Nucleic Acids (2LC1 and 2LCL1) (homeopathic)
"The doctors who pioneered their use in cancer have found that they "re-balance" the weakened immune system, and there are documented cases of recovery which include many advanced cancers – metastatic liver and breast cancer, [leukemias] etc. However, because nucleic acids are usually present in infinitesimal quantities, and high doses are toxic, the principles of homeopathy have been applied to their orthomolecular use."
ROLE OF THIAMIN (VITAMIN B-1) AND TRANSKETOLASE
Abstract: Metabolic control analysis predicts that stimulators of transketolase enzyme synthesis such as thiamin (vitamin B-1) support a high rate of nucleic acid ribose synthesis necessary for tumor cell survival, chemotherapy resistance, and proliferation. Metabolic control analysis also predicts that transketolase inhibitor drugs will have the opposite effect on tumor cells. This may have important implications in the nutrition and future treatment of patients with cancer.
Tumor cells have been shown to heavily utilize the nonoxidative transketolase pathway for ribose synthesis to build nucleic acid, in addition to the oxidative glucose-6-phosphate dehydrogenase (G-6-PD) pathway, the main ribose-producing reaction in the classical model of the pentose cycle ( 1-4). Metabolic substrate flux control coefficients of enzymes can be used to identify target sites that have maximal influence on the metabolic pathway flux, thereby assisting in the development of drugs that will have the highest possible efficacy ( 5). Transketolase not only predominates in the nucleic acid ribose synthesis process in mammalian cells but also has a remarkably high substrate flux control coefficient in liver cells and erythrocytes (approx 0.5) ( 6-7). It has recently been suggested that transketolase and its cofactor thiamin have a very high growth control coefficient in the Ehrlich tumor model ( 8). Therefore, modulation of transketolase has the potential to control substrate flow through the nonoxidative branch of the pentose cycle and control cell growth, a mechanism not yet targeted by anticancer drags.
Metabolic control analysis predicts that stimulators of transketolase enzyme synthesis such as dietary thiamin support tumor cell survival and proliferation. Unfortunately, thiamin, a regulator of transketolase mRNA synthesis in the transketolase pathway and a cofactor of transketolase, is a common supplement in a wide variety of foods and is also given in high amounts to cancer patients as a prophylactic against nutritional deficiencies ( 9). Because of the crucial role played by thiamin in enabling a high rate of ribose synthesis in tumor cells, supplementation of the cancer patient's diet with thiamin may adversely affect cancer treatment.
Role of Pentose Cycle Reactions in Tumor Cell Nucleic Acid Ribose Synthesis--the Standing Model in Light of New Discoveries
The classical role of the pentose cycle in mammalian cells is the production of NADPH through carbon flow from C6 sugars (hexoses) to C5 sugars (pentoses). Pentoses are subsequently recycled back into glycolysis through carbon interchange reactions within the nonoxidative branch of the pentose cycle by transaldolase, transketolase, aldolase, and isomerases. The classic model of hexose-pentose-hexose cycling allows the oxidative reactions to produce only a limited amount of ribose in mammalian cells ( 1), and the contribution of the nonoxidative pathway has been difficult to determine. It is widely accepted that carbon interchange reactions are responsible for the labeling pattern of ribose isolated from nucleic acid, which nevertheless strongly indicates the involvement of transketolase in ribose synthesis of tumors by its labeling pattern on C-1 and C-5 with 13C- or 14C-labeled glucose as the tracer ( 2-4). The underlying assumption used by Katz and Rognstad ( 1) for modeling the pentose cycle in a comprehensive study of 1967 was that ribose production through the irreversible oxidative pathway significantly exceeds the amount of ribose deposited into nucleic acid on the basis of the RNA and DNA content of tumor cells. Following this assumption, on the basis of what was known in 1967, it was further reasonable to conclude that transketolase is not involved in net ribose synthesis in tumor cells, but in carbon exchange, which is responsible for the heavy labeling of C-5 of ribose in nucleic acid.
