Investigation of extracellular metabolites in cancer and healthy colon cells in a time-dependent manner

Öz

Metabolite analysis is critical in the cancer field because of provides information about the metabolic status of cells. The profiling of extracellular metabolites presents technical advantages over intracellular metabolites, such as easier access to extracellular metabolites without a quenching method and the growth medium containing high biomass. This study aimed to investigate the extracellular level of metabolites in colon cancer cells in a time-dependent manner. One million cells were seeded in 4 Petri dishes and glucose, pyruvate, TCA cycle metabolites, and D2-HG in the conditioned medium were determined by DNS and HPLC method and pyruvate assay for 24-96th hours. The results showed that glucose is consumed, and pyruvate and TCA cycle intermediates are released in decreasing amounts in all cell lines. It was also observed that glucose was more consumed, and TCA cycle metabolites were less released in metastatic colon cancer cells (SW620) than in primary colon adenocarcinoma cells (Caco-2). Most importantly, D2-HG oncometabolite was released more into the growth medium of colon cancer cells than normal colon cells for four days. In conclusion, the D2-HG is highly produced and released to the growth medium of colon cancer cell lines in a cancer-type-specific manner.

Anahtar Kelimeler

• Colon cancer
• exametabolome analysis
• metabolite
• HPLC

D2-Hydroxyglutarate highly released from colon cancer cell lines in a time-dependent manner

Öz

Kaynakça

1. D. Hanahan, R.A. Weinberg, The hallmarks of cancer. Cell, 100, 2000, 57–70.
2. R. Twombly, Cancer surpasses heart disease as leading cause of death for all but the very elderly. J Natl Cancer Inst, 2005, 97, 330–331.
3. H. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray, Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA-Cancer J Clin, 71, 2021, 209–249.
4. C. Joachim, J. Macni, M. Drame, A. Pomier, P. Escarmant, J. Veronique-Baudin, V. Vinh-Hung, Overall survival of colorectal cancer by stage at diagnosis: Data from the Martinique Cancer Registry, Medicine, 98, 2019.
5. R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2019, CA- Cancer J Clin, 69, 2019, 7–34.
6. C. Neitzel, P. Demuth, S. Wittmann, J. Fahrer, Targeting altered energy metabolism in colorectal cancer: oncogenic reprogramming, the central role of the TCA cycle and therapeutic opportunities, Cancers, 12, 2020, 1731.
7. A. Judge, M.S. Dodd, Metabolism, Essays Biochem, 64, 2020, 607–647.
8. E. Holmes, I.D. Wilson, J.K. Nicholson, Metabolic phenotyping in health and disease, Cell, 134, 2008, 714–717.

