Inhibition Of Glyoxalase 1 Research Articles

CircMAN1A2_009 facilitates YBX1 nuclear localization to induce GLO1 activation for cervical adenocarcinoma cell growth.

The molecular mechanisms driving the development of cervical adenocarcinoma (CADC) and optimal patient management strategies remain elusive. In this study, we have identified circMAN1A2_009 as an oncogenic circular RNA (circRNA) in CADC. Clinically, circMAN1A2_009 showed significant upregulation in CADC tissues, with an impressive area under the curve value of 0.8075 for detecting CADC. Functional studies, involving both gain-of-function and loss-of-function experiments, revealed that circMAN1A2_009 suppressed reactive oxygen species accumulation and apoptosis, and boosted cell viability in CADC cells. Conversely, silencing circMAN1A2_009 reversed these effects. Further mechanistic investigations indicated that circMAN1A2_009 interacted with YBX1, facilitating the phosphorylation levels of YBX1 at serine 102 (p-YBX1S102) and facilitating YBX1 nuclear localization through sequence 245-251. This interaction subsequently increased the activity of the glyoxalase 1 (GLO1) promoter, leading to the activation of GLO1 expression. Consistently, inhibition of either YBX1 or GLO1 mirrored the biological effects of circMAN1A2_009 in CADC cells. Additionally, knockdown of YBX1 or GLO1 partially reversed the oncogenic behaviors induced by circMAN1A2_009. In conclusion, our findings propose circMAN1A2_009 as a potential oncogene and a promising indicator for diagnosing and guiding therapy in CADC patients.

Glyoxalase 1: Emerging biomarker and therapeutic target in cervical cancer progression.

Cervical cancer presents a significant global health challenge, disproportionately impacting underserved populations with limited access to healthcare. Early detection and effective management are vital in addressing this public health concern. This study focuses on Glyoxalase-1 (GLO1), an enzyme crucial for methylglyoxal detoxification, in the context of cervical cancer. We assessed GLO1 expression in cervical cancer patient samples using immunohistochemistry. In vitro experiments using HeLa cells were conducted to evaluate the impact of GLO1 inhibition on cell viability and migration. Single-cell RNA sequencing (scRNA-seq) and gene set variation analysis were utilized to investigate the role of GLO1 in the metabolism of cervical cancer. Additionally, public microarray data were analyzed to determine GLO1 expression across various stages of cervical cancer. Our analysis included 58 cervical cancer patients, and showed that GLO1 is significantly upregulated in cervical cancer tissues compared to normal cervical tissues, independent of pathological findings and disease stage. In vitro experiments indicated that GLO1 inhibition by S-p-bromobenzylglutathione cyclopentyl diester decreased cell viability and migration in cervical cancer cell lines. Analyses of scRNA-seq data and public gene expression datasets corroborated the overexpression of GLO1 and its involvement in cancer metabolism, particularly glycolysis. An examination of expression data from precancerous lesions revealed a progressive increase in GLO1 expression from normal tissue to invasive cervical cancer. This study highlights the critical role of GLO1 in the progression of cervical cancer, presenting it as a potential biomarker and therapeutic target. These findings contribute valuable insights towards personalized treatment approaches and augment the ongoing efforts to combat cervical cancer. Further research is necessary to comprehensively explore GLO1's potential in clinical applications.

Salicylate metal-binding isosteres as fragments for metalloenzyme inhibition.

