The hop-derived compounds xanthohumol,isoxanthohumol and 8-prenylnaringenin are tight-binding inhibitors of human aldo-keto reductases 1B1 and 1B10

Xanthohumol (XN), a prenylated chalcone unique to hops (Humulus lupulus) and two derived prenylflavanones, isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) gained increasing attention as potential anti-diabetic and cancer preventive compounds. Two enzymes of the aldo-keto reductase (AKR) superfamily are notable pharmacological targets in cancer therapy (AKR1B10) and in the treatment of diabetic complications (AKR1B1). Our results show that XN, IX and 8-PN are potent uncompetitive, tight-binding inhibitors of human aldose reductase AKR1B1 (K_i?=?15.08?μM, 0.34?μM, 0.71?μM) and of human AKR1B10 (K_i?=?20.11?μM, 2.25?μM, 1.95?μM). The activity of the related enzyme AKR1A1 was left unaffected by all three compounds. This is the first time these three substances have been tested on AKRs. The results of this study may provide a basis for further quantitative structure–activity relationship models and promising scaffolds for future anti-diabetic or carcinopreventive drugs.     

Kinetic studies of AKR1B10, human aldose reductase-like protein: Endogenous substrates and inhibition by steroids

A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, was identified as a biomarker of lung cancer, exhibiting high sequence identity with human aldose reductase (AKR1B1). Using recombinant AKR1B10 and AKR1B1, we compared their substrate specificity for biogenic compounds and inhibition by endogenous compounds and found the following unique features of AKR1B10. AKR1B10 efficiently reduced long-chain aliphatic aldehydes including farnesal and geranylgeranial, which are generated from degradation of prenylated proteins and metabolism of farnesol and geranylgeraniol derived from the mevalonate pathway. The enzyme oxidized aliphatic and aromatic alcohols including 20α-hydroxysteroids. In addition, AKR1B10 was inhibited by steroid hormones, bile acids and their metabolites, showing IC₅₀ values of 0.03-25 μM. Kinetic analyses of the alcohol oxidation and inhibition by the steroids and tolrestat, together with the docked model of AKR1B10-inhibitor complex, suggest that the inhibitory steroids and tolrestat bind to overlapping sites within the active site of the enzyme-coenzyme complex. Thus, we propose a novel role of AKR1B10 in controlling isoprenoid homeostasis that is important in cholesterol synthesis and cell proliferation through salvaging isoprenoid alcohols, as well as its metabolic regulation by endogenous steroids.     

Elevated Expression of AKR1C3 Increases Resistance of Cancer Cells to Ionizing Radiation via Modulation of Oxidative Stress

With the aim to elucidate the etiology of radioresistance, we explored the genetic alterations in non-radioresistant vs. resistant esophageal cancer cells acquired by long-term fractionated radiation. We found AKR1C3, an aldo-keto reductase expressed seldom in most human tissues, expressed higher in radioresistance-acquired cells. Suppression of AKR1C3 via RNAi or its chemical inhibitors restored the sensitivity of the acquired tumor cells and xenograft BALB/c nude mice to ionizing radiation (IR). Cellular monitoring of the oxidative stress in the AKR1C3-elevated cells indicated that IR-induced ROS accumulation and the concomitant DNA damage was significantly alleviated, and such protective consequence disappeared upon AKR1C3 knockdown. These findings uncover the potential involvement of AKR1C3 in removal of cellular ROS and explain, at least partially, the acquired radioresistance by AKR1C3 overexpression. A retrospective analysis of esophageal carcinomas also indicated a significant expression of AKR1C3 in radio-resistant but not radio-sensitive surgical samples. Our study may provide a potential biomarker for predicting prognosis of radiotherapy and even direct a targeted therapy for esophageal cancer and other tumors.     

