Flutamide (FLU), a nonsteroidal anti-androgen, is used for the treatment of prostate cancer but is also a cytochrome P450 (CYP) 1A inducer. Some CYP1A inducers are known to exert hepatocellular tumor-promoting activities in rodents, and reactive oxygen species (ROS) produced by CYP1A1 induction via a metabolism of FLU is probably involved in the liver tumor promotion. In the present study, to clarify the possible liver tumor promoting effect of FLU, a two-stage liver carcinogenesis assay was performed using male F344 rats. Rats received an intraperitoneal (ip) injection of 200 mg/kg body weight of N-diethylnitrosamine (DEN) and fed a diet containing 0, 0.1 or 0.2% FLU for 6 weeks. After 2 weeks of DEN treatment, all rats were subjected to two-thirds partial hepatectomy. Animals were killed 8 weeks after ip injection of DEN. Immunohistochemically, the number and area of glutathione S-transferase placental form (GST-P)-positive foci significantly increased in the liver of rats given 0.2% FLU as compared with the control. Ki-67-positive cell ratio also increased in rats given FLU at both concentrations. ROS generation in the microsomal fraction and production of thiobarbituric acid-reactive substance [TBARS] and 8-hydroxy-2'-deoxyguanosine (8-OHdG) content in the liver did not increase in any of the FLU-treated groups. The results of microarray and real-time RT-PCR revealed that phase 1 drug-metabolizing enzymes such as CYP1A1, Ugt1a61 and Nqo1 and phase II drug-metabolizing enzymes such as Yc2, Akr1b7, Akr1b8, Akr1b10, Aldh1a1, Gpx2 and Me1 were up-regulated in rats treated with FLU. In addition, the MAPK pathway family-related genes such as Prkcα, Mek1, Rafb, Myc, Mek2, Raf1 and Egfr were also up-regulated in FLU-treated groups. The results of the present study indicate that FLU is a CYP1A inducer but does not cause any production of microsomal ROS in the liver and suggest that microsomal ROS is not involved in the liver tumor promoting effect of FLU.

BACKGROUND

Aldo-keto reductase family 1 member B10 (AKR1B10) expression in oral squamous cell carcinoma (OSCC) tissue specimens is correlated with the progression and prognosis of OSCC.

METHODS

Saliva samples were obtained from 35 normal controls and 86 patients with OSCC before cancer surgery. The AKR1B10 levels were determined using enzyme-linked immunosorbent assay (ELISA).

RESULTS

The mean salivary AKR1B10 levels were significantly higher in the patients with OSCC than in the normal controls (P < .001). Higher salivary AKR1B10 levels were significantly associated with larger tumor size, more advanced clinical stage, and areca quid chewing habit. Patients with OSCC with a higher salivary AKR1B10 level (>646 pg/mL) had a significantly poorer survival than those with a lower (≤646 pg/mL) salivary AKR1B10 level (P = .026).

CONCLUSIONS

The salivary AKR1B10 level may be a promising biomarker for screening high-risk patients with OSCC and monitoring the progression of OSCC.

Objective: Aldo-keto reductase family 1 member B10 (AKR1B10) pathogenesis, early diagnosis and prognosis are closely related with hepatoma. Therefore, this study explores the effect and mechanism of AKR1B10 on cell cycle in hepatoma cells. Methods: HepG2 cells were infected with lentivirus LV-AKR1B10-shRNA or treated with epalrestat, an AKR1B10 inhibitor. The expression level of AKR1B10 was detected by Western blot assay and real-time fluorescence quantitative PCR (RT-qPCR). Decreased AKR1B10 activity was detected by reduced coenzyme II (NADPH) absorbance at 340 nm. The low expression of AKR1B10 and the effect of different concentrations of epalrestat on cell proliferation and cell cycle were detected by CCK-8 method and flow cytometry. The protein expression levels of p-rb, cyclin D1, E1, p27 in HepG2 cells were detected by Western blot. The mean of the two samples was tested using independent sample t-test. Results: AKR1B10 expression level in hepatoma cells was significantly increased compared to normal liver cells, and the relative expression level of AKR1B10 protein in HepG2 cells was 6.71 ± 1.11 (P = 0.012). Epalrestat was significantly inhibited with the enzymatic activity of AKR1B10 in a dose-dependent manner. AKR1B10 gene in HepG2 cells was effectively silenced. HepG2 cells treated with different concentrations of epalrestat (AKR1B10 inhibitor) for 24, 48 and 72 h had inhibited cell proliferation, promoted G0/G1 cell cycle arrest, reduced the expression of p-Rb, cyclin D1, and cyclin E1 and increased the expression of cyclin dependent kinase inhibitor p27 expression. Conclusion: AKR1B10 inhibitory expression and activity can promote G0/G1 cell cycle arrest in HepG2 cells through the p27 / p-Rb pathway.

