Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite

Abstract

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.

Keywords: GLUT3 redox state; PRDX1; SLC2A3; arsenite sensitivity.

Figure 1

PRDX1 depletion leads to arsenite sensitivity. (A,D,G) Western blot showing control and PRDX1 CRISPR knockout in HeLa, HEK293 and MDA-MB-231 cells, respectively. (B,E) H₂O₂ sensitivity using clonogenic survival assay in control and PRDX1 null HeLa and HEK293 cells, respectively. (C,F,H) Arsenite sensitivity of the control and PRDX1 null HeLa, HEK293 and MDA-MB-231 cells, respectively, using clonogenic survival assay. Survival fraction statistical analysis was performed using a two-way ANOVA test. The error bars represent the mean ± SD ** p < 0.01.

Figure 2

Arsenite upregulates GLUT3 in PRDX1-deleted cells. Briefly, cells were plated overnight in complete culture media; the following day, cells were treated with the indicated doses of arsenite in PBS. After treatment, cells were washed with PBS and collected by trypsinisation, then processed for RNA or protein extraction. (A,D) GLUT3 mRNA expression levels using RT-qPCR in the indicated control and PRDX1 null cells in the absence of treatment. GADPH was used for normalisation. (B,E) GLUT3 mRNA expression levels using RT-qPCR in the control and PRDX1 null cells treated with 5 μM and 100 μM arsenite for 10 min. (C,F) Western blot showing GLUT3 expression levels in the control and PRDX1 null cells treated with the indicated doses of arsenite. β-actin was used as a loading control. Student’s t-test was used in (A,D) to compare GLUT3 mRNA levels in control and PRDX1 knockouts. One-way ANOVA was used in (B,E). The error bars represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Modulating the GLUT3 level alters the response to arsenite. (A) Arsenite sensitivity according to clonogenic survival assay in HEK293 control and PRDX1 knockdown cells transfected with scrambled control or GLUT3 siRNA. (B) Clonogenic survival assay showing arsenite sensitivity in the control MDA-MB 231 cells and GLUT3 null cells. (C) Western blot showing GLUT3 CRISPR knockout in MDA-MB-231 cells. (D) MTT survival assay for arsenite sensitivity in HEK293 cells transfected with the vector control or the plasmid carrying GLUT3 tagged with EYFP. HEK293 cells were plated in 6-well plates overnight and transfected the following day with the vector control or GLUT3-EYFP overexpressing plasmid using the Fugene HD transfection reagent. After 48 h, cells were trypsinised and counted, and 2000 cells/well were re-plated into 96-well plates. After 24 h, cells were treated with the indicated doses of arsenite in PBS for 30 min. The cells were then washed with fresh culture media and incubated in culture media for another 72 h. Cell survival was analysed using the MTT reagent (see the Materials and Methods section). Statistical analysis was performed using two-way ANOVA. The error bars represent the mean ± SD. * p < 0.05. (E) Representative photomicrographic images of HEK293 transfected with the vector control or with GLUT3-EYFP overexpressing plasmid and either untreated or treated with 100 μM arsenite for 15 min. The magnification used was 63X. The red arrows indicate GLUT3 nuclear foci.

Figure 4

Re-localisation of GLUT3 upon arsenite treatment. (A) Representative photomicrographic images showing MDA-MB-231 control cells. (B–D) showing MDA-MB-231 control cells treated with 100 μM arsenite for 5, 10 and 15 min, respectively. (E) Representative photomicrographic images showing MDA-MB-231_PRDX1 null cells. (F–H) showing MDA-MB-231_PRDX1 null cells treated with 100 μM arsenite for 5, 10 and 15 min, respectively. (I,J) showing the quantification of GLUT3 nuclear fluorescence by ImageJ software in the control MDA-MB-231 cells and the MDA-MB-231_PRDX1 null cells in the absence and presence of arsenite treatment. Statistical analysis was performed using the one-way ANOVA test. The error bars represent the mean ± SD. * p < 0.05 and *** p < 0.001.

Figure 5

PRDX1 null cells exhibit increased glucose uptake and the accumulation of intracellular arsenic as compared to the control cells. (A) Relative glucose uptake in MDA-MB-231_PRDX1 null cells compared to the control cells. (B) Relative glucose uptake in MDA-MB-231_PRDX1 null cells treated with the indicated doses of arsenite as compared to the control cells. (C) Fold change in the intracellular arsenic level in MDA-MB-231 control and PRDX1 null cells treated with the indicated doses of arsenite. Cells were plated overnight and then treated with the indicated doses of arsenite in PBS. Cells were collected by trypsinisation; then, the pellets were resuspended in a non-denaturing lysis buffer and subjected to sonication to extract intracellular arsenite. Lysates were diluted in 5% nitric acid and analysed on ICP. (D) Arsenite sensitivity of the MDA-MB-231 control and MDA-MB-231_PRDX1 null cells pre-treated with the GLUT1 inhibitor Apigenin followed by clonogenic survival assay. Statistical analysis was performed using the one-way ANOVA test. The survival fraction statistical analysis was performed using two-way ANOVA. The error bars represent the mean ± SD. ** p < 0.01.

Figure 6

PRDX1 interacts with GLUT3 and protects it from arsenite-mediated degradation. (A,B) Immunoprecipitation analysis showing the interaction between PRDX1 and GLUT3 in MDA-MB 231 control cells and not in the PRDX1 null cells. (C) Western blot showing the GLUT3 protein level in MDA-MB-231 control and PRDX1 null cells following incubation with MG132 and treatment with arsenite. Cells were plated overnight and then treated with the proteasome inhibitor MG132 (25 μM) for 3 h. Cells were then treated with 100 μM arsenite for 30 min or left untreated. Cells were collected by trypsinisation for protein extraction and immunoblotting. (D,E) Quantification of GLUT3 protein levels by imageJ software in MDA-MB-231 controls and MDA-MB-231_PRDX1 null cells. Statistical analysis was performed using the one-way ANOVA test. The error bars represent the mean ± SD ** p < 0.01.

Figure 7

Non-reducing conditions show the multimeric forms of GLUT3 regulated by PRDX1 in response to arsenite. The HEK293_WT cells and HEK293_PRDX1 KO cells were treated with the indicated doses of arsenite or a fixed dose of H₂O₂. An amount of 20 μg of total extracts was processed for Western blot analysis under non-reducing conditions using 4–12% tris glycine gels. Lanes 1 and 6, untreated (UT); lanes 2, 3, 7 and 8, treated with 100 µM arsenite for the indicated times (15 or 30 min); and lanes 5 and 10, treated with 250 µM H₂O₂ for 15 min. Samples were heated at 70 °C for 5 min before processing on a non-reducing 4–12% gel and probed with anti-GLUT3 antibodies. Anti β–actin was used for assessing the total protein loaded in each well. M, molecular weight standards, kDa.