Cytosolic NADPH-dependent reductase AKR1B10 is a member of the aldo-keto reductase (AKR) superfamily. This enzyme is normally expressed in the gastrointestinal tract. However, it is overexpressed in many solid tumors, such as hepatocarcinoma, lung cancer and breast cancer. AKR1B10 may play a role in the formation and development of carcinomas through multiple mechanisms including detoxification of cytotoxic carbonyls, modulation of retinoic acid level, and regulation of cellular fatty acid synthesis and lipid metabolism. Studies have suggested that AKR1B10 may be a useful biomarker for cancer diagnosis and a potential target for cancer treatment. Over the last decade, a number of AKR1B10 inhibitors including aldose reductase inhibitors (ARIs), endogenous substances, natural-based derivatives and synthetic compounds have been developed, which could be novel anticancer drugs. This review provides an overview on related articles and patents about AKR1B10 inhibitors, with a focus on their inhibition selectivity and mechanism of function.

Radiation therapy is an important and effective treatment option for prostate cancer, but high-risk patients are prone to relapse due to radioresistance of cancer cells. Molecular mechanisms that contribute to radioresistance are not fully understood. Novel computational strategies are needed to identify radioresistance driver genes from hundreds of gene copy number alterations. We developed a network-based approach based on lasso regression in combination with network propagation for the analysis of prostate cancer cell lines with acquired radioresistance to identify clinically relevant marker genes associated with radioresistance in prostate cancer patients. We analyzed established radioresistant cell lines of the prostate cancer cell lines DU145 and LNCaP and compared their gene copy number and expression profiles to their radiosensitive parental cells. We found that radioresistant DU145 showed much more gene copy number alterations than LNCaP and their gene expression profiles were highly cell line specific. We learned a genome-wide prostate cancer-specific gene regulatory network and quantified impacts of differentially expressed genes with directly underlying copy number alterations on known radioresistance marker genes. This revealed several potential driver candidates involved in the regulation of cancer-relevant processes. Importantly, we found that ten driver candidates from DU145 (ADAMTS9, AKR1B10, CXXC5, FST, FOXL1, GRPR, ITGA2, SOX17, STARD4, VGF) and four from LNCaP (FHL5, LYPLAL1, PAK7, TDRD6) were able to distinguish irradiated prostate cancer patients into early and late relapse groups. Moreover, in-depth in vitro validations for VGF (Neurosecretory protein VGF) showed that siRNA-mediated gene silencing increased the radiosensitivity of DU145 and LNCaP cells. Our computational approach enabled to predict novel radioresistance driver gene candidates. Additional preclinical and clinical studies are required to further validate the role of VGF and other candidate genes as potential biomarkers for the prediction of radiotherapy responses and as potential targets for radiosensitization of prostate cancer.

The α-hydroxy ketones are used as building blocks for compounds of pharmaceutical interest (such as antidepressants, HIV-protease inhibitors and antitumorals). They can be obtained by the action of enzymes or whole cells on selected substrates, such as diketones. We have studied the enantiospecificities of several fungal (AKR3C1, AKR5F and AKR5G) and human (AKR1B1 and AKR1B10) aldo-keto reductases in the production of α-hydroxy ketones and diols from vicinal diketones. The reactions have been carried out with pure enzymes and with an NADPH-regenerating system consisting of glucose-6-phosphate and glucose-6-phosphate dehydrogenase. To ascertain the regio and stereoselectivity of the reduction reactions catalyzed by the AKRs, we have separated and characterized the reaction products by means of a gas chromatograph equipped with a chiral column and coupled to a mass spectrometer as a detector. According to the regioselectivity and stereoselectivity, the AKRs studied can be divided in two groups: one of them showed preference for the reduction of the proximal keto group, resulting in the S-enantiomer of the corresponding α-hydroxy ketones. The other group favored the reduction of the distal keto group and yielded the corresponding R-enantiomer. Three of the AKRs used (AKR1B1, AKR1B10 and AKR3C1) could produce 2,3-butanediol from acetoin. We have explored the structure/function relationships in the reactivity between several yeast and human AKRs and various diketones and acetoin. In addition, we have demonstrated the utility of these AKRs in the synthesis of selected α-hydroxy ketones and diols.