With the later discovery of continuously synthesized homologous nuclear RNA and nuclear ribosomal RNA precursors (half time 25 rain) ( 10), it is clear that mammalian cell nucleic acid synthesis and turnover rates cannot accurately be estimated by the nucleic acid content of cells, as was assumed by Katz and Rognstad ( 1). It is now known that intron sequences are continuously cut out from RNA, then rapidly degrade during posttranslational processing, which reduces the actual RNA amount by ~ 100-fold in the measured RNA content in the cytoplasm. Therefore, it cannot be assumed that continuous RNA synthesis, processing, and turnover with continuous DNA repair observed in mammalian cells are totally accounted for by the oxidative reactions of the pentose cycle. It has been recently shown that the nonoxidative reactions of the pentose cycle play a major role in cell proliferation by producing 5-phosphoribosyl-1-pyrophosphate directly from glycolytic metabolites in eight different tumor cell lines ( 11). The recent report that poor prognosis was found in nasopharyngeal cancer patients with low G-6-PD enzyme activity in their tumors clearly indicates that transketolase has a major role in tumor cell physiology as well as in determining cellular response to tumor therapy ( 12). Oxythiamin, the chemically modified form of thiamin and a noncompetitive inhibitor of transketolase, decreases in vitro and in vivo tumor cell proliferation and ribose synthesis directly through the nonoxidative steps of the pentose cycle ( 3). Oxythiamin induces a prominent cell cycle arrest in the G0-G1 phase, as demonstrated in tumor-bearing mice ( 13). Despite the new facts about RNA/DNA synthesis and reports demonstrating the importance of transketolase in the ribose synthesis process of tumors that compromise many of the critical assumptions used by Katz and Rognstad ( 1), the original model of the pentose cycle has not been revisited to address the potential underestimation of ribose synthesis as well as the contribution of transketolase and its cofactor thiamin pyrophosphate (thiamin-PP) to net ribose synthesis.
Metabolic Control Analysis and Its Application to Tumor Cell Nucleic Acid Ribose Synthesis Pathways
Metabolic control analysis is a relatively new field in biochemistry in which the contribution of enzymes to the control of a pathway substrate flux is determined and expressed quantitatively by means of substrate flux control coefficients. Essentially, substrate flow control through linear metabolic pathways is distributed among enzymes; therefore, linearly connected metabolic reactions of the pentose cycle individually regulate hexose, pentose, and triose substrate flow. For each participating enzyme in the pathway, the contribution to substrate flow regulation can be measured and described in a control coefficient with a value between 0 and 1. Key enzymes with a strong regulatory effect (formerly rate-limiting steps) have flux control coefficients approaching 1; enzymes with weak regulatory potential have lower flux control coefficients ( 5). The effects of regulatory drags on substrate flow are likely increased when they target enzymes with high flux control coefficients within the metabolic pathway.
Pentose cycle reactions have been characterized in terms of their flux control coefficients in liver tissue, erythrocytes, certain bacteria, and, very recently, in Ehrlich ascites tumor cells. In rat liver, it is determined that the enzymes that strongly regulate the oxidative path of the pentose cycle are glucokinase (EC 126.96.36.199) and G-6-PD (EC 188.8.131.52), with flux control coefficients achieving 0.6 and 0.42, respectively ( 6). In the same tissue, the nonoxidative pathway substrate flux control is dominated by transketolase (EC 184.108.40.206), with a flux control coefficient of 0.58. On the other hand, ribose phosphate isomerase (0.23), epimerase (0.14), transaldolase (0.08), and glucose phosphate isomerase (0.04) have limited strength in the regulation of carbon flow through the cycle. A similar substrate flux control role of transketolase in the nonoxidative pentose cycle (0.74) has been described in human erythrocytes ( 7). The growth control coefficient of transketolase in the Ehrlich tumor model was recently reported to be very high ( 14, 15). The strong tumor growth control properties of transketolase and thiamin-PP make nonoxidative nucleic acid ribose synthesis not only a promising new target for cancer treatment but also a tumor growth-promoting site with a known mechanism of action.
