In Vitro and In Silico Studies on Cytotoxic Properties of Oxythiamine and 2'-Methylthiamine.

It is important to search for cytostatic compounds in order to fight cancer. One of them could be 2'-methylthiamine, which is a thiamine antimetabolite with an additional methyl group at the C-2 carbon of thiazole. So far, the cytostatic potential of 2'-methylthiamine has not been studied. We have come forward with a simplified method of synthesis using commercially available substrates and presented a comparison of its effects, as boosted by oxythiamine, on normal skin fibroblasts and HeLa cancer cells, having adopted in vitro culture techniques. Oxythiamine has been found to inhibit the growth and metabolism of cancer cells significantly better than 2'-methylthiamine (GI50 36 and 107 µM, respectively), while 2'-methylthiamine is more selective for cancer cells than oxythiamine (SI = 180 and 153, respectively). Docking analyses have revealed that 2'-methylthiamine (ΔG -8.2 kcal/mol) demonstrates a better affinity with thiamine pyrophosphokinase than thiamine (ΔG -7.5 kcal/mol ) and oxythiamine (ΔG -7.0 kcal/mol), which includes 2'-methylthiamine as a potential cytostatic. Our results suggest that the limited effect of 2'-methylthiamine on HeLa arises from the related arduous transport as compared to oxythiamine. Given that 2'-methylthiamine may possibly inhibit thiamine pyrophosphokinase, it could once again be considered a potential cytostatic. Thus, research should be carried out in order to find the best way to improve the transport of 2'-methylthiamine into cells, which may trigger its cytostatic properties.

Targeting the pentose phosphate pathway increases reactive oxygen species and induces apoptosis in thyroid cancer cells.

The pentose phosphate pathway (PPP) plays an important role in the biosynthesis of ribonucleotide precursor and NADPH. Cancer cells frequently increase the flux of glucose into the PPP to support the anabolic demands and regulate oxidative stress. Consistently, metabolomic analyses indicate an upregulation of the PPP in thyroid cancer. In the present study, we found that the combination of glucose-6-phosphate dehydrogenase (G6PD) and transketolase inhibitors (6-aminonicotinamide and oxythiamine) exerted an additive or synergistic effect on cell growth inhibition in thyroid cancer cells. Targeting PPP significantly increased cellular reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress and apoptosis. Suppressed cell viability could be partially rescued with treatment with the ROS scavenger or apoptosis inhibitor but not ER-stress inhibitor. Taken together, dual PPP blockade leads to pharmacologic additivity or synergism and causes ROS-mediated apoptosis in thyroid cancer cells.

Dual effects for lovastatin in anaplastic thyroid cancer: the pivotal effect of transketolase (TKT) on lovastatin and tumor proliferation.

This study tested the hypothesis that the effects of lovastatin on anaplastic thyroid cancer cell growth are mediated by upregulation of transketolase (TKT) expression. The effects of lovastatin on TKT protein levels in ARO cells were determined using western blot and proteomic analyses. After treatment with lovastatin and oxythiamine, the in vitro and in vivo growth of ARO cells was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assays and tumor xenografts in nude mice. TKT protein expression in the ARO tumors was assessed using immunohistochemistry analysis. Proteomic analysis revealed that 25 µM lovastatin upregulated TKT expression. Co-treatment of ARO cells with 1 µM lovastatin + 1 µM oxythiamine increased TKT protein expression compared with control levels; however, no differences were observed with 10 µM lovastatin + 1 µM oxythiamine. Furthermore, treatment with either oxythiamine or lovastatin alone reduced ARO tumor expression of TKT, as well as decreased ARO cell proliferation in vitro and tumor growth in vivo. However, mice treated with both lovastatin and oxythiamine at the same time had tumor volumes similar to that of the untreated control group. We conclude that either lovastatin or oxythiamine reduced ARO cell growth; however, the combination of these drugs resulted in antagonism of ARO tumor growth.

EDIM-TKTL1/Apo10 Blood Test: An Innate Immune System Based Liquid Biopsy for the Early Detection, Characterization and Targeted Treatment of Cancer.