Tissue distribution of human AKR1C3 and rat homolog in the adult genitourinary system

Human aldo-keto reductase (AKR) 1C3 (type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase) catalyzes androgen, estrogen, and prostaglandin metabolism. AKR1C3 is therefore implicated in regulating ligand access to the androgen receptor, estrogen receptor, and peroxisome proliferator activating receptor gamma in hormone target tissues. Recent reports on close relationships between ARK1C3 and various cancers including breast and prostate cancers implicate the involvement of AKR1C3 in cancer development or progression. We previously described the characterization of an isoform-specific monoclonal antibody against AKR1C3 that does not cross-react with related, >86% sequence identity, human AKR1C1, AKR1C2, or AKR1C4, human aldehyde reductase AKR1A1, or rat 3alpha-hydroxysteroid dehydrogenase (AKR1C9). In this study, a clone of murine monoclonal antibody raised against AKR1C3 was identified and characterized for its recognition of rat homolog. Tissue distribution of human AKR1C3 and its rat homolog in adult genitourinary systems including kidney, bladder, prostate, and testis was studied by IHC. A strong immunoreactivity was detected not only in classically hormone-associated tissues such as prostate and testis but also in non-hormone-associated tissues such as kidney and bladder in humans and rats. The distribution of these two enzymes was comparable but not identical between the two species. These features warrant future studies of AKR1C3 in both hormone- and non-hormone-associated tissues and identification of the rodent homolog for establishing animal models.     

Murine aldo-keto reductase family 1 subfamily B: identification of AKR1B8 as an ortholog of human AKR1B10

Aldo-keto reductase family 1 member B10 (AKR1B10), over-expressed in multiple human cancers, might be implicated in cancer development and progression via detoxifying cytotoxic carbonyls and regulating fatty acid synthesis. In the present study, we investigated the ortholog of AKR1B10 in mice, an ideal modeling organism greatly contributing to human disease investigations. In the mouse, there are three aldo-keto reductase family 1 subfamily B (AKR1B) members, i.e., AKR1B3, AKR1B7, and AKR1B8. Among them, AKR1B8 has the highest similarity to human AKR1B10 in terms of amino acid sequence, computer-modeled structures, substrate spectra and specificity, and tissue distribution. More importantly, similar to human AKR1B10, mouse AKR1B8 associates with murine acetyl-CoA carboxylase-α and mediates fatty acid synthesis in colon cancer cells. Taken together, our data suggest that murine AKR1B8 is the ortholog of human AKR1B10.     

The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation

Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by human epoxide hydrolase (EH) and CYP1A1. Human dihydrodiol dehydrogenase isoforms (AKR1C1-AKR1C4), members of the aldo-keto reductase (AKR) superfamily, activate trans-dihydrodiols by converting them to reactive and redox-active o-quinones. We now show that the constitutively and widely expressed human AKR, aldehyde reductase (AKR1A1), will oxidize potent proximate carcinogen trans-dihydrodiols to their corresponding o-quinones. cDNA encoding AKR1A1 was isolated from HepG2 cells, overexpressed in Escherichia coli, purified to homogeneity, and characterized. AKR1A1 oxidized the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene with a higher utilization ratio (V(max)/K(m)) than any other human AKR. AKR1A1 also displayed a high V(max)/K(m) for the oxidation of 5-methylchrysene-7,8-diol, benz[a]anthracene-3,4-diol, 7-methylbenz[a]anthracene-3,4-diol, and 7,12-dimethylbenz[a]anthracene-3,4-diol. AKR1A1 displayed rigid regioselectivity by preferentially oxidizing non-K-region trans-dihydrodiols. The enzyme was stereoselective and oxidized 50% of each racemic PAH trans-dihydrodiol tested. The absolute stereochemistries of the reactions were assigned by circular dichroism spectrometry. AKR1A1 preferentially oxidized the metabolically relevant (-)-benzo[a]pyrene-7(R),8(R)-dihydrodiol. AKR1A1 also preferred (-)-benz[a]anthracene-3(R),4(R)-dihydrodiol, (+)-7-methylbenz[a]anthracene-3(S),4(S)-dihydrodiol, and (-)-7,12-dimethylbenz[a]anthracene-3(R),4(R)-dihydrodiol. The product of the AKR1A1-catalyzed oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was trapped with 2-mercaptoethanol and characterized as a thioether conjugate of benzo[a]pyrene-7,8-dione by LC/MS. Multiple human tissue expression array analysis showed coexpression of AKR1A1, CYP1A1, and EH, indicating that trans-dihydrodiol substrates are formed in the same tissues in which AKR1A1 is expressed. The ability of this general metabolic enzyme to divert trans-dihydrodiols to o-quinones suggests that this pathway of PAH activation may be widespread in human tissues.     