To clarify the relationship between aldo-keto reductase family 1 member B10 (AKR1B10) expression and early hepatocellular carcinoma (HCC) recurrence, this study detected AKR1B10 expression in tumor and adjacent non-tumor tissues from 110 patients with hepatitis B virus (HBV)-related HCC underwent liver resection and analyzed its correlations with clinicopathological characteristics and prognosis of these patients. Detected by quantitative reverse transcription polymerase chain reaction, AKR1B10 mRNA expression showed significantly higher in HCC tissues than in adjacent non-tumor tissues, with a low level in normal liver tissues. Similar results was confirmed at the protein level using immunohistochemistry and Western blotting. High AKR1B10 expression was negatively correlated with serum alpha-fetoprotein level and positively correlated with HBV-DNA level. Patients with high AKR1B10 expression had significantly higher disease-free survival than those with low expression within 2 years after liver resection. Multivariate analysis also confirmed high AKR1B10 expression to be a predictor of low risk of early HCC recurrence. In addition, high AKR1B10 expression was found to be a favorable factor of overall survival. These results suggest that AKR1B10 is involved in HBV-related hepatocarcinogenesis, but its high expression could predict low risk of early tumor recurrence in patients with HBV-related HCC after liver resection.

OBJECTIVE

Recent reports have indicated that aldo-keto reductase family 1 member B10 (AKR1B10), a cancer-related oxidoreductase, was upregulated in some chronic liver diseases. However, few studies have reported AKR1B10 expression in chronic hepatitis B virus (HBV)-infected patients. The aim of the present study was to analyze AKR1B10 expression and its relevance on hepatocellular carcinoma (HCC) development in patients with chronic HBV infection.

METHODS

Expression of AKR1B10 in the liver of 119 chronic HBV-infected patients was assessed and quantified immunohistochemically. A multivariate Cox model was used to estimate the hazard ratios of AKR1B10 expression for HCC development. The cumulative incidences of HCC were evaluated using Kaplan-Meier analysis.

RESULTS

Expression of AKR1B10 in the study cohort ranged from 0% to 84%. During the median follow-up time (6.2 years), 13 patients developed HCC. Multivariate analysis revealed that high AKR1B10 expression (≥15%) was an independent risk factor for HCC (hazard ratio, 10.8; 95% confidence interval, 3.0-38.6; P < 0.001). The 5-year cumulative incidences of HCC were 20.6% and 2.6% in patients with high and low AKR1B10 expression, respectively (P < 0.001). Patients with high AKR1B10 expression had significantly higher alanine aminotransferase levels during follow-up than those with low expression, even though antiviral treatment decreased HBV-DNA levels in both groups.

CONCLUSIONS

Chronic HBV-infected patients with high hepatic AKR1B10 expression had an increased risk of HCC development. This suggests that AKR1B10 upregulation might play a role in the early stages of HBV-related hepatocarcinogenesis.

Targeting the ubiquitin-proteasome pathway with the proteasome inhibitor bortezomib has emerged as a promising approach for the treatment of several malignancies. The cellular and molecular effects of this agent on colorectal cancer cells are poorly characterized. This study investigated the antiproliferative effect of bortezomib on colorectal cancer cell lines (Caco-2 and HRT-18). In order to define the proteins potentially involved in the mechanisms of action, proteome profiling was applied to detect the proteins altered by bortezomib. The in vitro efficacy of bortezomib as a single agent in colorectal cancer cell lines was confirmed. Proteome profiling with two-dimensional PAGE followed by mass spectrometry revealed the up-regulation of the major inducible isoform of heat shock protein 70 (hsp72) and lactate dehydrogenase B in both cell lines, as well as the induction of aldo-keto reductase family 1 member B10 (AKR1B10) in HRT-18 cells. Both AKR1B10 and hsp72 exert cell-protective functions. This study shows for the first time a bortezomib-induced up-regulation of AKR1B10. Small interfering RNA-mediated inhibition of this enzyme with known intracellular detoxification function sensitized HRT-18 cells to therapy with the proteasome inhibitor. To further characterize the relevance of AKR1B10 for colorectal tumors, immunohistochemical expression was shown in 23.2% of 125 tumor specimens. These findings indicate that AKR1B10 might be a target for combination therapy with bortezomib.

