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 α-oxoglutarate 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.

S RAY

S DUTTA

J HALDER

M RAY

Fulltext

University of Calcutta (informally known as Calcutta University or CU) is a collegiate public state university located in Kolkata, West Bengal, India.&nbsp;Within India it is recognized as a &quot;Five-Star University&quot; and accredited &quot;A&quot; Grade by National Assessment and Accreditation Council.

&nbsp;

The University of Calcutta has secured the Fifth position among the Universities of India in the prestigious &quot;Indian University Ranking 2019&quot; list, released by the NIRF of the Ministry of Human Resource Development, Government of India.

&nbsp;

It was established on 24 January 1857, and was one of the first institutions in Asia to be established as a multidisciplinary and Western-style university. Its alumni and faculty include several heads of state, heads of government, social reformers, prominent artists, only academy award winner and Dirac medal winner in India, many Fellows of the Royal Society and five Nobel laureates as of 2019 which is highest in South Asia.

University Of Calcutta

Biochemical Journal

Potent anticancer activity coupled with absence of toxicity at therapeutic dose established the glycolytic metabolite, methylglyoxal, as a promising candidate against malignant neoplasia. In this preclinical study we illustrate the applicability of methylglyoxal in formulating an optimally designed combination regimen with chemotherapeutic drugs against breast cancer. Results demonstrated a synergistic augmentation in doxorubicin and cisplatin mediated cytotoxicity in human breast cancer cell lines MDA MB 231 & MCF 7 with methylglyoxal co-treatment at metronomic concentrations. The cell death due to combination treatment was significantly prevented by N-Acetylcysteine and the synergistic effects were attenuated in presence of inhibitors for apoptosis and necroptosis, in MDA MB 231 and MCF 7 cells, respectively. Additionally, acridine orange staining and immunoblotting with LC3B antibody indicated the suppression of doxorubicin induced autophagy flux with methylglyoxal co-treatment. This report documents for the first time the preferential targeting of breast cancer stem cells by methylglyoxal. Combination treatment with doxorubicin or cisplatin hindered mammosphere forming efficiency and inclusively eliminated both cancer stem as well as non-stem cancer cells. The synergistic effect was validated in Ehrlich mammary carcinoma cell induced murine ascites model and the combination advantage in vivo was achieved without any additional deleterious effect to liver and kidney. Our present study evidences the implications of methylglyoxal inclusion in adjuvant multimodal chemotherapeutics against breast cancer and offers noteworthy insights into the possible outcome.

Biochemical Pharmacology

Methylglyoxal at metronomic doses sensitizes breast cancer cells to doxorubicin and cisplatin causing synergistic induction of programmed cell death and inhibition of sternness

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 fow 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 +21 mV 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. © 2015 Pal et al.

International Journal of Nanomedicine

Nanofabrication of methylglyoxal with chitosan biopolymer: A potential tool for enhancement of its anticancer effect

Previous in vivo studies from several laboratories had shown remarkable curative effect of methylglyoxal on cancer-bearing animals. In contrast, most of the recent in vitro studies have assigned a toxic role for methylglyoxal. The present study was initiated with the objective to resolve whether methylglyoxal is truly toxic in vivo and to reassess its therapeutic potential. Four species of animals, both rodent and non-rodent, were treated with different doses of methylglyoxal through oral, subcutaneous and intravenous routes. Acute (treatment for only 1 day) toxicity tests had been done with mouse and rat. These animals received 2, 1 and 0.3 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Chronic (treatment for around a month) toxicity test had been done with mouse, rat, rabbit and dog. Mouse, rat and dog received 1, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Rabbit received 0.55, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. It had been observed that methylglyoxal had no deleterious effect on the physical and behavioral pattern of the treated animals. Fertility and teratogenecity studies were done with rats that were subjected to chronic toxicity tests. It had been observed that these animals produced healthy litters indicating no damage of the reproductive systems as well as no deleterious effect on the offspring. Studies on several biochemical and hematological parameters of methylglyoxal-treated rats and dogs and histological studies of several organs of methylglyoxal-treated mouse were performed. These studies indicated that methylglyoxal had no apparent deleterious effect on some vital organs of these animals. A detailed pharmacokinetic study was done with mouse after oral administration of methylglyoxal. The effect of methylglyoxal alone and in combination with creatine and ascorbic acid on cancer-bearing animals had been investigated by measuring the increase in life span and tumor cell growth inhibition. The results indicated that anticancer effect of methylglyoxal was significantly augmented by ascorbic acid and further augmented by ascorbic acid and creatine. Nearly 80% of the animals treated with methylglyoxal plus ascorbic acid plus creatine were completely cured and devoid of any malignant cells within the peritoneal cavity. © 2005 Elsevier Inc. All rights reserved.

Toxicology and Applied Pharmacology

In vivo assessment of toxicity and pharmacokinetics of methylglyoxal: Augmentation of the curative effect of methylglyoxal on cancer-bearing mice by ascorbic acid and creatine

We had previously shown that creatine exerted a protective effect against inhibition of cardiac mitochondrial respiration by methylglyoxal (SinhaRoy S, Biswas S, Ray M, Ray S. Biochem J 372: 661-669, 2003). In the present study, we have investigated the mechanism of this protective effect by specific amino acid modifying reagent and by several compounds, which are structurally related to creatine. The results show that the compounds, which contain guanidine group such as arginine and guanidinopropionic acid, exert a protective effect, which is quantitatively similar to creatine. This result suggests the presence of carboxylic acid(s) such as glutamic and/or aspartic acid(s) in the creatine-binding site, which has been further supported by experiments with N-ethyl-5-phenyl isoxazolium-3′-sulfonate a reagent known to modify these amino acids. Both polarographic and spectrophotometric assays were performed with NADH as respiratory substrate by using a) submitochondrial particles by sonication, b) freeze-thawed mitochondria and c) mitochondria permeabilized by alamethicin treatment. The results of these studies as compared to that of intact mitochondria indicate that structural integrity of mitochondria is essential for the protective effect of creatine. © Springer 2005.

Molecular and Cellular Biochemistry

Possible involvement of glutamic and/or aspartic acid residue(s) and requirement of mitochondrial integrity for the protective effect of creatine against inhibition of cardiac mitochondrial respiration by methylglyoxal

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 supematant (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.

Protective effect of creatine against inhibition by methylglyoxal of mitochondrial respiration of cardiac 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 participate 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. Copyright © 1993 Wiley‐Liss, Inc., A Wiley Company

International Journal of Cancer

Inhibition of glycolysis and mitochondrial respiration of ehrlich ascites carcinoma cells by methylglyoxal

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. Copyright © 1991 Wiley‐Liss, Inc., A Wiley Company

Inhibition of respiration of tumor cells by methylglyoxal and protection of inhibition by lactaldehyde

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 89 000 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. © 1984.

BBA - General Subjects

Purification and partial characterization of a methylglyoxal reductase from goat liver

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. © 1984.

Journal	Biochemical Journal
Publisher	Portland Press Ltd
ISSN	0264-6021