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• W3013310134 abstract "Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis. Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis. The aerobic glycolysis (Warburg effect, WE) 2The abbreviations used are: WEWarburg effectG6Pglucose 6-phosphateF6Pfructose 6-phosphateFBPfructose 1,6-bisphosphateGA3Pglyceraldehyde 3-phosphate3-PG3-phosphoglycerate2-PG2-phosphoglyceratePEPphosphoenolpyruvatePyrpyruvateHKhexokinasePGIphosphohexose isomerasePFKphosphofructokinaseTPItriose-phosphate isomeraseGAPDHglyceraldehyde-3-phosphate dehydrogenasePGKphosphoglycerate kinasePKpyruvate kinaseLDHlactate dehydrogenaseFCCflux control coefficientNCnegative controlDHAPdihydroxyacetone phosphateMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt1,3-BPG1,3-bisphosphoglyceric acidPHGDHphosphoglycerate dehydrogenaseMGmethylglyoxalFBSfetal bovine serumG6PDHglucose-6-phosphate dehydrogenasePESphenazine ethosulfateAQC6-aminoquinoline N-succinimidyl ester. is a metabolic hallmark of cancer cells. Inhibiting WE is recognized as an approach to treat cancer (1Altman B.J. Stine Z.E. Dang C.V. 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Quantitative determinants of aerobic glycolysis identify flux through the enzyme GAPDH as a limiting step.eLife. 2014; 3 (25009227)10.7554/eLife.03342Crossref PubMed Scopus (163) Google Scholar, 11Locasale J.W. New concepts in feedback regulation of glucose metabolism.Curr. Opin. Syst. Biol. 2018; 8 (31602417): 32-3810.1016/j.coisb.2017.11.005Crossref PubMed Scopus (20) Google Scholar, 12Yun J. Mullarky E. Lu C. Bosch K.N. Kavalier A. Rivera K. Roper J. Chio I.I. Giannopoulou E.G. Rago C. Muley A. Asara J.M. Paik J. Elemento O. Chen Z. Pappin D.J. et al.Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.Science. 2015; 350 (26541605): 1391-139610.1126/science.aaa5004Crossref PubMed Scopus (582) Google Scholar). Warburg effect glucose 6-phosphate fructose 6-phosphate fructose 1,6-bisphosphate glyceraldehyde 3-phosphate 3-phosphoglycerate 2-phosphoglycerate phosphoenolpyruvate pyruvate hexokinase phosphohexose isomerase phosphofructokinase triose-phosphate isomerase glyceraldehyde-3-phosphate dehydrogenase phosphoglycerate kinase pyruvate kinase lactate dehydrogenase flux control coefficient negative control dihydroxyacetone phosphate 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt 1,3-bisphosphoglyceric acid phosphoglycerate dehydrogenase methylglyoxal fetal bovine serum glucose-6-phosphate dehydrogenase phenazine ethosulfate 6-aminoquinoline N-succinimidyl ester. Previous studies demonstrated that PGK1 was a rate-limiting enzyme in the glycolysis of cancer cells (13Hu H. Zhu W. Qin J. Chen M. Gong L. Li L. Liu X. Tao Y. Yin H. Zhou H. Zhou L. Ye D. Ye Q. Gao D. Acetylation of PGK1 promotes liver cancer cell proliferation and tumorigenesis.Hepatology. 2017; 65 (27774669): 515-52810.1002/hep.28887Crossref PubMed Scopus (132) Google Scholar, 14Zhang Y. Yu G. Chu H. Wang X. Xiong L. Cai G. Liu R. Gao H. Tao B. Li W. Li G. Liang J. Yang W. Macrophage-associated PGK1 phosphorylation promotes aerobic glycolysis and tumorigenesis.Mol. Cell. 2018; 71 (30029001): 201-215.e710.1016/j.molcel.2018.06.023Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 15Li X. Jiang Y. Meisenhelder J. Yang W. Hawke D.H. Zheng Y. Xia Y. Aldape K. He J. Hunter T. Wang L. Lu Z. Mitochondria-translocated PGK1 functions as a protein kinase to coordinate glycolysis and the TCA cycle in tumorigenesis.Mol. Cell. 2016; 61 (26942675): 705-71910.1016/j.molcel.2016.02.009Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 16Christofk H.R. Vander Heiden M.G. Wu N. Asara J.M. Cantley L.C. Pyruvate kinase M2 is a phosphotyrosine-binding protein.Nature. 