Determining the quantitative relationship between glycolysis and GAPDH in cancer cells exhibiting the Warburg effect

Abstract

Previous studies have identified GAPDH as a promising target for treating cancer and modulating immunity because its inhibition reduces glycolysis in cells (cancer cells and immune cells) with the Warburg effect, a modified form of cellular metabolism found in cancer cells. However, the quantitative relationship between GAPDH and the aerobic glycolysis remains unknown. Here, using siRNA-mediated knockdown of GAPDH expression and iodoacetate-dependent inhibition of enzyme activity, we examined the quantitative relationship between GAPDH activity and glycolysis rate. We found that glycolytic rates were unaffected by the reduction of GAPDH activity down to 19% ± 4.8% relative to untreated controls. However, further reduction of GAPDH activity below this level caused proportional reductions in the glycolysis rate. GAPDH knockdown or inhibition also simultaneously increased the concentration of glyceraldehyde 3-phosphate (GA3P, the substrate of GAPDH). This increased GA3P concentration countered the effect of GAPDH knockdown or inhibition and stabilized the glycolysis rate by promoting GAPDH activity. Mechanistically, the intracellular GA3P concentration is controlled by the Gibbs free energy of the reactions upstream of GAPDH. The thermodynamic state of the reactions along the glycolysis pathway was only affected when GAPDH activity was reduced below 19% ± 4.8%. Doing so moved the reactions catalyzed by GAPDH + PGK1 (phosphoglycerate kinase 1, the enzyme immediate downstream of GAPDH) away from the near-equilibrium state, revealing an important biochemical basis to interpret the rate control of glycolysis by GAPDH. Collectively, we resolved the numerical relationship between GAPDH and glycolysis in cancer cells with the Warburg effect and interpreted the underlying mechanism. Previous studies have identified GAPDH as a promising target for treating cancer and modulating immunity because its inhibition reduces glycolysis in cells (cancer cells and immune cells) with the Warburg effect, a modified form of cellular metabolism found in cancer cells. However, the quantitative relationship between GAPDH and the aerobic glycolysis remains unknown. Here, using siRNA-mediated knockdown of GAPDH expression and iodoacetate-dependent inhibition of enzyme activity, we examined the quantitative relationship between GAPDH activity and glycolysis rate. We found that glycolytic rates were unaffected by the reduction of GAPDH activity down to 19% ± 4.8% relative to untreated controls. However, further reduction of GAPDH activity below this level caused proportional reductions in the glycolysis rate. GAPDH knockdown or inhibition also simultaneously increased the concentration of glyceraldehyde 3-phosphate (GA3P, the substrate of GAPDH). This increased GA3P concentration countered the effect of GAPDH knockdown or inhibition and stabilized the glycolysis rate by promoting GAPDH activity. Mechanistically, the intracellular GA3P concentration is controlled by the Gibbs free energy of the reactions upstream of GAPDH. The thermodynamic state of the reactions along the glycolysis pathway was only affected when GAPDH activity was reduced below 19% ± 4.8%. Doing so moved the reactions catalyzed by GAPDH + PGK1 (phosphoglycerate kinase 1, the enzyme immediate downstream of GAPDH) away from the near-equilibrium state, revealing an important biochemical basis to interpret the rate control of glycolysis by GAPDH. Collectively, we resolved the numerical relationship between GAPDH and glycolysis in cancer cells with the Warburg effect and interpreted the underlying mechanism. Aerobic glycolysis (Warburg effect [WE]) is a prominent feature of cancer cells. The WE is crucial for the growth, survival, metastasis, and drug resistance of cancer cells (1Altman B.J. Stine Z.E. Dang C.V. From Krebs to clinic: Glutamine metabolism to cancer therapy.Nat. Rev. Cancer. 2016; 16: 619-634Crossref PubMed Scopus (790) Google Scholar, 2Cascone T. McKenzie J.A. Mbofung R.M. Punt S. Wang Z. Xu C. Williams L.J. Wang Z. Bristow C.A. Carugo A. Peoples M.D. Li L. Karpinets T. Huang L. Malu S. et al.Increased tumor glycolysis characterizes immune resistance to adoptive T cell therapy.Cell Metab. 2018; 27: 977-987.e974Abstract Full Text Full Text PubMed Scopus Google Scholar, and Rev. Cancer. 2016; 16: PubMed Scopus Google Scholar, and metabolism in Rev. Cancer. 2016; 16: PubMed Scopus Google glycolysis the in glycolysis as a promising to cancer Xu Huang inhibition for PubMed Scopus Google Scholar, C. A. C. A. and cancer cells by PubMed Scopus Google Scholar, Z. J.A. for of the Warburg effect GAPDH inhibition with a Metab. 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