Based On The Energy Metabolism Mediated By HIF-1? To Explore The Effect And Mechanism Of Cardamom In Inhibiting Breast Cancer

Objective:Cardamonin?CDN?,a chalcone isolated from Alpiniae katsumadai,has anti-inflammatory and anti-tumor activities.However,the effect and molecular mechanism by which cardamonin inhibits breast cancer progression largely remains to be determined.This study aims to investigate the inhibitory effect and the underlying mechanism of cardamonin on breast cancer growth based on HIF-1?-mediated cancer energy metabolism reprogramming,and to provide theoretical support for the development of cardamonin as a potential anti-tumor drug for breast cancer in clinic.Method:Cell viability was determined by CCK-8 method.Cell proliferation wasdetermined by Ed U staining.Cell mitochondrial membrane potential was detected by JC-1 probe.Cell migration was detected by laser holographic cell imaging and analysis system.The mitochondrial stress,glycolysis rate and ATP rate were performed on the Agilent's Seahorse Bioscience XFe96 Extracellular Flux Analyzer.2-NBDG was used to detect glucose uptake ability.L-Lactate Assay Kit was used to detect intracellular and extracellular lactate content.DCFH-DA fluorescent probe was used to detect ROS accumulation.Cobalt chloride induced high expression of HIF-1?.The nude mice model of breast cancer xenograft was constructed in vivo and HE staining was used to detect the pathological changes of tumor tissues.IHC method was used to detect the expression of CD31,LDHA and HIF-1?in tumor tissues.Western blot was used to detect protein expression.Results:1.Cardamonin had a significantly inhibitory effect on the survial oftriple-negative breast cancer MDA-MB-231 cells,further studies had found that cardamonin could observably inhibit the proliferation,reduce mitochondrial membrane potential,thus promote apoptosis and decrease the migration of breast cancer cells.Cardamonin could significantly inhibit the growth of breast cancer xenografts in nude mice and increase the protein expression of cleaved caspase 3 and Bax,and decrease the protein expression of Bcl2 in tumor tissues.In addition,the protein expression of HIF-1?,LDHA and CD31 in tumor tissues was also down-regulated by cardamonin.2.Cardamonin could inhibit the expression of HIF-1?and its energy metabolismrelated target genes,such as PDHK1?GLUT1?GLUT3?GLUT4?LDHA and MCT4in breast cancer cells,while had no significant effect on the protein expression of LDHB and MCT1.3.Before killed by cardamonin treatment,mitochondrial oxidativephosphorylation and Mito-ATP production were significantly enhanced by the stimulation of cardamonin for 3 hours in breast cancer cells,and the glycolysis and Glyco-ATP production were significantly reduced by cardamonin treatment as late as for 12 hours.Cardamonin could increase the accumulation of reactive oxygen species?ROS?from 4 h in a dose-dependent manner.NAC,reactive oxygen species scavenger,could abolish the inhibition of cardamonin on the cell viability of breast cancer cells.Cardamonin could significantly down-regulate the protein expression of Nrf2 and its antioxidant activity-related target genes NQO1 and HO-1 after 3h-treatment.4.Protein expression of HIF-1?was significantly induced by cobalt chloride?Co Cl₂?stimulation,and HIF-1?overexpression could counteract the inhibitory effect of cardamonin on the growth and the cell proliferation.Meanwhile,mitochondrial oxidative phosphorylation could be reduced after HIF-1?overexpression.And also,HIF-1?overexpression could reverse the enhanced mitochondrial oxidative phosphorylation and ROS accumulation induced by cardamonin.High expression of HIF-1?could abolish the inhibitory effect of cardamonin on the protein expression of HIF-1?and PDHK1.PDHK1overexpression could reverse the inhibitory effect of cardamonin on the viability of breast cancer cells.5.Before increasing the mitochondrial aerobic phosphorylation of breast cancercells,cardamonin could downregulate the protein expression of p-m TOR,p-P70S6K and p-e IF4E from 0.5 h,and the change of p-m TOR protein concent was the most obvious.MHY1485,the m TOR agonist,could promote the protein expression of p-m TOR,HIF-1?and PDHK1,and MHY1485 could also counteract the inhibitory effect of cardamonin on the protein expression of p-m TOR,HIF-1?and PDHK1 as well as the cell viability in breast cancer cells.6.Cardamonin could significantly inhibit glucose uptake ability,lactate production and efflux both at early and late stage after cardamonin treatment for 6-24 h in MDA-MB-231 cells.7.Cardamonin could obviously inhibit the protein expression of HIF-1?and its energy metabolism related target genes,such as,PDHK1,GLUT1,GLUT3 and LDHA in breast cancer xenograft tissues,while had no significant effect on the protein expression of LDHB,GLUT4 and MCT4.Cardamonin could significantly down-regulate the phosphorylation level of PI3K/AKT/m TOR/P70S6K signaling pathway in tumor tissues,but had no significant effect on the protein expression of the phosphorylation level of MAPK signaling pathway.Conclusion:By inhibiting the over-activation of m TOR/p70S6K/e IF4E signaling pathway in breast cancer MDA-MB-231 cells,cardamonin could down-regulate the expression and transcriptional regulation of HIF-1?,and then down-regulate the protein expression of PDHK1,GLUT1,GLUT3,GLUT4,LDHA and MCT4,thereby reduced the glucose uptake capacity of MDA-MB-231 cells and enhanced the mitochondrial oxidative phosphorylation and decreased glycolysis as well as lactic acid production and efflux in breast cancer cells.Furthermore,cardamonin could reduce the activity of Nrf₂ antioxidant system,promote the accumulation of ROS,then reduced the mitochondrial membrane potential of MDA-MB-231 cells,finally promoted the cell apoptosis and inhibited the cell proliferation of MDA-MB-231cells,thereby resulted in inhibition of breast cancer.