Metalloenzyme inhibitors typically share a common need to possess a metal-binding pharmacophore (MBP) for binding the active site metal ions. However, MBPs can suffer from physicochemical liabilities, impeding the pharmacological properties and drug-likeliness of inhibitors. To circumvent this, problematic features of the MBP can be identified and exchanged with isosteric replacements. Herein, the carboxylic and hydroxyl group of the salicylic acid MBP were replaced and a total of 27 salicylate metal-binding isosteres (MBIs) synthesized. Of these 27 MBIs, at least 12 represent previously unreported compounds, and the metal-binding abilities of >20 of the MBIs have not been previously reported. These salicylate MBIs were examined for their metal-binding features in model complexes, physicochemical properties, and biological activity. It was observed that salicylate MBIs can demonstrate a range of attractive physicochemical properties and bind to the metal in a variety of expected and unexpected binding modes. The biological activity of these novel MBIs was evaluated by measuring inhibition against two Zn2+-dependent metalloenzymes, human glyoxalase 1 (GLO1) and matrix metalloproteinase 3 (MMP-3), as well as a dinuclear Mn2+-dependent metalloenzyme, influenza H1N1 N-terminal endonuclease (PAN). It was observed that salicylate MBIs could maintain or improve enzyme inhibition and selectivity. To probe salicylate MBIs as fragments for fragment-based drug discovery (FBDD), an MBI that showed good inhibitory activity against GLO1 was derivatized and a rudimentary structure–activity relationship was developed. The resulting elaborated fragments showed GLO1 inhibition with low micromolar activity.

Tanshinone IIa protects retinal endothelial cells against mitochondrial fission induced by methylglyoxal through glyoxalase 1

Advanced glycation end products (AGEs) play an important role in the onset of diabetic retinopathy. Therefore, in the current study, we investigate whether and how Tanshinone IIa (Tan IIa) from Salvia miltiorrhiza protects bovine retinal endothelial cells (BRECs) against methylglyoxal (MGO) mediated cell dysfunction. The results showed that MGO reduced cell viability in dose dependent manner. The treatment of Tan IIa (50 μM) significantly improved cell viability induced by MGO in BRECs. MGO increased cellular reactive oxygen species formation and cellular nitric oxide (NO) level; enhanced nox1 and iNOS mRNA levels; inhibited prdx1 mRNA level. The treatment of Tan IIa effectually ameliorated cellular oxidative stress. Exposure of MGO resulted in mitochondrial fission and decrease of opa1 and mfn1. No significant difference in mRNA levels of mfn2 and drp1 was detected between MGO and medium. Tan IIa reduced mitochondrial fragmentation, enhanced the mRNA levels of mfn1 and opa1 in MGO cultured BRECs. The short time exposure of cellular antioxidatants, dimethylthiourea (10 mM) and tiron (10 mM) had no effect on mitochondrial fission although they ameliorated cellular reactive oxygen species level. Moreover, overexpression of glyoxalase 1 (GLO1) increased key proteins of mitochondrial fusion, including opa1 and mfn1 in BRECs cultured with MGO. However, inhibition of GLO1 by siRNA abolished the effect of Tan IIa on induction of mitochondrial fusion in MGO cultured BRECs. In conclusion, MGO caused the injury of retinal endothelial cells through induction of mitochondrial dysfunction and mitochondrial fission, the treatment of Tan IIa ameliorated mitochondrial dysfunction and fission induced by AGEs through enhancing GLO1.

Glyoxalase 1 gene is highly expressed in basal-like human breast cancers and contributes to survival of ALDH1-positive breast cancer stem cells.

Glyoxalase 1 (GLO1) is a ubiquitous enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis that induces apoptosis. In this study, we found that GLO1 gene expression correlates with neoplasm histologic grade (χ2 test, p = 0.002) and is elevated in human basal-like breast cancer tissues. Approximately 90% of basal-like cancers were grade 3 tumors highly expressing both GLO1 and the cancer stem cell marker ALDH1A3. ALDH1high cells derived from the MDA-MB 157 and MDA-MB 468 human basal-like breast cancer cell lines showed elevated GLO1 activity. GLO1 inhibition using TLSC702 suppressed ALDH1high cell viability as well as the formation of tumor-spheres by ALDH1high cells. GLO1 knockdown using specific siRNAs also suppressed ALDH1high cell viability, and both TLSC702 and GLO1 siRNA induced apoptosis in ALDH1high cells. These results suggest GLO1 is essential for the survival of ALDH1-positive breast cancer stem cells. We therefore conclude that GLO1 is a potential therapeutic target for treatment of basal-like breast cancers.

Glyoxalase 1 (GLO1) Inhibition or Genetic Overexpression Does Not Alter Ethanol's Locomotor Effects: Implications for GLO1 as a Therapeutic Target in Alcohol Use Disorders.