A rational approach to inhibit pentose synthesis in tumor cells is to target key enzymes with high flow control coefficients within the pentose cycle. Accordingly, the oxidative pathway is best controlled through G-6-PD and the nonoxidative pathway through transketolase. Although G-6-PD effectively regulates the oxidative pathway, its role in ribose production is limited in tumor cells, and therefore G-6-PD inhibitors such as dehydroepiandrosterone or its derivatives do not accomplish their mission in experimental tumor therapy ( 3).
Transketolase function is strictly dependent on the presence of its coenzyme thiamin (vitamin B-1), which strongly binds to the enzyme protein. Thiamin has been described as a regulator of transketolase enzyme protein synthesis in normal and tumor cells ( 16, 17). Consequently, excess thiamin has a major disadvantage in the nutrition and treatment of patients with cancer ( 9). The model of tumor cell ribose synthesis and the application of metabolic control analysis to its process indicate that excess thiamin, which promotes the synthesis of the transketolase enzyme protein and increases its activity, should be avoided. For example, if transketolase activity increases by 100% (doubled) because of increased thiamin intake, the rate of ribose synthesis and tumor cell growth will also increase by 58% (100% x 0.58, where 0.58 is the growth control coefficient of transketolase in human liver cells). If relative thiamin deficiency is present in tumor cells with excess transketolase apoenzyme protein waiting for thiamin to arrive through the bloodstream, the regulating effect of thiamin on nucleic acid synthesis as the critical cofactor for the synthesis of ribose is even more dramatic. Thiamin deficiency has been described in experimental tumors ( 18) as well as in patients undergoing chemotherapy ( 19), and it is also commonly observed in patients with early stages of breast and bronchial carcinoma ( 20). Therefore, significant modifications are necessary in the treatment of cancer with greater emphasis on more rational and discriminating use of thiamin supplementation, while new transketolase inhibitor drags need to be evaluated to limit transketolase activity and glucose use of tumor cells for nucleic acid synthesis and cell proliferation.
Tumor Cell Nucleic Acid Ribose Synthesis Pathways --Data Behind the Theory
Studies utilizing isotopically labeled glucose carbons recovered from in vivo hosted Yoshida tumors or in vitro cultured HeLa, Mia, H9, and Hep G2 cells unequivocally demonstrated that the nonoxidative part of the pentose cycle plays a significant role in tumor cell nucleic acid synthesis ( 1-4). More than 70% of nucleic acid ribose in these tumor cells was derived through transketolase, transaldolase, and triose phosphate isomerase reactions and only 10-15% was derived directly through the oxidative steps. In a recent experiment carried out in our laboratory, lung epithelial carcinoma (H411) cells demonstrated nucleic acid ribose synthesis almost entirely (99%) via the transketolase pathway, which indicates that there may be human tumors with poor prognosis that completely depend on transketolase and thiamin for de novo nucleic acid synthesis (20a).
Effect of Thiamin vs. Transketolase Inhibitors on Tumor Cell Nucleic Acid Synthesis and Proliferation
Although circumstantial links have been suspected between increased glucose utilization, pentose cycle activity, ribose synthesis, and tumor cell growth, few studies have directly addressed the mechanistic role of the oxidative and nonoxidative reactions in nucleic acid synthesis, cell transformation, and differentiation. Inasmuch as tumor growth is significantly stimulated during periods of nutrition support ( 21), the role of dietary supplements and the ramifications of thiamin supplementation to cancer patients are clinically relevant topics to address for scientists, dietitians, and care physicians who study, plan, and apply supportive treatments to cancer patients. Data in the medical literature lend wide support to the theory that thiamin-PP promotes mammalian cell nucleic acid synthesis and proliferation. Early reports from the late 1970s indicated that the reprogramming of the pentose cycle toward an increase in the nonoxidative path flux occurs in liver tumor cells ( 22). Liver tumor cells, but not regenerating or developing normal liver cells, showed a severalfold increase in 5-phospho-alpha-D-ribose-1-diphosphate synthase activity, which demonstrates the strong dependence of tumor cells on ribose phosphate production and nucleic acid synthesis ( 23). The intensive utilization of ribose and the salvage pathways for purine nucleotide synthesis have been demonstrated in leukemia cells of all four types (ALL, CLL, AML, and CML), which were rapidly saturated with low concentrations of 5-phosphoribosyl-1-pyrophosphate and less inhibited by the physiological feedback inhibitor adenosine 5'-monophosphate in culture ( 24). These findings were recently confirmed in human colon tumors and in chemically induced colon tumors in rats, where a severalfold increase in transketolase enzyme activity was demonstrated ( 25, 26).
