Deletion of yghZ in Escherichia coli promotes growth in presence of furfural with xylose as carbon source.

Thermo-acidic pretreatment of lignocellulosic biomass is required to make it amenable to microbial metabolism and results in generation of furfural due to breakdown of pentose sugars. Furfural is toxic to microbial metabolism and results in reduced microbial productivity and increased production costs. This study asks if deletion of yghZ gene which encodes a NADPH-dependent aldehyde reductase enzyme results in improved furfural tolerance in Escherichia coli host. The ∆yghZ strain-SSK201-was tested for tolerance to furfural in presence of 5% xylose as a carbon source in AM1 minimal medium. At 96 h and in presence of 1.0 g/L furfural, the culture harboring strain SSK201 displayed 4.5-fold higher biomass, 2-fold lower furfural concentration and 15.75-fold higher specific growth rate (µ) as compared to the parent strain SSK42. The furfural tolerance advantage of SSK201 was retained when the carbon source was switched to glucose in AM1 medium and was lost in rich LB medium. The findings have potential to be scaled up to a hydrolysate culture medium, which contains furan inhibitors and lack nutritionally rich components, under bioreactor cultivation and observe growth advantage of the ∆yghZ host. It harbors potential to generate robust industrial strains which can convert lignocellulosic carbon into metabolites of interest in a cost-efficient manner.

A novel aldo-keto reductase gene, OsAKR1, from rice confers higher tolerance to cadmium stress in rice by an in vivo reactive aldehyde detoxification.

Elevated levels of cadmium (Cd) have the ability to impede plant development. Aldo-keto reductases (AKRs) have been demonstrated in a number of plant species to improve tolerance to a variety of abiotic stresses by scavenging cytotoxic aldehydes; however, only a few AKRs have been identified to improve Cd tolerance. The OsAKR1 gene was extracted and identified from rice here. After being exposed to Cd, the expression of OsAKR1 dramatically rose in both roots and shoots, although more pronounced in roots. According to a subcellular localization experiment, the nucleus and cytoplasm are where OsAKR1 is primarily found. Mutants lacking OsAKR1 exhibited Cd sensitive phenotype than that of the wild-type (WT) Nipponbare (Nip), and osakr1 mutants exhibited reduced capacity to scavenge methylglyoxal (MG). Furthermore, osakr1 mutants exhibited considerably greater hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels, and increased catalase (CAT) activity in comparison to Nip. The expression of three isomeric forms of CAT was found to be considerably elevated in osakr1 mutants during Cd stress, as demonstrated by quantitative real-time PCR analysis, when compared to Nip. These results imply that OsAKR1 controlled rice's ability to withstand Cd by scavenging harmful aldehydes and turning on the reactive oxygen species (ROS) scavenging mechanism.

Gastric Cancer Immune Subtypes and Prognostic Modeling: Insights from Aging-Related Gene Analysis.

Gastric cancer (GC) is highly heterogeneous and influenced by aging-related factors. This study aimed to improve individualized prognostic assessment of GC by identifying aging-related genes and subtypes. Immune scores of GC samples from GEO and TCGA databases were calculated using ESTIMATE and scored as high immune (IS_high) and low immune (IS_low). ssGSEA was used to analyze immune cell infiltration. Univariate Cox regression was employed to identify prognosis-related genes. LASSO regression analysis was used to construct a prognostic model. GSVA enrichment analysis was applied to determine pathways. CCK-8, wound healing, and Transwell assays tested the proliferation, migration, and invasion of the GC cell line (AGS). Cell cycle and aging were examined using flow cytometry, ß-galactosidase staining, and Western blotting. Two aging-related GC subtypes were identified. Subtype 2 was characterized as lower survival probability and higher risk, along with a more immune-responsive tumor microenvironment. Three genes (IGFBP5, BCL11B, and AKR1B1) screened from aging-related genes were used to establish a prognosis model. The AUC values of the model were greater than 0.669, exhibiting strong prognostic value. In vitro, IGFBP5 overexpression in AGS cells was found to decrease viability, migration, and invasion, alter the cell cycle, and increase aging biomarkers (SA-ß-galactosidase, p53, and p21). This analysis uncovered the immune characteristics of two subtypes and aging-related prognosis genes in GC. The prognostic model established for three aging-related genes (IGFBP5, BCL11B, and AKR1B1) demonstrated good prognosis performance, providing a foundation for personalized treatment strategies aimed at GC.

An NADH/NAD+-favored aldo-keto reductase facilitates avilamycin A biosynthesis by primarily catalyzing oxidation of avilamycin C.