9. Y.J. Park, S.M. Han, J.Y. Huh, J.B. Kim, Emerging roles of epigenetic regulation in obesity and metabolic disease, J Biol Chem, 297, 2021.
10. M. Bulló, C. Papandreou, J. García-Gavilán, M. Ruiz-Canela, J. Li, M. Guasch-Ferré, E. Toledo, C. Clish, D. Corella, R. Estruch, E. Ros, M. Fitó, C.H. Lee, K. Pierce, C. Razquin, F. Arós, L. Serra-Majem, L. Liang, M.A. Martínez-González, F.B. Hu, J. Salas-Salvadó, Tricarboxylic acid cycle related-metabolites and risk of atrial fibrillation and heart failure, Metabolisim, 125, 2021.
11. D.R. Schmidt, R. Patel, D.G. Kirsch, C. Lewis, M.V. Vander Heiden, J. Locasale, Metabolomics in cancer research and emerging applications in clinical oncology, CA: Cancer J Clin, 71, 2021, 333–358.
12. A. Galal, M. Talal, A. Moustafa, Applications of machine learning in metabolomics: Disease modeling and classification, Front Genet, 13, 2022.
13. N. Paczia, A. Nilgen, T. Lehmann, J. Gätgens, W. Wiechert, S. Noack, Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms, Microb Cell Factories, 11, 2012, 1–14.
14. N. Granucci, F.R. Pinu, T.L. Han, S.G. Villas-Boas, Can we predict the intracellular metabolic state of a cell based on extracellular metabolite data?, Mol Biosyst, 11, 2015, 3297–3304.
15. D. Hunerdosse, D.K. Nomura, Activity-based proteomic and metabolomic approaches for understanding metabolism, Curr Opin Biotechnol, 28, 2014, 116–126.
16. D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell, 144, 2011, 646–674.
17. I. Martínez-Reyes, N.S. Chandel, Mitochondrial TCA cycle metabolites control physiology and disease, Nat Commun, 11, 2020, 1–11.
18. X.W. Hou, Y. Wang, C.W. Pan, Metabolomics in diabetic retinopathy: A systematic review, Investig Ophthalmol Vis Sci, 62, 2021, 4–4.
19. M. Sciacovelli, E. Gonçalves, T.I. Johnson, V.R. Zecchini, A.S. Henriques da Costa, E. Gaude, A.V. Drubbel, S.J. Theobald, S.R. Abbo, M.G.B. Tran, V. Rajeeve, S. Cardaci, S. Foster, H. Yun, P. Cutillas, A. Warren, V. Gnanapragasam, E. Gottlieb, K. Franze, B. Huntly, E.R Maher, P.H. Maxwell, J. Saez-Rodriguez, C. Frezza, Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition, Nature, 537, 2016, 544–547.
20. L. Dang, D.W. White, S. Gross, B.D. Bennett, M.A. Bittinger, E.M. Driggers, V.R. Fantin, H.G. Jang, S. Jin, M.C. Keenan, K.M. Marks, R.M. Prins, P.S. Ward, K.E. Yen, L.M. Liau, J.D. Rabinowitz, L.C. Cantley, C.B. Thompson, M.G. Vander Heiden, S.M. Su, Cancer-associated IDH1 mutations produce 2-hydroxyglutarate, Nature, 462, 2009, 739–744.
21. I.P. Tomlinson, N.A. Alam, A.J. Rowan, E. Barclay, E.E.M. Jaeger, D. Kelsell, I. Leigh, P. Gorman, H. Lamlum, S. Rahman, R.R. Roylance, S. Olpin, Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer, Nat Genet, 30, 2002, 406.
22. H.X. Hao, O. Khalimonchuk, M. Schraders, N. Dephoure, J.P. Bayley, H. Kunst, P. Devilee, C.W.R.J. Cremers, J.D. Schiffman, B.G. Bentz, S.P. Gygi, D.R. Winge, H. Kremer, J. Rutter, SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma, Science, 325, 2009, 1139–1142.
23. H. Colvin, N. Nishida, M. Konno, N. Haraguchi, H. Takahashi, J. Nishimura, T. Hata, K. Kawamoto, A. Asai, K. Tsunekuni, J. Koseki, T. Mizushima, T. Satoh, Y. Doki, M. Mori, H. Ishii, Oncometabolite D-2-hydroxyglurate directly induces epithelial-mesenchymal transition and is associated with distant metastasis in colorectal cancer, Sci Rep, 6, 2016, 36289.
24. E.B. Atalay, H.A. Kayali, The elevated D-2-hydroxyglutarate level found as a characteristic metabolic change of colon cancer in both in vitro and in vivo models, Biochem Biophys Res Commun, 627, 2022, 191–199.
25. S.K. Manna, N. Tanaka, K.W. Krausz, M. Haznadar, X. Xue, T. Matsubara, E.D. Bowman, E.R. Fearon, C.C. Harris, Y.M. Shah, F.J. Gonzalez, Biomarkers of coordinate metabolic reprogramming in colorectal tumors in mice and humans, Gastroenterology, 146, 2014, 1313–1324.
