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  • br Materials and methods br Results br Discussion

    2021-09-08


    Materials and methods
    Results
    Discussion Quercetin is a relatively abundant bioactive flavonoid with a wide variety of documented physiological effects, which has gained popularity as a nutritional supplement [40]. There is increasing interest in quercetin's anticancer properties (for recent reviews see Refs. [5,6,[40], [41], [42], [43]]). It is important to note that many cancers are highly glycolytic, have increased lactate production in spite of the presence of adequate oxygen, and the maintenance of normal oxidative metabolism. Known as aerobic fermentation, or the Warburg effect, this increased metabolism of glucose is often achieved by an increased expression of GLUT1 [21,44,45]. Thus, GLUT1 has emerged as a potential target for the development of new anticancer therapies in spite of the potential negative effects of GLUT1 inhibition on neural glucose transport [32,46]. The initial goal of this study was to investigate the effects quercetin on glucose uptake in L929 mouse fibroblast cells, which rely exclusively on GLUT1 for glucose uptake [34]. Quercetin inhibits 2DG uptake in L929 cells in a dose dependent manner with a maximum inhibition of about 90% inhibition achieved between 50 and 100 μM and a IC50 of 8.5 μM. We also showed quercetin increased the Km of 2DG uptake without a change in the Vmax of transport, which is consistent with competitive inhibition. These data are virtually identical to data from HL-60 cells where the IC50 was reported as 8 μM [14]. We also confirmed earlier studies that the oxidized form of quercetin does not inhibit 2DG uptake [12]. In our hands, the inhibitory activity of quercetin is immediate (concurrent with 2DG uptake), and is rapidly reversible upon simple washing of cells. This finding was different from what we had anticipated, since inhibitors that are transported into the GW441756 typically take time to diffuse back out of cells, and therefore exert prolonged inhibitory effects. The rapid recovery of 2DG uptake after a single washing step was our first hint that quercetin may be simply binding to the GLUT 1 and not transported by the GLUT as previously reported [[11], [12], [13]]. To approach the question of whether quercetin simply binds to GLUT1 or is also transported, we developed a novel assay for measuring quercetin in cells. We took advantage of the natural fluorescence of quercetin and the dramatic red shift of the emission spectra that occurs as quercetin binds to cells [39]. This shift allowed us to use flow cytometry to measure fluorescence of quercetin in individual cells. The dose dependent uptake of quercetin was maximized between 50 and 100 μM, with a Kd of 8.9 μM–values that are essentially identical to the inhibitory effects of quercetin on 2DG uptake. The time-course of quercetin uptake was rapid, with half max uptake time of 54 s that again matches the rapid onset and loss of its inhibition on 2DG uptake. The flow cytometry assay we developed, as well as previously employed quercetin uptake assays [13,14], are not able to distinguish between simple quercetin binding to the cell surface and its incorporation into the cell. The 2DG uptake results with quercetin, therefore, could be consistent with a transport process that rapidly comes to equilibrium, similar to what is observe in 3-O-methylglucose uptake assays. However, the observation that the total quercetin binding capacity of permeabilized cells is more than double that of live cells suggests that transported quercetin should be retained in the cell by binding to internalized GLUT1. This increased binding capacity of permeabilized cells is also consistent with an alternate interpretation of the time-dependent quercetin binding data. The binding of quercetin was modeled as a simple equilibrium binding process with a t1/2 = 54 s. However, these data are also consistent with a rapid initial binding of quercetin to GLUT1 followed by slow doubling of the binding over 40 min as GLUT1 cycles from and to the cell surface gradually ‘labeling’ the vesicular GLUT1 pool. It has been reported that the recycling time for GLUT1 after endocytosis is 45 min [47]. This model of GLUT1 regulation differs from what would be expected in erythrocytes, which do not traffic surface nutrient transporters in the same way as other cell types such as fibroblasts and epithelial cells.