Avilamycins, which possess potent inhibitory activity against Gram-positive bacteria, are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes. Among these structurally related oligosaccharide antibiotics, avilamycin A serves as the main bioactive component in veterinary drugs and animal feed additives, which differs from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. However, the mechanisms underlying assembly and modification of the oligosaccharide chain to diversify individual avilamycins remain poorly understood. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. Remarkably, the ratio of these two components produced by AviZ1 depends on the utilization of specific redox cofactors, namely NADH/NAD+ or NADPH/NADP+. These findings are inspired by gene disruption and complementation experiments and are further supported by in vitro enzymatic activity assays, kinetic analyses, and cofactor affinity studies on AviZ1-catalyzed redox reactions. Additionally, the results from sequence analysis, structure prediction, and site-directed mutagenesis of AviZ1 validate it as an NADH/NAD+-favored aldo-keto reductase that primarily oxidizes avilamycin C to form avilamycin A by utilizing abundant NAD+ in vivo. Building upon the biological function and catalytic activity of AviZ1, overexpressing AviZ1 in S. viridochromogenes is thus effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study represents, to our knowledge, the first characterization of biochemical reactions involved in avilamycin biosynthesis and contributes to the construction of high-performance strains with industrial value.IMPORTANCEAvilamycins are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes, which can be used as veterinary drugs and animal feed additives. Avilamycin A is the most bioactive component, differing from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. Currently, the biosynthetic pathway of avilamycins is not clear. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. More importantly, AviZ1 exhibits a unique NADH/NAD+ preference, allowing it to efficiently catalyze the oxidation of avilamycin C to form avilamycin A using abundant NAD+ in cells. Thus, overexpressing AviZ1 in S. viridochromogenes is effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study serves as an enzymological guide for rational strain design, and the resulting high-performance strains have significant industrial value.

Prunin from Poncirus trifoliata (L.) Rafin Inhibits Aldose Reductase and Glucose-Fructose-Mediated Protein Glycation and Oxidation of Human Serum Albumin.

Diabetes complications are associated with aldose reductase (AR) and advanced glycation end products (AGEs). Using bioassay-guided isolation by column chromatography, 10 flavonoids and one coumarin were isolated from Poncirus trifoliata Rafin and tested in vitro for an inhibitory effect against human recombinant AR (HRAR) and rat lens AR (RLAR). Prunin, narirutin, and naringin inhibited RLAR (IC50 0.48-2.84 µM) and HRAR (IC50 0.68-4.88 µM). Docking simulations predicted negative binding energies and interactions with the RLAR and HRAR binding pocket residues. Prunin (0.1 and 12.5 µM) prevented the formation of fluorescent AGEs and nonfluorescent Nε-(carboxymethyl) lysine (CML), as well as the fructose-glucose-mediated protein glycation and oxidation of human serum albumin (HSA). Prunin suppressed the formation of the ß-cross-amyloid structure of HSA. These results indicate that prunin inhibits oxidation-dependent protein damage, AGE formation, and AR, which may help prevent diabetes complications.

Decoding selectivity: computational insights into AKR1B1 and AKR1B10 inhibition.

Understanding selectivity mechanisms of inhibitors towards highly homologous proteins is of paramount importance in the design of selective candidates. Human aldo-keto reductases (AKRs) pertain to a superfamily of monomeric oxidoreductases, which serve as NADPH-dependent cytosolic enzymes to catalyze the reduction of carbonyl groups to primary and secondary alcohols using electrons from NADPH. Among AKRs, AKR1B1 is emerging as a promising target for cancer treatment and diabetes, despite its high structural similarity with AKR1B10, which leads to severe adverse events. Therefore, it is crucial to understand the selectivity mechanisms of AKR1B1 and AKR1B10 to discover safe anticancer candidates with optimal therapeutic efficacy. In this study, multiple computational strategies, including sequence alignment, structural comparison, Protein Contacts Atlas analysis, molecular docking, molecular dynamics simulation, MM-GBSA calculation, alanine scanning mutagenesis and pharmacophore modeling analysis were employed to comprehensively understand the selectivity mechanisms of AKR1B1/10 inhibition based on selective inhibitor lidorestat and HAHE. This study would provide substantial evidence in the design of potent and highly selective AKR1B1/10 inhibitors in future.

Computer-aided design to enhance the stability of aldo-keto reductase KdAKR.

