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Aparajita Pal,1,* Dipa Talukdar,1,* Anirban Roy,1 Subhankar Ray,2 Asish Mallick,3 Chitra Mandal,3 Manju Ray1 1Department of Biophysics, Bose Institute, Kolkata, India; 2Department of Biochemistry, University of Calcutta, Kolkata, India; 3Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute ofChemical Biology, Kolkata,India *These authors contributed equally tothis work Purpose: The normal metabolite methylglyoxal (MG) specifically kills cancer cells by inhibiting glycolysis and mitochondrial respiration without much adverse effect upon normal cells. Though the anticancer property of MG is well documented, its gradual enzymatic degradation in vivo has prompted interest in developing a nanoparticulate drug delivery system to protect it and also to enhance its efficacy. Materials and methods: MG-conjugated chitosan nanoparticles (Nano-MG) were prepared by conjugating the carbonyl group of MG with the amino group of chitosan polymer (Schiff’s base formation). Nano-MG were characterized in detail using the dynamic light scattering method, zeta potential measurement, Fourier transform infrared spectroscopy, and transmission electron microscopic analysis. Amount of MG anchored to Nano-MG, stability of Nano-MG, and in vitro release of MG from Nano-MG were estimated spectrophotometrically. Ehrlich ascites carcinoma (EAC) cells, human breast cancer cell line HBL-100, and lung epithelial adenocarcinoma cell line A549 were used as test systems to compare Nano-MG with bare MG in vitro. Cytotoxicity to EAC cells was evaluated by the trypan blue dye exclusion test, and cell viability of HBL-100 and A549 cells were studied using 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis of HBL-100 cells was assessed by flow cytometry and confocal microscopy. In vivo studies were performed on both EAC cells inoculated and also in sarcoma-180-induced solid tumor-bearing Swiss albino mice to assess the anticancer activity of Nano-MG in comparison to bare MG with varying doses, times, and administrative routes. Results: Fourier transform infrared spectroscopy revealed the presence of imine groups in Nano-MG due to conjugation of the amino group of chitosan and carbonyl group of MG with diameters of nanoparticles ranging from 50–100 nm. The zeta potential of Nano-MG was+21mV and they contained approximately 100 µg of MG in 1 mL of solution. In vitro studies with Nano-MG showed higher cytotoxicity and enhanced rate of apoptosis in the HBL-100 cell line in comparison with bare MG, but no detrimental effect on normal mouse myoblast cell line C2C12 at the concerned doses. Studies with EAC cells also showed increased cell death of nearly 1.5 times. Nano-MG had similar cytotoxic effects on A549 cells. In vivo studies further demonstrated the efficacy of Nano-MG over bare MG and found them to be about 400 times more potent in EAC-bearing mice and nearly 80 times more effective in sarcoma-180-bearing mice. Administration of ascorbic acid and creatine during in vivo treatments augmented the anticancer effect of Nano-MG.Conclusion: The results clearly indicate that Nano-MG may constitute a promising tool in anticancer therapeutics in the near future. Keywords: nano-methylglyoxal, anticancer agent, C2C12, HBL-100, A549, EAC, sarcoma-180, apoptosis

The aim of this study was to evaluate the influence of methylglyoxal (MG) on organogenesis and regeneration of tobacco (Nicotiana tabacum L.) plants from callus in media containing glycine or succinate. The best improvement in shoot proliferation and shoot length was obtained in the medium supplemented with 0.1 mM MG and 0.5 mM glycine or 0.25 mM succinate. The histological studies showed vigorous development of corm like structures and shoot organogenesis from callus tissues cultured in MG supplemented media. Biochemical studies also revealed higher content of δ-aminolaevulinic acid (a precursor of chlorophyll) and of chlorophyll.

Random deposition model with surface diffusion over several next nearest neighbours is studied. Several extensions of diffusion models to include multistep diffusion gives Family’s surface diffusion model in the nearest neighbour diffusion limit. The results for the various extensions agree with the results obtained by Family for the case of nearest neighbour diffusion. However, for larger allowed diffusion length, the growth exponent and roughness exponent show interesting dependence on diffusion length. The variation of values of exponents are fitted to empirical equations. The probable mechanism for dependence of exponents on the diffusion length is discussed.