26. E. Subasi, E. Bulut Atalay, D. Erdogan, B. Sen, B. Pakyapan, H.A. Kayali, Synthesis and characterization of thiosemicarbazone-functionalized organoruthenium (II)-arene complexes: Investigation of antitumor characteristics in colorectal cancer cell lines, Mater Sci Eng C Mater Biol Appl, 106, 2020, 110152.
27. S. Dietmair, N.E. Timmins, P.P. Gray, L.L. Nielsen, J.O. Krömer, Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol, Anal Biochem, 404, 2010, 155–164.
28. E.B. Atalay, S. Senturk, H.A. Kayali, Wild-type IDH1 Knockout Leads to G0/G1 Arrest, Impairs Cancer Cell Proliferation, Altering Glycolysis, and the TCA Cycle in Colon Cancer, Biochem Genet, 2023, 1–17.
29. G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal Chem, 31, 1959, 426–428.
30. K.S. Yoo, E.J. Lee, B.S. Patil, Underestimation of pyruvic acid concentrations by fructose and cysteine in 2, 4‐dinitrophenylhydrazine‐mediated onion pungency test, J Food Sci, 76, 2011, C1136–C1142.
31. H. Ayar-Kayali, L. Tarhan, Vancomycin antibiotic generation and TCA-glyoxalate pathways depending on the glucose concentration in Amycolatopsis orientalis, Enzyme Microb Technol, 38, 2006, 727–734.
32. G.M. Bodner, Metabolism Part II: The tricarboxylic acid (TCA), citric acid, or Krebs cycle, J Chem Educ, 63, 1986, 673.
33. P.H. Chen, L. Cai, K. Huffmanet, C. Yang, J. Kim, B. Faubert, L. Boroughs, B. Ko, J. Sudderth, E.A. McMillan, L. Girard, D. Chen, M. Peyton, M.D. Shields, B. Yao, D.S. Shames, H.S. Kim, B. Timmons, I. Sekine, R. Britt, S. Weber, L.A. Byers, J.V. Heymach, J. Chen, M.A. White, J.D. Minna, G. Xiao, R.J. DeBerardinis, Metabolic diversity in human non-small cell lung cancer cells, Mol Cell, 76, 2019, 838–851.
34. A.N. Lau, Z. Li, L.V. Danai, A.M. Westermark, A.M. Darnell, R. Ferreira, V. Gocheva, S. Sivanand, E.C. Lien, K.M. Sapp, J.R. Mayers, G. Biffi, C.R. Chin, S.M. Davidson, D.A. Tuveson, T. Jacks, N.J. Matheson, O. Yilmaz, M.G. Vander Heiden, Dissecting cell-type-specific metabolism in pancreatic ductal adenocarcinoma, Elife, 9, 2020, e56782.
35. R.A. Cairns, I.S. Harris, T.W. Mak, Regulation of cancer cell metabolism, Nat Rev Cancer, 11, 2011, 85–95.
36. E.D. Montal, R. Dewi, K. Bhalla, L. Ou, B.J. Hwang, A.E. Ropell, C. Gordon, W.J. Liu, R.J. DeBerardinis, J. Sudderth, W. Twaddel, L.G. Boros, K.R. Shroyer, S. Duraisamy, R. Drapkin, R.S. Powers, J.M. Rohde, M.B. Boxer, K.K. Wong, G.D. Girnun, PEPCK coordinates the regulation of central carbon metabolism to promote cancer cell growth, Mol Cell, 60, 2015, 571–583.
37. H. Krebs, W.A. Johnson, The role of citric acid in intermediate metabolism in animal tissues, FEBS letters, 117, 1980, K2-K10.
38. J. Eniafe, S. Jiang, The functional roles of TCA cycle metabolites in cancer, Oncogene, 40, 2021, 3351–3363.
39. K. Dettmer, F.C. Vogl, A.P. Ritter, W. Zhu, N. Nürnberger, M. Kreutz, P.J. Oefner, W. Gronwald, E. Gottfried, Distinct metabolic differences between various human cancer and primary cells, Electrophor, 34, 2013, 2836–2847.
40. D.A. Scott, A.D. Richardson, F.V. Filipp, C.A. Knutzen, G.G. Chiang, Z.A. Ronai, A.L. Osterman, J.W. Smith, Comparative metabolic flux profiling of melanoma cell lines beyond the warburg effect, J Biol Chem, 286, 2011, 42626–42634.
41. G. Czibik, V. Steeples, A. Yavari, H. Ashrafian, Citric acid cycle intermediates in cardioprotection, Circ Cardiovasc Genet, 7, 2014, 711–719.
42. K. Smolková, A. Dvořák, J. Zelenka, L. Vítek, P. Ježek, Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells, Int J Biochem Cell Biol, 65, 2015, 125–133.
43. P.K. Mukherjee, P. Funchain, M. Retuerto, R.J. Jurevic, N. Fowler, B. Burkey, C. Eng, M.A. Ghannouma, Metabolomic analysis identifies differentially produced oral metabolites, including the oncometabolite 2-hydroxyglutarate, in patients with head and neck squamous cell carcinoma, BBA Clin, 7, 2017, 8–15.