目的： 醛酮还原酶家族1成员B10（AKR1B10）与肝癌的发病机制、早期诊断和预后密切相关，故探讨AKR1B10在肝癌细胞中对细胞周期的影响及其作用机制。 方法： 采用慢病毒LV-AKR1B10-shRNA感染HepG2细胞，或AKR1B10抑制剂依帕司他处理HepG2细胞，蛋白质印迹法（Western blot）及实时荧光定量PCR法（RT-qPCR）检测AKR1B10的表达水平；通过测定还原型烟酰胺腺嘌呤二核苷酸磷酸在340 nm处吸光度值的下降检测AKR1B10的活性；CCK-8法及流式细胞术检测AKR1B10低表达及不同浓度依帕司他处理HepG2细胞后对细胞增殖和细胞周期的影响；Western blot检测HepG2细胞中p-Rb，细胞周期蛋白D1、E1，p27的蛋白表达水平；两样本均数采用独立样本t检验。 结果： AKR1B10在肝癌细胞中表达显著升高，与正常肝细胞相比，HepG2细胞中AKR1B10蛋白相对表达水平为6.71±1.11（P = 0.012）。依帕司他对AKR1B10的酶活性有明显抑制作用，呈剂量依赖性特点。HepG2细胞中AKR1B10基因被有效沉默，不同浓度AKR1B10抑制剂依帕司他处理HepG2细胞24、48、72 h后均可抑制细胞增殖，促进G(0)/G(1)细胞周期阻滞，使p-Rb、周期蛋白D1、E1表达降低，周期素依赖性激酶抑制蛋白p27表达升高。 结论： 抑制AKR1B10表达和活性可通过p27/p-Rb通路，促进HepG2细胞G(0)/G(1)细胞周期阻滞。.

Keywords: Aldo keto reductase1B10; Cell cycle arrest; Epalrestat; Hepatocellular carcinoma; shRNA.

Genome-wide association studies (GWASs) are unraveling the genetics of adult brain neuroanatomy as measured by cross-sectional anatomic magnetic resonance imaging (aMRI). However, the genetic mechanisms that shape childhood brain development are, as yet, largely unexplored. In this study we identify common genetic variants associated with childhood brain development as defined by longitudinal aMRI. Genome-wide single nucleotide polymorphism (SNP) data were determined in two cohorts: one enriched for attention-deficit/hyperactivity disorder (ADHD) (LONG cohort: 458 participants; 119 with ADHD) and the other from a population-based cohort (Generation R: 257 participants). The growth of the brain's major regions (cerebral cortex, white matter, basal ganglia, and cerebellum) and one region of interest (the right lateral prefrontal cortex) were defined on all individuals from two aMRIs, and a GWAS and a pathway analysis were performed. In addition, association between polygenic risk for ADHD and brain growth was determined for the LONG cohort. For white matter growth, GWAS meta-analysis identified a genome-wide significant intergenic SNP (rs12386571, P = 9.09 × 10-9 ), near AKR1B10. This gene is part of the aldo-keto reductase superfamily and shows neural expression. No enrichment of neural pathways was detected and polygenic risk for ADHD was not associated with the brain growth phenotypes in the LONG cohort that was enriched for the diagnosis of ADHD. The study illustrates the use of a novel brain growth phenotype defined in vivo for further study.

