Activation of HIF increases the susceptibility to Vc-induced cell toxicity. A, left panel, representative Western blot analysis showing normalization of HIF1α and HIF2α protein levels after reintroduction of VHL into RCC10 cells. An empty vector was used as a control (Ctrl). Tubulin was used as a loading control (also hereafter in similar experiments unless indicated otherwise). Center panel, VHL reintroduction protects RCC10 cells from Vc-induced toxicity. Vc was added for 1 h (also in A, right panel; B, center and right panels; C; D; E, right panel; and F, right panel). Cell survival (in percent) was measured by counting viable cells and is represented as relative to untreated cells (0 mm Vc; also in B, center panel; C; D; E, right panel; and F, right panel). The mean ± S.D. of four independent experiments is shown (also in B, center panel; C; D; and F, right panel). Right panel, a similar experiment as in A, center panel, but using an MTT assay and represented as relative to untreated cells (also in B, right panel). A representative experiment is shown (also in B, right panel). B, similar experiments as in A but using RCC4 cells. C and D, cell survival (in percent) was measured at four time points (12, 24, 36, and 48 h) after Vc treatment using RCC10 (C) and RCC4 (D). P, time post-treatment with Vc. E, left panel, representative Western blot analysis showing the specific reduction of HIF1α and HIF2α levels after transduction of VHL-defective RCC10 cells with two shRNA vectors (A and B). shRNA for firefly luciferase was used as a control. Right panel, shRNA for HIFα subunits reduces Vc-induced toxicity in VHL-defective RCC10 cells. The mean ± S.D. of three independent experiments is shown. F, left panel, representative Western blot analysis for HIFα subunits using lysates of RPTECs overexpressing constitutively active forms of HIF1α and HIF2α or treated with 0.5 mm DMOG for 24 h. GFP lentiviruses or untreated cells were used as a control. Right panel, induction of HIFα subunits by lentiviruses or DMOG increases the sensitivity of RPTECs to very high doses of Vc. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Activation of HIF renders non-renal VHL-competent cancer cells more sensitive to Vc-induced toxicity. A, representative Western blot analysis for HIFα subunits using lysates from non-renal VHL-competent cancer cell lines overexpressing constitutively active forms of HIF1α and HIF2α showing the expected increase in protein levels. Actin was used as a loading control (Ctrl). Lentiviruses producing GFP were used as a control. B, overexpression of HIF1α and HIF2α increases Vc-induced toxicity in non-renal VHL-competent cancer cell lines. Vc was added for 1 h to all cell lines (also in D). The concentrations were 2 mm for Bel-7402 and U251, 4 mm for HeLa, MDA-MB-435S and SK-OV-3, 8 mm for HCT116, and 16 mm for SW480 cells. Cell survival (percent) was measured by counting viable cells and is represented as relative to untreated cells (also in D). The mean ± S.D. of four independent experiments is shown (also in D). C, representative Western blot analysis for HIFα subunits using lysates from non-renal VHL-competent cancer cell lines treated or not treated with 0.5 mm DMOG for 24 h. D, pretreatment with 0.5 mm DMOG for 24 h increases Vc-induced toxicity in non-renal VHL-competent cancer cell lines. E, representative Western blot analysis comparing the basal levels (in normoxia) of HIF1α and HIF2α in a panel of non-renal VHL-competent cancer cell lines. Lysates from VHL-defective RCC10 cells were used as a control. **, p < 0.01; ***, p < 0.001.