Retinaldehyde is a substrate for human aldo-keto reductases of the 1C subfamily

Human AKR (aldo-keto reductase) 1C proteins (AKR1C1-AKR1C4) exhibit relevant activity with steroids, regulating hormone signalling at the pre-receptor level. In the present study, investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All of the enzymes except AKR1C2 showed retinaldehyde reductase activity with low Km values (~1 μM). The kcat values were also low (0.18-0.6 min-1), except for AKR1C3 reduction of 9-cis-retinaldehyde whose kcat was remarkably higher (13 min-1). Structural modelling of the AKR1C complexes with 9-cis-retinaldehyde indicated a distinct conformation of Trp227, caused by changes in residue 226 that may contribute to the activity differences observed. This was partially supported by the kinetics of the AKR1C3 R226P mutant. Retinol/retinaldehyde conversion, combined with the use of the inhibitor flufenamic acid, indicated a relevant role for endogenous AKR1Cs in retinaldehyde reduction in MCF-7 breast cancer cells. Overexpression of AKR1C proteins depleted RA (retinoic acid) transactivation in HeLa cells treated with retinol. Thus AKR1Cs may decrease RA levels in vivo. Finally, by using lithocholic acid as an AKR1C3 inhibitor and UVI2024 as an RA receptor antagonist, we provide evidence that the pro-proliferative action of AKR1C3 in HL-60 cells involves the RA signalling pathway and that this is in part due to the retinaldehyde reductase activity of AKR1C3.     

AKR1B10 confers resistance to radiotherapy via FFA/TLR4/NF-κB axis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is one kind of human head and neck cancers with high incidence in Southern China, Southeast Asia and North Africa. In spite of great innovations in radiation and chemotherapy treatments, the 5-year survival rate is not satisfactory. One of the main reasons is resistance to radiotherapy which leads to therapy failure and recurrence of NPC. The mechanism underlying remains to be fully elucidated. Aldo-keto reductase B10 (AKR1B10) plays a role in the formation and development of carcinomas. However, its role in resistance to radiotherapy of NPC is not clear. In this research, the relationships between AKR1B10 expression and the treatment effect of NPC patients, NPC cell survival, cell apoptosis, and DNA damage repair, as well as the effect and mechanism of AKR1B10 expression on NPC radioresistance were explored. A total of 58 paraffin tissues of NPC patients received radiotherapy were collected including 30 patients with radiosensitivity and 28 patients with radioresistance. The relationships between AKR1B10 expression and the treatment effect as well as clinical characteristics were analyzed by immuno-histochemical experiments, and the roles of AKR1B10 in cell survival, apoptosis and DNA damage repair were detected using the AKR1B10 overexpressed cell models. Furthermore the mechanism of AKR1B10 in NPC radioresistance was explored. Finally, the radioresistance effect of AKR1B10 expression was evaluated by the tumor xenograft model of nude mice and the method of radiotherapy. The results showed AKR1B10 expression level was correlated with radiotherapy resistance, and AKR1B10 overexpression promoted proliferation of NPC cells, reduced apoptosis and decreased cellular DNA damage after radiotherapy. The probable molecular mechanism is that AKR1B10 expression activated FFA/TLR4/NF-κB axis in NPC cells. This was validated by using the TLR4 inhibitor TAK242 to treat NPC cells with AKR1B10 expression, which reduced the phosphorylation of NF-κB. This study suggests that AKR1B10 can induce radiotherapy resistance and promote cell survival via FFA/TLR4/NF-κB axis in NPC, which may provide a novel target to fight against radiotherapy resistance of NPC.     

