Vitamin C activates pyruvate dehydrogenase (PDH) targeting the mitochondrial tricarboxylic acid (TCA) cycle in hypoxic KRAS mutant colon cancer

Abstract

Background: In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1α involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors. Methods: Annexin V-FITC and cell viability assays were carried out in order to verify vitamin C citotoxicity in KRAS mutant SW480 and DLD1 as well as in Immortalized Human Colonic Epithelial Cells (HCEC). HIF1a expression and activity were determined by western blot and functional analysis assays. HIF1a direct targets GLUT1 and PDK1 expression was checked using western blot and qRT-PCR. Inmunohistochemical assays were perfomed in tumors derived from murine xenografts in order to validate previous observations in vivo. Vitamin C dependent PDH expression and activity modulation were detected by western blot and colorimetric activity assays. Acetyl-Coa levels and citrate synthase activity were assessed using colorimetric/fluorometric activity assays. Mitochondrial membrane potential (Δψ) and cell ATP levels were assayed using fluorometric and luminescent test. Results: PDK-1 in KRAS mutant CRC cells and murine xenografts was downregulated using pharmacological doses of vitamin C through the proline hydroxylation (Pro402) of the Hypoxia inducible factor-1(HIF-1)α, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial Δψ dissipation and diminished pyruvate dehydrogenase E1-α phosphorylation at Serine 293, then boosting PDH and citrate synthase activity. Conclusion: We report a striking and previously non reported role of vitamin C in the regulation of the pyruvate dehydrogenase (PDH) activity, then modulating the TCA cycle and mitochondrial metabolism in KRAS mutant colon cancer. Potential impact of vitamin C in the clinical management of anti-EGFR chemoresistant colorectal neoplasias should be further considered.

Keywords: KRAS; PDK-1; cancer; chemoresistance; hypoxia; metabolism; vitamin C.

Figure 1

Vitamin C selectively kills KRAS mutant colon cancer cells A. Apoptosis-induced by vitamin C in normal human immortalized Colonocytes (HCEC), SW480 and DLD1 cancer cell lines were annalyzed by Annexin-PI assay. Each cell line (2,5 105 cells) was incubated with vitamin C (10 mM) and PBS for controls for 24 h. Apoptosis in each cell line was measured by staining with FITC-conjugated Annexin-V and Propidium Iodide (PI) using a Sigma-Aldrich Apoptosis kit. The populations of cells (annexin-V positive/PI negative) and late apoptotic cells (annexin-V positive/PI positive) as a percent of total cells were evaluated. Vitamin C displayed a selective killing effect on SW480 and DLD1. One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p< 0.05, **p < 0.001, n = 3. B.images show apoptotic activity in KRAS mutant SW480 and DLD1 cell lines after vitamin C treatment (10 mM) for 24 h. C. vitamin C treatment at different concentrations were carried out with HCEC and DLD1 and SW480 cancer lines. All cell lines were exposed to ascorbate at 1, 3 and 5mM, for 24 h. Then, cells were tripsinized and fixed with trypan blue solution (Sigma-Aldrich). Cell counting was carried out using a neubauer chamber (SigmaAldrich) One-way ANOVA followed by Dunnett's posttest for multiple comparisons < 0.05, **p < 0.001, n = 3. D. HCEC, SW480 and DLD1 cells were treated with 5Mm and 6Mm vitamin C respectively for 4,6, 12 and 24 h. Then, cells were tripsinized and fixed with trypan blue solution (SigmaAldrich). Cell counting was carried out using a neubauer chamber (SigmaAldrich) One-way ANOVA followed by Dunnett's posttest for multiple comparisons. *p < 0.05, **p < 0.001, n = 3.

