AKR1B10 Research Articles

Прогнозування розвитку діабетичної ретинопатії на основі визначення поліморфних локусів rs759853 І rs9640883 гена AKR1B1

Прогнозування розвитку діабетичної ретинопатії на основі визначення поліморфних локусів rs759853 І rs9640883 гена AKR1B1

Possible role of rs759853 and rs9640883 polymorphisms of the AKR1B1 gene in diabetic retinopathy and type 2 diabetes mellitus

Possible role of rs759853 and rs9640883 polymorphisms of the AKR1B1 gene in diabetic retinopathy and type 2 diabetes mellitus

Identification and characterization of functional antioxidant response elements in the promoter of the aldo-keto reductase AKR1B10 gene

AKR1B10 is a human-type aldo-keto reductase. The up-regulation of AKR1B10 has been associated with various cancers including non-small cell lung carcinoma, viral and bacterial infections, and skin diseases. However, the mechanisms underlying AKR1B10 gene regulation are not fully understood. We previously indicated the involvement of the transcription factor Nrf2 in AKR1B10 gene regulation. There are at least five potential Nrf2-responsive consensus sequences, so-called antioxidant response elements (AREs), and several ARE-like sequences in the 5’-flanking region up to −3282 bp of the AKR1B10 gene. In the present study, we attempted to identify functional AREs by luciferase reporter analyses using various mutants for each ARE. And we found that only those between −530 and −520 bp (ARE-A), which is the closest location to the translation start site, were functional among the five ARE consensus sites examined. Furthermore, ARE-A functioned co-operatively with the neighboring AP-1 site. Since the AP-1 site resembles ARE, the tandem arrangement of these two elements may be essential for augmented responsiveness to Nrf2 and plays an important role in AKR1B10 gene regulation by various Nrf2-mediating stimuli.

Combined Transcriptomic Analysis Revealed AKR1B10 Played an Important Role in Psoriasis through the Dysregulated Lipid Pathway and Overproliferation of Keratinocyte.

RNA-seq has enabled in-depth analysis of the pathogenesis of psoriasis on the transcriptomic level, and many biomarkers have been discovered to be related to the immune response, lipid metabolism, and keratinocyte proliferation. However, few studies have combined analysis from various datasets. In this study, we integrated different psoriasis RNA-seq datasets to reveal the pathogenesis of psoriasis through the analysis of differentially expressed genes (DEGs), pathway analysis, and functional annotation. The revealed biomarkers were further validated through proliferation phenotypes. The results showed that DEGs were functionally related to lipid metabolism and keratinocyte differentiation dysregulation. The results also showed new biomarkers, such as AKR1B10 and PLA2G gene families, as well as pathways that include the PPAR signaling pathway, cytokine-cytokine receptor interaction, alpha-linoleic acid metabolism, and glycosphingolipid biosynthesis. Using siRNA knockdown assays, we further validated the role that the AKR1B10 gene plays in proliferation. Our study demonstrated not only the dysfunction of the AKR1B10 gene in lipid metabolizing but also its important role in the overproliferation and migration of keratinocyte, which provided evidence for further therapeutic uses for psoriasis.

The association between methylation levels of targeted genes and albuminuria in patients with early diabetic kidney disease

Objective: The incidence of diabetes and its complications are greatly increasing world-wide. Diabeticnephropathy (DN) is the main cause of end-stage renal disease and is associated with high morbidity and mortality. It is important to predict patients with high risk for DN in the early stage. We selected the genes which have an important role on diabetic kidney disease. We aimed to investigate the association between DNA methylation levels of targeted genes and albuminuria in patients with early DN.Methods: We collected the clinical data of patients with type 2 diabetes mellitus. We measured spot urine albumin creatinine ratio to calculate albuminuria level. We divided patients into two groups based on albumin excretion as patients with (n = 69) and without DN (n = 27). We performed methylation profiling after bisulfite conversion by pyrosequencing method. The mean value of percent methylation level of each gene was calculated.Results: We compared targeted genes (TIMP-2, AKR1B1, MMP-2, MMP-9, MYL9, SCL2A4, SCL2A1, SCL4A3) methylation levels and albuminuria. We found significant negative correlation between TIMP-2 and AKR1B1 gene methylation levels and albuminuria levels.Conclusions: The present study provided evidence that hypomethylation of TIMP-2 and AKR1B1 genes can be associated with albuminuria in patients with early DN. We may speculate that the hypomethylation of TIMP-2 and AKR1B1 genes may be an early surrogate marker of DN.

