Vitamin C uncouples the Warburg metabolic switch in KRAS mutant colon cancer

Abstract

KRAS mutation is often present in many hard-to-treat tumors such as colon and pancreatic cancer and it is tightly linked to serious alterations in the normal cell metabolism and clinical resistance to chemotherapy.In 1931, the winner of the Nobel Prize in Medicine, Otto Warburg, stated that cancer was primarily caused by altered metabolism interfering with energy processing in the normal cell. Increased cell glycolytic rates even in the presence of oxygen is fully recognized as a hallmark in cancer and known as the Warburg effect.In the late 1970's, Linus Pauling and Ewan Cameron reported that vitamin C may have positive effects in cancer treatment, although deep mechanistic knowledge about this activity is still scarce.We describe a novel antitumoral mechanism of vitamin C in KRAS mutant colorectal cancer that involves the Warburg metabolic disruption through downregulation of key metabolic checkpoints in KRAS mutant cancer cells and tumors without killing human immortalized colonocytes.Vitamin C induces RAS detachment from the cell membrane inhibiting ERK 1/2 and PKM2 phosphorylation. As a consequence of this activity, strong downregulation of the glucose transporter (GLUT-1) and pyruvate kinase M2 (PKM2)-PTB dependent protein expression are observed causing a major blockage of the Warburg effect and therefore energetic stress.We propose a combination of conventional chemotherapy with metabolic strategies, including vitamin C and/or other molecules targeting pivotal key players involved in the Warburg effect which may constitute a new horizon in anti-cancer therapies.

Keywords: GLUT-1; Warburg; colon cancer; vitamin C.

Figure 1. Vitamin C selectively kills wild type and mutant KRAS colon cancer cells alone or in combination with cetuximab

A. apoptosis-inducing activities of vitamin C for Normal human immortalized Colonocytes (HCEC) and SW480 and LoVo cancer cell lines were annalyzed by Annexin-PI assay. Each cell line (2,3 10⁵ cells) was incubated with vitamin C (10 mM) and PBS for controls. for 20 hr, 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 (PI positive) as a percent of total cells were evaluated. Vitamin C displayed a selective killing effect on SW480 and LoVo. One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p< 0.05, **p < 0.001, n = 3. B. vitamin C treatment at different concentrations were carried out with LoVo and SW480 cancer lines. SW480 and LoVo CRC lines were exposed to ascorbate at 2, 5 and 10mM, for 20 hr. Then, cells were tripsinized and fixed with trypan blue solution (Sigma-Aldrich). The effective concentration that decreased survival 50% (EC50) was determined. EC50 was <10 mM for both tumor cells tested. Cell counting was carried out using a TC20™ Automated Cell Counter (Biorad). One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p < 0.05, **p < 0.001, n = 3. C. SW480 and LoVo cells were treated with vitamin C (7mM) for 5, 12 and 36 hr. Then, cells were tripsinized and fixed with trypan blue solution (Sigma-Aldrich). Cell counting was carried out using a TC20™ Automated Cell Counter (Biorad). One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p < 0.05, **p < 0.001, n = 3. D. HT29 harboring wild type KRAS, and the KRAS mutants LoVo and SW480 were treated with cetuximab (calculated IC50=0,4 μM), vitamin C (5mM) alone and combination for 12 hr. Then, cells were tripsinized and fixed with trypan blue solution (Sigma-Aldrich). Cell counting was carried out using a TC20™ Automated Cell Counter (Biorad). One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p < 0.05, **p < 0.001, n = 3. Combination of both vitamin C and the anti-EGFR antibody cetuximab displayed a higher killing effect in the three lines tested.

Figure 2. Vitamin C hampers the EGFR/MAPK signaling pathway through KRAS detachment from the cell plasma membrane