Figure 2

Vitamin C reduces HIF1a activity and stability. A. HIF1a expression in KRAS mutant cell lines SW480 and DLD1 treated with vitamin C (5 Mm; 6 mM) and NiCl2 (0.5 Mm) for 6, 8, 12 and 24 h. NiCl2 upregulates HIF1a expression mimicking tumor conditions which is downreguated when vitamin C is added. B. HIF1a activity in KRAS mutant cell lines SW480 and DLD1 treated with vitamin C (5 Mm; 6 mM) and NiCl2 (0.5 Mm). Vitamin C is able to downregulate the HIF1 increased transcriptional activity showed when cells are treated with Nicl2. C. Hidroxilated HIF1a expression in KRAS mutant cell lines SW480 and DLD1 treated with vitamin C (5 mM; 6 mM) and MG132 for 1, 3 and 6 h. Vitamin C induces hidroxilated HIF1a accumulation when proteasome is blocked at 6h.

Figure 3

Vitamin C interferes with PDK1-mediated phosphorylation of PDH. A GLUT1, PDK1, p-PDH (S293) and PDH expression in KRAS mutant SW480 and DLD1 cell lines treated with vitamin C (5 Mm; 6 mM) and NiCl2 (0.5 Mm) for 6 and 24 h. Beta-actin was employed as protein loading control. NiCL2 upregulates PDK1, GLUT1 expression simulating hypoxic tumor conditions while vitamin C treatment reduces GLUT1, PDK1 and p-PDH expression B. qPCR analysis of Glut1 and Pdk1 in KRAS mutant SW480 and DLD1 cell lines treated with vitamin C (5 Mm; 6 mM) and NiCl2 (0.5 mM) for 6 and 24 h. Vitamin C downregulates the expression of Glut1 and Pdk1 at mRNA level. C.immunohistochemistry assays in colon cancer patient derived xenografts (PDX) show increased PDK1 expression in Kras mutant patients, n=3. D. immunohistochemistry assays in SW480 derived tumors shows in vivo downregulation of GLUT1 and PDK1 in vitamin C-treated tumors. Suspensions of 2×106 cells were injected subcutaneously in control and experimental mice Animals were treated (i.p) with vehicle or vitamin C (4 gr/kg body weight) for 15 days once daily. Umpaired t-test with Welchs correction *p < 0.05, **p < 0.001, n = 3. E. PDH activity in colon cancer SW480 and DLD1 cell lines treated with vitamin C (5 mM; 6 mM) alone or in combination with NiCl2(0.5 mM). Vitamin C rescues the inactivation produced by NiCl2 treatment and is able to activate PDH activity when used alone. One-way ANOVA followed by Dunnett's posttest for multiple comparisons. *p < 0.05, ***p < 0.001, n = 3. F. Acetyl-CoA detection in KRAS mutant SW480 and DLD1 cell lines when treated with NiCl2 (0.5 Mm) alone or in combination with vitamin C (5 mM; 6 mM). Vitamin C induces acetyl-CoA accumulation One-way ANOVA followed by Dunnett's posttest for multiple comparisons. *p < 0.05, ***p < 0.001, n = 3. G. Ctrate synyhase (CS) activity in colon cancer SW480 and DLD1 cell lines treated with vitamin C (5 mM; 6 mM) alone or in combination with NiCl2(0.5 mM). Vitamin C rescues CS inactivation in hypoxic conditions. One-way ANOVA followed by Dunnett's posttest for multiple comparisons. *p < 0.05, n = 3.

Figure 4

Vitamin C reduces ATP production and dissipates mitochondrial potential. A. Mitochondrial membrane potential assay in KRAS mutant DLD1 and SW480 cells treated with vitamin C (5 Mm; 6 mM) and NiCl2 (0.5 Mm) for 2 and 6 h. Vitamin C reduces mitochondrial membrane potential in SW480 cell line. B. ATP detection assay in SW480 and DLD1 treated with vitamin C (5 Mm; 6 mM) and NiCl2 (0.5 Mm) for 2, 4 and 6 h. Vitamin C heavily reduces ATP production in the cells. One-way ANOVA followed by Dunnett's posttest for multiple comparisons. *p < 0.05, ***p < 0.001, n = 3.