In the preceding paper (A. Ghosh et al. (2011) Biochemistry (Moscow), 76, 1051-1060), using several comparable tissue materials, it has been convincingly demonstrated that methylglyoxal, a normal metabolite, inhibits mitochondrial complex I of specifically malignant cells. This suggests a distinct alteration of complex I, a highly important enzyme for energy (ATP) production, in malignancy. The present paper shows that as a consequence of this inhibition mitochondrial membrane potential is drastically reduced in sarcoma tissue but not in normal skeletal muscle. This was estimated spectrofluorimetrically using the dye rhodamine 123. As a consequence, cytochrome c was released from the sarcoma mitochondria as evidenced by Western blot analysis. Moreover, on treatment with methylglyoxal membrane potential collapse of sarcoma 180 cells was also indicated by fluorescence-activated cell sorter analysis. Atomic force microscopic study demonstrated gross structural alteration specifically of tumor mitochondria on methylglyoxal treatment. All these studies suggest that methylglyoxal might initiate an apoptotic event in malignant cells.

The creatine/creatine kinase (CK) system plays a key role in cellular energy buffering and transport. In vertebrates, CK has four isoforms expressed in a tissue-specific manner. In the process of creatine biosynthesis several other important metabolites are formed. The anticancer effect of creatine had been reported in the past, and recent literature has reported low creatine content in several types of malignant cells. Furthermore, creatine can protect cardiac mitochondria from the deleterious effects of some anticancer compounds. Previous work from our laboratory showed progressive decrease of phosphocreatine, creatine and CK upon transformation of skeletal muscle into sarcoma. It was convincingly demonstrated that prominent expression of creatine-synthesizing enzymes L-arginine: glycine amidinotransferase and N-guanidinoacetate methyltransferase occurs in sarcoma, Ehrlich ascites carcinoma and sarcoma 180 cells; whereas, both these enzymes are virtually undetectable in skeletal muscle. Creatine transporter also remained unaltered in malignant cells. The anticancer effect of methylglyoxal had been known for a long time. The present work shows that this anticancer effect of methylglyoxal is significantly augmented in presence of creatine. On creatine supplementation the effect of methylglyoxal plus ascorbic acid was further augmented and there was no visible sign of tumor. Moreover, creatine and CK, which were very low in sarcoma tissue, were significantly elevated with the concomitant regression of tumor.

Methylglyoxal (MG) is a highly reactive α-oxoaldehyde, demonstrating anticancer effect on plant neoplastic tumours. In in vivo studies it was observed that MG destroyed crown gall tumours in Nicotiana tabacum produced by Agrobacterium tumefaciens, without any adverse effect on the host. The efficacy of MG in comparison to other anticancer drugs viz. cisplatin and ellagic acid in the treatment of crown gall was investigated. A slight degeneration of galls was noted in plants treated with cisplatin and ellagic acid but the plants died subsequently. With MG however, crown galls were completely cured and the plants completed their usual life cycle by flowering and producing seeds. MG inhibited the respiration of crown gall calluses suggesting that energy depletion resulted in tumour destruction.

In various organisms, an array of enzymes is involved in the synthesis and breakdown of methylglyoxal. Through these enzymes, it is intimately linked to several other physiologically important metabolites, suggesting that methylglyoxal has some important role to play in the host organism. Several in vitro and in vivo studies showed that methylglyoxal acts specifically against different types of malignant cells. These studies culminated in a recent investigation to evaluate a methylglyoxal-based formulation in treating a small group of cancer patients, and the results were promising. Methylglyoxal acts against a number of pathogenic microorganisms. However, recent literature abounds with the toxic effects of methylglyoxal, which are supposed to be mediated through methylglyoxal-derived advanced glycation end products (AGE). Many diseases such as diabetes, cataract formation, hypertension, and uremia are proposed to be intimately linked with methylglyoxal-derived AGE. However methylglyoxal-derived AGE formation and subsequent pathogenesis might be a very minor event because AGE are nonspecific reaction products that are derived through the reactions of carbonyl groups of reducing sugars with amino groups present in the side chains of lysine and arginine and in terminal amino groups of proteins. Moreover, the results of some in vitro experiments with methylglyoxal under non-physiological conditions were extrapolated to the in vivo situation. Some experiments even showed contradictory results and were differently interpreted. For this reason conclusions about the potential beneficial effects of methylglyoxal have often been neglected, thus hindering the advancement of medical science and causing some confusion in fundamental understanding. Overall, the potential beneficial effects of methylglyoxal far outweigh its possible toxic role in vivo, and it should be utilized for the benefit of suffering humanity.