AKR1B10 is an NADPH-dependent reductase that plays an important function in several physiological reactions such as the conversion of retinal to retinol, reduction of isoprenyl aldehydes, and biotransformation of procarcinogens and drugs. A growing body of evidence points to the important role of the enzyme in the development of several types of cancer (e.g., breast, hepatocellular), in which it is highly overexpressed. AKR1B10 is regarded as a therapeutic target for the treatment of these diseases, and potent and specific inhibitors may be promising therapeutic agents. Several inhibitors of AKR1B10 have been described, but the area of natural plant products has been investigated sparingly. In the present study almost 40 diverse phenolic compounds and alkaloids were examined for their ability to inhibit the recombinant AKR1B10 enzyme. The most potent inhibitors-apigenin, luteolin, and 7-hydroxyflavone-were further characterized in terms of IC50, selectivity, and mode of action. Molecular docking studies were also conducted, which identified putative binding residues important for the interaction. In addition, cellular studies demonstrated a significant inhibition of the AKR1B10-mediated reduction of daunorubicin in intact cells by these inhibitors without a considerable cytotoxic effect. Although these compounds are moderately potent and selective inhibitors of AKR1B10, they constitute a new structural type of AKR1B10 inhibitor and may serve as a template for the development of better inhibitors.

All-trans-retinoic acid (ATRA) is the main biologically active metabolite of retinol (vitamin A) that is required for the regulation of such processes as embryogenesis, tissue differentiation, proliferation, and others. Multiple alcohol, retinol and retinaldehyde dehydrogenases (ADHs, RDHs and RALDHs) as well as aldo-keto reductases (AKRs) catalyze the biosynthesis of retinoic acid in humans. For many normal and neoplastic tissues, the key ATRA-synthesizing enzymes remain unknown. We identified ATRA-generating genes that are expressed in normal and malignant gastric tissues using the transcriptomic database analysis. Quantitative changes in the expression levels of these genes in gastric cancer were determined by semi-quantitative RT-PCR and real-time PCR. Significant decreases in the mRNA levels of genes encoding enzymes that catalyze the reversible oxidation/reduction of retinol and retinaldehyde (ADH4, ADH1B, ADH1C, RDHL, AKR1B10, AKR1B1, and RDH12), as well as the oxidation of retinaldehyde (RALDH1) were revealed in most of the tumor samples. The sharp reduction in the expression levels of genes encoding the key enzymes that convert retinol and retinaldehyde to retinoic acid could lead to a significant decrease in the content of ATRA--the transcriptional regulator of many genes, which in turn can lead to a dysregulation of cell proliferation/differentiation and initiate cancer development.

Continuous exposure to doxorubicin (DOX) accelerates hyposensitivity to the drug-elicited lethality of gastric cells, with increased risks of the recurrence and serious cardiovascular side effects. However, the detailed mechanisms underlying the reduction of DOX sensitivity remain unclear. In this study, we generated a DOX-resistant variant upon continuously treating human gastric cancer MKN45 cells with incremental concentrations of the drug, and investigated whether the gain of DOX resistance influences gene expression of four aldo-keto reductases (AKRs: 1B10, 1C1, 1C2 and 1C3). RT-PCR analysis revealed that among the enzymes AKR1B10 is most highly up-regulated during the chemoresistance induction. The up-regulation of AKR1B10 was confirmed by analyses of Western blotting and enzyme activity. The DOX sensitivity of MKN45 cells was reduced and elevated by overexpression and inhibition of AKR1B10, respectively. Compared to the parental MKN45 cells, the DOX-resistant cells had higher migrating and invasive abilities, which were significantly suppressed by addition of AKR1B10 inhibitors. Zymographic and real-time PCR analyses also revealed significant increases in secretion and expression of matrix metalloproteinase (MMP) 2 associated with DOX resistance. Moreover, the overexpression of AKR1B10 in the parental cells remarkably facilitated malignant progression (elevation of migrating and invasive potentials) and MMP2 secretion, which were lowered by the AKR1B10 inhibitors. These results suggest that AKR1B10 is a DOX-resistance gene in the gastric cancer cells, and is responsible for elevating the migrating and invasive potentials of the cells through induction of MMP2.