РАСПРЕДЕЛЕНИЕ ГЕНОТИПОВ И АЛЛЕЛЕЙ ПОЛИМОРФИЗМОВ rs759853 И rs9640883 ГЕНА AKR1B1 У БОЛЬНЫХ ДИАБЕТИЧЕСКОЙ РЕТИНОПАТИЕЙ, КАТАРАКТОЙ САХАРНЫМ ДИАБЕТОМ 2 ТИПА

Цель исследования – сравнение распределения генотипов и аллелей полиморфизмов rs759853 и rs9640883 гена AKR1B1 у больных диабетической ретинопатией (ДР), катарактой и сахарным диабетом 2 типа (СД2Т). В исследовании приняли участие 409 человек, которые были разделены на пять групп: 1-я – 98 пациентов с катарактой, которые не имели СД2Т; 2-я – 76 пациентов с катарактой и I стадией ДР (без видимых изменений на глазном дне); 3-я – 64 пациента с катарактой и непролиферативной ДР; 4-я – 64 пациента с катарактой и пролиферативной ДР; 5-я – контрольная группа – 107 пациентов без офтальмологической патологии соответствующих другим группам по возрасту и полу. Результаты работы показали, что по полиморфизму rs759853 гена AKR1B1 в группах больных было снижение частоты предкового генотипа G/G на фоне увеличения частоты гетерозиготы G/A при катаракте и при СД2Т без ретинопатии и увеличение частоты мутантной гомозиготы А/А при ДР (в 3,3–3,9 раза).В общем при ДР имело место снижение частоты предкового аллеля G и увеличение частоты мутантного аллеля А полиморфизма rs759853 гена AKR1B1. По полиморфизму rs9640883 гена AKR1B1показано существенное увеличение частоты предкового генотипа G/G у больных СД2Т (в 1,4–1,6 раза) на фоне уменьшения частоты гетерозиготы G/A (в 1,4–1,9 раза). Поскольку гомозиготный генотип А/А вообще оказывался у больных ДР, можно было обосновать предположение о проективной роли аллеля А rs9640883 в развитии ДР.

Prognostic role of expression levels of FABP3 and AKR1B10 genes in adenocarcinoma stage I patients.

7547 Background: In resected lung cancer, no reliable clinical or molecular predictors are currently available for identifying patients with high risk for recurrent disease. In a previous study, we...

Genetic variants in AKR1B10 associate with human eating behavior.

BackgroundThe 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.ResultsWe 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).ConclusionOur data suggest that genetic variants in the AKR1B10 locus may influence human eating behavior.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0189-9) contains supplementary material, which is available to authorized users.

Metabolomics reveals that aldose reductase activity due to AKR1B10 is upregulated in hepatitis C virus infection.

To understand the changes in the metabolome of hepatitis C virus (HCV)-infected persons, we conducted a metabolomic investigation in both plasma and urine of 30 HCV-positive individuals using plasmas from 30 HCV-negative blood donors and urines from 30 healthy volunteers. Samples were analysed by gas chromatography-mass spectrometry and data subjected to multivariate analysis. The plasma metabolomic phenotype of HCV-positive persons was found to have elevated glucose, mannose and oleamide, together with depressed plasma lactate. The urinary metabolomic phenotype of HCV-positive persons comprised reduced excretion of fructose and galactose combined with elevated urinary excretion of 6-deoxygalactose (fucose) and the polyols sorbitol, galactitol and xylitol. HCV-infected persons had elevated galactitol/galactose and sorbitol/glucose urinary ratios, which were highly correlated. These observations pointed to enhanced aldose reductase activity, and this was confirmed by real-time quantitative polymerase chain reaction with AKR1B10 gene expression elevated sixfold in the liver. In contrast, AKR1B1 gene expression was reduced 40% in HCV-positive livers. Interestingly, persons who were formerly HCV infected retained the metabolomic phenotype of HCV infection without reverting to the HCV-negative metabolomic phenotype. This suggests that the effects of HCV on hepatic metabolism may be long lived. Hepatic AKR1B10 has been reported to be elevated in hepatocellular carcinoma and in several premalignant liver diseases. It would appear that HCV infection alone increases AKR1B10 expression, which manifests itself as enhanced urinary excretion of polyols with reduced urinary excretion of their corresponding hexoses. What role the polyols play in hepatic pathophysiology of HCV infection and its sequelae is currently unknown.