Cystic pancreatic tumors account for 10% of cystic lesions in the pancreas. Evaluation focuses on identifying lesions that require surgical resection due to actual or potential malignancy. Cystic tumors with malignant potential include mucinous cystic neoplasms (MCNs), intraductal papillary mucinous neoplasms (IPMNs), and cystic neuroendocrine tumors (NETs). The sensitivity of endoscopic fine needle aspiration (FNA) to diagnose such lesions is low, and a more accurate marker of malignant potential is needed. Aldo-keto reductase 1B10 (AKR1B10) was originally found in human hepatocellular carcinoma. Since then, it has been identified in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia. Because there is difficulty in determining the malignant potential of cystic pancreatic tumors, we set out to examine the expression of AKR1B10 in these lesions as a potential biomarker of malignancy. AKR1B10 expression was analyzed in cell blocks from FNAs and surgical resection specimens using immunohistochemistry. We examined MCN (n=28), IPMN (n=18), and cystic NET (n=20) as well as nonmucinous cysts including pseudocysts (n=13) and serous cystadenomas (n=16). AKR1B10 expression was seen in 45 of 46 (98%) mucinous lesions evaluated. Strong staining (2+-3+/60%-100% staining) was seen in 16 of 18 (89%) IPMNs and 25 of 28 (90%) MCNs. No staining was seen in the nonmucinous lesions (n=49). In conclusion, AKR1B10 is upregulated in mucinous cystic pancreatic tumors, and this staining can be accomplished in cytology FNA material, making AKR1B10 a promising biomarker of malignant potential. Most importantly, this application could impact the clinical management of these patients by determining the best candidates for surgical resection.

AKR1B10 is a human member of the aldo-keto reductase (AKR) superfamily, and is considered to be a tumor biomarker because its expression is known to be significantly induced in the cells of various cancers such as lung non-small-cell carcinoma and hepatocellular carcinoma. However, the mechanisms underlying the regulation of its gene remain unclear. In the present study, we demonstrated that the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), down-regulated the expression of the AKR1B10 gene in the human lung cancer cell line, A549. The treatment of A549 cells with TPA for 24h significantly reduced the mRNA levels, protein levels, and promoter activity of AKR1B10 as well as the growth of A549 cells. TPA induced the phosphorylation of the MAP kinase, ERK, and U0126, an inhibitor of the MAP kinase kinase, MEK1, blocked the down-regulation of AKR1B10 by TPA, indicating that the MAP kinase ERK plays a role in regulating the expression of AKR1B10. TPA also induced c-jun gene expression in an ERK-dependent manner. The co-introduction of the c-Jun protein resulted in a decrease in the mRNA levels and promoter activity of AKR1B10 as well as A549 cell proliferation. These results suggested that the ERK/c-Jun signaling pathway may play an important role in the TPA-triggered down-regulation of AKR1B10 gene expression.

Docetaxel (DTX) is widely used for treatment of inveterate lung and prostate cancers, but its continuous administration elicits the hyposensitivity. Here, we established the DTX-resistant variants of human lung cancer A549 and androgen-independent prostate cancer Du145 cells and found that the resistance development provoked aberrant up-regulations of aldo-keto reductase (AKR) 1B10 and AKR1C3 in A549 and Du145 cells, respectively. In addition, the sensitivity to the DTX toxicity was significantly decreased and increased by overexpression and knockdown of the two AKR isoforms, respectively. Furthermore, the resistant cells exhibited a decreased level of reactive 4-hydroxy-2-nonenal formed during DTX treatment, and the decrease was alleviated by adding the AKR inhibitors, inferring that the two AKRs confer the chemoresistance through elevating the antioxidant properties. The development of DTX resistance was also associated with enhanced expression of an ATP-binding cassette (ABC) transporter ABCB1 among the ABC transporter isoforms. The combined treatment with inhibitors of the two AKRs and ABCB1 additively sensitized the resistant cells to DTX. Intriguingly, the AKR1B10 inhibitor also suppressed the lung cancer cross-resistance against cisplatin. The results suggest that combined treatment with AKRs (1B10 and 1C3) and ABCB1 inhibitors exerts overcoming effect against the cancer resistance to DTX and cisplatin, and can be used as the adjuvant therapy.

UVI2008, a retinoic acid receptor (RAR) β/γ agonist originated from C3 bromine addition to the parent RAR pan-agonist 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB), is also a selective inhibitor of aldo-keto reductase family member 1B10 (AKR1B10). Thus, it might become a lead drug for the design of compounds targeting both activities, as an AKR1B10 inhibitor and RAR agonist, which could constitute a novel therapeutic approach against cancer and skin-related diseases. Herein, the X-ray structure of the methylated Lys125Arg/Val301Leu AKR1B10 (i.e. AKME2MU) holoenzyme in complex with UVI2008 was determined at 1.5 Å resolution, providing an explanation for UVI2008 selectivity against AKR1B10 (IC50 = 6.1 μM) over the closely related aldose reductase (AR, IC50 = 70 μM). The carboxylic acid group of UVI2008 is located in the anion-binding pocket, at hydrogen-bond distance of catalytically important residues Tyr49 and His111. The inhibitor bromine atom can only fit in the wider active site of AKR1B10, mainly because of the native Trp112 side-chain orientation, not possible in AR. In AKR1B10, Trp112 native conformation, and thus UVI2008 binding, is facilitated through interaction with Gln114. IC50 analysis of the corresponding Thr113Gln mutant in AR confirmed this hypothesis. The elucidation of the binding mode of UVI2008 to AKR1B10, along with the previous studies on the retinoid specificity of AKR1B10 and on the stilbene retinoid scaffold conforming UVI2008, could indeed be used to foster the drug design of bifunctional antiproliferative compounds.