The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.

In vertebrates, phosphocreatine and ATP are continuously interconverted by the reversible reaction of creatine kinase in accordance with cellular energy needs. Sarcoma tissue and its normal counterpart, creatine-rich skeletal muscle, are good source materials to study the status of creatine and creatine kinase with the progression of malignancy. We experimentally induced sarcoma in mouse leg muscle by injecting either 3-methylcholanthrene or live sarcoma 180 cells into one hind leg. Creatine, phosphocreatine and creatine kinase isoform levels decreased as malignancy progressed and reached very low levels in the final stage of sarcoma development; all these parameters remained unaltered in the unaffected contralateral leg muscle of the same animal. Creatine and creatine kinase levels were also reduced significantly in frank malignant portions of human sarcoma and gastric and colonic adenocarcinoma compared with the distal nonmalignant portions of the same samples. In mice, immunoblotting with antibodies against cytosolic muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase showed that both of these isoforms decreased as malignancy progressed. Expressions of mRNA of muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase were also severely downregulated. In human sarcoma these two isoforms were undetectable also. In human gastric and colonic adenocarcinoma, brain-type creatine kinase was found to be downregulated, whereas ubiquitous mitochondrial creatine kinase was upregulated. These significantly decreased levels of creatine and creatine kinase isoforms in sarcoma suggest that: (a) the genuine muscle phenotype is lost during sarcoma progression, and (b) these parameters may be used as diagnostic marker and prognostic indicator of malignancy in this tissue.

Background For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. Methods GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. Result Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. Conclusion PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2172-x) contains supplementary material, which is available to authorized users.

A pharmaceutical composition and treatment method to reduce the proliferation of cancerous or tumor cells, in which the combined active agents are methylglyoxal, ascorbic acid, creatine and melatonin.

It is well known that cytokinins, a group of plant hormones are absolutely required for the differentiation of calluses for the regeneration of plantlets through organogenesis. In the present work, it had been observed that methylglyoxal could completely replace kinetin to initiate differentiation of plantlets from calluses of Solanum nigrum and Daucus carota. Moreover, the effect of methylglyoxal was more pronounced compared to that of kinetin and the optimum concentration for methylglyoxal had been determined to be 0.5Â mM. Parallel with the differentiation of calluses to plantlets, the chlorophyll contents increased whereas the endogenous level of methylglyoxal remained unchanged. This remarkable effect of methylglyoxal in plant differentiation had been found out to be specific because some related compounds such as pyruvate and -lactate could not replace the requirement for methylglyoxal in the differentiation process. The activities of several enzymes were monitored during both methylglyoxal and kinetin-induced differentiation. The activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase (NADP-dependent) involved in energy generation process in photosynthesis, increased as the differentiation proceeded. Whereas, the activities of both glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase (NAD-dependent) decreased with differentiation. The activity of glyoxalase I, which catalyzes the conversion of methylglyoxal and glutathione to S--lactoylglutathione, decreased with differentiation. The endogenous level of glutathione showed an initial decrease followed by an increase. It appears from the results presented above, that the effect of kinetin and methylglyoxal are similar in nature, the significance of which has been discussed.