Down-regulation of aldo–keto reductase AKR1B10 gene expression by a phorbol ester via the ERK/c-Jun signaling pathway

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.

Molecular mechanisms of Nano-selenium in mitigating hepatocellular carcinoma induced by N-nitrosodiethylamine (NDEA) in rats

The possible molecular mechanisms of Nano-selenium (nano-se) in attenuating hepatocellular carcinoma (HCC) was investigated in this study. To achieve this target, the apoptotic/necrotic rate in hepatic cells was investigated morphologically by double staining with acridine orange/ethidium bromide to address the type of cell death induced by nano-Se in HCC-bearing rats. To predict the oxidative stress and DNA damage, the generation of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 2-deoxyguanosine (2-dG) was examined. Moreover, the expression of some HCC-related genes was investigated such as aldo-keto reductase 1B10 (Akr1b10), ING3 and Foxp1 genes. As well as the histopathological study of liver tissue sections was performed. The results obtained from this study revealed that (HCC+Nano Se) group shows the highest number of damaged cancerous cells. Furthermore, the necrotic/apoptotic rate was significantly higher in (nano-Se+HCC), (HCC+Doxo) and (HCC+Doxo+nano-se) compared to that in the untreated HCC group. Treatment of HCC group with nano-se decreased the ratio of 8-OHdG/2-dG generation significantly with respect to the untreated HCC group. The opposite was observed regarding the gene expression of AKr1b10 and ING3. The treatment of HCC group with nano-se elicited significant increase in the expression of Akr1b10 and ING3 genes compared with untreated HCC group. On the other hand, the expression of Foxp1 gene was significantly decreased in HCC group treated with nano-se in comparison with the untreated HCC group. The histopathological study provided a supportive evidence for the molecular genetics study. Our data shed light on the molecular mechanisms of nano-se in attenuating HCC in the experimental model.

Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms.

In addition to its well-characterized role in the regulation of drug metabolism and transport by xenobiotics, pregnane X receptor (PXR) critically impacts on lipid homeostasis. In mice, both ligand-dependent activation and knockout of PXR were previously shown to promote hepatic steatosis. To elucidate the respective pathways in human liver, we generated clones of human hepatoma HepG2 cells exhibiting different PXR protein levels, and analyzed effects of PXR activation and knockdown on steatosis and expression of lipogenic genes. Ligand-dependent activation as well as knockdown of PXR resulted in increased steatosis in HepG2 cells. Activation of PXR induced the sterol regulatory element-binding protein (SREBP) 1-dependent lipogenic pathway via PXR-dependent induction of SREBP1a, which was confirmed in primary human hepatocytes. Inhibiting SREBP1 activity by blocking the cleavage-dependent maturation of SREBP1 protein impaired the induction of lipogenic SREBP1 target genes and triglyceride accumulation by PXR activation. On the other hand, PXR knockdown resulted in up-regulation of aldo-keto reductase (AKR) 1B10, which enhanced the acetyl-CoA carboxylase (ACC)-catalyzed reaction step of de novo lipogenesis. In a cohort of human liver samples histologically classified for non-alcoholic fatty liver disease, AKR1B10, SREBP1a and SREBP1 lipogenic target genes proved to be up-regulated in steatohepatitis, while PXR protein was reduced. In summary, our data suggest that activation and knockdown of PXR in human hepatic cells promote de novo lipogenesis and steatosis by induction of the SREBP1 pathway and AKR1B10-mediated increase of ACC activity, respectively, thus providing mechanistic explanations for a putative dual role of PXR in the pathogenesis of steatohepatitis.

Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2 synthase activity of aldo-ketoreductase 1B1

Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1β (IL-1β). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1β. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.