Glyoxalase 1 (GLO1) is an enzyme that metabolizes methylglyoxal (MG), which is a competitive partial agonist at GABAA receptors. Inhibition of GLO1 increases concentrations of MG in the brain and decreases binge-like ethanol (EtOH) drinking. This study assessed whether inhibition of GLO1, or genetic overexpression of Glo1, would also alter the locomotor effects of EtOH, which might explain reduced EtOH consumption following GLO1 inhibition. We used the prototypical GABAA receptor agonist muscimol as a positive control. Male C57BL/6J mice were pretreated with either the GLO1 inhibitor S-bromobenzylglutathione cyclopentyl diester (pBBG; 7.5mg/kg; Experiment 1) or muscimol (0.75mg/kg; Experiment 2), or their corresponding vehicle. We then determined whether locomotor response to a range of EtOH doses (0, 0.5, 1.0, 1.5, 2.0, and 2.5) was altered by either pBBG or muscimol pretreatment. We also examined the locomotor response to a range of EtOH doses in FVB/NJ wild-type and transgenic Glo1 overexpressing mice (Experiment 3). Anxiety-like behavior (time spent in the center of the open field) was assessed in all 3 experiments. The EtOH dose-response curve was not altered by pretreatment with pBBG or by transgenic overexpression of Glo1. In contrast, muscimol blunted locomotor stimulation at low EtOH doses and potentiated locomotor sedation at higher EtOH doses. No drug or genotype differences were seen in anxiety-like behavior after EtOH treatment. The dose of pBBG used in this study is within the effective range shown previously to reduce EtOH drinking. Glo1 overexpression has been previously shown to increase EtOH drinking. However, neither manipulation altered the dose-response curve for EtOH's locomotor effects, whereas muscimol appeared to enhance the locomotor sedative effects of EtOH. The present data demonstrate that reduced EtOH drinking caused by GLO1 inhibition is not due to potentiation of EtOH's stimulant or depressant effects.

Methylglyoxal-induced glycation changes adipose tissue vascular architecture, flow and expansion, leading to insulin resistance

Microvascular dysfunction has been suggested to trigger adipose tissue dysfunction in obesity. This study investigates the hypothesis that glycation impairs microvascular architecture and expandability with an impact on insulin signalling. Animal models supplemented with methylglyoxal (MG), maintained with a high-fat diet (HFD) or both (HFDMG) were studied for periepididymal adipose (pEAT) tissue hypoxia and local and systemic insulin resistance. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to quantify blood flow in vivo, showing MG-induced reduction of pEAT blood flow. Increased adipocyte size and leptin secretion were observed only in rats feeding the high-fat diet, without the development of hypoxia. In turn, hypoxia was only observed when MG was combined (HFDMG group), being associated with impaired activation of the insulin receptor (Tyr1163), glucose intolerance and systemic and muscle insulin resistance. Accordingly, the adipose tissue angiogenic assay has shown decreased capillarization after dose-dependent MG exposure and glyoxalase-1 inhibition. Thus, glycation impairs adipose tissue capillarization and blood flow, hampering its expandability during a high-fat diet challenge and leading to hypoxia and insulin resistance. Such events have systemic repercussions in glucose metabolism and may lead to the onset of unhealthy obesity and progression to type 2 diabetes.

Abstract 3355: Inhibiting glyoxylase 1 as a strategy to target highly glycolytic cancer cells