In terms of drug disposal and metabolism SDR21C1 (carbonyl reductase 1; CBR1) exerts an assorted substrate spectrum among a large variety of clinically relevant substances. Additionally, this short-chain dehydrogenase/reductase is extensively expressed in most tissues of the human body, thus underpinning its role in xenobiotic metabolism. Reduction of the chemotherapeutic daunorubicin (DAUN) to daunorubicinol (DAUNol) is a prominent example of its metabolic properties in terms of chemoresistance and cardiotoxicity. The hop-derived prenylated chalcone xanthohumol (XN) and its physiological metabolites isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) have previously been reported to inhibit other DAUN reducing reductases and dehydrogenases including AKR1B1 and AKR1B10. Also with regard to their effects by means of interacting with cancer-related molecular pathways, XN and related prenylated flavonoids in particular have been in the focus of recent studies. In this study, inhibitory properties of these substances were examined with CBR1-mediated 2,3-hexanedione and DAUN reduction. All substances tested in this study turned out to efficiently inhibit recombinant human CBR1 within a low micromolar to submicromolar range. Among the substances tested, 8-PN turned out to be the most effective inhibitor when using 2,3-hexanedione as a substrate (K_i(app) = 180 ± 20 nM). Inhibition rates of recombinant CBR1-mediated DAUN reduction were somewhat weaker with IC50-values ranging from 11 to 20 μM. XN, IX and 8-PN also efficiently inhibited DAUN reduction by SW480 colon adenocarcinoma cytosol (IC₅₀ = 3.71 ± 0.26 μM with 8-PN as inhibitor). This study identifies prenylated inhibitors, which might potentially interact with endogenous CBR1-driven (de-)toxication systems.

Mouse aldo-keto reductase family 1 member B8 (AKR1B8) has the highest similarity to human aldo-keto reductase family 1 member B10 (AKR1B10), a secretory protein through lysosomes-mediated non-classical secretory pathway. To identify whether AKR1B8 is secreted through the same pathway, we carried out this study. Self-developed sandwich ELISA and western blot were used to detect AKR1B8 in cells and culture medium of CT-26 murine colon carcinoma cells. AKR1B8 releases in an independent manner to Brefeldin A, an inhibitor of ER-to-Golgi classical secretion pathway. Several factors, which are involved in the non-classical secretion pathway, such as temperature, ATP and calcium ion, regulated AKR1B8 secretion from mouse colorectal cancer cells CT-26. Lysosomotropic NH4Cl increased AKR1B8 secretion, and AKR1B8 was located in isolated lysosomes. Therefore, AKR1B8 is a new secretory protein through the lysosomes-mediated non-classical pathway.

Malignant pleural mesothelioma (MPM) is a universally lethal type of cancer that is increasing in incidence worldwide; therefore, the development of new drugs for MPM is an urgent task. Bullfrog sialic acid‑binding lectin (cSBL) is a multifunctional protein that has carbohydrate‑binding and ribonuclease activities. cSBL exerts marked antitumor activity against numerous types of cancer cells, with low toxicity to normal cells. Although in vitro and in vivo studies revealed that cSBL was effective against MPM, the mechanism by which cSBL exerts antitumor effects is not fully understood. To further understand the mechanism of action of cSBL, the present study aimed to identify the key molecules whose expression was affected by cSBL. The present study established cSBL‑resistant MPM cells. Microarray analyses revealed that there were significant pleiotropic changes in the expression profiles of several genes, including multiple genes involved in metabolic pathways in cSBL‑resistant cells. Furthermore, the expression of some members of the aldo‑keto reductase family was revealed to be markedly downregulated in these cells. Among these, it was particularly interesting that cSBL action reduced the level of AKR1B10, which has been reported as a biomarker candidate for MPM prognosis. These findings revealed novel aspects of the effect of cSBL, which may contribute to the development of new therapeutic strategies for MPM.