Oxythiamin, a thiamin derivative and noncompetitive inhibitor of transketolase, decreases DNA/RNA levels and proliferation of in vivo hosted Ehrlich tumor cells. Oxythiamin also reduced the ribose fraction in nucleic acid that arrived directly through the transketolase pathway in MIA pancreatic adenocarcinoma cells in culture. These changes in the nucleic acid ribose moiety were accompanied by a significant decrease in tumor cell proliferation in cultures of MIA cells as well as oxythiamin-treated mice hosting Ehrlich tumors ( 3). Decreased tumor growth was the result of a prominent G1 cycle arrest without signs of direct tumor cell or host toxicity after oxythiamin treatment ( 13). On the other hand, thiamin increases the proliferation of human endothelial cells ( 27) and was shown to be trophic in neuroblastoma cell cultures ( 28). It has also been shown that substrate flux through the nonoxidative pentose cycle is rapid and large in cultured liver tumor cells compared with the oxidative reactions and glucose intake of the cell ( 29).
High-affinity thiamin transport deficiency in cultured fibroblasts results in the cessation of cell proliferation followed by increased cell death. Excess thiamin promptly rescues these cells and reestablishes normal cell proliferation in culture ( 30). High-affinity thiamin transporter protein deficiency clinically manifests with thiamin-responsive megaloblastic anemia, which points to DNA synthesis disturbances in proliferating bone marrow cells. Although the link between thiamin and DNA synthesis in erythropoietic cells has been known since 1969 ( 31), no studies in the medical literature have attempted to elucidate the mechanism by which thiamin directly affects DNA synthesis and cell proliferation. The fact that thiamin transporter protein-deficient cultured cells did not proliferate in the presence of glucose, which bypasses the thiamin-dependent steps in nucleic acid synthesis, demonstrates the limited contribution of G-6-PD to nucleic acid ribose synthesis and proliferation. It is apparent from these results that thiamin and ribose are equally important and required for rapid cell proliferation and that the metabolic control on this process is exerted by transketolase and thiamin-PP together. On the other hand, the limited synthesis of ribose phosphate accomplished through G-6-PD in normal cells makes future nonoxidative ribose synthesis inhibitors both selective and effective primarily on tumor cells.
This review highlights the crucial role of transketolase and its cofactor thiamin-PP in nonoxidative ribose synthesis, which predominates in all tumor cells studied so far. The strong involvement of the transketolase pathway in tumor cell nucleic acid synthesis is not emphasized in medical textbooks, although it is a crucial process in tumor cell nucleic acid synthesis, proliferation, and growth. Nutritional supplements and prepared food additives that include thiamin need to be reevaluated for cancer patients and for the population as a whole in countries with high cancer rates. The possible role of thiaminase, the natural thiamine-degrading enzyme found abundantly in fish, meat, and vegetables, which seems to be beneficial for cancer patients, needs further investigations. The varying cancer rates between countries where diets containing natural thiaminases are regularly consumed in the form of raw or fermented fish, vegetables, or roasted insects (Asia and Africa) compared with those in countries where thiamin-enriched food products are preferred (United States and Western Europe) need to be explored.
Acknowledgments and Notes
The authors acknowledge help from Owen Johnson, a cancer research advocate, who volunteered in our laboratory and assisted in the preparation of the manuscript in memory of his wife Marilyn, who died of cancer. The text of the manuscript was coedited by Dale Chenoweth (Austin, TX). This work was supported by Scientific and Education Office of the Spanish Government Direccion General de Investigacion Cientifica y Tecnica Health Program Grant BIO98-0365, European Commission INCOCOPERNICUS Grant ERBIC 15CT960307, and the National Institutes of Health. Address reprint requests to Laszlo G. Boros, MD, Endocrinology and Metabolism, UCLA School of Medicine, Harbor-UCLA Research and Education Institute, RB-I, 1124 West Carson St., Torrance, CA 90502. E-mail: email@example.com.
