosis was detected by Hoechst33258 staining.Meanwhile,the microvessels formation by HUVECs was detected using angiogenesis experiment.Finally,the expression levels of VEGF,MMP-9,β-catenin and Vimentin in HUVECs were determined by Western blot.Results: The derivative caycosin CA028 significantly decreased lactic acid secretion from MCF-7 and MDA-MB-231 cells in co-culture model in a concentration-dependent manner(p<0.05 or 0.01).Moreover,this inhibitory effects could last at least 48 h after the removal of CA028.The declined lactate concentrations led to decreased migration,invasion and angiogenesis ability of HUVECs in co-culture model,while promoted apoptosis of endothelial cells(p<0.05 or 0.01).However,there was no significant change in HUVECs proliferation.Meanwhile,the expression levels of angiogenesis-related factors VEGF,MMP-9,β-catenin and Vimentin were significantly reduced in HUVECs(p <0.05 or 0.01).Conclusions: The calycosin derivative CA028 could reduce the production and release of lactic acid,which next inhibits metastasis and angiogenesis of HUVECs in co-culture model by down-regulation of angiogenesis-related gene expression.This may help to prevent breast cancer matastasis.