Epitope detection in monocytes (EDIM) represents a liquid biopsy exploiting the innate immune system. Activated monocytes (macrophages) phagocytose unwanted cells/cell fragments from the whole body including solid tissues. As they return to the blood, macrophages can be used for a non-invasive detection of biomarkers, thereby providing high sensitivity and specificity, because the intracellular presence of biomarkers is due to an innate immune response. Flow cytometry analysis of blood enables the detection of macrophages and phagocytosed intracellular biomarkers. In order to establish a pan-cancer test, biomarkers for two fundamental biophysical mechanisms have been exploited. The DNaseX/Apo10 protein epitope is a characteristic of tumor cells with abnormal apoptosis and proliferation. Transketolase-like 1 (TKTL1) is a marker for an anaerobic glucose metabolism (Warburg effect), which is concomitant with invasive growth/metastasis and resistant to radical and apoptosis inducing therapies. The detection of Apo10 and TKTL1 in blood macrophages allowed a sensitive (95.8%) and specific (97.3%) detection of prostate, breast and oral squamous cell carcinomas. Since TKTL1 represents a drugable target, the EDIM based detection of TKTL1 enables a targeted cancer therapy using the vitamin derivatives oxythiamine or benfo-oxythiamine.

Mechanical insights of oxythiamine compound as potent inhibitor for human transketolase-like protein 1 (TKTL1 protein).

Transketolase is a connecting link between glycolytic and pentose phosphate pathway, which is considered as the rate-limiting step due to synthesis of large number of ATP molecule and it can be proposed as a plausible target facilitating the growth of cancerous cells suggesting its potential role in cancer. Oxythiamine, an antimetabolite has been proved to be an efficient anticancerous compound in vitro, but its structural elucidation of the inhibitory mechanism has not yet been done against the human transketolase-like 1 protein (TKTL1). The three-dimensional (3D) structure of TKTL1 protein was modeled and subjected for refinement, stability and validation. Based on the reported homologs of transketolase (TKT), the active site residues His46, Ser49, Ser52, Ser53, Ile56, Leu82, Lys84, Leu123, Ser125, Glu128, Asp154, His160, Thr216 and Lys218 were identified and considered for molecular-modeling studies. Docking studies reveal the H-bond interactions with residues Ser49 and Lys218 that could play a major role in the activity of TKTL1. Molecular dynamics (MD) simulation study was performed to reveal the comparative stability of both native and complex forms of TKTL1. MD trajectory at 30 ns, confirm the role of active site residues Ser49, Lys84, Glu128, His160 and Lys218 in suppressing the activity of TKTL1. Glu128 is observed to be the most important residue for deprotonation state of the aminopyrimidine moiety and preferred to be the site of inhibitory action. Thus, the proposed mechanism of inhibition through in silico studies would pave the way for structure-oriented drug designing against cancer.

Metabolic response of LLC xenografted mice to oxythiamine, as measured by [¹H] NMR spectroscopy.

Oxythiamine (OT) has been proven to be a potential anticancer drug. With the help of NMR-based metabonomics, we studied the metabolic changes within tumor-bearing mice with different levels of OT administration using a C57BL/6 mouse Lewis lung carcinoma tumor transplantation model. We administered different concentrations of OT (75, 150, 300, and 600 mg∙kg(-1)∙day(-1)) to the mice orally for 2 weeks, recorded animal weights and tumor volumes, sacrificed the animals, and collected blood and tumor mass samples for nuclear magnetic resonance determination. Compared with the findings for the control (untreated) group, the tumor weights and volumes of the 150, 300, and 600 mg∙kg-1∙day-1 groups decreased with no difference among these OT groups. A large metabolite difference was observed in plasma metabolites between the blank and control groups, which indicated the success of the tumor-bearing model. The metabolites in tumor associated with thiamine-dependent enzymes (TDEs) underwent considerable change between the OT and control groups, exhibiting concentration dependence and enzyme specificity. The restriction of TDEs by OT may be a major mechanism underlying its anticancer effect. The role of OT as a potential anticancer drug and a dehydrogenase inhibitor should therefore be taken into consideration in future tumor research.

JC virus T-antigen regulates glucose metabolic pathways in brain tumor cells.

Recent studies have reported the detection of the human neurotropic virus, JCV, in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as ß-catenin and IRS-1, plays a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells and in glioblastoma xenografts that both endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by 5'-activated AMP kinase (AMPK), an important sensor of the AMP/ATP ratio in cells. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen prevents G1 arrest and sustains cells in the G2 phase during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on reactive oxygen species (ROS) production during glucose deprivation, and T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. Additionally, we have found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamide and oxythiamine, and that T-antigen modulates expression of the glycolytic enzyme, hexokinase 2 (HK2), and the pentose phosphate enzyme, transaldolase-1 (TALDO1), indicating a potential link between T-antigen and metabolic regulation. These studies point to the possible involvement of JCV T-antigen in medulloblastoma proliferation and the metabolic phenotype and may enhance our understanding of the role of viral proteins in glycolytic tumor metabolism, thus providing useful targets for the treatment of virus-induced tumors.

Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media.