2008; 452 (18337815): 181-18610.1038/nature06667Crossref PubMed Scopus (787) Google Scholar, 17Anastasiou D. Poulogiannis G. Asara J.M. Boxer M.B. Jiang J.K. Shen M. Bellinger G. Sasaki A.T. Locasale J.W. Auld D.S. Thomas C.J. Vander Heiden M.G. Cantley L.C. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses.Science. 2011; 334 (22052977): 1278-128310.1126/science.1211485Crossref PubMed Scopus (826) Google Scholar, 18Chaneton B. Hillmann P. Zheng L. Martin A.C.L. Maddocks O.D.K. Chokkathukalam A. Coyle J.E. Jankevics A. Holding F.P. Vousden K.H. Frezza C. O'Reilly M. Gottlieb E. Serine is a natural ligand and allosteric activator of pyruvate kinase M2.Nature. 2012; 491 (23064226): 458-46210.1038/nature11540Crossref PubMed Scopus (422) Google Scholar, 19Anastasiou D. Yu Y. Israelsen W.J. Jiang J.K. Boxer M.B. Hong B.S. Tempel W. Dimov S. Shen M. Jha A. Yang H. Mattaini K.R. Metallo C.M. Fiske B.P. Courtney K.D. et al.Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis.Nat. Chem. Biol. 2012; 8 (22922757): 839-84710.1038/nchembio.1060Crossref PubMed Scopus (510) Google Scholar). PGK1 catalyzes a step at the middle of glycolysis and produces ATP and 3-PG (a precursor for serine). Given the high glycolytic rate, PGK1 activity accounts for a large part in maintaining the energy homeostasis and serine biosynthesis. Clinically, PGK1 was overexpressed in many types of tumors (20Hwang T.L. Liang Y. Chien K.Y. Yu J.S. Overexpression and elevated serum levels of phosphoglycerate kinase 1 in pancreatic ductal adenocarcinoma.Proteomics. 2006; 6 (16493704): 2259-227210.1002/pmic.200500345Crossref PubMed Scopus (111) Google Scholar, 21Yan H. Yang K. Xiao H. Zou Y.J. Zhang W.B. Liu H.Y. Over-expression of cofilin-1 and phosphoglycerate kinase 1 in astrocytomas involved in pathogenesis of radioresistance.CNS Neurosci. Ther. 2012; 18 (22742733): 729-73610.1111/j.1755-5949.2012.00353.xCrossref PubMed Scopus (42) Google Scholar, 22Ahmad S.S. Glatzle J. Bajaeifer K. Bühler S. Lehmann T. Königsrainer I. Vollmer J.P. Sipos B. Ahmad S.S. Northoff H. Königsrainer A. Zieker D. Phosphoglycerate kinase 1 as a promoter of metastasis in colon cancer.Int. J. Oncol. 2013; 43 (23727790): 586-59010.3892/ijo.2013.1971Crossref PubMed Scopus (74) Google Scholar, 23Zieker D. Königsrainer I. Tritschler I. Löffler M. Beckert S. Traub F. Nieselt K. Bühler S. Weller M. Gaedcke J. Taichman R.S. Northoff H. Brücher B.L. Königsrainer A. Phosphoglycerate kinase 1 a promoting enzyme for peritoneal dissemination in gastric cancer.Int. J. Cancer. 2010; 126 (19688824): 1513-152010.1002/ijc.24835PubMed Google Scholar, 24Ai J. Huang H. Lv X. Tang Z. Chen M. Chen T. Duan W. Sun H. Li Q. Tan R. Liu Y. Duan J. Yang Y. Wei Y. Li Y. Zhou Q. FLNA and PGK1 are two potential markers for progression in hepatocellular carcinoma.Cell. Physiol. Biochem. 2011; 27 (21471709): 207-21610.1159/000327946Crossref PubMed Scopus (75) Google Scholar). Experimentally, it is found that this enzyme was dynamically modulated in cells. This enzyme was transcriptionally up-regulated by HIF1 (25Kress S. Stein A. Maurer P. Weber B. Reichert J. Buchmann A. Huppert P. Schwarz M. Expression of hypoxia-inducible genes in tumor cells.J. Cancer Res. Clin. Oncol. 1998; 124 (9692838): 315-32010.1007/s004320050175Crossref PubMed Scopus (78) Google Scholar) but down-regulated by PPAR-γ (26Shashni B. Sakharkar K.R. Nagasaki Y. Sakharkar M.K. Glycolytic enzymes PGK1 and PKM2 as novel transcriptional targets of PPARγ in breast cancer pathophysiology.J. Drug Target. 