BACKGROUND

The human Aldoketoreductase 1B10 gene (AKR1B10) encodes one of the enzymes belonging to the family of aldoketoreductases and may be involved in detoxification of nutrients during digestion. Further, AKR1B10 mRNA (messenger ribonucleic acid) expression was diminished in brain regions potentially involved in the regulation of eating behavior in rats which are more sensitive to cocaine and alcohol. We hypothesized that the human AKR1B10 gene may also play a role in the regulation of human eating behavior.

RESULTS

We investigated the effects of 5 genetic variants of AKR1B10 on human eating behavior among 548 subjects from a German self-contained population, the Sorbs, and in 350 subjects from another independent German cohort. Among the Sorbs, we observed nominal associations with disinhibition at the 5' untranslated region (5' UTR) variant rs10232478 and the intragenic variants rs1834150 and rs782881 (all P ≤ 0.05). Further, we detected a relationship of rs1834150 and rs782881 with waist, smoking consumption (rs782881) and coffee consumption (rs1834150) (all P ≤ 0.05). Albeit non-significant, replication analyses revealed similar effect directions for disinhibition at rs1834150 (combined P = 0.0096). Moreover, in the replication cohort we found rs1834150 related to increased restraint scores with a similar direction as in the Sorbs (combined P = 0.0072).

CONCLUSIONS

Our data suggest that genetic variants in the AKR1B10 locus may influence human eating behavior.

We performed a transcriptome-based bioassay (TSB assay) using human hepatoma HepG2 cells to evaluate the potential toxicity of whole wastewater effluents from two membrane bioreactors (MBRs) and a conventional activated sludge process (AS). The biologically active agent(s) in the wastewater effluents were characterized based on expression of the marker genes (i.e., CYP1A1, AKR1B10, GCLM and GPX2) selected by DNA microarray analysis, after the wastewater effluent samples were concentrated by a reverse osmosis (RO) membrane and further fractionated by various manipulations. The qPCR assay of marker genes demonstrated that the induction of CYP1A1 and GPX2 was mitigated after passing through C18 and chelate columns. In addition, clear induction of CYP1A1 was observed in the smallest size fraction with 1 k Da or smaller organic molecules in all the tested effluents. These results together with the water quality data of the fractionated samples suggested that responsible constituents for potentially adverse and abnormal transcriptomic responses in HepG2 could have hydrophobic nature and act with metal-dissolved organic matter (DOM) complexes in 1 k Da or smaller size fraction. Although DOM is known to play two contradictory roles as a protector and an inducer of toxicants, our present study indicated the DOM in wastewater effluent, particularly humic substances with acidic nature, functioned as a toxicity inducer of residual chemicals in the effluents. This study provided a new insight into the nature of "toxic unknowns" in the wastewater effluents, which should be monitored whole through the reclamation process and prioritized for removal.

<AbstractText><em>AKR1B10</em>, first cloned from liver cancer tissues, has recently been reported to be up-regulated significantly in hepatocellular carcinoma (HCC) tissues, but the relationship between serum level of <em>AKR1B10</em> and the risk of HCC is not understood.</AbstractText><AbstractText>170 HCC patients and 120 health donors from October 2014 to March 2017 were recruited in the affiliated hospital of Guilin Medical University. Serum <em>AKR1B10</em> in all cases were detected and in 30 HCC patients were analyzed preoperatively and postoperatively by Time-resolved fluoroimmunoassay.</AbstractText><p><div><b>RESULTS</b></div>The level of serum <em>AKR1B10</em> was significantly higher in HCC patients (1800.24±2793.79) than in health donors (129.34±194.129), and downregulation of serum <em>AKR1B10</em> in HCC patients was observed after hepatectomy. When samples were grouped according to the serum level of <em>AKR1B10</em> (≥232.7pg/ml), serum <em>AKR1B10</em> positively correlated to serum AFP (χ²=6.295, P=0.012), ALT (χ²=18.803, P=0.000), AST (χ²=33.421, P=0.000), tumor nodule number (χ²=6.777, P=0.009), cirrhosis (χ²=43.458, P=0.000), and tumor size (χ²=6.042, P=0.014) in the Chi-square test.</p><AbstractText>Diagnosis of HCC could be improved using the both predictors of serum <em>AKR1B10</em> and AFP. <em>AKR1B10</em> was thus considered to be a new serological biomarker for HCC.</AbstractText>