BACKGROUND

Aldo-keto reductases (AKRs) modify carbonyl groups on aldehyde or ketones to form primary or secondary alcohols, which are then conjugated with sulfates or glucuronide for excretion. The AKR1B10 gene encodes a member of the AKR superfamily. Overexpression of AKR1B10 plays an important role in the tumorigenesis of lung cancer cells; however, the prognostic value of AKR1B10 expression in patients with lung adenocarcinoma has not been well demonstrated.

METHODS

A total of 96 patients with resected lung adenocarcinoma were included in the study. AKR1B10 expression was determined by immunohistochemistry in tumor specimens. The prognostic value of AKR1B10 overexpression and its relationship with clinicopathological variables were investigated.

RESULTS

AKR1B10 overexpression was identified in 22 (22.9%) of the 96 patients and tended to be significantly associated with N1 or N2 status (P = 0.055). AKR1B10 overexpression was not a significant prognostic factor for overall survival (P = 0.301) but was a significant prognostic factor for poor recurrence-free survival (P = 0.015). T status (T3 or T4 vs. T1 or T2; P = 0.020), N1 or N2 (vs. N0; P = 0.019), predominant pattern group (lepidic/acinar/papillary vs. micropapillary/solid; P = 0.023), and AKR1B10 overexpression (P = 0.013) were significant prognostic factors for poor recurrence-free survival in multivariate analysis.

CONCLUSIONS

AKR1B10 overexpression was a significant prognostic factor for poor recurrence-free survival in patients with resected lung adenocarcinoma. This information is useful to stratify patients at high-risk of recurrence after lung adenocarcinoma resection.

The intrinsic heterogeneity of hepatocellular carcinoma (HCC) represents a great challenge for its molecular classification and for detecting predictive biomarkers. Aldo-keto reductase (Akr) family members have shown differential expression in human HCC, while AKR1B10 overexpression is considered a biomarker; AKR7A3 expression is frequently reduced in HCC.

To investigate the time-course expression of Akr members in the experimental hepatocarcinogenesis.

Using DNA-microarray data, we analyzed the time-course gene expression profile from nodules to tumors (4-17 months) of 17 Akr members induced by the resistant hepatocyte carcinogenesis model in the rat.

The expression of six members (Akr1c19, Akr1b10, Akr7a3, Akr1b1, Akr1cl1, and Akr1b8) was increased, comparable to that of Ggt and Gstp1, two well-known liver cancer markers. In particular, Akr7a3 and Akr1b10 expression also showed a time-dependent increment at mRNA and protein levels in a second hepatocarcinogenesis model induced with diethylnitrosamine. We confirmed that aldo-keto reductases 7A3 and 1B10 were co-expressed in nine biopsies of human HCC, independently from the presence of glypican-3 and cytokeratin-19, two well-known HCC biomarkers. Because it has been suggested that expression of Akr members is regulated through NRF2 activity at the antioxidant response element (ARE) sequences, we searched and identified at least two ARE sites in Akr1b1, Akr1b10, and Akr7a3 from rat and human gene sequences. Moreover, we observed higher NRF2 nuclear translocation in tumors as compared with non-tumor tissues.

Our results demonstrate that Akr7a3 mRNA and protein levels are consistently co-expressed along with Akr1b10, in both experimental liver carcinogenesis and some human HCC samples. These results highlight the presence of AKR7A3 and AKR1B10 from early stages of the experimental HCC and introduce them as a potential application for early diagnosis, staging, and prognosis in human cancer.

Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most effective chemotherapeutic drugs that are used for treatment of patients with gastrointestinal cancer cells, but its continuous administration often evokes the development of chemoresistance. In this study, we investigated alterations in antioxidant molecules and functions using a newly established CDDP-resistant variant of gastric cancer MKN45 cells, and found that aldo-keto reductase 1B10 (AKR1B10) is significantly up-regulated with acquisition of the CDDP resistance. In the nonresistant MKN45 cells, the sensitivity to cytotoxic effect of CDDP was decreased and increased by overexpression and silencing of AKR1B10, respectively. In addition, the AKR1B10 overexpression markedly suppressed accumulation and cytotoxicity of 4-hydroxy-2-nonenal that is produced during lipid peroxidation by CDDP treatment, suggesting that the enzyme acts as a crucial factor for facilitation of the CDDP resistance through inhibiting induction of oxidative stress by the drug. Transient exposure to CDDP and induction of the CDDP resistance decreased expression of peroxisome proliferator-activated receptor-γ (PPARγ) in MKN45 and colon cancer LoVo cells. Additionally, overexpression of PPARγ in the cells elevated the sensitivity to the CDDP toxicity, which was further augmented by concomitant treatment with a PPARγ ligand rosiglitazone. Intriguingly, overexpression of AKR1B10 in the cells resulted in a decrease in PPARγ expression, which was recovered by addition of an AKR1B10 inhibitor oleanolic acid, inferring that PPARγ is a downstream target of AKR1B10-dependent mechanism underlying the CDDP resistance. Combined treatment with the AKR1B10 inhibitor and PPARγ ligand elevated the CDDP sensitivity, which was almost the same level as that in the parental cells. These results suggest that combined treatment with the AKR1B10 inhibitor and PPARγ ligand is an effective adjuvant therapy for overcoming CDDP resistance of gastrointestinal cancer cells.

Aldo-keto reductase family 1 member B10 (AKR1B10) is implicated in xenobiotic detoxification and has disparate functions in tumorigenesis that are dependent on the cell types. The purpose of this study was to investigate the clinicopathological significance of AKR1B10 as a prognostic marker for oral squamous cell carcinomas (OSCCs).

AKR1B10 protein expression was analyzed by immunohistochemistry in 77 patients with OSCC.

The AKR1B10 labeling score for OSCCs (1.16 ± 1.14) was significantly higher than that for normal oral mucosa (0.10 ± 0.23; p < .0001). High expression of AKR1B10 significantly correlated with large tumor size (p = .041), advanced TNM classification (p = .037), and patient's areca quid chewing habit (p = .025). Multivariate analysis revealed that high AKR1B10 labeling score >1.16 (hazard ratio, 3.647; p = .001) significantly correlated with mortality.

AKR1B10 overexpression is an independent poor prognostic biomarker for OSCC. AKR1B10 inhibitors may be promising in clinical trials against OSCC. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1327-1332, 2017.

Endometrial cancer is associated with enhanced cell proliferation due to high concentrations of estrogens, and decreased cell differentiation due to low levels of progesterone and retinoic acid. It is also associated with aberrant inflammatory responses and concomitant increased production of prostaglandins. The human members of the aldo-keto reductase 1B (AKR1B) subfamily, AKR1B1 and AKR1B10, have roles in these processes and can thus be implicated in endometrial cancer. To date, there have been no reports on the expression of AKR1B1 in endometrial cancer, while AKR1B10 has only been studied at the cellular level. To evaluate the roles of these AKR1B enzymes, we investigated expression of AKR1B1 and AKR1B10 in 47 paired samples of cancerous and adjacent control endometrium at the mRNA and protein levels, by quantitative PCR, Western blotting and immunohistochemistry staining. There were significantly lower mRNA and protein levels of AKR1B1 in cancerous tissues compared to adjacent endometrium. The gene expression of AKR1B10 at the mRNA level was significantly increased, while there were significantly decreased protein levels. Immunohistochemistry revealed that both of these enzymes were present in all of the samples, and are located in epithelial cells of cancerous and control endometrial glands. Elevated levels in adjacent non-cancerous tissues imply that these enzymes are more important in the initiation of endometrial cancer than in its progression. To the best of our knowledge, this is the first report on the expression of AKR1B1 and AKR1B10 in endometrial cancer. Further studies are needed to define the precise roles of these enzymes in the pathogenesis of endometrial cancer.

