Inhibition of the pentose phosphate pathway by dichloroacetate unravels a missing link between aerobic glycolysis and cancer cell proliferation

Abstract

Glucose fermentation through glycolysis even in the presence of oxygen (Warburg effect) is a common feature of cancer cells increasingly considered as an enticing target in clinical development. This study aimed to analyze the link between metabolism, energy stores and proliferation rates in cancer cells. We found that cell proliferation, evaluated by DNA synthesis quantification, is correlated to glycolytic efficiency in six cancer cell lines as well as in isogenic cancer cell lines. To further investigate the link between glycolysis and proliferation, a pharmacological inhibitor of the pentose phosphate pathway (PPP) was used. We demonstrated that reduction of PPP activity decreases cancer cells proliferation, with a profound effect in Warburg-phenotype cancer cells. The crucial role of the PPP in sustaining cancer cells proliferation was confirmed using siRNAs against glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the PPP. In addition, we found that dichloroacetate (DCA), a new clinically tested compound, induced a switch of glycolytic cancer cells to a more oxidative phenotype and decreased proliferation. By demonstrating that DCA decreased the activity of the PPP, we provide a new mechanism by which DCA controls cancer cells proliferation.

Keywords: bioenergetics; dichloroacetate; glycolysis; pentose phosphate pathway; proliferation.

Figure 1. Glycolytic efficiency is positively linked to proliferation but not to ATP levels in cancer cells

A. Correlation plots between metabolic parameters (glycolytic efficiency and mitochondrial oxygen consumption rate [mitoOCR]), intracellular ATP content and proliferation of six human and murine cancer cell lines. Measurements were performed after 24 h incubation in the presence of a culture medium containing only glucose as energetic fuel. The mitoOCR was determined by the oligomycin-sensitive OCR of viable whole cells and the glycolytic efficiency (glucose consumption/lactate production ratio) was measured from cells supernatant. Total ATP was quantified from lysed cells and normalized to protein content. Proliferation rates were analyzed by the incorporation of a nucleotid analog (5-bromo-2′-deoxyuridine [BrdU]). A significant correlation was found between glycolytic efficiency and proliferation (p-value = 0.04, Pearson r = 0.82). Non-significant correlations were found between mitoOCR and ATP content (p-value = 0.06, Pearson r = 0.78), mitoOCR and proliferation (p-value = 0.053, Pearson r = −0.80) and between glycolytic efficiency and ATP content (p-value = 0.11, Pearson r = 0.72). B.-F. Comparison of the metabolic profile (B-C-D), intracellular ATP content E. and proliferation F. between wild-type (WT) and mitochondria-depleted (ρ0) isogenic SiHa cancer cells. Two-sided t test. *p < 0.05, **p < 0.01, ***p < 0.001. Results are expressed as means ± SEM.

Figure 2. DCA significantly influences the metabolism and proliferation of glycolytic but not oxidative cancer cells

A. Mitochondrial oxygen consumption rate, B. glucose consumption, C. intracellular ATP content and D. proliferation of MDA-MB-231 (glycolytic) and SiHa (oxidative) human cancer cells after 48 h dichloroacetate (DCA) 5 mM treatment. E. Proliferation of mitochondria-depleted (ρ0) SiHa cancer cells after 48 hours DCA 5 mM treatment. Medium containing no FBS was used as positive control in proliferation experiments. Two-sided t test A.-C. or one-way ANOVA with Bonferroni post-hoc test D.-E. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. Results are expressed as the relative change from control cells and as means ± SEM.

Figure 3. The PPP differentially supports proliferation in glycolytic and oxidative cancer cells

Intracellular NADP⁺ and NADPH levels measured individually in viable glycolytic MDA-MB-231 A. and oxidative SiHa B. cancer cells. 6-aminonicotinamide (6-AN), a specific inhibitor of the PPP, was used (100 μM, 48 h treatment) to highlight NADPH production from the PPP (NADPH_ppp). C. NADPH_PPP/NADP⁺ ratios in MDA-MB-231 and SiHa cancer cells. D. Proliferation measured by the incorporation of BrdU in MDA-MB-231 and SiHa cancer cells after exposure to 6-AN (100 μM, 48 h treatment). Medium containing no FBS was used as positive control in proliferation experiments. Two-sided t test A.-C. or one-way ANOVA with Bonferroni post-hoc test D.. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. Results are expressed as means ± SEM.

Figure 4. Glucose-6-phosphate dehydrogenase inhibition with siRNA reduces proliferation of glycolytic cancer cells

MDA-MB-231 cancer cells were transfected with siRNAs targeting G6PD (siG6PD) or non-targeting siRNAs (siCTL). 48 hours after transfection, cells were subjected to A. immunoblot analysis using Hsp90 as loading control, B. NADP⁺ and NADPH levels quantification and C. DNA synthesis measurement. Two-sided t test. *p < 0.05, ***p < 0.001. Results are expressed as means ± SEM.

Figure 5. DCA decreases PPP activity

A. Intracellular NADP⁺ and NADPH levels measured in MDA-MB-231 cancer cells treated with ± DCA (5 mM, 48 h) and ± 6-AN (100 μM, 48 h). B. Intracellular NADP⁺ and NADPH levels measured in MDA-MB-231 cancer cells transfected with siRNAs against G6PD (siG6PD) or non-targeting siRNAs (siCTL) and treated with ± DCA. One-way ANOVA with Bonferroni post-hoc test. *p < 0.05, ns, not significant. Results are expressed as means ± SEM.

Figure 6. Mechanism by which DCA controls proliferation of glycolytic cancer cells

Highly glycolytic cancer cells exhibiting a Warburg phenotype ferment large amounts of glucose into lactate even in the presence of oxygen. This phenomenon allows rapid ATP production and provides precursors for biosynthetic processes promoting cell proliferation. By alleviating PDH inhibition by PDK, DCA fosters the conversion of pyruvate into acetyl-CoA and activates mitochondrial respiration. The consequent allosteric inhibition of glycolysis by ATP reduces glucose consumption and glycolytic intermediates levels, thereby decreasing the flux of the pentose phosphate pathway along with cellular proliferation. PDH, Pyruvate Dehydrogenase. PDK, Pyruvate Dehydrogenase Kinase. G6P, Glucose-6-phosphate. NAD, Nicotinamide Adenine Dinucleotide. NADP, Nicotinamide Adenine Dinucleotide Phosphate. Glut, Glucose Transporter. MCT4, Monocarboxylate Transporter 4.