Expression Of Aldo-keto Reductase Research Articles

Phylogenetic and transcriptomic study of aldo-keto reductases in Haemonchus contortus and their inducibility by flubendazole

Aldo-keto reductases (AKRs), a superfamily of NADP(H)-dependent oxidoreductases, catalyze the oxidoreduction of a wide variety of eobiotic and xenobiotic aldehydes and ketones. In mammals, AKRs play essential roles in hormone and xenobiotic metabolism, oxidative stress, and drug resistance, but little is known about these enzymes in the parasitic nematode Haemonchus contortus. In the present study, 22 AKR genes existing in the H. contortus genome were investigated and a phylogenetic analysis with comparison to AKRs in Caenorhabditis elegans, sheep and humans was conducted. The constitutive transcription levels of all AKRs were measured in eggs, larvae, and adults of H. contortus, and their expression was compared in a drug-sensitive strain (ISE) and a benzimidazole-resistant strain (IRE) previously derived from the sensitive strain by imposing benzimidazole selection pressure. In addition, the inducibility of AKRs by exposure of H. contortus adults to benzimidazole anthelmintic flubendazole in vitro was tested. Phylogenetic analysis demonstrated that the majority of AKR genes in H. contortus lack orthologues in the sheep genome, which is a favorable finding for considering AKRs as potential drug targets. Large differences in the expression levels of individual AKRs were observed, with AKR1, AKR3, AKR8, and AKR10 being the most highly expressed at most developmental stages. Significant changes in the expression of AKRs during the life cycle and pronounced sex differences were found. Comparing the IRE and ISE strains, three AKRs were upregulated, and seven AKRs were downregulated in adults. In addition, the expression of three AKRs was induced by flubendazole exposure in adults of the ISE strain. Based on these results, AKR1, AKR2, AKR3, AKR5, AKR10 and AKR19 in particular merit further investigation and functional characterization with respect to their potential involvement in drug biotransformation and anthelmintic resistance in H. contortus.

Aldo-keto reductases: Role in cancer development and theranostics.

Aldo-keto reductases (AKRs) are a superfamily of enzymes that play crucial roles in various cellular processes, including the metabolism of xenobiotics, steroids, and carbohydrates. A growing body of evidence has unveiled the involvement of AKRs in the development and progression of various cancers. AKRs are aberrantly expressed in a wide range of malignant tumors. Dysregulated expression of AKRs enables the acquisition of hallmark traits of cancer by activating oncogenic signaling pathways and contributing to chemoresistance. AKRs have emerged as promising oncotherapeutic targets given their pivotal role in cancer development and progression. Inhibition of aldose reductase (AR), either alone or in combination with chemotherapeutic drugs, has evolved as a pragmatic therapeutic option for cancer. Several classes of synthetic aldo-keto reductase (AKR) inhibitors have been developed as potential anticancer agents, some of which have shown promise in clinical trials. Many AKR inhibitors from natural sources also exhibit anticancer effects. Small molecule inhibitors targeting specific AKR isoforms have shown promise in preclinical studies. These inhibitors disrupt the activation of oncogenic signaling by modulating transcription factors and kinases and sensitizing cancer cells to chemotherapy. In this review, we discuss the physiological functions of human AKRs, the aberrant expression of AKRs in malignancies, the involvement of AKRs in the acquisition of cancer hallmarks, and the role of AKRs in oncogenic signaling, and drug resistance. Finally, the potential of aldose reductase inhibitors (ARIs) as anticancer drugs is summarized.

In-silico Investigations of quinine and quinidine as potential Inhibitors of AKR1B1 and AKR1B10: Functional and structural characterization.

The aberrant expression of aldo keto reductases (AKR1B1 & AKR1B10) has been extensively studied in different types of cancer especially the colon cancer but a very few studies have yet been reported regarding the discovery of inhibitors for the treatment of colon cancer by targeting these isozymes. Therefore, there is a need of selective inhibitors of both targets for the eradication of colon cancer. Currently, the study is focused on the exploration of two quinolone compounds i.e., (S)-(6-Methoxyquinolin-4-yl)[(1S,2R,4S,5R)-5-vinylquinuclidin-2-yl]methanol (Quinidine) and (R)-(6-Methoxyquinolin-4-yl)[(1S,2S,4S,5R)-5-vinylquinuclidin-2-yl]methanol (Quinine) as the potential inhibitors of AKR1B1 and AKR1B10 via detailed in-silico approach. The structural properties including vibrational frequencies, dipole moment, polarizability and the optimization energies were estimated using density functional theory (DFT) calculations; where both compounds were found chemically reactive. After that, the optimized structures were used for the molecular docking studies and here quinidine was found more selective towards AKR1B1 and quinine exhibited maximum inhibition of AKR1B10. The results of molecular docking studies were validated by molecular dynamics simulations which provided the deep insight of stability of protein ligand complex. At the end, the ADMET properties were determined to demonstrate the druglikeness properties of both selected compounds. These findings suggested further exploration of both compounds at molecular level using different in-vivo and in-vitro approaches that will lead to the designing of potential inhibitor of AKR1B1/AKR1B10 for curing colon cancer and related malignancies.

