Glyoxalase-I Is Upregulated in Acute Cerulein-Induced Pancreatitis: A New Mechanism in Pancreatic Inflammation?

Inflammation caused by oxidative stress (ROS) demonstrates an essential mechanism in the pathogenesis of acute pancreatitis (AP). Important sources for ROS comprise the reactive compound methylglyoxal (MGO) itself and the MGO-derived formation of advanced glycation end-products (AGEs). AGEs bind to the transmembrane receptor RAGE and activate NF-κB, and lead to the production of pro-inflammatory cytokines. MGO is detoxified by glyoxalase-I (Glo-I). The importance of Glo-I was shown in different models of inflammation and carcinogenesis. Nevertheless, the role of Glo-I and MGO in AP has not been evaluated so far. This study analyzed Glo-I in cerulein-(CN)-induced AP and determined the effects of Glo-I knockdown, overexpression and pharmacological modulation. Methods: AP was induced in C57BL6/J mice by i.p. injection of CN. Glo-I was analyzed in explanted pancreata by Western Blot, qRT-PCR and immunohistochemistry. AR42J cells were differentiated by dexamethasone and stimulated with 100 nM of CN. Cells were simultaneously treated with ethyl pyruvate (EP) or S-p-bromobenzylglutathione-cyclopentyl-diester (BrBz), two Glo-I modulators. Knockdown and overexpression of Glo-I was achieved by transient transfection with Glo-I siRNA and pEGFP-N1-Glo-I-Vector. Amylase secretion, TNF-α production (ELISA) and expression of Glo-I, RAGE and NF-κB were measured. Results: Glo-I was significantly upregulated on protein and mRNA levels in CN-treated mice and AR42J cells. Dexamethasone-induced differentiation of AR42J cells increased the expression of Glo-I and RAGE. Treatment of AR42J cells with CN and EP or BrBz resulted in a significant reduction of CN-induced amylase secretion, NF-κB, RAGE and TNF-α. Overexpression of Glo-I led to a significant reduction of CN-induced amylase levels, NF-κB expression and TNF-α, whereas Glo-I knockdown revealed only slight alterations. Measurements of specific Glo-I activity and MGO levels indicated a complex regulation in the model of CN-induced AP. Conclusion: Glo-I is overexpressed in a model of CN-induced AP. Pharmacological modulation and overexpression of Glo-I reduced amylase secretion and the release of pro-inflammatory cytokines in AP in vitro. Targeting Glo-I in AP seems to be an interesting approach for future in vivo studies of AP.

Revisiting Szent-Györgyi's Cancer Hypothesis

Rationale for this communication • -The promine/retine hypothesis on the control of cancer never reached a definite conclusion.• -The chemical natures of promine and retine remain unsettled though usually assumed to be glyoxalases and methylglyoxal.• -Many years ago we published some data that indicated the hypothesis may be operating in plants in which glyoxalase I may exist in normal cells in an inhibited state rather than compartmentalized as in early versions of the hypothesis.• -Manju Ray in India has published many papers claiming that methylglyoxal can be used to successfully treat cancer in humans.• -We present here previously unpublished data that shows depriving glyoxalase I of GSH allows methylglyoxal to kill mouse lymphoma cells. During treatment of two human cancer cell lines, killing of one line was enhanced by blocking thioredoxin as well as GSH.It is hoped that what is conveyed here may reignite interest in the near term. Nuclear methodology and statistics can be found in Figure 1. The data show a strong interaction between the hypothesis and thiols. It is concluded that the hypothesis has yet to be thoroughly investigated.

Identification of the first “two digit nano-molar” inhibitors of the human glyoxalase-I enzyme as potential anticancer agents

Background: Glyoxalase-I (Glo-I) enzyme is recognized as an indispensable druggable target in cancer treatment. Its inhibition will lead to the accumulation of toxic aldehyde metabolites and cell death. Paramount efforts were spent to discover potential competitive inhibitors to eradicate cancer. Objective: Based on our previously work on this target for discovering potent inhibitors of this enzyme, herein, we address the discovery of the most potent Glo-I inhibitors reported in literature with two digits nano-molar activity. Methods: Molecular docking and in vitro assay were performed to discover these inhibitors and explore the active site's binding pattern. A detailed SAR scheme was generated, which identifies the significant functionalities responsible for the observed activity. Results: Compound 1 with an IC50 of 16.5 nM exhibited the highest activity, catechol moiety as an essential zinc chelating functionality. It has been shown by using molecular modeling techniques that the catechol moiety is responsible for the chelation zinc atom at the active site, an essential feature for enzyme inhibition. Conclusion: Catechol derivatives are successful zinc chelators in the Glo-I enzyme while showing exceptional activity against the enzyme to the nanomolar level.