Abstract Cancer cells typically display altered glucose metabolism characterized by a preference of aerobic glycolysis (Warburg effect) resulting in higher levels of glycolytic waste including methylglyoxal (MG). GLO1 (Glyoxalase 1) functions in the detoxification of MG: MG reacts with glutathione to form hemithioacetal, which is converted into S-D-Lactoylglutathione by GLO1 and further metabolised into D-lactate by GLO2. When not detoxified, MG acts as a cytotoxic reagent by forming DNA- and protein-adducts (advanced glycation end products = AGEs) that subsequently lead to cell death. Therefore, GLO1 has been discussed as a potential anti-tumor target for highly glycolytic cancers. We confirmed previous reports that GLO1 expression is elevated in several tumor entities as compared to normal tissue. Tumor cells with high GLO1 expression levels are proposed to be highly dependent on GLO1 for removal of toxic MG. Therefore sensitivity to GLO1 inhibition was probed in a panel of cell lines covering lung, colon, breast, prostate and skin cancer with varying GLO1 amplification and expression levels. siRNA-mediated knock down of GLO1 did neither cause significant reduction of cellular proliferation nor significant induction of apoptosis in any of the tested tumor cells. Furthermore, treatment of these cells with a potent GLO1 inhibitor (Chiba et al. Bioorg Med Chem Lett. 2012, IC50=11 nM) did not affect proliferation or induce apoptosis at sub-µM concentrations. In addition, no increase in AGEs upon GLO1 inhibition could be detected by western blot. Activity of the GLO1 inhibitor was further tested under conditions expected to increase endogenous MG levels such as hypoxia and elevated glucotriose (via GAPDH inhibition). Furthermore, a spheroid assay was developed to test the GLO1 inhibitor in a 3D assay system, better reflecting in vivo tumor growth. Even under hypoxic or spheroidal assay conditions, proliferation of cancer cells could not be reduced upon GLO1 inhibition. We could demonstrate however that GLO1 inhibition in A375 melanoma and SW620 colon adenocarcinoma cells sensitized the cells to exogenous MG. Taken together we could not confirm earlier published data that GLO1 inhibition leads to reduced proliferation and induction of apoptosis in cancer cells that have elevated GLO1 expression levels. Being aware that cellular systems may not reflect the situation in vivo, we speculate that GLO1 function alone may be redundant in detoxifying MG. Other possible enzymes which have been linked to MG removal include aldose reductase. Simultaneous inhibition of both enzymes might be a strategy to target highly glycolytic cancers in the future. Citation Format: Sylvia Gruenewald, Michael Steckel, Andreas Timmermann, Hartmut Rehwinkel, Patrick Steigemann, Sylvia Zacharias, Alexander Walter, Marcus Bauser, Andrea Haegebarth. Inhibiting glyoxylase 1 as a strategy to target highly glycolytic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3355. doi:10.1158/1538-7445.AM2014-3355

Aging-Dependent Reduction in Glyoxalase 1 Delays Wound Healing

Methylglyoxal (MG), the major dicarbonyl substrate of the enzyme glyoxalase 1 (GLO1), is a reactive metabolite formed via glycolytic flux. Decreased GLO1 activity in situ has been shown to result in an accumulation of MG and increased formation of advanced glycation endproducts, both of which can accumulate during physiological aging and at an accelerated rate in diabetes and other chronic degenerative diseases. To determine the physiological consequences which result from elevated MG levels and the role of MG and GLO1 in aging, wound healing in young (≤12 weeks) and old (≥52 weeks) wild-type mice was studied. Old mice were found to have a significantly slower rate of wound healing compared to young mice (74.9 ± 2.2 vs. 55.4 ± 1.5% wound closure at day 6; 26% decrease; p < 0.0001). This was associated with decreases in GLO1 transcription, expression and activity. The importance of GLO1 was confirmed in mice by inhibition of GLO1. Direct application of MG to the wounds of young mice, decreased wound healing by 24% compared to untreated mice, whereas application of BSA modified minimally by MG had no effect. Treatment of either young or old mice with aminoguanidine, a scavenger of free MG, significantly increased wound closure by 16% (66.8 ± 1.6 vs. 77.2 ± 3.1%; p < 0.05) and 64% (40.4 ± 7.9 vs. 66.4 ± 5.2%; p < 0.05), respectively, by day 6. As a result of the aminoguanidine treatment, the overall rate of wound healing in the old mice was restored to the level observed in the young mice. These findings were confirmed in vitro, as MG reduced migration and proliferation of fibroblasts derived from young and old, wild-type mice. The data demonstrate that the balance between MG and age-dependent GLO1 downregulation contributes to delayed wound healing in old mice.