OBJECTIVES: A new method of determining protein turnover by labeling protein with N amino acids was used in conjunction with serum-free cell culture to profile secreted proteins that are released by MIA PaCa-2 pancreatic cancer cells in culture. METHODS: MIA PaCa-2 cells were first cultured in Dulbecco modified Eagle medium (Gibco by Invitrogen, Carlsbad, Calif) with 10% fetal bovine serum, then in serum-free modified Eagle medium with or without 50% N algal amino acid mixture. The effect of oxythiamine chloride on secreteome was studied. Secreteome from cell culture media was analyzed by 2-dimensional (2D) gel electrophoresis. Differentially expressed proteins were detected and identified. Protein turnover rates were calculated according to the newly established method. Western blot and enzyme-linked immunosorbent assay were used to validate identified proteins. RESULTS: Among the 14 differentially expressed proteins after oxythiamine treatment, tissue inhibitor of metalloproteases-1 and cytokeratin-10 were identified as 2 newly synthesized secreted proteins caused by substantial N incorporation. The inhibition of tissue inhibitor of metalloproteases-1 expression in MIA PaCa-2 cells by oxythiamine treatment was first demonstrated by 2D gel electrophoresis and further validated by Western blotting and enzyme-linked immunosorbent assay analyses. CONCLUSIONS: Our method of labeling protein with N amino acids in conjunction with serum-free cell culture allows the identification of actively secreted proteins from pancreatic cancer cells and is a useful method for serum biomarker discovery.

Is transketolase like 1 a target for the treatment of differentiated thyroid carcinoma? A study on thyroid cancer cell lines.

Radioactive iodine-refractory [(18)F] fluorodeoxy-glucose-positron emission tomography-positive thyroid carcinomas represent especially aggressive tumors. Targeting glucose metabolism by the transketolase isoenzyme transketolase like 1 (TKTL-1) which is over-expressed in various neoplasms, may be effective. The correlation of TKTL-1 expression and the response to oxythiamine as the currently best-characterized inhibitor of transketolases was studied in differentiated thyroid cancer cell lines. We determined TKTL-1 expression, proliferation, glucose uptake and GLUT-1 expression in non-treated thyroid cells and recorded the effect of oxythiamine on iodide uptake and on thymidine uptake. TKTL 1 was highest expressed in cell lines derived from more invasive tumors but the expression level was not strongly correlated to proliferation rate, to GLUT-1 expression or to the response to oxythiamine. Oxythiamine showed only a weak effect in the TKTL-1 expressing cell lines. Over-expression of TKTL-1 is not an indicator for responsiveness to oxythiamine. More specific inhibitors should be tested.

Detection of transketolase in bone marrow-derived insulin-producing cells: benfotiamine enhances insulin synthesis and glucose metabolism.

Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus.

Comparative characteristic of thiamine antagonists on apoptosis induction in different types of nerve cell lines.

Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. The mechanisms of neurodegeneration induced by thiamine deficiency remain incompletely elucidated. The susceptibility of various types of nerve cells to thiamine (vitamin B) antagonists--oxythiamine (OT), pyrithiamine (PT) and amprolium (Am) was investigated. Four cell lines (neuronally differentiated rat PC-12, rat astrocytes DITNC, neuronally differentiated human SH-SY5Y and human astrocytic cells 1321N1) were used for experiments as neural cell models. When different cell types were cultivated with thiamine antagonists, a significant decrease of viability was detected in a time- and dose-dependent manner as demonstrated by the WST-1 colorimetric assay. These data were similar to those of caspase 3 activity and DNA fragmentation induced by thiamine antagonists. All tested cell lines were more vulnerable to OT and PT than to Am. Am displayed a pronounced damaging action on neuronal cells and had a modest influence on astrocytes. The last observation gives the basis to suppose, that neuronal cells need external arrival of thiamine more than astrocytes. Thus, the results testify that various types of nerve cells have different susceptibility to the thiamine antagonists and this relates to extent of apoptosis development.

Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells.

Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.

Pentose phosphate cycle oxidative and nonoxidative balance: A new vulnerable target for overcoming drug resistance in cancer.

The metabolic network of cancer cells confers adaptive mechanisms against many chemotherapeutic agents, but also presents critical constraints that make the cells vulnerable to perturbation of the network due to drug therapy. To identify these fragilities, combination therapies based on targeting the nucleic acid synthesis metabolic network at multiple points were tested. Results showed that cancer cells overcome single hit strategies through different metabolic network adaptations, demonstrating the robustness of cancer cell metabolism. Analysis of these adaptations also identified the maintenance of pentose phosphate cycle oxidative and nonoxidative balance to be critical for cancer cell survival and vulnerable to chemotherapeutic intervention. The vulnerability of cancer cells to the imbalance on pentose phosphate cycle was demonstrated by phenotypic phase plane analysis.