Submitted 13 October 1999; accepted in final form 23 November 1999.
1. Katz, J, and Rognstad, R: The labeling of pentose phosphate from glucose-14C and estimation of the rates of transaldolase, transketolase, the contribution of the pentose cycle, and ribose phosphate synthesis. Biochemistry 6, 2227-2247, 1967.
2. Horecker, BL, Domagk, G, and Hiatt, HH: A comparison of 14C labeling patterns in deoxyribose and ribose in mammalian cells. Arch Biochem Biophys 78, 510-517, 1958.
3. Bores, LG, Puigjaner, J, Cascante, M, Lee, WNP, Brandes, JL, et al.: Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation. Cancer Res 57, 4242-4248, 1997.
4. Macallan, DC, Fullerton, CA, Neese, RA, Haddock, K, Park, SS, et al.: Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: studies in vitro, in animals, and in humans. Proc Natl Acad Sci USA 95, 708-713, 1998.
5. Schuster, S, Dandekar, T, and Fell, AD: Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering. Trends Biotech 17, 53-60, 1999.
6. Sabate, L, Franco, R, Canela, El, Centelles, JJ, and Cascante, M: A model of the pentose phosphate pathway in rat liver cells. Mol Cell Biochem 142, 9-17, 1995.
7. Berthon, HA, Kuchel, PW, and Nixon, PF: High control coefficient of transketolase in the nonoxidative pentose phosphate pathway of human erythrocytes: NMR, antibody, and computer simulation studies. Biochemistry 31, 12792-12798, 1992.
8. Cascante, M, Comin, B, Rais, B, Boren, J, Centelles, JJ, et al.: Application of metabolic control analysis to the design of a new strategy for cancer therapy. In Technological and Medical Implications of Metabolic Control Analysis, A Cornish-Bowden and ML Cardenas (eds). Dordrecht, The Netherlands: Kluwer Academic, 2000, pp 173-180.
9. Boros, LG, Brandes, JL, Lee, WN, Cascante, M, Puigjaner, J, et al.: Thiamine supplementation to cancer patients: a double-edged sword. Anticancer Res 18, 595-602, 1998.
10. Brandhorst, PB, and McConkey, EH: Relationship between nuclear and cytoplasmic poly(adenylic acid). Proc Natl Acad Sci USA 72, 3580-3584, 1975.
11. Chesney, J, Mitchell, R, Benigni, F, Bacher, M, Spiegel, L, et al.: An inducible gene product for 6-phosphofructo-2-kinase with an AU-rich instability element: role in tumor cell glycolysis and the Warburg effect. Proc Natl Acad Sci USA 96, 3047-3052, 1999.
12. Cheng, AJ, Chang, JT, and Chiu, D: Poor prognosis was found in nasopharyngeal cancer patients with low glucose-6-phosphate-dehydrogenase (abstr). Proc Am Assoc Cancer Res 40, 2099, 1999.
13. Rais, B, Comin, B, Puigjaner, J, Brandes, JL, Creppy, E, et al.: Oxythiamine and dehydroepiandrosterone induce a G1 cycle arrest in Ehrlich tumor cells through inhibition of the pentose cycle. FEBS Lett 456, 113-118, 1999.
14. Cascante, M: Application of metabolic control analysis to the design of a new strategy for cancer therapy (abstr). MCA 99, NATO Advanced Research Workshop--Technical and Medical Implications of Metabolic Control Analysis. Budapest, Hungary, 10-16 April 1999, p 14.
15. Cornish-Bowden, A: Metabolic control analysis in biotechnology and medicine. Nat Biotechnol 17, 641-643, 1999.
16. Pekovich, SR, Martin, PR, and Singleton, CK: Thiamine deficiency decreases steady-state transketolase and pyruvate dehydrogenase but not et-ketoglutarate dehydrogenase mRNA levels in three human cell types. J Nutr 128, 683-687, 1998.
17. Sheu, KF, Calingasan, NY, Dienel, GA, Baker, H, Jung, EH, et al.: Regional reductions of transketolase in thiamine-deficient rat brain. J Neurochem 67, 684-691, 1996.