A. western blot analysis of ERK1/2 and p-ERK1/2 expression in Normal Human immortalized Colonocytes (HCEC) and the KRAS mutant cell lines SW480 and LoVo treated with vitamin C (8 mM) for 20 hr. Beta-actin was employed as protein loading control. B. Western blot analysis of MEK1 and p-MEK-1 expression in Normal Human immortalized Colonocytes (HCEC) and the KRAS mutant cell lines SW480 and LoVo treated with vitamin C for 20 hr. C. LoVo cells were treated with treated with vitamin C for 20 hr. Double immunofluorescence with anti RAS (red) and cytokeratin 20 (green) raised antibodies shows cytoplasmic colocalization of both proteins after vitamin C treatment (8 mM). D. activity of vitamin C in quenching intracellular reactive oxygen species (ROS) was measured using a Cellular ROS/Superoxide Detection Assay Kit (Abcam). SW480 and LoVo CRC lines were exposed to ascorbate (7 mM) and PBS for controls for 20 hr. After treatment, cells were washed twice with PBS solution and then incubated in medium containing 10 μmol/l H2DCF-DA at 37°C for 30 minutes. After incubation, cells were harvested by trypsinization, and intracellular levels of ROS were measured by fluorescence emission. Relative intracellular ROS levels after treatment with vitamin C were calculated in relation to control, which was given a value of 100. One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p < 0.05, **p < 0.001, n = 3.

Figure 3. Vitamin C impairs the Warburg effect in KRAS mutant cells through downregulation of GLUT-1 and PKM2

A. immunofluorescence analysis of GLUT-1 expression in SW480 and LoVo cells after vitamin C treatment (8 mM) for 20 hr. Glucose receptor 1 (GLUT-1) is shown in green. Cell nucleus is depicted in blue after DAPI staining. B. western blot and Real Time Quantitative PCR analysis of GLUT-1 protein and mRNA expression in SW480 and LoVo cells after vitamin C treatment for 20 hr. C. immunofluorescence analysis of PKM2 and p-PKM2 expression in SW480 and cells after vitamin C treatment (5 mM) for 20 hr. PKM2 and p-PKM2 is shown in green. Cell nucleus is depicted in blue after DAPI staining. Western-Blot analysis of PKM2 and p-PKM2 expression in SW480 cells after vitamin C treatment for 20 hr. D. immunofluorescence analysis of PKM2 and p-PKM2 expression in LoVo cells after vitamin C treatment (8 mM) for 20 hr. PKM2 and p-PKM2 is shown in green. Cell nucleus is depicted in blue after DAPI staining. Western-Blot analysis of PKM2 and p-PKM2 expression in LoVo cells after vitamin C treatment for 20 hr.

Figure 4. Vitamin C inhibits tumor growth in SW480 xenografts generated in athymic nude mice Foxn1nu though downregulation of GLUT-1, p-ERK and PKM2

A. immunohistochemistry carried out in human normal colon mucosa (upper and bottom) and colon adenocarcinoma shows differential expression of GLUT-1 and PKM2. Vitamin C receptor (SLC23A1) shows no differential expression. Box-plot shows the relative expression of SLC23A1, GLUT-1 and PKM2 in normal colon mucosa and adenocarcinoma (upper and bottom crypt). Box represents 25th and 75th percentile expression and bar representsmedian. Whiskers corresponds to 5th and 95th percentile and outliers aremarked separately. B. tumor growth of SW480 KRAS (G12V) xenograft in female athymic nude mice (Foxn1nu, 7 weeks old). Suspensions of 2×10⁶cells 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. Tumors were measured twice weekly in three perpendicular dimensions using a Vernier caliper and tumor volume was calculated. C. immunohistochemistry assays in SW480 tumors shows in vivo downregulation of PKM2, GLUT-1 and p-ERK in vitamin C-treated tumors. Box-plot shows the relative expression of GLUT-1, PKM2 and p-ERK in normal colon mucosa and adenocarcinoma (upper and bottom crypt). Box represents 25th and 75th percentile expression and bar Whiskers correspond to 5th and 95th percentile and outliers are marked separately.

Figure 5. Vitamin C impairs the Warburg effect in KRAS mutant cells through downregulation of GLUT-1 and PKM2

A. in absence of vitamin C, PKM2 is phosphorylated at ser 37 and translocates to the cell nucleus. Then, p-PKM2 binds to β-catenin and TCF/LEF transcriptional complex promoting c-Myc transcription that, in turn enhances the expression of GLUT-1 and PTB that participates in the splicing of PKM2 mRNA. B. vitamin C enters into the cell via GLUT-1 and SVCT1 inducing RAS detachment from plasma membrane, blocking downstream phosphorylation of PKM2 phosphorylation at ser 37. Disruption of the transcriptional complex formed by p-PKM2, β-catenin and TCF/LEF leads to downregulation of c-Myc, GLUT-1 and PTB expression. Absence of PTB activity stalls the splicing of PKM2 mRNA.