2013; 21 (23130662): 161-17410.3109/1061186X.2012.736998Crossref PubMed Scopus (42) Google Scholar). Hu et al. (13Hu H. Zhu W. Qin J. Chen M. Gong L. Li L. Liu X. Tao Y. Yin H. Zhou H. Zhou L. Ye D. Ye Q. Gao D. Acetylation of PGK1 promotes liver cancer cell proliferation and tumorigenesis.Hepatology. 2017; 65 (27774669): 515-52810.1002/hep.28887Crossref PubMed Scopus (132) Google Scholar) reported that PGK1 could be acetylated by PCAF and Sirtuin 7, and the acetylation of PGK1 enhanced its activity and promoted glycolysis and liver cancer tumorigenesis. Zhang et al. (14Zhang Y. Yu G. Chu H. Wang X. Xiong L. Cai G. Liu R. Gao H. Tao B. Li W. Li G. Liang J. Yang W. Macrophage-associated PGK1 phosphorylation promotes aerobic glycolysis and tumorigenesis.Mol. Cell. 2018; 71 (30029001): 201-215.e710.1016/j.molcel.2018.06.023Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) demonstrated that polarized M2 macrophages secreted IL-6, which enhances PGK1 phosphorylation in tumor cells and promoted glycolysis, and this phosphorylation is associated with malignance and prognosis of human GBM. Li et al. (15Li X. Jiang Y. Meisenhelder J. Yang W. Hawke D.H. Zheng Y. Xia Y. Aldape K. He J. Hunter T. Wang L. Lu Z. Mitochondria-translocated PGK1 functions as a protein kinase to coordinate glycolysis and the TCA cycle in tumorigenesis.Mol. Cell. 2016; 61 (26942675): 705-71910.1016/j.molcel.2016.02.009Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) revealed that, apart from its canonical activity, PGK1 could translocate into mitochondria to phosphorylate and to activate PDHK1, which in turn phosphorylated and inhibited PDH, impairing the TCA cycle and enhancing glycolysis. In contrast, Tanner et al. (27Tanner L.B. Goglia A.G. Wei M.H. Sehgal T. Parsons L.R. Park J.O. White E. Toettcher J.E. Rabinowitz J.D. Four key steps control glycolytic flux in mammalian cells.Cell Syst. 2018; 7 (29960885): 49-62.e810.1016/j.cels.2018.06.003Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) reported that a perturbation of PGK1 did not significantly affect glycolysis. The mixed reports indicated a vague understanding of the mechanism by which PGK1 regulates glycolysis. As the rate control of glycolysis is fundamentally a question of kinetics and thermodynamics of glycolysis, we sought to investigate the effect of perturbing PGK1 on glycolysis with respect to thermodynamics and related kinetics. To investigate the above-mentioned questions, we sought to know the kinetics of PGK1. PGK1 catalyzes a reversible reaction, and its reverse-reaction kinetics is known, but its forward reaction kinetics is unknown because there are no available methods for research. Some studies reported that the forward reaction activity of PGK1 could be measured by the production of NADH, with a reaction mixture containing GA3P, β-NAD, and ADP (13Hu H. Zhu W. Qin J. Chen M. Gong L. Li L. Liu X. Tao Y. Yin H. Zhou H. Zhou L. Ye D. Ye Q. Gao D. Acetylation of PGK1 promotes liver cancer cell proliferation and tumorigenesis.Hepatology. 2017; 65 (27774669): 515-52810.1002/hep.28887Crossref PubMed Scopus (132) Google Scholar, 28Chen X. Zhao C. Li X. Wang T. Li Y. Cao C. Ding Y. Dong M. Finci L. Wang J.H. Li X. Liu L. Terazosin activates Pgk1 and Hsp90 to promote stress resistance.Nat. Chem. Biol. 2015; 11 (25383758): 19-2510.1038/nchembio.1657Crossref PubMed Scopus (57) Google Scholar). The forward reaction of PGK1 can promote GAPDH to produce NADH (13Hu H. Zhu W. Qin J. Chen M. Gong L. Li L. Liu X. Tao Y. Yin H. Zhou H. Zhou L. Ye D. Ye Q. Gao D. Acetylation of PGK1 promotes liver cancer cell proliferation and tumorigenesis.Hepatology. 