The aldo-keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) to t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer-aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKRM6 . Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The "best" mutant KdAKRM10 (KdAKRM6 -S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T50 15 and Tm values were increased by 10.2 and 10.0°C, respectively, and half-life (t1/2 ) at 40°C was increased by 17.6 h. Additionally, KdAKRM10 demonstrated improved resistance to organic solvents compared to that of KdAKRM6 . Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKRM10 , thus improving its stability. The results validated the feasibility of the computer-aided design strategy in improving the stability of AKRs.

Aldehyde Reductase Activity of Carboxylic Acid Reductases.

Carboxylic acid reductase enzymes (CARs) are well known for the reduction of a wide range of carboxylic acids to the respective aldehydes. One of the essential CAR domains - the reductase domain (R-domain) - was recently shown to catalyze the standalone reduction of carbonyls, including aldehydes, which are typically considered to be the final product of carboxylic acid reduction by CAR. We discovered that the respective full-length CARs were equally able to reduce aldehydes. Herein we aimed to shed light on the impact of this activity on aldehyde production and acid reduction in general. Our data explains previously inexplicable results and a new CAR from Mycolicibacterium wolinskyi is presented.

Aldo-keto reductases 7A subfamily: A mini review.

Aldo-keto reductase-7A (AKR7A) subfamily belongs to the AKR superfamily and is associated with detoxification of aldehydes and ketones by reducing them to the corresponding alcohols. So far five members of ARK7A subfamily are identified: two human members-AKR7A2 and AKR7A3, two rat members-AKR7A1 and AKR7A4, and one mouse member-AKR7A5, which are implicated in several diseases including neurodegenerative diseases and cancer. AKR7A members share similar crystal structures and protein functional domains, but have different substrate specificity, inducibility and biological functions. This review will summarize the research progress of AKR7A members in substrate specificity, tissue distribution, inducibility, crystal structure and biological function. The significance of AKR7A members in the occurrence and development of diseases will also be discussed.

An aldose reductase inhibitor, WJ-39, ameliorates renal tubular injury in diabetic nephropathy by activating PINK1/Parkin signaling.

Renal tubular injury is a critical factor during the early stages of diabetic nephropathy (DN). Proximal tubular epithelial cells, which contain abundant mitochondria essential for intracellular homeostasis, are susceptible to disruptions in the intracellular environment, making them especially vulnerable to diabetic state disorders, which may be attributed to their elevated energy requirements and reliance on aerobic metabolism. It is widely thought that overactivation of the polyol pathway is implicated in DN pathogenesis, and inhibition of aldose reductase (AR), the rate-limiting enzyme in this pathway, represents a promising therapeutic avenue. WJ-39, a novel aldose reductase inhibitor, was investigated in this study for its protective effects on renal tubules in DN and the underlying mechanisms. Our findings revealed that WJ-39 significantly ameliorated the renal tubular morphology in high-fat diet (HFD)/streptozotocin (STZ)-induced DN rats, concurrently inhibiting fibrosis. Notably, WJ-39 safeguarded the structure and function of renal tubular mitochondria by enhancing mitochondrial dynamics. This involved the regulation of mitochondrial fission and fusion proteins and the promotion of PTEN-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy. Furthermore, WJ-39 demonstrated the inhibition of endogenous apoptosis by mitigating the production of mitochondrial reactive oxygen species (ROS). The protective effects of WJ-39 on mitochondria and apoptosis were countered in high glucose-treated HK-2 cells upon transfection with PINK1 siRNA. Overall, our findings suggest that WJ-39 protects the structural and functional integrity of renal tubules in DN, which may be attributed to its capacity to inhibit aldose reductase activity, activate the PINK1/Parkin signaling pathway, promote mitophagy, and alleviate apoptosis.

High incidence of sebaceous gland inflammation in aldose reductase-deficient mice.

Aldose reductase is a member of the 1B1 subfamily of aldo-keto reductase gene superfamily. The action of aldose reductase (AR) has been implicated in the pathogenesis of a variety of disease states, most notably complications of diabetes mellitus including neuropathy, retinopathy, nephropathy, and cataracts. To explore for mechanistic roles for AR in disease pathogenesis, we established mutant strains produced using Crispr-Cas9 to inactivate the AKR1B3 gene in C57BL6 mice. Phenotyping AR-knock out (ARKO) strains confirmed previous reports of reduced accumulation of tissue sorbitol levels. Lens epithelial cells in ARKO mice showed markedly reduced epithelial-to-mesenchymal transition following lens extraction in a surgical model of cataract and posterior capsule opacification. A previously unreported phenotype of preputial sebaceous gland swelling was observed frequently in male ARKO mice homozygous for the mutant AKR1B3 allele. This condition, which was shown to be accompanied by infiltration of proinflammatory CD3+ lymphocytes, was not observed in WT mice or mice heterozygous for the mutant allele. Despite this condition, reproductive fitness of the ARKO strain was indistinguishable from WT mice housed under identical conditions. These studies establish the utility of a new strain of AKR1B3-null mice created to support mechanistic studies of cataract and diabetic eye disease.

