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

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. 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GAPDH attracted much attention recently (6Yun J. Mullarky E. Lu C. Bosch K.N. Kavalier A. Rivera K. Roper J. Chio II, Giannopoulou E.G. Rago C. Muley A. Asara J.M. Paik J. Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.Science. 2015; 350: 1391-1396Crossref PubMed Scopus (480) Google Scholar, 7Liberti M.V. Dai Z. Wardell S.E. Baccile J.A. Liu X. Gao X. Baldi R. Mehrmohamadi M. Johnson M.O. Madhukar N.S. Shestov A.A. Chio I.I.C. Elemento O. A predictive model for selective targeting of the Warburg effect through GAPDH inhibition with a natural product.Cell Metab. 2017; 26: 648-659Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 8Kornberg M.D. Bhargava P. Kim P.M. Putluri V. Snowman A.M. Putluri N. Calabresi P.A. Snyder S.H. Dimethyl fumarate targets GAPDH and aerobic glycolysis to modulate immunity.Science. 2018; 360: 449-453Crossref PubMed Scopus (238) Google Scholar, 15Shestov A.A. Liu X. Ser Z. Cluntun A.A. Hung Y.P. 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We sought to investigate the quantitative relationship between GAPDH and aerobic glycolysis to delineate the biochemical insight into this relationship. We used five cancer cells lines for this work, cervical cancer cell line HeLa, gastric cancer cell line MGC80-3, colon cancer cell line RKO, liver cancer cell line SK-HEP-1, and lung cancer cell line A549. These cell lines exhibited the WE, as described by us recently (20Jin C. Zhu X. Wu H. Wang Y. Hu X. Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells.J. Biol. Chem. 2020; 295: 6425-6446Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). In this study, the GAPDH specific activity (GAPDH-SA) refers to the activity assayed at the saturating substrate concentration. The concentration of the GAPDH is derived from the equation Vspecfic-activity ≈ Vmax = kcat[E]: we set the relative concentration of GAPDH in the control cells as 100%, then the relative concentration of GAPDH in the cells treated with siRNA or GAPDH inhibitor would be the following.[GAPDH]treated=GAPDH−SAtreatedGAPDH−SActrl[GAPDH]ctrl(1) The GAPDH actual activity (GAPDH-AA) refers to its activity at the actual concentration of its substrates in the glycolysis. siGAPDH specifically knocked down GAPDH-SA with no or marginal effect on the activities of other glycolytic enzymes (Fig. 1). The knockdown efficiency ranged from 48% (RKO) to 72% (HeLa) (Fig. 2, A and B). GAPDH knockdown marginally affected glucose (Glc) consumption and lactate generation (Fig. 2C) in HeLa, RKO, MGC80-3, and SK-HEP-1 cells. In A549 cells, GAPDH knockdown moderately reduced lactate production by about 15% but with a marginal reduced Glc consumption, in comparison with the control (Fig. 2C). The data suggested that the effect of GAPDH knockdown may vary from cell to cell, but the variation was moderate.Figure 2Knockdown of GAPDH marginally perturbed glycolysis. A, GAPDH activity and (B) Western blot of GAPDH in cells with or without GAPDH knockdown. C, the glucose consumption rate and lactate generation rate in cells with or without GAPDH knockdown (left and middle panels); the right panels compare the relative rates of glucose consumption, lactate generation, and GAPDH, showing that the reduction of GAPDH is virtually irrelevant with the rates of glucose consumption and lactate generation; the relative glucose consumption rate and relative lactate generation rate are based on the data in the left and middle panels. Data are from Table S1. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 by two-tailed Student's t test. Data are the mean ± SEM from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although GAPDH-knockdown cells produced a lactate amount comparable with control cells, it could be that a fraction of lactate produced by GAPDH-knockdown cells was from other metabolic pathways, so that the total lactate amount was not less than that in control cells. To exclude this possibility, we used [13C6]glucose to trace lactate in cells and demonstrated that about 95% lactate (m + 3) generated by control and GAPDH knockdown cells was from Glc (Fig. S1). However, siRNA knockdown could not generate serial clones with a stepwise decrement of GAPDH activity, so that the quantitative relationship between GAPDH and aerobic glycolysis could not be established. Iodoacetate (IA) is a GAPDH inhibitor (21Williamson J.R. Glycolytic Control Mechanisms III. Effects of iodoacetamide and fluoroacetate on glucose metabolism in the perfused rat heart.J. Biol. 