Stress-Induced Detoxification Enzymes in Rice Have Broad Substrate Affinity.

Reactive carbonyl compounds (RCCs) such as hydroxynonenol, malondialdehyde, acrolein, crotonaldehyde, methylglyoxal, and glyoxal accumulate at higher levels under stress in plants and damage the cell metabolic activities. Plants have evolved several detoxifying enzymes such as aldo–keto reductases (AKRs), aldehyde/alcohol dehydrogenases (ALDH/ADH), and glyoxalases. We report the phylogenetic relationship of these proteins and in silico analysis of rice-detoxifying protein structures and their substrate affinity with cofactors using docking and molecular simulation studies. Molecular simulations with nicotinamide adenine dinucleotide phosphate or glutathione cofactor docking with commonly known reactive substrates suggests that the AKRs, ALDH, and ADH proteins attain maximum conformational changes, whereas glyoxalase has fewer conformational changes with cofactor binding. Several AKRs showed a significant binding affinity with many RCCs. The rice microarray studies showed enhanced expression of many AKRs in resistant genotypes, which also showed higher affinity to RCCs, signifying their importance in managing carbonyl stress. The higher expression of AKRs is regulated by stress-responsive transcription factors (TFs) as we identified stress-specific cis-elements in their promoters. The study reports the stress-responsive nature of AKRs, their regulatory TFs, and their best RCC targets, which may be used for crop improvement programs.

Long-chain fatty acids inhibit human members of the aldo-keto reductase 1C subfamily.

Four human hydroxysteroid dehydrogenases in the aldo-keto reductase (AKR) superfamily, AKR1C1-AKR1C4, are involved in the metabolism of steroids and other carbonyl compounds including drugs, and altered expression of AKRs (1C1, 1C2 and/or 1C3) is related to the pathogenesis of several extrahepatic cancers. Here, we report that unsaturated fatty acids (FAs) are potent competitive inhibitors of the AKR enzymes. The sensitivities to the FAs were different among the enzymes, especially between AKR1C1 and AKR1C2. The most potent inhibitors for AKR1C1, AKR1C2 and AKR1C4 were docosahexaenoic acid (Ki 0.77 µM), palmitoleic acid (Ki 0.41 µM) and linoleic acid (Ki 0.33 µM), respectively. AKR1C3 was the most sensitive to FA inhibition, showing low Ki values (0.23-0.29 µM) for oleic, linoleic, eicosapentaenoic and docosahexaenoic acids. Linoleic and oleic acids also inhibited AKR1C3-mediated metabolism of 9,10-phenanthrenequinone in colon DLD1 cells. Molecular docking and site-directed mutagenesis studies suggested upon FA binding to AKR1C1 and AKR1C3: (i) the carboxyl group of the FA binds to the oxyanion-binding site in the active site; (ii) the difference in FA sensitivity between AKR1C1 and AKR1C2 is due to their residue difference at position 54; (iii) Ser118, Phe306 and Phe311 of AKR1C3 are important for determining the inhibitory potency of FAs.

Alterations in estrogen signalling pathways upon acquisition of anthracycline resistance in breast tumor cells