Glyoxalase 1 and its substrate methylglyoxal are novel regulators of seizure susceptibility

Epilepsy is a complex disease characterized by a predisposition toward seizures. There are numerous barriers to the successful treatment of epilepsy. For instance, current antiepileptic drugs have adverse side effects and variable efficacies. Furthermore, the pathophysiologic basis of epilepsy remains largely elusive. Therefore, investigating novel genes and biologic processes underlying epilepsy may provide valuable insight and enable the development of new therapeutic agents. We previously identified methylglyoxal (MG) as an endogenous γ-aminobutyric acid (GABAA ) receptor agonist. Here, we investigated the role of MG and its catabolic enzyme, glyoxalase 1 (GLO1), in seizures. We pretreated mice with MG before seizure induction with picrotoxin or pilocarpine and then assessed seizures behaviorally or by electroencephalography (EEG). We then investigated the role of GLO1 in seizures by treating mice with a pharmacologic inhibitor of GLO1 before seizure induction with pilocarpine and measured subsequent seizure phenotypes. Next, we explored the genetic relationship between Glo1 expression and seizures. We analyzed seizure phenotypes among C57BL/6J × DBA/2J (BXD) recombinant inbred (RI) mice with differential Glo1 expression. Lastly, we investigated a causal role for Glo1 in seizures by administering pilocarpine to transgenic (Tg) mice that overexpress Glo1. Pretreatment with MG attenuated pharmacologically-induced seizures at both the behavioral and EEG levels. GLO1 inhibition, which increases MG concentration in vivo, also attenuated seizures. Among BXD RI mice, high Glo1 expression was correlated with increased seizure susceptibility. Tg mice overexpressing Glo1 displayed reduced MG concentration in the brain and increased seizure severity. These data identify MG as an endogenous regulator of seizures. Similarly, inhibition of GLO1 attenuates seizures, suggesting that this may be a novel therapeutic approach for epilepsy. Furthermore, this system may represent an endogenous negative feedback loop whereby high metabolic activity increases inhibitory tone via local accumulation of MG. Finally, Glo1 may contribute to the genetic architecture of epilepsy, as Glo1 expression regulates both MG concentration and seizure severity.

C. elegans as Model for the Study of High Glucose– Mediated Life Span Reduction

OBJECTIVEEstablishing Caenorhabditis elegans as a model for glucose toxicity–mediated life span reduction.RESEARCH DESIGN AND METHODSC. elegans were maintained to achieve glucose concentrations resembling the hyperglycemic conditions in diabetic patients. The effects of high glucose on life span, glyoxalase-1 activity, advanced glycation end products (AGEs), and reactive oxygen species (ROS) formation and on mitochondrial function were studied.RESULTSHigh glucose conditions reduced mean life span from 18.5 ± 0.4 to 16.5 ± 0.6 days and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.4 days, independent of glucose effects on cuticle or bacterial metabolization of glucose. The formation of methylglyoxal-modified mitochondrial proteins and ROS was significantly increased by high glucose conditions and reduced by mitochondrial uncoupling and complex IIIQo inhibition. Overexpression of the methylglyoxal–detoxifying enzyme glyoxalase-1 attenuated the life-shortening effect of glucose by reducing AGE accumulation (by 65%) and ROS formation (by 50%) and restored mean (16.5 ± 0.6 to 20.6 ± 0.4 days) and maximum life span (23.2 ± 0.4 to 27.7 ± 2.3 days). In contrast, inhibition of glyoxalase-1 by RNAi further reduced mean (16.5 ± 0.6 to 13.9 ± 0.7 days) and maximum life span (23.2 ± 0.4 to 20.3 ± 1.1 days). The life span reduction by glyoxalase-1 inhibition was independent from the insulin signaling pathway because high glucose conditions also affected daf-2 knockdown animals in a similar manner.CONCLUSIONSC. elegans is a suitable model organism to study glucose toxicity, in which high glucose conditions limit the life span by increasing ROS formation and AGE modification of mitochondrial proteins in a daf-2 independent manner. Most importantly, glucose toxicity can be prevented by improving glyoxalase-1–dependent methylglyoxal detoxification or preventing mitochondrial dysfunction.