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Cells were incubated in a serial of concentrations of IA (1–100 μM) for 7 h. The cellular GSH content did not change significantly at the IA concentration between 0 and 10 μM (Fig. 3B). At 25 μM IA, there was a moderate reduction of the cellular GSH concentration (by 23% and 15% for HeLa and RKO, respectively) but no statistical significance. When the IA concentration increased to 50 μM, the cellular GSH concentration was significantly reduced. The result was consistent with the previous report (24Schmidt M.M. Dringen R. Differential effects of iodoacetamide and iodoacetate on glycolysis and glutathione metabolism of cultured astrocytes.Front. inneuroenergetics. 2009; 1-1Google Scholar). In contrast, when cells were treated with IA at the concentration range between 0 and 25 μM, the GAPDH activity was inversely proportional to the IA concentration (Fig. 3C). GAPDH activity was significantly inhibited even by 1 μM IA. When IA was 25 μM, the residual activity of GAPDH only retained 2.6% and 1.1% for HeLa and RKO, respectively. Moreover, IA at the effective concentration inactivating GAPDH did not significantly affect other glycolytic enzymes (Fig. 3A). We prepared cell lysate from HeLa and RKO and measured the inhibition of GAPDH-SA by IA. The inhibition curve was similar between HeLa and RKO (Fig. 3D). Therefore, the inhibition is cell-type independent. We measured the stability of GAPDH-SA in the cell lysate prepared from HeLa and RKO pretreated with IA (Fig. 3E). The data showed that GAPDH-SA was stable. This stable inhibition is important for real-time monitor of the GAPDH-SA in the subsequent study, otherwise the quantitative relationship between GAPDH and the aerobic glycolysis in cells could not be established. We also did the substrate-dependent kinetics of GAPDH in the cell lysate from HeLa and RKO cells with or without IA treatment. When converting the kinetic rate to percentage, they were identical (Fig. 3F), indicating that IA inhibition only reduced the concentration of functional GAPDH without affecting the kinetic feature of the enzyme. Finally, we checked if cell density would affect the inhibition efficacy of IA. The results showed that IA efficacy decreased with the increase in the cell number (Fig. 3G). Therefore, in the subsequent experiments, we fixed the seeding cell number. We then treated HeLa cells with IA. At the indicated intervals, we collected cells and medium, measured GAPDH-SA (Fig. 4A), Glc consumption (Fig. 4B), and lactate generation (Fig. 4C). We then deduced the instantaneous rate of change of Glc consumption (Vi-glucose) (Fig. 4D) and lactate generation (Vi-lactate) (Fig. 4E) by differentiating the data of Figure 4, B and C and correlated the GAPDH-SA with Vi-glucose (Fig. 4F) and Vi-lactate (Fig. 4G). The quantitative relationship between GAPDH-SA and Vi-lactate appeared to be composed of two functions (Fig. 4G). When GAPDH-SA% was between 23.8% ± 3% and 100% (Table 1), the Vi-lactate was a constant, the function could be expressed byf(x) = c(2) Table 1The aerobic glycolysis (V) as a function of the GAPDH specific activity as a variableGAPDH-SA%HeLaRKOMGC80-3SK-HEP-1A549CombinedaThe mean ± SD of five cell lines. Data of HeLa, RKO, MGC80-3, SK-HEP-1, and A549 are the mean ± SEM from three independent experiments.V = c = VmaxbThe glycolytic rate (V) in the control cells whose GAPDH-SA is 100%.100–23.8 ± 3100–15.3 ± 1.8100–18.7 ± 1.6100–13.5 ± 3.4100–23.9 ± 2.3100–19 ± 4.8V = a(GAPDH−SA%)b+(GAPDH−SA%)<23.8 ± 3<15.3±1.8<18.7 ± 1.6<13.5 ± 3.4<23.9 ± 2.3<19 ± 4.8Critical GAPDH-SA% point that saturates the flux23.8 ± 315.3 ± 1.818.7 ± 1.613.5 ± 3.423.9 ± 2.319 ± 4.8Critical GAPDH-SA% point that corresponds to 1/2 Vmax8.1 ± 0.73.5 ± 0.46.7 ± 0.44.1 ± 0.64.8 ± 0.75.4 ± 1.9GAPDH-SA, GAPDH specific activity.a The mean ± SD of five cell lines. Data of HeLa, RKO, MGC80-3, SK-HEP-1, and A549 are the mean ± SEM from three independent experiments.b The glycolytic rate (V) in the control cells whose GAPDH-SA is 100%. Open table in a new tab GAPDH-SA, GAPDH specific activity. When GAPDH-SA% was below 23.8% ± 3%, Vi-lactate was positively correlated with GAPDH-SA%, the relationship