Intrinsic or acquired drug resistance is a major impediment to the successful treatment of women with breast cancer using chemotherapy. We have observed that MCF-7 breast tumor cells selected for resistance to doxorubicin or epirubicin (MCF-7DOX2 and MCF-7EPI cells, respectively) exhibited increased expression of several members of the aldo-keto reductase (AKR) gene family (in particular AKR1C3 and AKR1B10) relative to control MCF-7CC cells selected by propagation in the absence of drug. Normal cellular roles for the AKRs include the promotion of estrogen (E2) synthesis from estrone (E1) and the hydroxylation and detoxification of exogenous xenobiotics such as anthracycline chemotherapy drugs. While hydroxylation of anthracyclines strongly attenuates their cytotoxicity, it is unclear whether the enhanced AKR expression in the above anthracycline-resistant cells promotes E2 synthesis and/or alterations in E2 signalling pathways and whether such changes contribute to enhanced survival and anthracycline resistance. To determine the role of AKRs and E2 pathways in doxorubicin resistance, we examined changes in the expression of E2-related genes and proteins upon acquisition of doxorubicin resistance. We also assessed the effects of AKR overexpression or downregulation or the effects of activators or inhibitors of E2-dependent pathways on previously acquired resistance to doxorubicin. In this study we observed that the enhanced AKR expression upon acquisition of anthracycline resistance was, in fact, associated with enhanced E2 production. However, the expression of estrogen receptor α (ERα) was reduced by 2- to 5-fold at the gene transcript level and 2- to 20-fold at the protein level upon acquisition of anthracycline resistance. This was accompanied by an even stronger reduction in ERα phosphorylation and activity, including highly suppressed expression of two proteins under E2-dependent control (Bcl-2 and cyclin D1). The diminished Bcl-2 and cyclin D1 expression would be expected to reduce the growth rate of the cells, a hypothesis which was confirmed in subsequent cell proliferation experiments. AKR1C3 or AKR1B10 overexpression alone had no effect on doxorubicin sensitivity in MCF-7CC cells, while siRNA-mediated knockdown of AKR1C3 and/or AKR1B10 expression had no significant effect on sensitivity to doxorubicin in MCF-7DOX2 or MCF-7EPI cells. This suggested that enhanced or reduced AKR expression/activity is insufficient to confer anthracycline resistance or sensitivity to breast tumor cells, respectively. Rather, it would appear that AKR overexpression acts in concert with other proteins to confer anthracycline resistance, including reduced E2-dependent expression of both an important apoptosis inhibitor (Bcl-2) and a key protein associated with activation of cell cycle-dependent kinases (cyclin D1).

Aldo-keto reductases are biomarkers of NRF2 activity and are co-ordinately overexpressed in non-small cell lung cancer.

Background:Although the nuclear factor-erythroid 2-related factor 2 (NRF2) pathway is one of the most frequently dysregulated in cancer, it is not clear whether mutational status is a good predictor of NRF2 activity. Here we utilise four members of the aldo-keto reductase (AKR) superfamily as biomarkers to address this question.Methods:Twenty-three cell lines of diverse origin and NRF2-pathway mutational status were used to determine the relationship between AKR expression and NRF2 activity. AKR expression was evaluated in lung cancer biopsies and Cancer Genome Atlas (TCGA) and Oncomine data sets.Results:AKRs were expressed at a high basal level in cell lines carrying mutations in the NRF2 pathway. In non-mutant cell lines, co-ordinate induction of AKRs was consistently observed following activation of NRF2. Immunohistochemical analysis of lung tumour biopsies and interrogation of TCGA data revealed that AKRs are enriched in both squamous cell carcinomas (SCCs) and adenocarcinomas that contain somatic alterations in the NRF2 pathway but, in the case of SCC, AKRs were also enriched in most other tumours.Conclusions:An AKR biomarker panel can be used to determine NRF2 status in tumours. Hyperactivation of the NRF2 pathway is far more prevalent in lung SCC than previously predicted by genomic analyses.

Quantitative Evaluation of Aldo–keto Reductase Expression in Hepatocellular Carcinoma (HCC) Cell Lines

The involvement of aldo–keto reductases (AKRs) in tumorigenesis is widely reported, but their roles in the pathological process are not generally recognized due to inconsistent measurements of their expression. To overcome this problem, we simultaneously employed real-time PCR to examine gene expression and multiple reaction monitoring (MRM) of mass spectrometry (MS) to examine the protein expression of AKRs in five different hepatic cell lines. These include one relatively normal hepatic cell line, L-02, and four hepatocellular carcinoma (HCC) cell lines, HepG2, HuH7, BEL7402 and SMMC7721. The results of real-time PCR showed that expression of genes encoding the AKR1C family members rather than AKR1A and AKR1B was associated with tumor, and most of genes encoding AKRs were highly expressed in HuH7. Similar observations were obtained through MRM. Different from HuH7, the protein abundance of AKR1A and AKR1B was relatively consistent among the other four hepatic cell lines, while protein expression of AKR1C varied significantly compared to L-02. Therefore, we conclude that the abundant distribution of AKR1C proteins is likely to be associated with liver tumorigenesis, and the AKR expression status in HuH7 is completely different from other liver cancer cell lines. This study, for the first time, provided both overall and quantitative information regarding the expression of AKRs at both mRNA and protein levels in hepatic cell lines. Our observations put the previous use of AKRs as a biomarker into question since it is only consistent with our data from HuH7. Furthermore, the data presented herein demonstrated that quantitative evaluation and comparisons within a protein family at both mRNA and protein levels were feasible using current techniques.

