Andrographolide Regulates Chondrosarcoma Growth By Targeting The GLUT1-mediated Glucose Uptake Under The Use Of A Dual-fluorescence Reporter

ObjectiveThe purpose of this study is to investigate the subcellular localization of GLUT1 in chondrosarcoma cells and to elucidate the mechanism of Andro regulating chondrosarcoma cells energy metabolism reprogramming.Methods1.The subcellular localization of GLUT1 in chondrosarcoma cells was observed by constructing mCherry-EGFP-GLUT1,a dual-fluorescence reporter.2.The dual-fluorescence GLUT1 translocation coefficient calculation model was established by the formula.3.Constructing GLUT1 mutant mCherry-EGFP-GLUT1-S226D and mCherry-EGFP-GLUT1-ΔC4 to determine the specificity of mCherry-EGFP-GLUT1 dual fluorescence plasmid to detect the transport of GLUT1 in cells.4.The effect of glucose deprivation on GLUT1 transport in chondrosarcoma cells was detected by immunofluorescence and dual-fluorescence tracer.5.To confirmed the role of andrographolide on glucose metabolism in chondrosarcoma cells,the glucose uptake was determined by glucose uptake assay(2-NBDG)and flow cytometry and the extracellular acidification rate(ECAR)was detected by glycolysis pressure test.6.In order to test the effect of andrographolide in chondrosarcoma,the subcellular localization of GLUT1 was observed under the dual fluorescent tracer,plasma membrane protein isolation and Western Blotting(WB).Results1.The mCherry-EGFP-GLUT1 dual-fluorescent plasmid can be used to observe the transport of GLUT1 from neutral endosomes to acidic lysosomes.2.The TC value can well reflect the subcellular transport of GLUT1 between plasma membrane and acidic lysosomes in chondrosarcoma.3.The mutant mCherry-EGFP-GLUT1-S226D produces a predominant localization of mCherry-EGFP-GLUT1-S226D at the PM in both the glucose+ and glucose-groups.While mCherry-EGFP-GLUT1-ΔC4 was unable to recycle back to the PM and was instead sequestered in the endolysosomal system.In a word,the specificity of mCherry-EGFP-GLUT1 was confirmed by mutations.4.Glucose deprivation can induce the transport of GLUT1 in chondrosarcoma cells and has a high degree of co-localization with lysosomal marker LAMP1 and late endocytic marker Rab7.5.Andrographolide can reduce the expression of glycolytic rate-limiting enzyme HK2,autophagy signaling factor p-p70S6K and SOX9 protein and upregulation the Nrf-2 which suggests that andrographolide can inhibit the growth of chondrosarcoma by regulating autophagy and glucose metabolism.6.Andrographolide reduced glucose uptake and glycolysis metabolism in chondrosarcoma cells.7.Andrographolide suppresses GLUT1 expression in the PM of chondrosarcoma cells mainly by targeting GLUT1 for lysosomal degradation.Conclution1.The dual-fluorescence-based GLUT1 tracking tool can be used to monitor GLUT1 trafficking in real-time during the endocytosis process.2.Andrographolide can reduce glucose uptake and glycolysis to inhibit the growth of chondrosarcoma cells.3.Andrographolide inhibits GLUT1 expression by targeting GLUT1 for lysosomal degradation.4.pmCherry-EGFP-GLUT1 can be utilized as a biosensor for GLUT1-dependent functional studies and potential small molecule screening.