2017; 65 (27774669): 515-52810.1002/hep.28887Crossref PubMed Scopus (132) Google Scholar, 28Chen X. Zhao C. Li X. Wang T. Li Y. Cao C. Ding Y. Dong M. Finci L. Wang J.H. Li X. Liu L. Terazosin activates Pgk1 and Hsp90 to promote stress resistance.Nat. Chem. Biol. 2015; 11 (25383758): 19-2510.1038/nchembio.1657Crossref PubMed Scopus (57) Google Scholar). This method may tell the difference of the NADH generation rate with or without PGK1, but it cannot accurately measure the activity of PGK1 nor the kinetic parameters. Therefore, we developed a method to accurately measure the forward reaction kinetics of PGK1. We designed a coupled-enzyme assay to measure the forward-reaction rate of PGK1. In a reaction mixture containing GA3P, NAD, and Pi, by adding excessive amounts of GAPDH, Reaction 1 would rapidly reach equilibrium.GA3P+NAD+Pi↔GAPDH1,3-BPG+NADHReaction 1 The equilibrium of the reactions is monitored at 340 nm, which increases at first and then remains stable (Fig. 1), which is indicative of the equilibrium state. By adding ADP and PGK1 into the reaction mixture, we initiate Reaction 2 (Fig. 1).1,3-BPG+ADP↔PGK13-PG+ATPReaction 2 Consumption of 1,3-BPG immediately disrupts the equilibrium state of Reaction 1, thus driving GA3P and NAD to 1,3-BPG and NADH. The rate of NADH generation can be readily spectrophotometrically monitored. According to Reactions 1 and 2, we could derive the following equations: 1) the number of 3-PG molecules generated = the numbers of 1,3-BPG molecules consumed; 2) the number of 1,3-BPG molecules consumed = the numbers of GA3P molecules consumed; 3) the number of GA3P molecules generated = the numbers of NADH molecules generated; and 4) therefore, NADH generation rate = turnover rate of 1,3-BPG to 3-PG catalyzed by PGK1. Therefore, the initial rate of PGK1 could be accurately measured. For measurement, based on the equilibrium of Reaction 1, we set serial concentrations of GA3P, which gave rise to the serial concentrations of 1,3-BPG (Fig. 2A). Then we determined the Km and Vmax values of human and yeast PGK1 (Fig. 2, B and C). The Km values for 1,3-BPG were 4.36 μm (yeast) and 6.86 μm (human); the Km values for 3-PG were 146 μm (yeast) and 186 μm (human), and the Vmax of the forward reaction (from 1,3-BPG to 3PG) was about 3.5-fold (human) and 5-fold (yeast) higher than that of the reverse reaction (Fig. 2D), indicating that the enzyme favors the forward reaction. By setting 1,3-BPG at a saturating concentration (about 50 μm) and varying the ADP concentrations (0.02 to 2 mm), we obtained the Km value for ADP (Fig. 2D). We sought to explore the physiological meaning of the kinetic parameters of PGK1 in glycolysis. The low Km for 1,3-BPG, the high Km for 3-PG, and the much higher rates of the forward reaction than those of the reverse reaction are the biochemical bases to maintain a low concentration of 1,3-BPG and a high concentration of 3-PG in cells. Indeed, cellular 3-PG concentrations were kept at relatively high concentrations, between 0.18 and 0.55 mm (depending on the cell lines) (Table 1). 