Endometrial cancer is the sixth most common cancer in women worldwide. It is associated with aberrant actions of steroid hormones, estrogens and progesterone, but also with enhanced inflammation and reduced cellular differentiation. Here, we show data on demographic and histopathological characteristics of 51 patients with endometrial cancer, together with data on correlations between the expression of 38 genes involved in estrogen and progesterone actions, inflammation and differentiation, and demographic characteristics. We also show data on changes in gene expression of these 38 genes according to histopathological and clinical characteristics of these patients. This article includes data referenced in the manuscript entitled »STAR and AKR1B10 are down-regulated in high-grade endometrial cancer by Sinreih et al. (in press) [1].

Background: Numbers of studies have reported that the expression of aldo-keto reductase family 1 member B10 (AKR1B10) is abnormal in digestive system cancers, and could be used as a prognostic biomarker. However, the results are argued. Therefore, we conduct a meta-analysis to comprehensively evaluate the prognostic value of high AKR1B10 expression for overall survival (OS), disease specific survival (DSS), and disease-free survival/recurrence-free survival (DFS/PFS) in digestive system cancers.

Methods: Hazard ratios (HRs) with its 95% confidence intervals (CIs) were calculated to assess the prognostic value of AKR1B10 by using the random effects model. The STATA version 12.0 software were used to perform all the analyses.

Results: Eleven articles including 1428 patients involved in this meta-analysis. The pooled analysis suggested that high AKR1B10 expression was not associated with OS (HR: 1.18; 95% CI: 0.69-2.00) and DFS/PFS (HR: 1.08, 95% CI: 0.67-1.76) in digestive system cancers. However, Further analysis revealed that high AKR1B10 expression indicated poor OS in oral squamous cell carcinomas (OSCC) (HR: 2.92, 95% CI: 1.86-4.58) and favorable DSS in hepatocellular carcinoma (HCC) (HR: 0.71, 95% CI: 0.52-0.97).

Conclusions: The prognostic value of high AKR1B10 expression varied in different types of digestive system cancers. Further studies exploring the prognostic role of AKR1B10 in digestive system cancers are needed.

New substituted (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids were designed as the inhibitor of AKR1B1 based upon the structure of rhetsinine, a minor alkaloidal component of Evodia rutaecarpa, and twenty derivatives were synthesized and evaluated. The most active compound of the series was (2-benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7m), which showed comparable inhibitory activity for AKR1B1 (IC(50)=0.15μM) with clinically used epalrestat (IC(50)=0.1μM). In the view of activity and selectivity, the most potent compound was (2-benzyl-6-carboxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7t), which showed strong inhibitory effect (IC(50)=0.17μM) and very high selectivity for AKR1B1 against AKR1A1 (311:1) and AKR1B10 (253:1) compared with epalrestat.

Aldo-keto reductase 1B10 (AKR1B10), a member of aldo-keto reductase superfamily, contributes to detoxification of xenobiotics and metabolization of physiological substrates. Although increased expression of AKR1B10 was found in hepatocellular carcinoma (HCC), the role of AKR1B10 in the development of HCC remains unclear. This study aims to illustrate the role of AKR1B10 in hepatocarcinogenesis based on its intrinsic oxidoreduction abilities. HCC cell lines with AKR1B10 overexpression or knockdown were treated with doxorubicin or hydrogen peroxide to determinate the influence of aberrant AKR1B10 expression on cells' response to oxidative stress. Using Akr1b8 (the ortholog of human AKR1B10) knockout mice, diethylnitrosamine (DEN) induced liver injury, chronic inflammation and hepatocarcinogenesis were explored. Clinically, the pattern of serum AKR1B10 relevant to disease progression was investigated in a patient cohort with chronic hepatitis B (n=30), liver cirrhosis (n=30) and HCC (n=40). AKR1B10 expression in HCC tissues was analyzed using both the TCGA database (n=371) and our collected HCC samples (n=67). AKR1B10 overexpression reduced hepatocyte injury while AKR1B10 knockdown augmented reactive oxygen species (ROS) accumulation and apoptotic cell death. Consistently, Akr1b8 deficiency in mice promoted DEN-induced hepatocyte damage and liver inflammation characterized by increased phospho-H2AX, serum alanine aminotransferase, interleukin-6 and tumor necrosis factor alpha level, myeloid cell infiltration and led to more severe hepatocarcinogenesis and metastasis compared with wild type mice due to significant alteration on detoxification and oxidoreduction. AKR1B10 was compensatory expressed and gradually upregulated in the process of liver disease progression in HCC and increased oxidative stress upregulated AKR1B10 through NRF2. Our results here suggested that through oxidoreduction and detoxification, AKR1B10 played an important role in protecting hepatocytes from damage induced by ROS. Deficiency of AKR1B10 might accelerate hepatotoxin and inflammation-associated hepatocarcinogenesis. AKR1B10 expression elevation in HCC could be a result of compensatory upregulation, rather than a driver of malignant transformation during the development of HCC.