Increased DHA transport through GLUT1 underlies Vc-induced toxicity in VHL-defective renal cancer cells. A, HPLC shows increased intracellular Vc levels in VHL-defective RCC10 cells overexpressing VHL or the empty vector. Vc was added for 10 min or 50 min (also in B). The mean ± S.D. of four independent experiments is shown. Ctrl, control. B, liquid scintillation values after adding isotope-labeled Vc to VHL-defective RCC10 cells overexpressing VHL or the empty vector. The mean ± S.D. of three independent experiments is shown. C, representative quantitative PCR analysis showing the relative expression of different Vc transporters in VHL-defective RCC10 and RCC4 overexpressing VHL or the empty vector. D, left panel, representative Western blot analysis showing reduction of GLUT1 protein after transduction of VHL-defective RCC10 cells with three shRNA constructs (A, B, and C). Right panel, shRNA for GLUT1 reduces Vc-induced toxicity in VHL-defective RCC10 cells. Vc was added for 1 h (also in E and F). Cell survival (percent) was measured by counting viable cells and is represented as relative to untreated cells (also in E right and F). The mean ± S.D. of three independent experiments is shown. E, similar experiments as in D using two shRNA vectors (A and B) for GLUT3, which fail to reduce Vc-induced toxicity in VHL-defective RCC10 cells. The mean ± S.D. of four independent experiments is shown (also in F). F, 2-deoxy-d-glucose and d-glucose reduce Vc-induced toxicity in VHL-defective RCC10 cells. The two compounds were added simultaneously with Vc. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Toxicity of three different forms of Vc in VHL-defective renal cancer cells. A–C, differential sensitivity of VHL-defective RCC10 cells overexpressing VHL or the empty vector to DHA (administered for 4 h), As-2P (administered for 4 h), and AA6P (administered for 30 min). Cell survival (percent) was measured by counting viable cells and is represented as relative to untreated cells. The mean ± S.D. of four independent experiments is shown. Ctrl, control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

High intracellular levels of Vc increase ROS and trigger necrosis in VHL-defective renal cancer cells. A, dichlorofluorescin (DCF) labeling shows increased ROS production in VHL-defective RCC10 cells treated with Vc. Vc was added for 1 h (also in B–H). The mean ± S.D. of three independent experiments is shown (also in B and C). B and C, the indicated antioxidants or iron chelators reduce Vc-induced toxicity in VHL-defective RCC10 cells. Cells were pretreated for 2 h before adding Vc. Cell survival (percent) was measured by counting viable cells and is represented as relative to untreated cells. DTPA, diethylenetriaminepentaacetic acid. D, representative immunofluorescence shows DNA damage after Vc treatment of VHL-defective RCC10 cells. Cells were either stained for γH2A (left column) or using the comet assay (right column). E, representative Western blot analysis for γH2A after Vc treatment of VHL-defective RCC10 cells overexpressing VHL or the empty vector. F, quantification of the comet assay after Vc treatment of VHL-defective RCC10 cells overexpressing VHL or the empty vector. A representative experiment is shown (also in H). G, representative Western blot analysis for γH2A after Vc treatment of VHL-defective RCC10 cells pretreated or not treated with NAC and TCEP. Ctrl, control. H, dual labeling with Hoechst 33342 and PI shows increased necrosis in VHL-defective RCC10 cells treated with Vc. P, time post-treatment with Vc. **, p < 0.01; ***, p < 0.001.

Oxidative DNA damage kills VHL-defective renal cancer cells by exhausting the cellular pool of ATP. A, reduced NAD⁺ levels at different time points after treating VHL-defective RCC10 cells overexpressing VHL or the empty vector with Vc. Vc was added for 1 h (also in B–E). The mean ± S.D. of four independent experiments is shown (also in C–E). Ctrl, control; P, time post-treatment with Vc. B, the PARP inhibitors 3-aminobenzamide and nicotinamide (added 2 h before Vc) reduce Vc-induced toxicity in VHL-defective RCC10 cells. Cell survival (percent) was measured by counting viable cells and is represented as relative to untreated cells (also in D and E). The mean ± S.D. of three independent experiments is shown. C, reduced ATP levels at different time points after treating VHL-defective RCC10 cells overexpressing VHL or the empty vector with Vc. D, DCA (added 48 h before Vc) reduces Vc-induced toxicity in VHL-defective RCC10 cells. E, oligomycin (added 3 h before Vc) increases Vc-induced toxicity in VHL-defective RCC10 cells overexpressing VHL. *, p < 0.05; **, p < 0.01; ***, p < 0.001.