Previous publications from our laboratory have shown that methylglyoxal inhibits mitochondrial respiration of malignant and cardiac cells, but it has no effect on mitochondrial respiration of other normal cells [Biswas, Ray, Misra, Dutta and Ray (1997) Biochem. J. 323, 343–348; Ray, Biswas and Ray (1997) Mol. Cell. Biochem. 171, 95–103]. However, this inhibitory effect of methylglyoxal is not significant in cardiac tissue slices. Moreover, post-mitochondrial supernatant (PMS) of cardiac cells could almost completely protect the mitochondrial respiration against the inhibitory effect of methylglyoxal. A systematic search indicated that creatine present in cardiac cells is responsible for this protective effect. Glutathione has also some protective effect. However, creatine phosphate, creatinine, urea, glutathione disulphide and β-mercaptoethanol have no protective effect. The inhibitory and protective effects of methylglyoxal and creatine respectively on cardiac mitochondrial respiration were studied with various concentrations of both methylglyoxal and creatine. Interestingly, neither creatine nor glutathione have any protective effect on the inhibition by methylglyoxal on the mitochondrial respiration of Ehrlich ascites carcinoma cells. The creatine and glutathione contents of several PMS, which were tested for the possible protective effect, were measured. The activities of two important enzymes, namely glyoxalase I and creatine kinase, which act upon glutathione plus methylglyoxal and creatine respectively, were also measured in different PMS. Whether mitochondrial creatine kinase had any role in the protective effect of creatine had also been investigated using 1-fluoro-2,4-dinitrobenzene, an inhibitor of creatine kinase. The differential effect of creatine on mitochondria of cardiac and malignant cells has been discussed with reference to the therapeutic potential of methylglyoxal.

The involvement of the lysine residue present at the active site of Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (Gra3PDH) was investigated by using the lysine specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal phosphate (PP). Both TNBS and PP inactivated EAC cell Gra3PDH with pseudo-first-order kinetics with the rate dependent on modifier concentration. Kinetic analysis, including a Tsou plot, indicated that both TNBS and PP apparently react with one lysine residue per enzyme molecule. Two of the substrates, d-glyceraldehyde-3-phosphate and NAD, and also NADH, the product and competitive inhibitor, almost completely protected the enzyme from inactivation by TNBS. A comparative study of Gra3PDH of EAC cell and rabbit muscle indicates that the nature of active site of the enzyme is significantly different in these two cells. A double inhibition study using 5,5'-dithiobis(2-nitrobenzoic acid) and TNBS and subsequent reactivation of only the rabbit muscle enzyme by dithiothreitol suggested that a cysteine residue of this enzyme possibly reacts with TNBS. These studies on the other hand, confirm that an essential lysine residue is involved in the catalytic activity of the EAC cell enzyme. This difference in the nature of the active site of EAC cell Gra3PDH that may be related to the high glycolysis of malignant cells has been discussed.

Glyceraldehyde-3-phosphate dehydrogenase has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells. The enzyme is quite active over a pH range of 7.5-9.0 with an optimum pH of 8.4-8.7. The specific activity of the enzyme is much higher than that from other normal sources. In contrast to enzyme obtained from rabbit muscle, the EAC cell enzyme is not significantly inhibited by physiological concentrations of ATP at physiological pH. Kinetic studies using different substrates and inhibitors indicate that the properties of the EAC cell enzyme are significantly different from those of glyceraldehyde-3-phosphate dehydrogenase obtained from other normal sources. The striking dissimilarity of the malignant cell glyceraldehyde-3-phosphate dehydrogenase compared with this enzyme from other normal sources, particularly in respect to the interaction with ATP, may in part explain the high glycolysis of malignant cells.

The effect of methylglyoxal on the oxygen consumption of mitochondria of both normal and leukaemic leucocytes was tested by using different respiratory substrates and complex specific artificial electron donors and inhibitors. The results indicate that methylglyoxal strongly inhibits mitochondrial respiration in leukaemic leucocytes, whereas, at a much higher concentration, methylglyoxal fails to inhibit mitochondrial respiration in normal leucocytes. Methylglyoxal strongly inhibits ADP-stimulated α-oxoglutarate and malate plus NAD+-dependent respiration, whereas, at a higher concentration, methylglyoxal fails to inhibit succinate and α-glycerophosphate-dependent respiration. Methylglyoxal also fails to inhibit respiration which is initiated by duroquinone and cannot inhibit oxygen consumption when the N,N,N´,N´-tetramethyl-p-phenylenediamine by-pass is used. NADH oxidation by sub-mitochondrial particles of leukaemic leucocytes is also inhibited by methylglyoxal. Lactaldehyde, a catabolite of methylglyoxal, can exert a protective effect on the inhibition of leukaemic leucocyte mitochondrial respiration by methylglyoxal. Methylglyoxal also inhibits l-lactic acid formation by intact leukaemic leucocytes and critically reduces the ATP level of these cells, whereas methylglyoxal has no effect on normal leucocytes. We conclude that methylglyoxal inhibits glycolysis and the electron flow through mitochondrial complex I of leukaemic leucocytes. This is strikingly similar to our previous studies on mitochondrial respiration, glycolysis and ATP levels in Ehrlich ascites carcinoma cells [Ray, Dutta, Halder and Ray (1994) Biochem. J. 303, 69–72; Halder, Ray and Ray (1993) Int. J. Cancer 54, 443–449], which strongly suggests that the inhibition of electron flow through complex I of the mitochondrial respiratory chain and inhibition of glycolysis by methylglyoxal may be common characteristics of all malignant cells.