Carbonyl reduction is an important metabolic pathway for endogenous and xenobiotic substances. The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, nicotine-derived nitrosamine ketone) is classified as carcinogenic to humans (IARC, Group 1) and considered to play the most important role in tobacco-related lung carcinogenesis. Detoxification of NNK through carbonyl reduction is catalyzed by members of the AKR- and the SDR-superfamilies which include AKR1B10, AKR1C1, AKR1C2, AKR1C4, 11β-HSD1 and CBR1. Because some reductases are also involved in steroid metabolism, five different hormones were tested for their inhibitory effect on NNK carbonyl reduction. Two of those hormones were estrogens (estradiol and ethinylestradiol), another two hormones belong to the gestagen group (progesterone and drospirenone) and the last tested hormone was an androgen (testosterone). Furthermore, one of the estrogens (ethinylestradiol) and one of the gestagens (drospirenone) are synthetic hormones, used as hormonal contraceptives. Five of six NNK reducing enzymes (AKR1B10, AKR1C1, AKR1C2, AKR1C4 and 11β-HSD1) were significantly inhibited by the tested sex hormones. Only NNK reduction catalyzed by CBR1 was not significantly impaired. In the case of the other five reductases, gestagens had remarkably stronger inhibitory effects at a concentration of 25 μM (progesterone: 66-88% inhibition; drospirenone: 26-87% inhibition) in comparison to estrogens (estradiol: 17-51% inhibition; ethinylestradiol: 14-79% inhibition) and androgens (14-78% inhibition). Moreover, in most cases the synthetic hormones showed a greater ability to inhibit NNK reduction than the physiologic derivatives. These results demonstrate that male and female sex hormones have different inhibitory potentials, thus indicating that there is a varying detoxification capacity of NNK in men and women which could result in a different risk for developing lung cancer.

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily, reduces anthracycline anticancer drugs to their less potent anticancer C-13 hydroxy metabolites, which are linked with pathogenesis of cardiotoxicity, a side effect of the drugs. CBR1 inhibitors are thought to be promising agents for adjuvant therapy with a twofold beneficial effect in prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity. In order to search for new potential inhibitors of CBR1, we synthesized a series of des-methoxyphenyl derivatives of (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) that was developed previously as a potent inhibitor of aldo-keto reductase (AKR) 1B10 and AKR1B1. Among the newly synthesized inhibitors, 8-hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-chlorophenyl)amide (13h) was the most potent competitive inhibitor of CBR1, showing a Ki value of 15 nM. 13h also showed high selectivity to CBR1 over its isozyme CBR3 and other enzymes with CBR activity (AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C4, DXCR and DHRS4). Furthermore, 13h inhibited the cellular metabolism by CBR1 at its concentration of 4 μM. The structure-activity relationship of the derivatives, site-directed mutagenesis of putative binding residues (Met141 and Trp229) and molecular docking of 13h in CBR1 revealed that the interactions of 13h with the substrate-binding residues (Ser139, Met141, Tyr193 and Trp229) are important for the tight binding.