Mining and tailor-made engineering of a novel keto reductase for asymmetric synthesis of structurally hindered γ- and δ-lactones.

A novel carbonyl reductase from Hyphopichia burtoni (HbKR) was discovered by gene mining. HbKR is a NADPH-dependent dual function enzyme with reduction and oxidation activity belonging to SDR superfamily. HbKR strictly follows Prelog priority in the reduction of long-chain aliphatic keto acids/esters containing remote carbonyl groups, such as 4-oxodecanoic acid and 5-oxodecanoic acid, producing (S)-γ-decalactone and (S)-δ-decalactone in >99 % e.e. Tailor-made engineering of HbKR was conducted to improve its catalytic efficiency. Variant F207A/F86M was obtained with specific activity of 8.37 U/mg toward 5-oxodecanoic acid, which was 9.7-fold of its parent. Employing F207A/F86M, 100 mM 5-oxodecanoic acid could be reduced into optically pure (S)-δ-decalactone. Molecular docking analysis indicates that substitution of aromatic Phe with smaller residues renders sufficient space for accommodating substrates in a more stable conformation. This study offers an efficient biocatalyst for the biosynthesis of (S)-lactones, and provides guidance for engineering carbonyl reductases toward structurally hindered substrates.

Novel spiroindoline derivatives targeting aldose reductase against diabetic complications: Bioactivity, cytotoxicity, and molecular modeling studies.

Despite significant developments in therapeutic strategies, Diabetes Mellitus remains an increasing concern, leading to various complications, e.g., cataracts, neuropathy, retinopathy, nephropathy, and several cardiovascular diseases. The polyol pathway, which involves Aldose reductase (AR) as a critical enzyme, has been focused on by many researchers as a target for intervention. On the other hand, spiroindoline-based compounds possess remarkable biological properties. This guided us to synthesize novel spiroindoline oxadiazolyl-based acetate derivatives and investigate their biological activities. The synthesized molecules' structures were confirmed herein, using IR, NMR (1H and 13C), and Mass spectroscopy. All compounds were potent inhibitors with KI constants spanning from 0.186 ± 0.020 µM to 0.662 ± 0.042 µM versus AR and appeared as better inhibitors than the clinically used drug, Epalrestat (EPR, KI: 0.841 ± 0.051 µM). Besides its remarkable inhibitory profile compared to EPR, compound 6k (KI: 0.186 ± 0.020 µM) was also determined to have an unusual pharmacokinetic profile. The results showed that 6k had less cytotoxic effect on normal mouse fibroblast (L929) cells (IC50 of 569.58 ± 0.80 µM) and reduced the viability of human breast adenocarcinoma (MCF-7) cells (IC50 of 110.87 ± 0.42 µM) more than the reference drug Doxorubicin (IC50s of 98.26 ± 0.45 µM and 158.49 ± 2.73 µM, respectively), thus exhibiting more potent anticancer activity. Moreover, molecular dynamic simulations for 200 ns were conducted to predict the docked complex's stability and reveal significant amino acid residues that 6k interacts with throughout the simulation.

Hyperglycemia exacerbates cerebral ischemia/reperfusion injury by up-regulating autophagy through p53-Sesn2-AMPK pathway.

