Fig. 4: GLUT3 promotes the uptake of extracellular temozolomide and capecitabine.
From: GLUT3 enhances chemosensitivity in glioblastoma by transporting temozolomide and capecitabine
A Representative chromatograms of IS (internal standard). IS is added to each sample as a mixture of 2H3-Temozolomide (upper panel) and 2H11-Capecitabine (lower panel). The sample’s final concentrations of 2H3-Temozolomide and 2H11-Capecitabine were 60 ng/ml and 12 ng/ml, respectively. B Representative chromatograms of TMZ in blank (left panel), blank with TMZ (middle panel, the final concentration of TMZ was 0.64 μM), and test sample (right panel). Cells were treated with drug-free transport buffer (blank), transport buffer containing 200 μM TMZ (test sample) at 37 °C for 15 min. The harvested cells were extracted with 80% methanol and prepared for LC-MS/MS analysis. C Representative chromatograms of CAPE in blank (left panel), blank with CAPE (middle panel, the final concentration of CAPE was 0.64 μM), and test sample of CAPE (right panel). Cells were treated with drug-free transport buffer (blank), transport buffer containing 200 μM CAPE (test sample) at 37 °C for 15 min. D Calibration curves for TMZ (upper panel) and CAPE (lower panel). E The uptake of TMZ (upper panel) and CAPE (lower panel) by GLUT3 KD (U343-MG) or GLUT3 OE (U251-MG) cells. Cells were treated with transport buffer or transport buffer containing 200 μM TMZ or CAPE at 37 °C for 15 min. The cell lysates were used for LC-MS/MS for intracellular TMZ and CAPE levels. F The levels of remaining extracellular TMZ (upper panel) and CAPE (lower panel) after uptake by GLUT3 KD (U343-MG) or GLUT3 OE (U251-MG) cells. G Inhibition of glucose uptake of TMZ or CAPE. 2-NBDG fluorescence assays were performed in GLUT3-OE vs. vector control U251-MG cells with TMZ (1, 10, 100 μM) or CAPE (1, 10, 100 μM) for 24 h. H Glucose concentration-dependent inhibition of chemotherapeutic drug uptake. U251-MG cells (GLUT3-OE and control vector) were exposed to TMZ (upper panel) and CAPE (lower panel) in presence of varying glucose concentration (1, 10, 25 mM). I Extracellular drug accumulation quantified by LC-MS/MS: Upper panel, TMZ levels under glucose modulation; Lower panel, CAPE levels under glucose modulation. Data are presented as mean ± SEM (n ≥ 5 biological replicates). Statistical analysis was performed using two-tailed unpaired t-tests E, F, or one-way ANOVA with Tukey’s post-hoc test (G–I) using SPSS 20 (IBM). Significance thresholds: *P < 0.05, **P < 0.01, ***P < 0.001.