Altered Expression of Multiple Genes Involved in Retinoic Acid Biosynthesis in Human Colorectal Cancer

All-trans-retinoic acid (atRA), the oxidized form of vitamin A (retinol), regulates a wide variety of biological processes, such as cell proliferation and differentiation. Multiple alcohol, retinol and retinaldehyde dehydrogenases (ADHs, RDHs, RALDHs) as well as aldo-keto reductases (AKRs) catalyze atRA production. The reduced atRA biosynthesis has been observed in several human tumors, including colorectal cancer. However, subsets of atRA-synthesizing enzymes have not been determined in colorectal tumors. We investigated the expression patterns of genes involved in atRA biosynthesis in normal human colorectal tissues, primary carcinomas and cancer cell lines by RT-PCR. These genes were identified using transcriptomic data analysis (expressed sequence tags, RNA-sequencing, microarrays). Our results indicate that each step of the atRA biosynthesis pathway is dysregulated in colorectal cancer. Frequent and significant decreases in the mRNA levels of the ADH1B, ADH1C, RDHL, RDH5 and AKR1B10 genes were observed in a majority of colorectal carcinomas. The expression levels of the RALDH1 gene were reduced, and the expression levels of the cytochrome CYP26A1 gene increased. The human colon cancer cell lines showed a similar pattern of changes in the mRNA levels of these genes. A dramatic reduction in the expression of genes encoding the predominant retinol-oxidizing enzymes could impair atRA production. The most abundant of these genes, ADH1B and ADH1C, display decreased expression during progression from adenoma to early and more advanced stage of colorectal carcinomas. The diminished atRA biosynthesis may lead to alteration of cell growth and differentiation in the colon and rectum, thus contributing to the progression of colorectal cancer.

Proteomics analysis of hepatitis B virus and aflatoxin B1 related hepatocellular carcinoma in Guangxi,China

Objective To explore the effects and the molecular mechanism of synergistic hepatocarcinogenesis of HBV and AFB1 in the development of HCC by studying the difference in protein expression profiles in hepatocellular carcinoma exposed to hepatitis B virus and Aflatoxin B1.Method 32 HCC specimens were labeled under four categories based on their biomarkers of HBV and AFB1 exposure:group A:HBV(+)/AFB1 (+),10 cases,group B:HBV(+)/AFB1 (-),10 cases,group C:HBV(-)/AFB1(+),6 cases,groupD:HBV(-)/AFB1(-),6 cases.Normal hepatic tissues from 10 cases of hepatic hemangioma,liver resection and liver transplant donor were chosen as the normal control group.Isobaric Tagging Reagent Quantitative (iTRAQ) Proteomics together with 2DLC MS/MS were applied to analyze the differentially expressed proteins among the 4 groups.Result (1) A total of 117 unique differentially expressed proteins including 53 up-regulated proteins and 64 down-regulated proteins were identified in the four groups.The number of unique differentially expressed proteins,including up-regulated and down-regulated proteins,in group A,group B,group C and group D were 106,97,104 and 74 respectively.(2) Among the 117 differentially expressed proteins,9 proteins were heat shock proteins or chaperones,and they were up regulated in group A,B and C.Besides,15 proteins were detoxification and drug metabolism pathway related proteins,12 of them were down-regulated in group A,and more than half of them were also down-regulated in group B and C.(3) The Reverse Transcriptase PCR result showed the mean expression level of AKR1B10 mRNA in group A was significantly higher than group B,C and D (all P＜0.05,respectively).Group C also showed significantly a higher expression level of AKR1B10 mRNA than group D (P＜0.05).The Western-blot results showed the mean expression level of AKR1B10 protein in group A was significantly higher than group B and D (all P＜0.05,respectively).Conclusions The up-regulated expression of heat shock protein and the down-regulated expression of most protein enzymes related to detoxification and drug metabolism were the common molecular biological events of HCC associated with exposure to HBV and AFB1.This suggested the synergistic hepatocarcinogenesis effects of HBV and AFB1 may be related to dysregulation of protein enzymes related to detoxification and drug metabolism.The overexpression of AKR1B10 may be involved in the AFB1-related hepatocarcinogenesis process. Key words: Hepatitis B virus; Aflatoxin B1; Hepatocellular carcinoma; Proteomics; Guangxi

