Calycosin Inhibites Migration And Invasion Of Breast Cancer Cells By HIF-1α-mediated Regulation Of The “Warburg Effect”

Objective: The effects of phytoestrogen calycosin on migration and invasion of ER-positive and ER-negative breast cancer cells were compared and analyzed.Also,the involvement of HIF-1α-mediated regulation of "Warburg effect" in metastasis inhibition of breast cancer cells by calycosin was explored.Methods: ER-positive breast cancer cells MCF-7 and ER-negative breast cancer cells MDA-MB-231 were treated with different concentrations of calycosin(0,25,50,100,150μM),and then cell migration and invasion were detected by wound healing assay and transwell assay.Glucose intake,lactate dehydrogenase(LDH)activity,pyruvate and lactate concentrations in breast cancer cells were respectively evaluated.q RT-PCR and Western blot assays were performed to determine HIF-1α expression and activities of glycolysis-related enzymes(PDK1 and PKM2).Next,HIF-1α was overexpressed and knocked down in MDA-MB-231 cells by transfection with pc DNA-HIF-1α and pc DNA-si RNA-HIF-1α.Transfected cells were treated with or without calycosin,and then the capability of cell migration and invasion was detected,along with changes in glycolysis.Meanwhile,the expression of HIF-1α and glycolysis-related enzymes in transfected cells was determined by Western blot.Results: It was demonstrated that calycosin significantly inhibited the migration and invasion of ER(+)breast cancer cells MCF-7 and ER(-)breast cancer cells MDA-MB-231 in a concentration-dependent manner,and decreased glucose uptake,LDH activity,pyruvate and lactate content in the two cell lines(P<0.05 or P<0.01).Notably,these alterations were more prominent in ER(-)breast cancer cells MDA-MB-231.Meanwhile,calycosin exerted inhibitory effects on HIF-1α expression and activities of glycolysis-related enzymes.However,the decrease of HIF-1α levels in MCF-7 cells was more pronounced than in MDA-MB-231 cells,while the activities of PDK1 and PKM2 in MDA-MB-231 cells were further suppressed by calycosin(P<0.05 or P<0.01).When HIF-1αwas overexpressed in MDA-MB-231 cells,the invasion and migration ability of cells was promoted,accompanied with enhanced glycolysis and increased phosphorylation of PDK1 and PKM2(P<0.01).By contrast,the knock-down of HIF-1α had the opposite effect on cell metastasis and Warburg effect(P<0.01).Otherwise,HIF-1α overexpression attenuated the inhibitory effect of calycosin on migration and invasion of breast cancer cells,and blocked the decrease of glucose intake,LDH activity,pyruvate and lactate content as well as the activity of glycolysis-related enzymes by calycosin.On the other hand,calycosin-induced inhibitory effects on breast cancer cells were further enhanced by knock-down of HIF-1α(P<0.05 or P<0.01).Conclusions: HIF-1α-mediated regulation of glycolysis could promote migration and invasion of breast cancer cells.By targeting HIF-1α and reversing "Warburg effect",calycosin could inhibit breast cancer cell metastasis,which may provide new targets and ideas for the treatment of breast cancer by phytoestrogens.