Zinc(II), cadmium(II) and mercury(II) complexes of the vitamin B1 antagonist oxythiamine.

The reaction of oxythiamine chloride hydrochloride (HOxTCl x HCl) with ZnCl2, CdCl2 and HgCl2 in ethanol yielded the complexes [ZnCl3(HOxT)], [CdCl3(HOxT)] and [HgCl3(HOxT)]. In water, the reaction with CdCl2 afforded [CdCl2(OxT)], but reaction with ZnCl2 or HgCl2 yielded unidentified products. The four new complexes were characterized by mass spectrometry and IR spectroscopy in the solid state and by 1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy in hexadeuterated dimethylsulfoxide (DMSO-d6), and three were also studied by X-ray diffractometry. In [ZnCl3(HOxT)] and [HgCl3(HOxT)] the oxythiamine ligand is bound to the metal via N(1') and adopts the V conformation exhibited by thiamine in biological contexts. The infrared (IR) spectrum of [CdCl3(HOxT)] suggests a similar coordination mode. In [CdCl2(OxT)] each OxT zwitterion coordinates to one Cd(II) ion via its N(1') atom and to another via its N(3') and O atoms, giving rise to a polymeric chain along the x-axis. The coordination number of the metal is made up to six by Cdc...Cl interactions, two of which link the polymeric chains in pairs. This seems to be the first metal complex containing the oxythiamine ligand as a zwitterion, with the N(3')-H/O(4'alpha)-H group deprotonated. Neither HOxTCl nor its zinc(II) complex showed any significant activity in vitro against HeLa cells.

The effect of thiamine supplementation on tumour proliferation. A metabolic control analysis study.

Thiamine deficiency frequently occurs in patients with advanced cancer and therefore thiamine supplementation is used as nutritional support. Thiamine (vitamin B1) is metabolized to thiamine pyrophosphate, the cofactor of transketolase, which is involved in ribose synthesis, necessary for cell replication. Thus, it is important to determine whether the benefits of thiamine supplementation outweigh the risks of tumor proliferation. Using oxythiamine (an irreversible inhibitor of transketolase) and metabolic control analysis (MCA) methods, we measured an in vivo tumour growth control coefficient of 0.9 for the thiamine-transketolase complex in mice with Ehrlich's ascites tumour. Thus, transketolase enzyme and thiamine clearly determine cell proliferation in the Ehrlich's ascites tumour model. This high control coefficient allows us to predict that in advanced tumours, which are commonly thiamine deficient, supplementation of thiamine could significantly increase tumour growth through transketolase activation. The effect of thiamine supplementation on tumour proliferation was demonstrated by in vivo experiments in mice with the ascites tumour. Thiamine supplementation in doses between 12.5 and 250 times the recommended dietary allowance (RDA) for mice were administered starting on day four of tumour inoculation. We observed a high stimulatory effect on tumour growth of 164% compared to controls at a thiamine dose of 25 times the RDA. This growth stimulatory effect was predicted on the basis of correction of the pre-existing level of thiamine deficiency (42%), as assayed by the cofactor/enzyme ratio. Interestingly, at very high overdoses of thiamine, approximately 2500 times the RDA, thiamine supplementation had the opposite effect and caused 10% inhibition of tumour growth. This effect was heightened, resulting in a 36% decrease, when thiamine supplementation was administered from the 7th day prior to tumour inoculation. Our results show that thiamine supplementation sufficient to correct existing thiamine deficiency stimulates tumour proliferation as predicted by MCA. The tumour inhibitory effect at high doses of thiamine is unexplained and merits further study.

Oxythiamine and dehydroepiandrosterone induce a G1 phase cycle arrest in Ehrlich's tumor cells through inhibition of the pentose cycle.

Transketolase (TK) reactions play a crucial role in tumor cell nucleic acid ribose synthesis utilizing glucose carbons, yet, current cancer treatments do not target this central pathway. Experimentally, a dramatic decrease in tumor cell proliferation after the administration of the TK inhibitor oxythiamine (OT) was observed in several in vitro and in vivo tumor models. Here, we demonstrate that pentose cycle (PC) inhibitors, OT and dehydroepiandrosterone (DHEA), efficiently regulate the cell cycle and tumor proliferation processes. Increasing doses of OT or DHEA were administered by daily intraperitoneal injections to Ehrlich's ascites tumor hosting mice for 4 days. The tumor cell number and their cycle phase distribution profile were determined by DNA flow histograms. Tumors showed a dose dependent increase in their G0-G1 cell populations after both OT and DHEA treatment and a simultaneous decrease in cells advancing to the S and G2-M cell cycle phases. This effect of PC inhibitors was significant, OT was more effective than DHEA, both drugs acted synergistically in combination and no signs of direct cell or host toxicity were observed. Direct inhibition of PC reactions causes a G1 cell cycle arrest similar to that of 2-deoxyglucose treatment. However, no interference with cell energy production and cell toxicity is observed. PC inhibitors, specifically ones targeting TK, introduce a new target site for the development of future cancer therapies to inhibit glucose utilizing pathways selectively for nucleic acid production.

Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation.

This study investigates the significance of the glucose-6-phosphate dehydrogenase (G6PD) catalyzed oxidative and the transketolase (TK) catalyzed nonoxidative pentose cycle (PC) reactions in the tumor proliferation process by characterizing tumor growth patterns and synthesis of the RNA ribose moiety in the presence of respective inhibitors of G6PD and TK. Mass spectra analysis of 13C-labeled carbons revealed that these PC reactions contribute to over 85% of de novo ribose synthesis in RNA from [1,2-(13)C]glucose in cultured Mia pancreatic adenocarcinoma cells, with the fraction synthesized through the TK pathway predominating (85%). Five days of treatment with the TK inhibitor oxythiamine (OT) and the G6PD inhibitor dehydroepiandrosterone-sulfate (0.5 microM each) exerted a 39 and a 23% maximum inhibitory effect on cell proliferation in culture, which was increased to 60% when the two drugs were administered in combination. In vivo testing of 400 mg/kg OT or dehydroepiandrosterone-sulfate in C57BL/6 mice hosting Ehrlich's ascitic tumor cells revealed a 90.4 and a 46% decrease in the final tumor mass after 3 days of treatment. RNA ribose fractional synthesis through the TK reaction using metabolites directly from glycolysis declined by 9.1 and 23.9% after OT or the combined treatment, respectively. Nonoxidative PC reactions play a central regulating role in the carbon-recruiting process toward de novo nucleic acid ribose synthesis and cell proliferation in vitro and in vivo. Therefore, enzymes or substrates regulating the nonoxidative synthesis of ribose could also be the sites to preferentially target tumor cell proliferation by new anticancer drugs.

Ascorbate as a substrate for glycolysis or gluconeogenesis: evidence for an interorgan ascorbate cycle.

Ascorbate catabolism was investigated in murine and human cells unable to synthesize ascorbate due to the missing gulonolactone oxidase activity. In HepG2 cells the addition of ascorbate or dehydroascorbate resulted in high glucose production, while human erythrocytes, MCF7 cells and the cellular elements of the murine blood were able to metabolize ascorbate or dehydroascorbate to lactate. The oxidative agent menadione stimulated, while the transketolase inhibitor oxythiamine inhibited, the metabolism of dehydroascorbate in each of these three cell types. Our results suggest that ascorbate breakdown through the pentose phosphate pathway can reach the glycolytic/gluconeogenic route in different cells. In ascorbate synthesizing species the ascorbate-lactate route in peripheral cells may form a catabolic branch of an interorgan ascorbate cycle, where hepatocytes are responsible for ascorbate synthesis. The catabolic part of this cycle using exogenous ascorbate could be demonstrated even in humans cells.

Antitumor activity of hydroxythiamine and methotrexate immobilized on monocarboxycellulose.

The aim of the present study was to examine the antitumor and antivitamin activities of some new combinations of methotrexate (MT) and hydroxythiamine (HT), antagonists of folic acid and thiamine, respectively, immobilized on monocarboxycellulose (MCC). Ehrlich ascites carcinoma and sarcoma 180 were used as test malignant tumors in mice. It has been shown, that the compounds studied decreased significantly the amount of mitotically dividing tumor cells and increased the percentage of dead cells, inhibited the tumor growth (up to 10-30 fold at the early stage of neoplasm development) and elongated the life-span of tumor-bearing animals (by 1.6-3.3-fold) as compared to the control. All MCC-immobilized HT and MT complexes studied demonstrated higher antitumor efficacy compared to the mixture of these drugs or each of the agents applied individually. The specific feature of the newly synthesized substances was strong and prolonged antitumor effect following single administration. The severity of thiamine and folic acid deficiencies essentially depended on the amount of HT and MT in the preparations. A close correlation was found between the inhibition of transketolase in the tumors and the antiblastoma properties of the preparations. It enables us to suggest, that the antithiamine action of these preparations is one of the important factors in the mechanism of their antitumor effect.