Expression of aldo-keto reductase family 1 member B10 in gastric cancer tissues and its clinical significance

To investigate the association of AKR1B10 expression in gastric cancer tissues with clinicopathologic features and prognosis of gastric cancer patients. Real-time polymerase chain reaction (RT-PCR) was conducted to detect AKR1B10 mRNA expression in gastric cancer and adjacent gastric mucosa tissues (n=36). AKR1B10 protein expression was measured by immunohistochemistry in primary gastric cancer tissues (n=100) and non-tumorous gastric mucosa tissues (n=70). RT-PCR results confirmed that AKR1B10 was significantly down-regulated in gastric cancer tissues compared with that in paired adjacent mucosa [8.3% (3/36) vs. 91.7% (33/36), P=0.000]. Immunohistochemistry revealed that the percentage of AKR1B10 positive specimens in gastric carcinoma was lower than that in normal specimens [33.0% (33/100) vs. 92.9% (65/70), P=0.000]. The frequencies of positive AKR1B10 in patients was significantly correlated with tumor size (P=0.000), invasive depth (P=0.004), lymph node metastasis (P=0.028), distant metastasis (P=0.031) and TNM stages (P=0.000). The 5-year survival rate of positive AKR1B10 group was significantly higher as compared to negative group (60.6% vs. 32.8%, P<0.01). The down-regulation of AKR1B10 expression in gastric cancer may be associated with the progress of gastric cancer is suggestive of poor prognosis.

The prostaglandin F synthase activity of the human aldose reductase AKR1B1 brings new lenses to look at pathologic conditions

Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1β in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1β is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1β particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies.

Reduction of Cytotoxic p-Quinone Metabolites of tert-Butylhydroquinone by Human Aldo-keto Reductase (AKR) 1B10

2-tert-Butylhydroquinone (BHQ), an antioxidant used as a food additive, exhibits an anticancer effect, whereas it is carcinogenic in rodents at high doses. BHQ is metabolized into cytotoxic tert-butylquinone (BQ), which is further converted to 6-tert-butyl-2,3-epoxy-4-hydroxy-5-cyclohexen-1-one (TBEH) through 6-tert-butyl-2,3-epoxy-4-benzoquinone (TBE), which induces chromosomal aberration. The reductases for BQ and TBE may be protective against the toxicity of the two p-quinones, but the responsible human enzymes remain unidentified. In this study, we compared the ability of 12 human recombinant enzymes in the aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase superfamilies to reduce BQ and TBE. Among them, AKR1B10 was the most efficient catalyst of the stoichiometric two-electron reduction of BQ and TBE into BHQ and TBEH, respectively. BQ and TBE are more cytotoxic towards endothelial cells than BHQ and TBEH, and their cytotoxicity was decreased by the overexpression of AKR1B10 in the cells. Additionally, AKR1B10 gene expression in human HCT116 cells was up-regulated by treatments with BHQ, BQ and TBE. These results suggest a role for the enzyme in protection at least against the toxicity of the two p-quinone metabolites of BHQ.

Regulation of aldo–keto reductase AKR1B10 gene expression: Involvement of transcription factor Nrf2

Aldo–keto reductase 1B10 (AKR1B10) is an aldose reductase-like oxidoreductase of human origin. The expression of AKR1 B10 is highly induced in the cells of various cancers such as lung non-small-cell carcinoma and hepatocellular carcinoma. Since the enzyme exhibits broad substrate specificities toward various xenobiotics such as anti-tumor drugs or various endogenous compounds such as retinaldehyde, AKR1B10 may play an important role in tumor progression or drug resistance. However, very little is known about its gene regulation. In this study, we investigated the regulation of AKR1 B10 expression. A −3282 bp of the 5′-flanking fragment of AKR1 B10 gene was isolated from A549 lung carcinoma cells. This region contains several putative regulatory motifs such as AP-1, NF-κB and antioxidant response element. In addition, a complex polymorphic microsatellite with repetitive sequences enriched with C and T was found. However, luciferase reporter assay revealed that the microsatellite polymorphism did not influence the basal promoter activity. We found that an antioxidant ethoxyquin induced the AKR1 B10 expression based on RT-PCR analysis and luciferase reporter assay. Since ethoxyquin is known to activate the gene expression mediated through transcription factor Nrf2, the involvement of Nrf2 was examined. Forced expression of dominant-negative Nrf2 mutant suppressed the ethoxyquin-induced AKR1 B10 expression, and co-introduction of Nrf2 expression plasmid into the cells significantly augmented the luciferase reporter activity. Deletion analysis revealed that Nrf2-regulating cis-element(s) lay within −539 bp of the 5′-flanking region. These results suggest that Nrf2 is one of the major factors involved in the AKR1 B10 gene regulation.