Abstract 2066: Identification of NRF2 target genes in human cells: Aldoketo reductases as a potential biomarker for NRF2 activity

Abstract The transcription factor NF-E2-related factor 2 (NRF2) plays a critical role in cellular defense system by up-regulating multiple antioxidant genes. Therefore, the identification of biomarker genes reflecting NRF2 activity would be important for the prediction of high-risk populations to environmental stresses. In the current study, we have investigated human genes of which expression is highly dependent on NRF2 to establish biomarker genes for NRF2 activity. For this purpose, NRF2-specific interfering RNA was stably introduced in normal human renal epithelial cells (HK-2) and human bronchial epithelial cells (NL-20), and changes in inducible genes were determined following treatment with 4 types of NRF2 activators: thiol-reacting sulforaphane, free-radical generating tert-butylhydroquinone, Michael acceptor cinnamic aldehyde and pro-oxidant hydrogen peroxide. These treatments showed relatively common alterations in gene expression, and among these, the expression of aldoketo reductase (AKR) 1C1 was highly inducible by all of these treatments in an NRF2-depedent manner. The levels for AKRs were found to be constitutively high in renal carcinoma A498 cells, of which NRF2 activity is elevated compared to normal renal epithelial HK-2 cells. In addition, the expression of AKRs was greatly enhanced by NRF2 activator treatments in human monocytes (U937), implying the potential utility of AKRs as a peripheral NRF2 marker. In conclusion, our results indicate that the expression of AKRs is highly dependent on NRF2 in human cells; therefore AKRs can be an effective biomarker for predicting NRF2 activity in human cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2066. doi:1538-7445.AM2012-2066

Proteasome inhibitors MG-132 and bortezomib induce AKR1C1, AKR1C3, AKR1B1, and AKR1B10 in human colon cancer cell lines SW-480 and HT-29

Aldo-keto reductases (AKRs) play central roles in the reductive metabolism of endogenous signaling molecules and in the detoxification of xenobiotics. AKRC1–1C3, AKR1B1 and AKR1B10 have been shown to be regulated via nuclear factor-erythroid 2 related factor 2 (Nrf2), a transcription factor that is activated upon oxidative stress. Proteasome inhibitors bortezomib and MG-132 produce mild oxidative stress that activates Nrf2-mediated gene expression that in turn may have cytoprotective effects. Bortezomib is clinically approved to treat haematological malignancies and it has also proven activity in solid tumors such as colon cancer. The present study investigated the effect of bortezomib and MG-132 on the expression of AKR1C1–1C4, AKR1B1, and AKR1B10 in colon cancer cell lines HT-29 and SW-480. Human cancer cell lines derived from different organs (lung, colon, pancreas, skin, liver, ovary) were initially assayed for the expression of the AKRs, showing a very unequal distribution. Even among the colon cell lines HT-29, Caco-2, HCT116 and SW-480, the AKRs were expressed quite non-uniformly. HT-29 cells expressed all AKRs on the mRNA level including liver-specific AKR1C4, but AKR1B1 was almost undetectable. In SW-480 cells, treatment with bortezomib (50 nM, 48 h) dramatically increased mRNA levels of AKR1B10 (32-fold), AKR1B1 (5.5-fold), and, to a lesser extent, AKR1C1 and AKR1C3. Drug-efflux transporter MRP2 (ABCC2) and Cox-2 were induced as well. AKR1C2 mRNA was down-regulated in SW-480 but induced in HT-29 cells. MG-132 increased mRNA amounts of AKR1C1, 1C3, 1B1, and 1B10 in a concentration-dependent manner. AKR1B10 and AKR1B1 protein expression was inducible by bortezomib in HT-29 cells, but not detectable in SW-480 cells. In conclusion, treatment with proteasome inhibitors increased the expression of several AKRs as well as of MRP2. It remains to be investigated whether this enzyme induction may contribute to enhanced cell survival and thereby supporting the phenomenon of multidrug resistance upon cancer chemotherapy.