The effect of methylglyoxal on the oxygen consumption of mitochondria of heart and of several other organs of normal animals of different species has been tested. The results indicate that methylglyoxal (3.5 mM) strongly inhibits ADP-stimulated α-oxoglutarate and malate plus pyruvate-dependent respiration of exclusively heart mitochondria of normal animals of different species. Whereas, with the same substrates, but at a higher concentration of methylglyoxal (7.5 mM), the respiration of mitochondria of other organs of normal animals is not inhibited. Methylglyoxal also inhibits the respiration of slices of rat and toad hearts. But this inhibition is less pronounced. However, methylglyoxal (15 mM) fails to have any effect on perfused toad heart. Using rat heart mitochondria as a model, the effect of methylglyoxal on the oxygen consumption was also tested with different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-spe inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal. The results strongly suggest that methylglyoxal inhibits the electron flow through complex I of rat heart mitochondrial respiratory chain. Moreover, lactaldehyde (0.6 mM), a catabolite of methylglyoxal, can exert a protective effect on the inhibition of rat heart mitochondrial respiration by methylglyoxal (2.5 mM). The effect of methylglyoxal on heart mitochondria as described in the present paper is strikingly similar to the results of our previous work with mitochondria of Ehrlich ascites carcinoma cells and leukemic leukocytes. We have recently proposed a new hypothesis on cancer which suggests that excessive ATP formation in cells may lead to malignancy. The above mentioned similarity apparently provides a solid experimental foundation for the proposed hypothesis which has been discussed.

The effect of methylglyoxal on the activity of glyceraldehyde-3-phosphate dehydrogenase (GA3PD) of several normal human tissues and benign and malignant tumors has been tested. Methylglyoxal inactivated GA3PD of all the malignant cells (47 samples) and the degree of inactivation was in the range of 25-90%, but it had no inhibitory effect on this enzyme from several normal cells (24 samples) and benign tumors (13 samples). When the effect of methylglyoxal on other two dehydrogenases namely glucose 6-phosphate dehydrogenase (G6PD) and L-lactic dehydrogenase (LDH) of similar cells was tested as controls it has been observed that methylglyoxal has some inactivating effect on G6PD of all the normal, benign and malignant samples tested, whereas, LDH remained completely unaffected. These studies indicate that the inactivating effect of methylglyoxal on GA3PD specifically of the malignant cells may be a common feature of all the malignant cells, and this phenomenon can be used as a simple and rapid device for the detection of malignancy.

Methylglyoxal strongly inhibited mitochondrial respiration of a wide variety of malignant tissues including sarcoma of mice, whereas no such significant effect was noted on mitochondrial respiration of normal tissues with the exception of cardiac cells. This inhibition by methylglyoxal was found to be at the level of mitochondrial complex I (NADH dehydrogenase) of the electron transport chain. L-Lactaldehyde, which is structurally and metabolically related to methylglyoxal, could protect against this inhibition. NADH dehydrogenase of submitochondrial particles of malignant and cardiac cells was inhibited by methylglyoxal. This enzyme of these cells was also inactivated by methylglyoxal. The possible involvement of lysine residue(s) for the activity of NADH dehydrogenase was also investigated by using lysine-specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal 5' phosphate (PP). Inactivation of NADH dehydrogenase by both TNBS and PP convincingly demonstrated the involvement of lysine residue(s) for the activity of the sarcoma and cardiac enzymes, whereas both TNBS and PP failed to inactivate the enzymes of skeletal muscle and liver. Together these studies demonstrate a specific effect of methylglyoxal on mitochondrial complex I of malignant cells and importantly some distinct alteration of this complex in cancer cells.