AKR1B10 is induced by hyperglycaemia and lipopolysaccharide in patients with diabetic nephropathy

Aldose reductase family member B10 (AKR1B10) belongs to the aldo–keto reductase gene superfamily and is closely related to aldose reductase (AKR1B1). It has been shown that AKR1B10 is present in many of the same human tissues as AKR1B1. The objective of this study was to investigate whether AKR1B10 has a role in diabetic nephropathy (DN) by investigating its response to high glucose and inflammation, both of which have been associated with the development and progression of DN. Expression levels of AKR1B10 were determined in peripheral blood mononuclear cells (PBMCs) obtained from 25 patients with type 1 diabetes and nephropathy, 25 without DN and 25 normal healthy controls that were exposed to high glucose (25 mM d-glucose) and also the inflammatory stressor lipopolysaccharide (LPS, 10 μm). Under high glucose and LPS conditions, there was a significant increase in the expression of AKR1B10 in the PBMCs from patients with DN compared to those without DN and the normal controls. In conclusion, these results suggest that AKR1B10 may have an important role in the development and progression of DN.     

Chromene-3-carboxamide derivatives discovered from virtual screening as potent inhibitors of the tumour maker,AKR1B10

A human aldose reductase-like protein, AKR1B10 in the aldo-keto reductase (AKR) superfamily, was recently identified as a therapeutic target in the treatment of several types of cancer. In order to identify potential leads for new inhibitors of AKR1B10, we adopted the virtual screening approach using the automated program icm, which resulted in the discovery of several chromene-3-carboxamide derivatives as potent competitive inhibitors. The most potent (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide inhibited the reductase activity of AKR1B10 with a K_i value of 2.7 nM, and the metabolism of farnesal and 4-hydroxynonenal in the AKR1B10-overexpressed cells from 0.1 μM with an IC₅₀ value equal to 0.8 μM.     

Aldo-keto reductase family 1, member B10 is secreted through a lysosome-mediated non-classical pathway

AKR1B10 (aldo-keto reductase family 1, member B10) protein is primarily expressed in normal human small intestine and colon, but overexpressed in several types of human cancers and considered as a tumour marker. In the present study, we found that AKR1B10 protein is secreted from normal intestinal epithelium and cultured cancer cells, as detected by a newly developed sandwich ELISA and Western blotting. The secretion of AKR1B10 was not affected by the protein-synthesis inhibitor cycloheximide and the classical protein-secretion pathway inhibitor brefeldin A, but was stimulated by temperature, ATP, Ca(2+) and the Ca(2+) carrier ionomycin, lysosomotropic NH(4)Cl, the G-protein activator GTPγS and the G-protein coupling receptor N-formylmethionyl-leucyl-phenylalanine. The ADP-ribosylation factor inhibitor 2-(4-fluorobenzoylamino)-benzoic acid methyl ester and the phospholipase C inhibitor U73122 inhibited the secretion of AKR1B10. In cultured cells, AKR1B10 was present in lysosomes and was secreted with cathepsin D, a lysosomal marker. In the intestine, AKR1B10 was specifically expressed in mature epithelial cells and secreted into the lumen at 188.6-535.7 ng/ml of ileal fluids (mean=298.1 ng/ml, n=11). Taken together, our results demonstrate that AKR1B10 is a new secretory protein belonging to a lysosome-mediated non-classical protein-secretion pathway and is a potential serum marker.     

Characterization of enzymes participating in carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human placenta

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) has been identified as one of the strongest nitrosamine carcinogens in tobacco products in all species tested. Carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronosylation is considered to be the main detoxification pathway in humans. In previous investigations, we have identified a microsomal NNK carbonyl reductase as being identical to 11ß-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Recently, we provided evidence that carbonyl reduction of NNK does also take place in cytosol from mouse and human liver and lung. In human liver cytosol, carbonyl reductase, a SDR enzyme, and AKR1C1, AKR1C2 and AKR1C4 from the aldo-keto reductase (AKR) superfamily were demonstrated to be responsible for NNK reduction. Since NNK and/or its metabolites can diffuse through the placenta and reach fetal tissues, we now investigated NNK carbonyl reduction in the cytosolic fraction of human placenta in addition to that in microsomes. Concluding from the sensitivity to menadione, ethacrynic acid, rutin and quercitrin as specific inhibitors, mainly carbonyl reductase (EC 1.1.1.184) seems to perform this reaction in human placenta cytosol. The presence of carbonyl reductase was confirmed by RT-PCR. This is the first report to provide evidence that NNAL formation in placenta is mediated by carbonyl reductase.     