Brain metastasis (BM) is a leading cause of mortality in patients with non-small cell lung cancer (NSCLC). However, the molecular mechanisms underlying BM of NSCLC remain largely unknown because of the lack of models to accurately investigate such a dynamic and complex process. Here we developed a multi-organ microfluidic chip as a new methodological platform to study BM. The chip consisted of two bionic organ units - an upstream "lung" and a downstream "brain" characterized by a functional "blood-brain barrier (BBB)" structure, allowing real-time visual monitoring of the entire BM process, from the growth of primary tumor to its breaking through the BBB, and finally reaching the brain parenchyma. The chip was verified by lung cancer cell lines with differing metastatic abilities and then applied for the BM research where we first demonstrated that the protein expression of Aldo-keto reductase family 1 B10 (AKR1B10) was significantly elevated in lung cancer BM. Silencing AKR1B10 in brain metastatic tumor cells suppressed their extravasation through the BBB in the in vitro Transwell model, in our ex vivo microfluidic chip, as well as the in vivo model of brain metastasis in nude mice. Moreover, AKR1B10 downregulated the expression of matrix metalloproteinase (MMP)-2 and MMP-9 via MEK/ERK signaling in metastatic lung cancers. These data suggest that our multi-organ microfluidic chip is a practical alternative to study BM pathogenesis, and AKR1B10 is a diagnostic biomarker and a prospective therapeutic target for NSCLC BM. STATEMENT OF SIGNIFICANCE: Brain metastasis (BM) of non-small cell lung cancer (NSCLC) is a complex cascade, and in particular, the process of lung cancer cells penetrating the blood-brain barrier (BBB) is very unique. However, due to the lack of reliable models that can faithfully mimic the dynamic process of BBB breaking, its molecular mechanisms have not well elucidated so far. In addition, although Aldo-keto reductase family 1 B10 (AKR1B10) has been implicated to the tumor development of liver cancer and many other cancers, little is known on its roles in the BM. Here, we established a multi-organ microfluidic bionic chip platform to recapitulate the entire BM process, and applied it to the BM pathology research, especially BBB extravasation. By using the chip and traditional models synergistically, we first demonstrated that AKR1B10 was significantly elevated in lung cancer BM, and defined the value of AKR1B10 as a diagnostic serum biomarker for lung cancer patients suffering from BM. Further, we investigated the role and mechanisms of AKR1B10 in BM that it promotes the extravasation of cancer cells through the BBB.

OBJECTIVE

To clarify the association between aldo-keto reductase family 1 member B10 (AKR1B10) expression and hepatocarcinogenesis after hepatitis C virus eradication.

METHODS

In this study, we enrolled 303 chronic hepatitis C patients who had achieved sustained virological response (SVR) through interferon-based antiviral therapy. Pretreatment AKR1B10 expression in the liver was immunohistochemically assessed and quantified as a percentage of positive staining area by using image-analysis software. A multivariate Cox analysis was used to estimate the hazard ratios (HRs) of AKR1B10 expression for hepatocellular carcinoma (HCC) development after achieving SVR. The cumulative incidences of HCC development were evaluated using Kaplan-Meier analysis and the log-rank test.