The effect of methylglyoxal (MG) on the aerobic glycolysis of Ehrlich ascites carcinoma (EAC) cells has been tested. Methylglyoxal inhibited glucose utilization and glucose 6-phosphate (G6P) and L-lactate formation in whole EAC cells. Methylglyoxal strongly inactivated glyceraldehyde 3-phosphate dehydrogenase (GA3PD) of the malignant cells, whereas MG has little inactivating effect on this enzyme from several normal sources. Methylglyoxal also inactivated only the particulate hexominase of the EAC cells, but this inactivation was less pronounced than the effect on GA3PD. Methylglyoxal has little inactivating effect on glucose 6-phosphate dehydrogenase (G6PD), and no effect on L-lactate dehydrogenase (LDH) of the malignant cells. Glucose-dependent L-lactic acid formation of EAC-cell-free homogenate was strongly inhibited by MG, but when GA3PD of normal cells was added to this homogenate, significant lactate formation was observed even in the presence of MG. Methylglyoxal also inhibited the respiration of EAC-cell mitochondria. Respiration of mitochondria isolated from liver and kidney of normal mice, however, remained unaffected. As a consequence of the inhibition of glycolysis and mitochondrial respiration, the ATP level of the EAC cells was drastically reduced. Studies reported herein strongly suggest that the tumoricidal effect of MG is mediated at least in part through the inhibition of mitochondrial respiration and inactivation of GA3PD, and this enzyme may play an important role in the high glycolytic capacity of the malignant cells.

We have purified a novel protein from mice muscle, which through N-terminal amino acid sequencing was identified as a truncated form of mouse albumin. The protein was found to be a monomer of ∼64 kDa and located in the cytosol. The purified protein strongly crossreacted with commercial albumin antibody. Presence of this protein was observed in different mouse organs. Further biochemical studies as well as CD spectroscopy indicated that the protein binds D-glyceraldehyde-3-phosphate limiting the availability of the substrate to the enzyme D-glyceraldehyde-3-phosphate dehydrogenase, thereby inhibiting its catalytic activity. The implication of this protein in the control of glycolysis has been discussed. © 2009 IUBMB IUBMB Life, 61(10): 995–1000, 2009

The arginine-specific reagents phenylglyoxal and butane-2,3-dione inactivated goat liver aminoacetone synthase with pseudo-first-order kinetics, with the rate dependent on modifier concentration. Phenylglyoxal and butane-2,3-dione appeared to react with one arginine residue per enzyme molecule. The inactivated enzyme could be re-activated by Tris, suggesting additional evidence of modification of the arginine residue. Acetyl-CoA, one of the substrates, completely protected the enzyme from inactivation. Glycine gave partial protection. Protection by substrates against inactivation by phenylglyoxal and butane-2,3-dione suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of acetyl-CoA showed that only the arginine residue at the active site could be modified by phenylglyoxal. The stability of the enzyme is dependent on the presence of both EDTA and Mg2+.

In biological systems, adenosine triphosphate (ATP) is the principal contributor of free energy necessary for anabolic reactions and is also a precursor of nucleic acids. Moreover, active transport of metabolites into cells is also driven by hydrolysis of ATP. So, a cell may grow, multiply and ultimately turn malignant when it has been transformed in such a manner that it produces excess ATP as compared with its usual metabolic demand. Recent studies have indicated that mitochondrial complex I and the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GA3PD) may be critically altered specifically in malignant cells. So, we further propose that this excessive ATP formation may be due to altered mitochondrial complex I and GA3PD of malignant cells.