Objective To prepare rabbit anti-aldo-keto reductase family 1 member B10 (AKR1B10) polyclonal antibody and identify its specificity. Methods AKR1B10 cDNA was amplified by reverse transcription PCR (RT-PCR) and inserted into prokaryotic expression vector pET-15b to form recombinant plasmid pET-15b-AKR1B10. The recombinant plasmid pET-15b-AKR1B10 was transformed into E.coli DH5α. Isopropylthio-β-D-galactoside (IPTG) was used to induce the expression of the recombinant protein His-tagged AKR1B10 in E.coli DH5α. The expression products from different clones of E.coli DH5α were identified by SDS-PAGE. The positive bacteria were picked out and amplified. His-Tag-AKR1B10 protein was purified from the expression product of the positive clones by His-tagged purification column. The purified recombinant protein His-Tag-AKR1B10 was used to immunize New Zealand white rabbits. Antisera were acquired after two months. Anti-AKR1B10 polyclonal antibodies were purified by antigen purification column with AKR1B10 recombinant protein. Lastly, the purified polyclonal antibodies were identified by SDS-PAGE, ELISA, Western blotting. Results The recombinant plasmid pET-15b-AKR1B10 was constructed successfully, and the recombinant protein His-Tag-AKR1B10 with high purity was acquired. The purified polyclonal antibodies were able to specifically recognize AKR1B10 protein. Conclusion The rabbit anti-AKR1B10 polyclonal antibodies is prepared successfully with high specificity.

Human aldo-keto reductases (AKRs) are enzymes involved in the reduction, among other substrates, of all-trans-retinal to all-trans-retinol (vitamin A), thus contributing to the control of the levels of retinoids in organisms. Structure-activity relationship studies of a series of C11-to-C14 methyl-shifted (relative to natural C13-methyl) all-trans-retinal analogues as putative substrates of AKRs have been reported. The synthesis of these retinoids was based on the formation of a C10-C11 single bond of the pentaene skeleton starting from a trienyl iodide and the corresponding dienylstannanes and dienylsilanes, using the Stille-Kosugi-Migita and Hiyama-Denmark cross-coupling reactions, respectively. Since these reagents differ by the location and presence of methyl groups at the dienylorganometallic fragment, the study also provided insights into the ability of the different positional isomers to undergo cross-coupling and the sensitivity of these processes to steric hindrance. The resulting C11-to-C14 methyl-shifted all-trans-retinal analogues were found to be active substrates when tested with AKR1B1 and AKR1B10 enzymes, although relevant differences in substrate specificities were noted. For AKR1B1, all analogues exhibited higher catalytic efficiency (kcat/Km) than parent all-trans-retinal. In addition, only all-trans-11-methylretinal, the most hydrophobic derivative, showed a higher value of kcat/Km = 106 000 ± 23 200 mM-1 min-1 for AKR1B10, which is in fact the highest value from all known retinoid substrates of this enzyme. The novel structures, identified as efficient AKR substrates, may serve in the design of selective inhibitors with potential pharmacological interest.

Aldo-keto reductases (AKRs) are distributed in three families and multiple subfamilies in mammals. The mouse Akr1b3 gene is clearly orthologous to human AKR1B1, both coding for aldose reductase, and their gene products show similar tissue distribution, regulation by osmotic stress and kinetic properties. In contrast, no unambiguous orthologs of human AKR1B10 and AKR1B15.1 have been identified in rodents. Although two more AKRs, AKR1B7 and AKR1B8, have been identified and characterized in mouse, none of them seems to exhibit properties similar to the human AKRs. Recently, a novel mouse AKR gene, Akr1b16, was annotated and the respective gene product, AKR1B16 (sharing 83% and 80% amino acid sequence identity with AKR1B10 and AKR1B15.1, respectively), was expressed as insoluble and inactive protein in a bacterial expression system. Here we describe the expression and purification of a soluble and enzymatically active AKR1B16 from E. coli using three chaperone systems. A structural model of AKR1B16 allowed the estimation of its active-site pocket volume, which was much wider (402 Å3) than those of AKR1B10 (279 Å3) and AKR1B15.1 (60 Å3). AKR1B16 reduced aliphatic and aromatic carbonyl compounds, using NADPH as a cofactor, with moderate or low activity (highest kcat values around 5 min-1). The best substrate for the enzyme was pyridine-3-aldehyde. AKR1B16 showed poor inhibition with classical AKR inhibitors, tolrestat being the most potent. Kinetics and inhibition properties resemble those of rat AKR1B17 but differ from those of the human enzymes. In addition, AKR1B16 catalyzed the oxidation of 17β-hydroxysteroids in a NADP+-dependent manner. These results, together with a phylogenetic analysis, suggest that mouse AKR1B16 is an ortholog of rat AKR1B17, but not of human AKR1B10 or AKR1B15.1. These human enzymes have no counterpart in the murine species, which is evidenced by forming a separate cluster in the phylogenetic tree and by their unique activity with retinaldehyde.