RESULTS

Of the 303 chronic hepatitis C patients, 153 (50.5%) showed scarce hepatic AKR1B10 expression, quantified as 0%, which was similar to the expression in control normal liver tissues. However, the remaining 150 patients (49.5%) exhibited various degrees of AKR1B10 expression in the liver, with a maximal AKR1B10 expression of 73%. During the median follow-up time of 3.6 years (range 1.0-10.0 years), 8/303 patients developed HCC. Multivariate analysis revealed that only high AKR1B10 expression (≥ 8%) was an independent risk factor for HCC development (HR = 15.4, 95%CI: 1.8-132.5, P = 0.012). The 5-year cumulative incidences of HCC development were 13.7% and 0.5% in patients with high and low AKR1B10 expression, respectively (P < 0.001). During the follow-up period after viral eradication, patients expressing high levels of AKR1B10 expressed markedly higher levels of alanine aminotransferase and α-fetoprotein than did patients exhibiting low AKR1B10 expression.

CONCLUSIONS

Chronic hepatitis C patients expressing high levels of hepatic AKR1B10 had an increased risk of HCC development even after SVR.

Inhibitors of a human member (AKR1B10) of the aldo-keto reductase superfamily are regarded as promising therapeutics for the treatment of cancer. Recently, we have discovered (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) as the potent competitive inhibitor using the virtual screening approach, and proposed its 4-methoxy group on the 2-phenylimino moiety as an essential structural prerequisite for the inhibition. In this study, 18 derivatives of 1 were synthesized and their inhibitory potency against AKR1B10 evaluated. Among them, 7-hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic acid benzylamide (5n) was the most potent inhibitor showing a Ki value of 1.3nM. The structure-activity relationship of the derivatives indicated that the 7-hydroxyl group on the chromene ring, but not the 4-methoxy group, was absolutely required for inhibitory activity, The molecular docking of 5n in AKR1B10 and site-directed mutagenesis of the enzyme residues suggested that the hydrogen-bond interactions between the 7-hydroxyl group of 5n and the catalytic residues (Tyr49 and His111) of the enzyme, together with a π-stacking interaction of the benzylamide moiety of 5n with Trp220, are important for the potent inhibition.

14-3-3ε is overexpressed in hepatocellular carcinoma (HCC) and its expression significantly associates with a poor prognostic outcome. To uncover how 14-3-3ε contributes to the tumor progression of HCC, we investigated the potential downstream targets regulated by 14-3-3ε. We found that 14-3-3ε increases expression and nuclear translocation of β-catenin and that 14-3-3ε-induced cell proliferation is attenuated by β-catenin silencing in HCC cells. Moreover, 14-3-3ε induces aldo-keto reductase family 1 member B10 (AKR1B10) expression through the activation of β-catenin signaling. Knockdown of AKR1B10 by siRNAs abolished 14-3-3ε-induced in vitro cell proliferation, anchorage-independent growth as well as in vivo tumor growth. Furthermore, AKR1B10 silencing increased retinoic acid (RA) levels in the serum of tumor-bearing mice and RA treatment attenuated 14-3-3ε-induced HCC cell proliferation. We further examined 14-3-3ε and AKR1B10 expression and clinicopathological characteristics of HCC tumors. Although the expression of AKR1B10 was significantly correlated with 14-3-3ε, an increase of AKR1B10 expression in 14-3-3ε positive patients paradoxically had better overall survival and disease-free survival rates as well as lower metastatic incidence than those without an AKR1B10 increase. Finally, we found a loss of AKR1B10 expression in cells exhibiting a high capacity of invasiveness. Silencing of AKR1B10 resulted in inducing snail and vimentin expression in HCC cells. These results indicate that AKR1B10 may play a dual role during HCC tumor progression. Our results also indicate that 14-3-3ε regulates AKR1B10 expression by activating β-catenin signaling. A combination of 14-3-3ε with AKR1B10 is a potential therapeutic target and novel prognostic biomarker of HCC.

Aldo-keto reductase family 1 B10 (AKR1B10) is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily, and has been considered to be a potential cancer therapeutic target. Total extract from the bark of Rhus verniciflua (Toxicodendron vernicifluum (Stokes)) showed AKR1B10 inhibitory activity. To identify the active compounds from R. verniciflua responsible for AKR1B10 inhibition, nine compounds were isolated via bioactivity-guided isolation and tested for their effects against recombinant human AKR1B10 (rhAKR1B10). Results showed that butein, isolated from the ethyl acetate fraction, was most able to inhibit rhAKR1B10. The inhibitory rate of butein against rhAKR1B10 was 42.86% at 1 microM with an IC(50) value of 1.47 microM, and enzyme kinetic analysis revealed its inhibition mode to be uncompetitive.