18. Trebukhina, RV, Ostrovsky, YM, Shapot, VS, Petushok, VG, Velichko, MG, et al.: Thiamine metabolism in the liver of mice with Ehrlich's ascites carcinoma. Neoplasma 29, 257-268, 1982.
19. Trebuhkina, R: Metabolism of thiamin-diphosphate in animals with Ehrlich's ascites carcinoma and patients after treatment with cyclophosphamide treatment. In Biochemistry and Physiology of Thiamin Diphosphate Enzymes. Blauberen, Germany: Heinrich-Fabri Inst, 1996, pp 438-445.
20. Basu, TK, and Dickerson, JW: The thiamine status of early cancer patients with particular reference to those with breast and bronchial carcinomas. Oncology 33, 250-252, 1976.
20a. Boros, LG, Torday, JS, Lim, S, Bassilian, S, Cascante, M, et al.: Transforming growth factor-beta2 promotes glucose carbon incorporation into nucleic acid ribose through the nonoxidative pentose cycle in lung epithelial carcinoma cells. Cancer Res 60, 1183-1185, 2000.
21. Torosian, MH: Stimulation of tumor growth by nutrition support. J Parent Enteral Nutr 16, 76S-82S, 1992.
22. Heinrich, PC, Morris, HP, and Weber, G: Behavior of transaldolase (EC 220.127.116.11) and transketolase (EC 18.104.22.168). Activities in normal, neoplastic, differentiating, and regenerating liver. Cancer Res 36, 3189-3197, 1976.
23. Balo-Banga, JM, and Weber, G: Increased 5-phospho-alpha-D-ribose-1-diphosphate synthetase (ribosephosphate pyrophosphokinase, EC 22.214.171.124) activity in rat hepatomas. Cancer Res 44, 5004-5009, 1984.
24. Becher, H, Weber, M, and Lohr, GW: Purine nucleotide synthesis in normal and leukemic blood cells. Kiln Wochenschr 56, 275-283, 1978.
25. Butler, RN, Antoniou, D, Butler, W, Porter, S, and McIntosh, G: Maximal catalytic activity of the nonoxidative pentose pathway: a new marker for colonic transformation (abstr). Gastroenterology 14, G2341, 1998.
26. Antoniou, D, Porter, SN, Thomson-Roberts, IC, and Butler, R: Elevated nonoxidative pentose pathway activity: a new risk marker for colon cancer (abstr). Gastroenterology 14, G2280, 1998.
27. La Selva, M, Beltramo, E, Pagnozzi, F, Bena, E, Molinatti, PA, et al.: Thiamine corrects delayed replication and decreases production of lactate and advanced glycation end-products in bovine retinal and human umbilical vein endothelial cells cultured under high glucose conditions. Diabetologia 39, 1263-1268, 1996.
28. Bettendorff, L: A non-cofactor role of thiamine derivatives in excitable cells? Arch Physiol Biochem 104, 745-751, 1996.
29. Lee, WN, Boros, LG, Puigjaner, J, Bassilian, S, Lim, S, et al.: Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose. Am J Physiol Endocrinol Metab 274, E843-E851, 1998.
30. Stagg, AR, Fleming, JC, Baker, MA, Sakarnoto, M, Cohen, N, et al.: Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts. J Clin Invest 103, 723-729, 1999.
31. Rogers, LE, Porter, SF, and Sidbury, JB: Thiamine-responsive mcgaloblastic anemia. J Pediatr 74, 494-504, 1969.
By Marta Cascante; Josep J. Centelles; Richard L. Veech; Wai-Nang Paul Lee and Laszlo G. Boros
M. Cascante and J.J. Centelles are affiliated with the Department of Biochemistry and Molecular Biology, Institut d'Investigacions Biomediques August Pi i Sunyer, University of Barcelona, Barcelona, Catalonia 08028, Spain. R. L. Veech is affiliated with The National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852. W.-N. P. Lee and L. G. Boros are affiliated with the University of California, Los Angeles, School of Medicine, Harbor-UCLA Research and Education Institute, Torrance, CA 90502.