3-PG is a precursor for serine synthesis. Phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first step for de novo serine synthesis, has the Km value of 0.26 mm for 3-PG (29Fan J. Teng X. Liu L. Mattaini K.R. Looper R.E. Vander Heiden M.G. Rabinowitz J.D. Human phosphoglycerate dehydrogenase produces the oncometabolite d-2-hydroxyglutarate.ACS Chem. Biol. 2015; 10 (25406093): 510-51610.1021/cb500683cCrossref PubMed Scopus (126) Google Scholar). Keeping a high cellular 3-PG concentration is very important for this molecule to shuttle to serine synthesis, because the specific activity (at saturating concentration of 3-PG) of PHGDH in cancer cell is very low. The activity was undetectable even using 0.15 mg of cell lysate protein in our assay system, whereas the HK activity could be accurately determined using 0.03 mg of cell lysate protein.Table 1Intracellular Glc and glycolytic intermediate concentration (mm) in cells with or without PGK1 knockdownMetabolites (mm)GlcG6PF6PFBPDHAPGA3P3PG2PGPEPPyrADPATPNADNADHHeLa-NC5.56 ± 0.0370.56 ± 0.070.12 ± 0.080.38 ± 0.0350.75 ± 0.040.05 ± 0.020.28 ± 0.040.09 ± 0.030.10 ± 0.0140.20 ± 0.030.71 ± 0.123.80 ± 0.580.68 ± 0.110.033 ± 0.005HeLa-siPGK15.52 ± 0.030.57 ± 0.10.14 ± 0.110.65 ± 0.002***1.28 ± 0.08***0.09 ± 0.03*0.28 ± 0.030.07 ± 0.020.11 ± 0.020.14 ± 0.030.61 ± 0.073.10 ± 0.540.66 ± 0.120.035 ± 0.008MGC80–3-NC3.9 ± 0.070.43 ± 0.040.12 ± 0.030.28 ± 0.010.58 ± 0.050.05 ± 0.020.18 ± 0.050.10 ± 0.0660.15 ± 0.0040.27 ± 0.020.59 ± 0.0143.74 ± 0.210.58 ± 0.040.039 ± 0.004MGC80–3-siPGK13.96 ± 0.110.44 ± 0.050.11 ± 0.020.55 ± 0.014***1.17 ± 0.1***0.096 ± 0.04*0.14 ± 0.070.078 ± 0.040.15 ± 0.0010.21 ± 0.02*0.83 ± 0.0624.54 ± 0.80.78 ± 0.120.048 ± 0.005RKO-NC5.2 ± 0.470.31 ± 0.0150.11 ± 0.0110.37 ± 0.0340.47 ± 0.040.043 ± 0.0030.55 ± 0.070.066 ± 0.0280.06 ± 0.030.56 ± 0.046.7 ± 0.1911.3 ± 0.541.45 ± 0.030.16 ± 0.01RKO-siPGK15.2 ± 0.140.29 ± 0.10.13 ± 0.0370.50 ± 0.04*0.77 ± 0.091**0.073 ± 0.005**0.43 ± 0.120.085 ± 0.0170.049 ± 0.0130.48 ± 0.076.9 ± 0.2511.5 ± 0.641.45 ± 0.060.16 ± 0.003SK-HEP-1-NC3.88 ± 0.240.29 ± 0.0280.078 ± 0.0220.24 ± 0.0370.55 ± 0.0470.045 ± 0.0060.49 ± 0.090.13 ± 0.0450.23 ± 0.0640.51 ± 0.0960.77 ± 0.059.3 ± 0.070.93 ± 0.0010.025 ± 0.003SK-HEP-1-siPGK13.4 ± 0.180.35 ± 0.050.11 ± 0.0320.39 ± 0.035**0.8 ± 0.045**0.066 ± 0.009*0.52 ± 0.110.13 ± 0.0540.29 ± 0.0220.51 ± 0.100.77 ± 0.059.9 ± 0.420.72 ± 0.550.029 ± 0.003A549-NC4.2 ± 0.040.21 ± 0.040.038 ± 0.0030.14 ± 0.030.28 ± 0.030.021 ± 0.0020.22 ± 0.0440.071 ± 0.030.084 ± 0.040.64 ± 0.0660.84 ± 0.05710.7 ± 0.251.78 ± 0.0330.28 ± 0.009A549-siPGK13.7 ± 0.05***0.177 ± 0.010.05 ± 0.0130.20 ± 0.035*0.54 ± 0.08***0.042 ± 0.003***0.21 ± 0.0520.069 ± 0.0180.085 ± 0.0310.625 ± 0.0140.9 ± 0.06911.1 ± 0.561.78 ± 0.0380.28 ± 0.01 Open table in a new tab HK2 knockdown reduced HK activity by ∼50%, glucose consumption by ∼40%, and lactate generation by ∼50% (Fig. 3A). However, HK2 knockdown did not significantly reduce the 3-PG concentration (Fig. 3B) and serine synthesis, as manifested by the analysis of the serine isotopologues (Fig. 3C). The m + 0 serine species is provided by the culture medium, and m + 3 serine isotopologue was generated from [13C6]glucose through 3-PG. The consumption rate of m + 0 serine was comparable between control cells and HK2 knockdown cells (Fig. 3C, left panel), and the percentages of extracellular and intracellular m + 0 and m + 3 serine species were also comparable between control cells and HK2 knockdown (Fig. 3C, middle and right panels). A fraction of serine was further used for glycine synthesis. The consumption rate of glycine (m + 0, which is provided by culture medium) is comparable between control and HK2 knockdown cells (Fig. 3D, left panel) and so were the percentages of extracellular and intracellular glycine (m + 2, which is derived from m + 3 serine) (Fig. 3D, middle and right panels). These data support that glucose carbon shuttling to serine and glycine was not significantly affected by HK2 knockdown, and even HK2 knockdown reduced the glycolysis rate to lactate by 