In this study, we identified Nrf2 as a molecular target of [6]-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography-tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2(-/-) mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms.

OBJECTIVE

α1-Antitrypsin deficiency (A1ATD) is an autosomal recessive disorder caused by mutations in the SERPINA1 gene. Individuals with the Z variant (Gly342Lys) retain polymerised protein in the endoplasmic reticulum (ER) of their hepatocytes, predisposing them to liver disease. The concomitant lack of circulating A1AT also causes lung emphysema. Greater insight into the mechanisms that link protein misfolding to liver injury will facilitate the design of novel therapies.

METHODS

Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes provide a novel approach to interrogate the molecular mechanisms of A1ATD because of their patient-specific genetic architecture and reflection of human physiology. To that end, we utilised patient-specific hiPSC hepatocyte-like cells (ZZ-HLCs) derived from an A1ATD (ZZ) patient, which faithfully recapitulated key aspects of the disease at the molecular and cellular level. Subsequent functional and "omics" comparisons of these cells with their genetically corrected isogenic-line (RR-HLCs) and primary hepatocytes/human tissue enabled identification of new molecular markers and disease signatures.

RESULTS

Our studies showed that abnormal A1AT polymer processing (immobilised ER components, reduced luminal protein mobility and disrupted ER cisternae) occurred heterogeneously within hepatocyte populations and was associated with disrupted mitochondrial structure, presence of the oncogenic protein AKR1B10 and two upregulated molecular clusters centred on members of inflammatory (IL-18 and Caspase-4) and unfolded protein response (Calnexin and Calreticulin) pathways. These results were validated in a second patient-specific hiPSC line.

CONCLUSIONS

Our data identified novel pathways that potentially link the expression of Z A1AT polymers to liver disease. These findings could help pave the way towards identification of new therapeutic targets for the treatment of A1ATD.

UNASSIGNED

This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease α1-antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human-induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance.

As a transcriptional coactivator, PGC-1α contributes to the regulation of a broad range of metabolic processes in skeletal muscle health and disease; however, there is limited information about the genes it transcriptionally regulates. To identify new potential gene targets of PGC-1α regulation, mouse C2C12 myotubes were screened by microarray analysis following PGC-1α overexpression. Genes with an mRNA expression of 2.5-fold or more (P < 0.001) were identified. From these, further genes were singled out if they had no previous connection to PGC-1α regulation or characterization in skeletal muscle, or were unannotated with no known function. Following confirmation of their regulation by PGC-1α using qPCR analysis, eight genes were focused on for further investigation (Akr1b10, Rmnd1, 1110008P14Rik, 1700021F05Rik, Mtfp1, Mrm1, Oxnad1 and Cluh). Bioinformatics indicated a number of the genes were linked to a range of metabolic-related functions including fatty acid oxidation, oxido-reductase activity, and mitochondrial remodeling and transport. Treating C2C12 myotubes for 6 h with AICAR, a known activator of AMP kinase and inducer of Pgc-1α gene expression, increased the mRNA levels of both Pgc-1α (P < 0.001) and of Mtfp1, Mrm1, Oxnad1 and Cluh (P < 0.05). Screening of the promoter and intron 1 regions also revealed all genes to contain either a consensus or near consensus response elements for the estrogen-related receptor α (ERRα), a key transcription factor-binding partner of PGC-1α in skeletal muscle. Furthermore, knockdown of endogenous ERRα levels partially or completely blocked the induction of gene expression of all genes by PGC-1α, while each gene was significantly upregulated in the presence of a constitutively active form of ERRα (P < 0.05) except for Akr1b10. These findings provide preliminary evidence for the novel regulation of these genes by PGC-1α and its signaling pathway in skeletal muscle.