could be expressed by the following:f(x)=ax/(b+x)(3) where x refers the GAPDH-SA%. The half maximal Vi-lactate corresponded to 8.1% ± 0.7% GAPDH-SA in HeLa cells (Table 1). The relationship between GAPDH-SA% and Vi-glucose exhibited a similar pattern (Fig. 4F) to that between GAPDH-SA% and Vi-lactate. Using the same approach, we obtained the curves between Glc consumption or lactate generation and the GAPDH-SA in RKO, SK-HEP-1, MGC80-3, and A549. (Figs. S2–S5). Again, the quantitative relationship between Glc consumption or lactate generation and the GAPDH-SA could be grouped into two functions, as described for HeLa cells. The half maximal Vi-lactate corresponded to 3.5% ± 0.4% GAPDH-SA for RKO cells, 6.7% ± 0.4% GAPDH-SA for MGC80-3 cells, 4.1% ± 0.6% GAPDH-SA for SK-Hep-1 cells, and 4.8% ± 0.7% GAPDH-SA for A549 cells (Table 1). The data revealed a high similarity of the quantitative relationship between the glycolysis and GAPDH in the five cancer cell lines. We summarized the quantitative relationship between GAPDH-SA% and the glycolysis rate derived from the five cell lines in Table 1. The equations numerically correlate the glycolysis rate to GAPDH-SA% and introduce two critical values of the glycolysis rate that were achieved at two critical points of GAPDH-SA%. The first critical point of GAPDH-SA% is 19% ± 4.8%; above this point, the glycolysis rate is a constant, and below this point, the glycolysis rate decreased according to Equation 3. The second critical point of GAPDH-SA% is 5.4% ± 1.9%, at which the glycolysis rate is decreased by half. Numerically, the glycolysis rate versus GAPDH-SA% is equivalent to the glycolysis rate versus the concentration of GAPDH. We treated HeLa cells with serial concentrations of IA and monitored the cell growth, Glc consumption, lactate generation, and GAPDH-SA in a time course (Fig. 5, A–D). The data demonstrated an association between the variables (IA, GAPDH-SA, glycolysis, and cell growth). To better correlate the quantitative nature of these variables, we extracted the data of the 24 h point. We first correlated IA with the glycolysis rate and cell growth (Fig. 5, E and F). The IC50 of IA for Glc consumption and lactate generation were 9.0 and 9.5 μM, respectively, which were close to the IC50 (8.2 μM) of IA for cell growth inhibition. The cell number were positively correlated with the glycolysis rate (Fig. 5, G and H) Then, we correlated IA with GAPDH. The IC50 of IA for GAPDH-SA was 2.5 μM (Fig. 5I), which was much lower than the IC50 for glycolysis and cell growth. By plotting GAPDH-SA with the glycolysis rate, the half inhibition of Glc consumption and lactate generation corresponded to 6.8% and 6.5% GAPDH-SA, respectively (Fig. 5, J and K), which were consistent to the data in Figure 4. Cell growth versus GAPDH-SA conveyed the following information (Fig. 5L). We dissected the line into three segments. Cell growth was not affected by perturbing GAPDH-SA from 100% and 42.9% (the black segment); cell growth was inhibited by further reducing GAPDH-SA from 42.9% and 9.5% (green segment); and cell death occurred by further inhibiting GAPDH-SA% down to <9.5% (red segment). We then used RKO to repeat the same experiments, and we obtained the similar results (Fig. S6). We were curious about if the cells treated with 8 μM IA could resume proliferation. We treated HeLa and RKO cells with 8 μM IA for 48 h. The residual GAPDH-SA only retained 2.3% and 4.5% in HeLa and RKO, respectively, and cell growth was completely inhibited. We then removed the drug by displacing the medium. GAPDH activity recovered by 75% and 72%, respectively, in HeLa and RKO cells at the 72 h point after displacing the medium. Cells partially resumed proliferation (Fig. S7). It is noted that when GAPDH-SA% in cells varied from 100% to 19% ± 4.8%, the lactate generation rate kept constant. As the lactate generation rate kept constant, the rate through GAPDH in the glycolysis must also keep constant. Then why GAPDH-SA reduced from 100% to 19% does not affect the rate through GAPDH in the glycolysis? We found that GAPDH knockdown was associated with an increase of [GA3P] in cells, consistent in five cell lines (Fig. 6A). In IA titration experiments, [GA3P] was incrementally increased, accompanied with a corresponding decrement of GAPDH-SA (Fig. 6B). The reciprocal relationship between [GAPDH] and [GA3P] could be expressed quantitatively by Equation 4 (Fig. 6C):f(x)= C/(Ax+B)(4) where f(x) and x refer [GA3P] and GAPDH-SA%, respectively. The most fundamental principle of enzyme kinetics is that the catalytic rate of an enzyme is the function of the concentrations of both the enzyme and its substrate. The Km values of GAPDH were 0.065 to 0.11 mM (Fig. S8), consistent with the reported value (25Lambeir A.M. Loiseau A.M. Kuntz D.A. Vellieux F.M. Michels P.A. Opperdoes F.R. The cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei Kinetic properties and comparison with homologous enzymes.Eur. J. Biochem. 