50%. Taken together, the kinetic parameters of PGK1 are associated with a stable steady-state concentration of 3-PG in cancer cells, which is around the Km value of PHGDH, underlying a sound biochemical basis for glycolysis to shuttle to the de novo serine synthesis pathway. We used five cell lines from different tissue origins (cervical cancer cell line HeLa, gastric cancer cell line MGC80-3, colon cancer cell line RKO, lung cancer cell line A549, and liver cancer cell line SK-HEP-1). These cells were derived from different organs from different patients, representing five types of cancer cell lines. They exhibited widespread mutations (30Ahmed D. Eide P.W. Eilertsen I.A. Danielsen S.A. Eknæs M. Hektoen M. Lind G.E. Lothe R.A. 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Mutat. 2009; 30 (19472407): 1199-120610.1002/humu.21028Crossref PubMed Scopus (100) Google Scholar). HeLa cells had WT TP53, but p53 protein was repressed because of overexpression of human papillomavirus type 16 E6 (34Yaginuma Y. Westphal H. Analysis of the p53 gene in human uterine carcinoma cell lines.Cancer Res. 1991; 51 (1660340): 6506-6509PubMed Google Scholar, 35Hoppe-Seyler F. Butz K. Repression of endogenous p53 transactivation function in HeLa cervical carcinoma cells by human papillomavirus type 16 E6, human mdm-2, and mutant p53.J. Virol. 1993; 67 (8388491): 3111-311710.1128/JVI.67.6.3111-3117.1993Crossref PubMed Google Scholar). In MGC80-3, decreased expression of TWSG1 was detected compared with normal gastric cells (36Yuan J. Zeng J. Shuai C. Liu Y. TWSG1 is a novel tumor suppressor in gastric cancer.DNA Cell Biol. 2018; 37 (29756996): 574-58310.1089/dna.2018.4188Crossref PubMed Scopus (7) Google Scholar). In RKO cells, TP53 was WT, whereas PTEN, KRAS, BRAF, and PIK3CA were mutated (30Ahmed D. Eide P.W. Eilertsen I.A. Danielsen S.A. Eknæs M. Hektoen M. Lind G.E. Lothe R.A. Epigenetic and genetic features of 24 colon cancer cell lines.Oncogenesis. 2013; 2 (24042735): e7110.1038/oncsis.2013.35Crossref PubMed Scopus (574) Google Scholar, 32Berg K.C.G. Eide P.W. Eilertsen I.A. Johannessen B. Bruun J. Danielsen S.A. Bjørnslett M. Meza-Zepeda L.A. Eknaes M. Lind G.E. Myklebost O. Skotheim R.I. Sveen A. Lothe R.A. Multi-omics of 34 colorectal cancer cell lines–a resource for biomedical studies.Mol. Cancer. 2017; 16 (28683746): 11610.1186/s12943-017-0691-yCrossref PubMed Scopus (171) Google Scholar). WT TP53 was detected in both SK-HEP-1 and A549. BRAF was mutated in SK-HEP-1, whereas CDKN2A and KRAS were mutated in A549 cells (31Zhao Y. Chen Y. Hu Y. Wang J. Xie X. He G. Chen H. Shao Q. Zeng H. Zhang H. Genomic alterations across six hepatocellular carcinoma cell lines by panel-based sequencing.Transl. Cancer Res. 2018; 7: 231-23910.21037/tcr.2018.02.14Crossref Scopus (9) Google Scholar, 33Blanco R. Iwakawa R. Tang M. Kohno T. Angulo B. Pio R. Montuenga L.M. Minna J.D. Yokota J. Sanchez-Cespedes M. A gene-alteration profile of human lung cancer cell lines.Hum. Mutat. 2009; 30 (19472407): 1199-120610.1002/humu.21028Crossref PubMed Scopus (100) Google Scholar). Despite the differences, they shared the same metabolic feature, the Warburg effect. They exhibited a similar pattern of glycolytic enzymes (Fig. 4A), and they converted most incoming glucose to lactate (Fig. 4B). If the glucose consumption rate is expressed by kilograms of glucose/kg of cells per day, the number would be larger than 1.5 kg per kg of cancer cells per day (Fig. 4C). Thus, the data demonstrated that these cells shared the feature of the Warburg effect. 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