Ellagic acid: A potent glyoxalase-I inhibitor with a unique scaffold

The glyoxalase system, particularly glyoxalase-I (GLO-I), has been approved as a potential target for cancer treatment. In this study, a set of structurally diverse polyphenolic natural compounds were investigated as potential GLO-I inhibitors. Ellagic acid was found, computationally and experimentally, to be the most potent GLO-I inhibitor among the tested compounds which showed an IC50 of 0.71 mmol L−1. Its binding to the GLO-I active site seemed to be mainly driven by ionic interaction via its ionized hydroxyl groups with the central Zn ion and Lys156, along with other numerous hydrogen bonding and hydrophobic interactions. Due to its unique and rigid skeleton, it can be utilized to search for other novel and potent GLO-I inhibitors via computational approaches such as pharmacophore modeling and similarity search methods. Moreover, an inspection of the docked poses of the tested compounds showed that chlorogenic acid and dihydrocaffeic acid could be considered as lead compounds worthy of further optimization.

Establishment of Glyoxalase I gene transformation and regeneration of cotton (Gossypium hirsutum L.)

Aim: The current study was carried out to develop transgenic cotton plantlets with glyoxalase I (gly I) gene using Agrobacterium-mediated transformation. Methodology: Seeds of cotton were inoculated on MS medium and the explants such as shoot tip (3-5 mm), hypocotyl and leaf were aseptically removed from in vitro plantlets. The pre-cultured and infected explants with Agrobacterium harboring gly I gene and the shoot tip were inoculated on MS media for shoot initiation and subcultured on elongation medium with growth hormones, and antibiotics. Healthy and well-grown shoots were subcultured on medium with indole butyric acid (IBA) (0.3 mgl-1) for root formation and the plantlets were hardened in plastic cups with sterile soil. The putative transgenic plantlets were analyzed histochemically for gus gene and the PCR analysis was performed for gly 1 gene. Results: The transformation efficiency of cotton ranged 48.57 to 64.53 %. The regenerated plantlets showed the presence of gus gene in terms of blue coloration in shoots, whole leaf and leaf segments. The PCR was performed in putative transgenic plant lets with both gus gene as well as gly I gene primers. The PCR results showed the presence of 1031 bp DNA band with gus gene primers and 800 bp DNA band with the gly I gene primers. Interpretation: The current study has proven the reproducible procedure for the Agrobacterium-mediated gene transfer and regeneration of Indian cotton varieties. The PCR results revealed the presence of glyoxalase I gene in the transformants. Key words: Cotton varieties, Glyoxalase I gene, PCR analysis, Regeneration, Transformation

Polymorphisms in Glyoxalase I Gene Are Not Associated with Glyoxalase I Expression in Whole Blood or Markers of Methylglyoxal Stress: The CODAM Study

Glyoxalase 1 (Glo1) is the rate-limiting enzyme in the detoxification of methylglyoxal (MGO) into D-lactate. MGO is a major precursor of advanced glycation endproducts (AGEs), and both are associated with development of age-related diseases. Since genetic variation in GLO1 may alter the expression and/or the activity of Glo1, we examined the association of nine SNPs in GLO1 with Glo1 expression and markers of MGO stress (MGO in fasting plasma and after an oral glucose tolerance test, D-lactate in fasting plasma and urine, and MGO-derived AGEs CEL and MG-H1 in fasting plasma and urine). We used data of the Cohort on Diabetes and Atherosclerosis Maastricht (CODAM, n = 546, 60 ± 7 y, 25% type 2 diabetes). Outcomes were compared across genotypes using linear regression, adjusted for age, sex, and glucose metabolism status. We found that SNP4 (rs13199033) was associated with Glo1 expression (AA as reference, standardized beta AT = −0.29, p = 0.02 and TT = −0.39, p = 0.3). Similarly, SNP13 (rs3799703) was associated with Glo1 expression (GG as reference, standardized beta AG = 0.17, p = 0.14 and AA = 0.36, p = 0.005). After correction for multiple testing these associations were not significant. For the other SNPs, we observed no consistent associations over the different genotypes. Thus, polymorphisms of GLO1 were not associated with Glo1 expression or markers of MGO stress, suggesting that these SNPs are not functional, although activity/expression might be altered in other tissues.