1991; 198: 429-435Crossref PubMed Scopus (74) Google Scholar, 26Mountassif D. Baibai T. Fourrat L. Moutaouakkil A. Iddar A. El Kebbaj M.S. Soukri A. Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes.Acta Biochim. Biophys. Sinica. 2009; 41: 399-406Crossref PubMed Scopus (14) Google Scholar). The cellular [GA3P] in untreated cells was 0.031 to 0.061 mM (Table S4), which were not saturating GAPDH. Therefore, the increased [GA3P] could increase GAPDH activity and countered the effect of GAPDH knockdown or inhibition. Based on the information, we propose that the rate through GAPDH in the glycolysis is balanced by the concentration of both GAPDH and glyceraldehyde 3-phosphate (GA3P), that is, the reciprocal relationship of the concentrations between GAPDH and GA3P could compensate each other and stabilize the rate through GAPDH in the glycolysis. The next question is to what extent the increased GA3P concentration could compensate the decreased GAPDH activity induced by GAPDH knockdown or inhibition. This issue requires determination of the GAPDH-AA. However, as the GAPDH in cells is compartmented (27Daniela A.S. Ellen K.S. Leonor C.A.S. Calderon-Aranda E.S. Role of nitric oxide produced by inos through nf-κb pathway in migration of cerebellar granule neurons induced by lipopolysaccharide.Cell. Signal. 2011; 23: 425-435Crossref PubMed Scopus (69) Google Scholar, 28Hara M.R. 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We prepared cell lysate from HeLa-siGAPDH and added pure GAPDH into cell lysate to titrate GAPDH-SA. The titrated cell lysate was used for glycolysis assay. In the given range of relative concentration of GAPDH ([GAPDH]) from 0.6 to 1.4, we did not observe a rate of increase of lactate generation (Fig. 7A). [GA3P] was inversely proportional to relative [GAPDH] (Fig. 7B), and the data fit Equation 4. We then calculated the GAPDH-AA, which were virtually the same in the [GAPDH] range between 0.6 and 1.4 (Fig. 7C), quantitatively explaining why titration of GAPDH did not change the lactate generation rate. We then prepared cell lysate from HeLa cells pretreated with IA, which inhibited GAPDH-SA by 95%. The cell lysate was titrated by GAPDH and was used for cell-free glycolysis assay. We correlated the lactate generation with GAPDH-SA (Fig. 7D). The quantitative relationship is similar to that in the cells, that is, it could be expressed by two functions, f(x) = c when GAPDH-SA% is between 40% and 100% and f(x) = ax/(b + x) when GAPDH-SA% was below 40%. The reciprocal quantitative relationship between [GA3P] and GAPDH-SA% fits Equation 4 very well (Fig. 7E). Then, we calculated the GAPDH-AA and correlated GAPDH-AA with GAPDH-SA (Fig. 7F). From Figure 7, D and E, we could see that when GAPDH-SA% ranged from 100% to 40%, the lactate generation rate was a constant and GAPDH-AA was nearly constant. The half lactate generation rate corresponded to 4.3% GAPDH-SA and the estimated 50% GAPDH-AA corresponded to 10% GAPDH-SA. The data demonstrated that GAPDH-AA was in parallel with the lactate generation rate, indicating that the rate through GAPDH in the glycolysis was balanced by both [GAPDH] and [GA3P]. The thermodynamic state of glycolysis in five different cancer cell lines share the same pattern (Fig. S9A). The Q values (Table S3) were far smaller than Keq in the reactions catalyzed by HK, PFK1, and PK, generating a large and negative ΔG, which drives the forward flux of glycolysis. The ΔG of the lactate dehydrogenase (LDH)-catalyzed reaction was between −3.4 and −4.1 kJ/mol, favoring the forward reaction, that is, converting pyruvate (Pyr) and NADH to lactate and NAD. This underlies the thermodynamic basis for the WE. The concentrations of the glycolytic intermediates were controlled by ΔG. The pattern of the glycolytic intermediates could be defined by the Q values or by the concentrations. The inter