The possible effect of several physiologically important aldehydes has been tested on partially purified glyoxalase I of Ehrlich ascites carcinoma (EAC) cells. The results indicate that D, and L-lactaldehyde are strong non-competitive inhibitors of glyoxalase I and the effect with the D-isomer is more pronounced, whereas both D,L-glyceraldehyde and acetaldehyde are moderately inhibitory and the nature of inhibition is strictly competitive. Moreover, D,L-glyceraldehyde strongly inhibits the utilization of methylglyoxal by intact EAC cells. A search for the presence of several aldehyde metabolizing enzymes in EAC cells indicates that non-specific aldehyde reductase, methylglyoxal reductase, aldehyde dehydrogenase and alcohol dehydrogenase are apparently absent in this rapidly growing, highly de-differentiated malignant cell.

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K12 cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K(m) for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8-6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 +/- 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.

A single novel enzyme, glyoxalase III, which catalyses the conversion of methylglyoxal into D-lactate without involvement of GSH, has been detected in and purified from Escherichia coli. Of several carbonyl compounds tested, only the alpha-ketoaldehydes methylglyoxal and phenylglyoxal were found to be substrates for this enzyme. Glyoxalase III is active over a wide range of pH with no sharp pH optimum. In its native form it has an M(r) of 82000 +/- 2000, and it is composed of two subunits of equal M(r). Glutathione analogues, which are inhibitors of glyoxalase I, do not inhibit glyoxalase III. Glyoxalase III is found to be sensitive to thiol-blocking reagents. The p-hydroxymercuribenzoate-inactivated enzyme could be almost completely re-activated by dithiothreitol and other thiol-group-containing compounds, indicating the possible involvement of thiol group(s) at or near the active site of the enzyme.

The effect of methylglyoxal on the oxygen consumption of Ehrlich-ascites-carcinoma (EAC)-cell mitochondria was tested by using different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-specific inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal on the oxygen consumption by these cells. The results indicate that methylglyoxal strongly inhibits ADP-stimulated alpha-oxo-glutarate and malate plus pyruvate-dependent respiration, whereas, at a much higher concentration, methylglyoxal fails to inhibit succinate-dependent respiration. Methylglyoxal also fails to inhibit respiration which is initiated by duroquinol, an artificial electron donor. Moreover, methylglyoxal cannot inhibit oxygen consumption when the NNN'N′-tetramethyl-p-phenylenediamine by-pass is used. The inhibitory effect of methylglyoxal is identical on both ADP-stimulated and uncoupler-stimulated respiration. Lactaldehyde, a catabolite of methylglyoxal, can exert a protective effect on the inhibition of EAC-cell mitochondrial respiration by methylglyoxal. We suggest that methylglyoxal possibly inhibits the electron flow through complex I of the EAC-cell mitochondrial respiratory chain.

The effect of methylglyoxal (MG), ascorbic acid and lactaldehyde has been tested on the in vitro respiration of Ehrlich ascites carcinoma (EAC) cells and several normal and malignant human tissues. Methylglyoxal inhibited the respiration of each type of malignant cell and tissue tested, but it had practically no inhibitory effect on the respiration of any of the normal cells and tissues. Ascorbic acid exhibited a synergistic effect with MG in inhibiting the respiration of all the neoplastic cells. In the presence of lactaldehyde, a catabolite of MG, the inhibitory effect of MG on the respiration of tumor cells was significantly reduced. Lactaldehyde can exert a similar protective effect on the loss of viability and transplantability of MG-treated EAC cells.

L-Threonine dehydrogenase, which forms aminoacetone from L-threonine and NAD, has been extensively purified from goat liver. A feedback inhibition of this enzyme has been observed with methylglyoxal. Kinetic data and other experiments indicate that methylglyoxal acts at a site other than the active site of the enzyme. The enzyme contains a single subunit of Mr 89,000. The apparent Km values of the enzyme for L-threonine and NAD were found to be 5.5 and 1 mM, respectively.