Hyperglycemia exacerbates ischemic brain injury by up-regulating autophagy. However, the underlying mechanisms are unknown. This study aims to determine whether hyperglycemia activates autophagy through the p53-Sesn2-AMPK signaling pathway. Rats were subjected to 30-min middle cerebral artery occlusion (MCAO) with reperfusion for 1- and 3-day under normo- and hyperglycemic conditions; and HT22 cells were exposed to oxygen deprivation (OG) or oxygen-glucose deprivation and re-oxygenation (OGD/R) with high glucose. Autophagy inhibitors, 3-MA and ARI, were used both in vivo and in vitro. The results showed that, compared with the normoglycemia group (NG), hyperglycemia (HG) increased infarct volume and apoptosis in penumbra area, worsened neurological deficit, and augmented autophagy. after MCAO followed by 1-day reperfusion. Further, HG promoted the conversion of LC-3I to LC-3II, decreased p62, increased protein levels of aldose reductase, p53, P-p53ser15, Sesn2, AMPK and numbers of autophagosomes and autolysosomes, detected by transmission electron microscopy and mRFP-GFP-LC3 molecular probe, in the cerebral cortex after ischemia and reperfusion injury in animals or in cultured HT22 cells exposed to hypoxia with high glucose content. Finally, experiments with autophagy inhibitors 3-MA and aldose reductase inhibitor (ARI) revealed that while both inhibitors reduced the number of TUNEL positive neurons and reversed the effects of hyperglycemic ischemia on LC3 and p62, only ARI decreased the levels of p53, P-p53ser15. These results suggested that hyperglycemia might induce excessive autophagy to aggravate the brain injury resulted from I/R and that hyperglycemia might activate the p53-Sesn2-AMPK signaling pathway, in addition to the classical PI3K/AKT/mTOR autophagy pathway.

Mitigation of lens opacification by a functional food in a diabetic rodent model.

The current study was designed to test a functional food (FF) mixture containing aldose reductase inhibitors and antiglycation bioactive compounds for suppressing the onset and progression of cataracts in a diabetic rat model. Two-month-old Sprague Dawley rats were grouped as control (C), diabetes untreated (D), and diabetic rats treated with FF at two doses (FF1 = 1.35 g and FF2 = 6.25 g/100g of diet). Diabetes was induced by a single injection of streptozotocin. The FF is a mixture of amla, turmeric, black pepper, cinnamon, ginger, and fenugreek added to the rodent diet. The status of cataracts was monitored weekly by a slit lamp examination for 20 weeks, after which animals were sacrificed to collect eye lenses. Feeding FF1 and FF2 to diabetic rats yielded a significant anti-hyperglycaemic effect and marginally prevented body weight loss. FF delayed cataract progression, and FF2 showed better efficacy than FF1. FF prevented the loss of lens crystallins and their insolubilization in diabetic rats. The antioxidant potential of FF was evident with the lowered protein carbonyls, lipid peroxidation, and prevention of altered antioxidant enzyme activities induced by diabetes. These studies demonstrate the efficacy of plant-derived dietary supplements against the onset and progression of cataracts in a well-established rat model of diabetic eye disease.

Proteomics-based screening of AKR1B1 as a therapeutic target and validation study for sepsis-associated acute kidney injury.

Background: Sepsis and sepsis-associated acute kidney injury (SA-AKI) pose significant global health challenges, necessitating the development of innovative therapeutic strategies. Dysregulated protein expression has been implicated in the initiation and progression of sepsis and SA-AKI. Identifying potential protein targets and modulating their expression is crucial for exploring alternative therapies. Method: We established an SA-AKI rat model using cecum ligation perforation (CLP) and employed differential proteomic techniques to identify protein expression variations in kidney tissues. Aldose reductase (AKR1B1) emerged as a promising target. The SA-AKI rat model received treatment with the aldose reductase inhibitor (ARI), epalrestat. Blood urea nitrogen (BUN) and creatinine (CRE) levels, as well as IL-1ß, IL-6 and TNF-α levels in the serum and kidney tissues, were monitored. Hematoxylin-eosin (H-E) staining and a pathological damage scoring scale assessed renal tissue damage, while protein blotting determined PKC (protein kinase C)/NF-κB pathway protein expression. Result: Differential proteomics revealed significant downregulation of seven proteins and upregulation of 17 proteins in the SA-AKI rat model renal tissues. AKR1B1 protein expression was notably elevated, confirmed by Western blot. ARI prophylactic administration and ARI treatment groups exhibited reduced renal injury, low BUN and CRE levels and decreased IL-1ß, IL-6 and TNF-α levels compared to the CLP group. These changes were statistically significant (P < 0.05). AKR1B1, PKC-α, and NF-κB protein expression levels were also lowered in the ARI prophylactic administration and ARI treatment groups compared to the CLP group (P < 0.05). Conclusions: Epalrestat appeared to inhibit the PKC/NF-κB inflammatory pathway by inhibiting AKR1B1, resulting in reduced inflammatory cytokine levels in renal tissues and blood. This mitigated renal tissue injuries and improved the systemic inflammatory response in the severe sepsis rat model. Consequently, AKR1B1 holds promise as a target for treating sepsis-associated acute kidney injuries.