Characterization of enzymes participating in carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human placenta

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) has been identified as one of the strongest nitrosamine carcinogens in tobacco products in all species tested. Carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronosylation is considered to be the main detoxification pathway in humans. In previous investigations, we have identified a microsomal NNK carbonyl reductase as being identical to 11beta-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Recently, we provided evidence that carbonyl reduction of NNK does also take place in cytosol from mouse and human liver and lung. In human liver cytosol, carbonyl reductase, a SDR enzyme, and AKR1C1, AKR1C2 and AKR1C4 from the aldo-keto reductase (AKR) superfamily were demonstrated to be responsible for NNK reduction. Since NNK and/or its metabolites can diffuse through the placenta and reach fetal tissues, we now investigated NNK carbonyl reduction in the cytosolic fraction of human placenta in addition to that in microsomes. Concluding from the sensitivity to menadione, ethacrynic acid, rutin and quercitrin as specific inhibitors, mainly carbonyl reductase (EC 1.1.1.184) seems to perform this reaction in human placenta cytosol. The presence of carbonyl reductase was confirmed by RT-PCR. This is the first report to provide evidence that NNAL formation in placenta is mediated by carbonyl reductase.     

Mouse AKR1E1 is an ortholog of pig liver NADPH dependent 1,5-anhydro-D-fructose reductase

In many organisms, glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is reduced to 1,5-anhydro-D-glucitol (AG). AF reductase, which catalyzes the latter reaction, was purified from pig liver, but mouse ortholog has not yet been reported. In the database, aldo-keto reductase family 1, member E1 (AKR1E1) showed highest homology to pig enzyme. We confirmed that cloned AKR1E1 is mouse ortholog based on enzymatic properties of purified recombinant protein.     

Characterization of a monoclonal antibody for human aldo-keto reductase AKR1C3 (type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase); immunohistochemical detection in breast and prostate

Human aldo-keto reductase AKR1C3 (type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase) catalyzes the reduction of Delta(4)-androstene-3,17-dione to yield testosterone, the reduction of 5alpha-dihydrotestosterone to yield 3alpha- and 3beta-androstanediol, and the reduction of estrone to yield 17beta-estradiol. Relatively, high mRNA expression of AKR1C3 was found in human prostate and mammary gland where it is implicated in regulating ligand access to the androgen and estrogen receptor, respectively. AKR1C3 shares high sequence identity >86% with related plastic human 20alpha-hydroxysteroid dehydrogenases (AKR1C1), type 3 3alpha-hydroxysteroid dehydrogenase (AKR1C2) and type 1 3alpha-hydroxysteroid dehydrogenase (AKR1C4), and reagents are urgently needed to discriminate between these enzymes at the mRNA, protein and functional level. We describe the characterization of a high-titer isoform specific monoclonal antibody (Ab) for AKR1C3. It does not cross react with human AKR1C1, AKR1C2 or AKR1C4, human aldehyde reductase AKR1A1 or rat 3alpha-hydroxysteroid dehydrogenase (AKR1C9) on immunoblot analysis. The monoclonal Ab can be used to detect AKR1C3 expression by immunohistochemistry in sections of paraffin-embedded mammary gland and prostate. In the breast enzyme staining was detected in ductal carcinoma in situ where the cancerous cells were strongly immunoreactive. In normal prostate immunoreactivity was limited to stromal cells with only faint staining in the epithelial cells. In adenocarcinoma of the prostate elevated staining was observed in the endothelial cells and carcinoma cells. The reagent thus has utility to access the localized expression of AKR1C3 in hormonal dependent malignancies of the breast and prostate.     

Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals

Aldehyde reductase (AKR1A), a member of the aldo–keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.