Gammasecretase Inhibition Results in Both Caspase Dependant and Independent Apoptosis in Breast Cancer Cell Lines.

Abstract BackgroundNotch signalling is normally involved in lateral inhibition during embryogenesis and is found to be de regulated in many breast cancers. Gamma-secretase is an enzyme complex formed by constitution of Nicastrin, APH1, Presenilin and Pen2 and is involved in the cleavage of notch proteins. We investigated the effects of γ-secretase inhibition and its mechanism of action in breast cancer cell lines.Materials and MethodsGrowth assay was performed using Gammasecretase inhibitor-1(Z-Leu-Leu-Nle-CHO) in MCF-7, MDA-MB-231, T47D and ZR-75 breast cancer cell lines and 226 U19 and MCF10A non tumourigenic epithelial cell lines at concentrations between10nM and 60uM. Expression of Bcl-2, Bcl-XL, P-Bad and XIAP were measured by Western blot and cell cycle analysis was done by FACS in MCF-7 and MDA-MB-231 cells treated with GSI-1 at 0.75μM, 2μM and 5μM for 48 hours. Caspase 3 and 7 activity was measured in MCF-7 and MDA-MB-231 cells treated with GSI-1 at 0.75μM, 2μM and 5μM for 24 and 48 hours using Promega assay kit. MCF-7 cells were also treated with 0.75μM GSI-1 and DMSO in five replicates for 48 hours and gene array was performed using 44K Agilent array. Fold changes of AKR1B10 and Osteopontin genes were confirmed by Real time PCR at 0.75μM of GSI-1 in MCF-7 and they were also measured at concentrations of 2μM and 5μM in MCF-7 and MDA MB 231 cells using Taqman.ResultsThe 4 cancer cell lines MCF-7, MDA-MB231, ZR-75 and T47D showed growth inhibition in a dose dependant manner and were killed at 5uM. However this concentration did not affect the non tumourigenic cell lines. Expression of Bcl-2, Bcl-XL, inhibitor of apoptosis (XIAP) and P-BAD went down with escalating doses of GSI-1 at 48 hours in both MCF-7 and MDA MB 231 cells. Cell cycle analysis confirmed increase in the percentage of cells in G2M arrest leading to apoptosis at 48 hours in the same cell lines. However we observed increase in Caspase 3 and 7 activity only in MDA-MB-231 not in MCF-7 cells. Gene array in MCF-7 cells treated with GSI1 showed 15 and 4 fold changes in the expression of AKR1B10 and Osteopontin. There was also dose dependant increase in the expression these two genes with GSI-1 in MCF-7 but no such change was observed in MDA MB 231 cells.DiscussionThis is the first time we are reporting the effect of GSI-1 and its possible mechanism of action in breast cancer cell lines. Apoptosis appears to be the main mechanism of cell death as evidenced by our Western blots and FACS analysis in MCF-7 and MDA MB 231 cell lines. However, MCF-7 did not show any Caspase 3/7 activity implying other mechanisms may be involved in the induction of apoptosis. Production of reactive oxygen species (ROS) triggers the damage to mitochondrial membrane and release of cytochrome c, Apoptosis inducing Factor and Endonuclease G resulting in the initiation of Caspase independent apoptosis. AKR1B10 and Osteopontin were shown to be highly expressed in conditions resulting in increased production of ROS. Thus inhibition of γ-secretase enzyme results in breast cancer cell death in a dose which was non lethal to non tumourigenic cell lines. Both Caspase dependant and independent apoptosis appear to be involved in the mechanism of cell death. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3129.