Aldose reductase is a potential therapeutic target for neurodegeneration.

Neurodegeneration is a complex process involving various inflammatory mediators and cellular responses. Aldose reductase (AR) is a key enzyme in the polyol pathway, which converts glucose to sorbitol. Beyond its metabolic role, AR has also been found to play a significant role in modulating neuroinflammation. This review aims to provide an overview of the current knowledge regarding the involvement of AR inhibition in attenuating neuroinflammation and complications from diabetic neuropathies. Here, we review the literature regarding AR and neuropathy/neurodegeneration. We discuss the mechanisms underlying the influence of AR inhibitors on ocular inflammation, beta-amyloid-induced neurodegeneration, and optic nerve degeneration. Furthermore, potential therapeutic strategies targeting AR in neurodegeneration are explored. The understanding of AR's role in neurodegeneration may lead to the development of novel therapeutic interventions for other neuroinflammatory disorders.

The polyol pathway and nuclear ketohexokinase A signaling drive hyperglycemia-induced metastasis of gastric cancer.

Diabetes might be associated with increased cancer risk, with several studies reporting hyperglycemia as a primary oncogenic stimulant. Since glucose metabolism is linked to numerous metabolic pathways, it is difficult to specify the mechanisms underlying hyperglycemia-induced cancer progression. Here, we focused on the polyol pathway, which is dramatically activated under hyperglycemia and causes diabetic complications. We investigated whether polyol pathway-derived fructose facilitates hyperglycemia-induced gastric cancer metastasis. We performed bioinformatics analysis of gastric cancer datasets and immunohistochemical analyses of gastric cancer specimens, followed by transcriptomic and proteomic analyses to evaluate phenotypic changes in gastric cancer cells. Consequently, we found a clinical association between the polyol pathway and gastric cancer progression. In gastric cancer cell lines, hyperglycemia enhanced cell migration and invasion, cytoskeletal rearrangement, and epithelial-mesenchymal transition (EMT). The hyperglycemia-induced acquisition of metastatic potential was mediated by increased fructose derived from the polyol pathway, which stimulated the nuclear ketohexokinase-A (KHK-A) signaling pathway, thereby inducing EMT by repressing the CDH1 gene. In two different xenograft models of cancer metastasis, gastric cancers overexpressing AKR1B1 were found to be highly metastatic in diabetic mice, but these effects of AKR1B1 were attenuated by KHK-A knockdown. In conclusion, hyperglycemia induces fructose formation through the polyol pathway, which in turn stimulates the KHK-A signaling pathway, driving gastric cancer metastasis by inducing EMT. Thus, the polyol and KHK-A signaling pathways could be potential therapeutic targets to decrease the metastatic risk in gastric cancer patients with diabetes.

In vitro evaluation of the reductive carbonyl idarubicin metabolism to evaluate inhibitors of the formation of cardiotoxic idarubicinol via carbonyl and aldo-keto reductases.

The most important dose-limiting factor of the anthracycline idarubicin is the high risk of cardiotoxicity, in which the secondary alcohol metabolite idarubicinol plays an important role. It is not yet clear which enzymes are most important for the formation of idarubicinol and which inhibitors might be suitable to suppress this metabolic step and thus would be promising concomitant drugs to reduce idarubicin-associated cardiotoxicity. We, therefore, established and validated a mass spectrometry method for intracellular quantification of idarubicin and idarubicinol and investigated idarubicinol formation in different cell lines and its inhibition by known inhibitors of the aldo-keto reductases AKR1A1, AKR1B1, and AKR1C3 and the carbonyl reductases CBR1/3. The enzyme expression pattern differed among the cell lines with dominant expression of CBR1/3 in HEK293 and MCF-7 and very high expression of AKR1C3 in HepG2 cells. In HEK293 and MCF-7 cells, menadione was the most potent inhibitor (IC50 = 1.6 and 9.8 µM), while in HepG2 cells, ranirestat was most potent (IC50 = 0.4 µM), suggesting that ranirestat is not a selective AKR1B1 inhibitor, but also an AKR1C3 inhibitor. Over-expression of AKR1C3 verified the importance of AKR1C3 for idarubicinol formation and showed that ranirestat is also a potent inhibitor of this enzyme. Taken together, our study underlines the importance of AKR1C3 and CBR1 for the reduction of idarubicin and identifies potent inhibitors of metabolic formation of the cardiotoxic idarubicinol, which should now be tested in vivo to evaluate whether such combinations can increase the cardiac safety of idarubicin therapies while preserving its efficacy.