An enzyme which catalyzes the reduction of methylglyoxal to lactaldehyde has been isolated and purified from goat liver to apparent homogeneity. NADH was found to be a better substrate than NADPH for methylglyoxal reduction. Stoichiometrically equivalent amounts of lactaldehyde and NAD are formed from methylglyoxal and NADH. Enzyme activity was located only in the soluble supernatant fractions of liver cells. Of the various carbonyl compounds tested, methylglyoxal was found to be the best substrate. The pH optimum of the enzyme was found to be 6.5, and Km for methylglyoxal was 0.4 mM. The molecular weight of the enzyme was found to be 89000 by gel filtration on a Sephadex G-200 column. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel revealed that the enzyme is composed of two subunits. The enzyme is highly sensitive to sulfhydryl group reagents. The inactivation by p-chloromercuribenzoate could be substantially protected by methylglyoxal in combination with NADH, indicating a possible involvement of one or more sulfhydryl group(s) at the active site of the enzyme.

It has been reported earlier that nucleotides, nucleosides and a series of structurally related compounds as well as compounds based on transition state analogy inhibit yeast glyoxalase I. In our study on the metabolic regulation of glyoxalase I, we have found that nucleotides such as ATP, GTP and different classes of other reagents based on transition state analogy (D-isoascorbate, dihydroxyfumaric acid, rhodizonic acid) do not inhibit yeast or goat liver glyoxalase I. The reported inhibition of glyoxalase I by these compounds has been found to be due to the interference of these compounds with the absorbancy at 240 nm of S-D-lactoylglutathione formed by the glyoxalase I reaction.

An enzyme which oxidizes aminoacetone to methylglyoxal has been purified from the particulate fraction of goat liver. Polyamines, such as spermidine and spermine, are also good substrates for this enzyme. The pH optimum for aminoacetone oxidation was found to be 8.2. The apparent Km values of the enzyme for aminoacetone and spermidine were 0.009 and 0.095 mM, respectively. The subunit molecular weight of the enzyme was 93,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of the native enzyme was 186,000 by gel filtration. The enzyme is highly sensitive to carbonyl group reagents. The enzyme is not inhibited by monoamine and diamine oxidase inhibitors.

An enzyme fraction which specifically catalyzes the formation of methylglyoxal from dihydroxyacetone phosphate has been isolated and partially purified from goat liver. The enzyme fraction appears to be substantially free from glyoxalase I, reduced glutathione, and triosephosphate isomerase. Approximately equimolar quantities of methylglyoxal and inorganic phosphate were obtained from dihydroxyacetone phosphate. Formation of methylglyoxal was confirmed by colorimetric and enzymatic estimations as well as by paper chromatography and its spectrum. Glyceraldehyde-3-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone, and glyceraldehyde, failed to act as substrates. The enzyme is inhibited by some phosphorylated compounds and inorganic phosphates.

Methylglyoxal is a toxic metabolite with growth inhibitory properties against Leishmania donovani promastigotes. We have shown in the present study that both log and stationary phase promastigotes of L. donovani can catabolize methylglyoxal to D-lactate as the major end product. The specific activity of methylglyoxal reductase was found to be the highest of all the catabolic enzymes. In contrast, the anabolic pathway for methylglyoxal could not be detected. Moreover, when control promastigotes or promastigotes in which the glycolytic pathway was inhibited were incubated with glucose, glycerol or dihydroxyacetone phosphate as energy source, neither methylglyoxal nor D-lactate could be detected.

An enzyme fraction which oxidizes lactaldehyde to lactic acid has been purified from goat liver. This enzyme was found to be identical with the cytosolic aldehyde dehydrogenase. Lactaldehyde was found to be primarily oxidized by this enzyme. Almost 90% of the total lactaldehyde-oxidizing activity is located in the cytosol. Methylglyoxal and glyceraldehyde 3-phosphate were found to be strong competitive inhibitors of this enzyme. Aldehyde dehydrogenase from goat liver mitochondria has also been partially purified and found to be strongly inhibited by these metabolites. The inhibitory effects of these metabolites on both these enzymes are highly pH dependent. The inhibitory effects of both the metabolites have been found to be stronger for the cytosolic enzyme at pH values higher than the physiological pH. For the mitochondrial enzyme, the inhibition with methylglyoxal was more pronounced at higher pH values, whereas stronger inhibition was observed with glyceraldehyde 3-phosphate at physiological pH.

An enzyme from goat plasma which catalyzes the oxidation of aminoacetone to methylglyoxal has been isolated and purified to apparent homogeneity. This enzyme has been found to be identical with the well known amine oxidase. Several properties of the enzyme have also been described.