Abstract 3546: Role of the “1C” Aldo-Keto Reductases in Resistance to Doxorubicin in MCF-7 breast cancer cells

Abstract Metabolism of anthracyclines by members of the aldo-keto reductase (AKR) family of enzymes presents a potential mechanism of drug resistance by converting anthracyclines such as daunorubicin, doxorubicin, or epirubicin to less cytotoxic 13′-OH metabolites. The exact mechanism and relative role of AKRs in anthracycline resistance remains unclear. Changes in anthracycline sensitivity may, in part, be the result of altered expression of AKRs in anthracycline-resistant cancers. Anthracycline-resistant cell lines were created in our laboratory by selection of MCF-7 cells for survival in increasing doses of doxorubicin (MCF-7DOX-2) or epirubicin (MCF-7EPI). “Co-cultured control” MCF-7CC cell lines were also created by an identical selection procedure in the absence of drug. Drug resistance relative to MCF-7CC cells was established at a specific threshold dose, after which the magnitude of resistance increased with increasing selection dose. Microarray and confirmatory Q-PCR studies revealed that AKR1C2 and AKR1C3 transcripts were overexpressed in MCF-7DOX2 and MCF-7EPI lines relative to MCF-7CC cells at or above the threshold dose. At the highest selection dose, the AKR1C2/3 inhibitor 5β-cholanic acid (5βC) almost completely restored sensitivity to doxorubicin in MCF-7DOX2 cells, but had little effect on MCF-7EPI and MCF-7CC cells. HPLC analysis showed altered intracellular levels of doxorubicin in MCF-7DOX2 and MCF-7EPI relative to MCF-7CC. Upon addition of 5βC, intracellular levels of doxorubicin increased in MCF-7CC and MCF-7DOX2 but not in MCF-7EPI cells (p < 0.05), likely due to the strong overexpression of the Abcb1 drug exporter in the latter cell line. Surprisingly, cellular levels of doxorubicinol (the 13′-OH metabolite of doxorubicin) were undetectable by HPLC in all cell lines. Laser scanning confocal microscopy further revealed that, in contrast to its nuclear location in MCF-7CC cells, doxorubicin was found primarily outside the nucleus when added to MCF-7DOX-2 and MCF-7EPI cells. Doxorubicinol, on the other hand, remained extra-nuclear in all three cell lines (in the absence or presence of 5βC). Doxorubicin localization to the nucleus was restored in MCF-7DOX-2 cells (but not MCF-7EPI cells) upon addition of 5βC. Doxorubicin stained isolated nuclei from MCF-7CC cells much more strongly than doxorubicinol; consistent with this finding, doxorubicin showed a higher DNA binding affinity than doxorubicinol in a fluorescent intercalator displacement assay. Taken together, our findings suggest that selection of breast tumour cells for anthracycline resistance results in overexpression of specific AKR isoforms. These AKRs convert the parent compound entering the cytoplasm to a less toxic 13-OH metabolite, with a lower affinity for DNA. The metabolite may then diffuse from cells or be further metabolized, preventing its detection by HPLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3546.

Overexpression of aldo-keto reductase 1C2 as a high-risk factor in bladder cancer

Intravesical adjuvant chemotherapy and neoadjuvant chemotherapy has been respectively administered for superficial transitional cell carcinoma (TCC) of urinary bladder and advanced TCC for years. However, the therapeutic efficacy is limited. Recently, overexpression of aldo-keto reductase (AKR) in lung, esophageal, uterine cervical and ovarian cancers was shown to be closely associated with disease progression and drug resistance. In this study, we used immunohistochemistry to determine AKR expression in pathological specimens of 347 patients with urinary bladder cancer (UBC). Some of these patients were from areas with a high risk of black foot disease (BFD), a disease that is closely associated with arsenic contamination of drinking water. The presence of AKR was confirmed by immunoblotting, matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF) and reverse transcription-polymerase chain reaction (RT-PCR). AKR isotype was determined by cDNA sequencing. Our results showed overexpression of AKR1C2 in 226 (65.1%) patients. BFD areas had a higher frequency of patients expressing AKR1C2 in UBC. Among AKR1C2-positive UBC, 148 (65.5%) were invasive, 70 (31.0%) were non-invasive and 8 (3.5%) were carcinoma in situ (CIS). These data indicated that AKR1C2 expression could be significantly associated with cancer invasiveness (p<0.001) and disease progression. Because BFD has been closely related to arsenic ingestion, our results suggested that continual intake of arsenic in drinking water might provoke AKR1C2 expression that could in turn induce drug resistance in UBC, and AKR1C2 could be a tumor marker for UBC.

Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype

A duplex 21 nucleotide small interfering RNA (siRNA) against human Keap1 is described that represents a unique class of cancer chemopreventive agent. This siRNA can knockdown Keap1 mRNA and thereby relieve negative regulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-mediated gene expression. The siRNA lowered endogenous Keap1 mRNA to <30% of control levels between 24 and 72 h after transfection in human HaCaT keratinocyte cells and was capable of blocking ectopic expression of FLAG-tagged human Keap1 protein but not that of ectopic V5-tagged mouse Keap1 protein. Transfection of human HaCaT cells with Keap1 siRNA markedly enhanced endogenous levels of nuclear factor erythroid 2 p45-related factor 2 (Nrf2) protein and increased transcription of an antioxidant response element-driven reporter gene by 2.3-fold. Furthermore, 48 h after transfection of these cells with Keap1 siRNA, expression of aldo-keto reductase 1C1/2 and the glutamate cysteine ligase catalytic and modifier subunits was elevated between 5- and 14-fold. A modest increase of 3-fold in NAD(P)H:quinone oxidoreductase 1 was also observed. The Keap1 siRNA produced a 1.75-fold increase in intracellular glutathione 48 h after transfection. Thus, antagonism of Keap1 by siRNA can be used to preadapt human cells to oxidative stress without the need to expose them to redox stressors.

The biochemical basis for increased testosterone production in theca cells propagated from patients with polycystic ovary syndrome.

Ovarian theca cells propagated from patients with polycystic ovary syndrome (PCOS) convert steroid precursors into T more efficiently than normal theca cells. To identify the basis for increased T production by PCOS theca cells, we examined type I-V 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isoform expression in long-term cultures of theca and granulosa cells isolated from normal and PCOS ovaries. RT-PCR analysis demonstrated that theca cells express type V 17 beta HSD a member of the aldo-keto reductase (AKR) superfamily (17 beta HSDV, AKR1C3), whereas expression of type I, II, and IV 17 beta HSD, which are members of the short-chain dehydrogenase/reductase superfamily, was limited to granulosa cells. Type III 17 beta HSD, the testicular isoform, was not detected in either granulosa or theca cells. Northern and real-time PCR analyses demonstrated that 17 beta HSDV transcripts were not significantly increased in PCOS theca cells compared with normal theca cells. RT-PCR analysis revealed that theca cells also express another AKR, 20 alpha HSD (AKR1C1). Both basal and forskolin-stimulated 20 alpha HSD mRNA levels were increased in PCOS theca cells compared with normal theca cells. However, 17 beta HSD enzyme activity per theca cell was not significantly increased in PCOS, suggesting that neither AKR1C3 nor AKR1C1 contributes to the formation of T in this condition. In contrast, 17 alpha-hydroxylase/C17,20 lyase and 3 beta HSD enzyme activities were elevated in PCOS theca cells, driving increased production of T precursors. These findings indicate that 1) increased T production in PCOS theca cells does not result from dysregulation of "androgenic" 17 beta HSD activity or altered expression of AKRs that may express 17 beta HSD activity; and 2) increased synthesis of T precursors is the primary factor driving enhanced T secretion in PCOS.

Stable expression of rat dihydrodiol dehydrogenase (AKR1C9) in human breast MCF-7 cells results in the formation of PAH-o-quinones and enzyme mediated cell death.

Dihydrodiol dehydrogenase members of the aldo-keto reductase (AKR) superfamily have been implicated in the metabolic activation of PAH trans-dihydrodiols because they convert these proximate carcinogens to reactive and redox-active o-quinones. In this study, rat liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (AKR1C9) was stably transfected into human breast carcinoma (MCF-7) cells, which represent a null-environment for AKR expression, to detect the formation of PAH o-quinones in a cellular context and the cellular consequences of o-quinone formation. The heterologous transfected cells expressed AKR1C9 mRNA and protein. Immunotitration of the enzyme activity indicated that the expressed protein constituted 1.0% of the soluble protein. The specific activity of the expressed enzyme was also comparable to that observed in rat liver cytosol. The transfectants were found to convert (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) to benzo[a]pyrene-7,8-dione (BPQ). The identity of this metabolite was confirmed by co-chromatography and by UV-Vis diode-array spectrometry. This conversion was not evident in mock-transfected cells. The cytotoxic consequences of BPQ formation was also examined. Transfectants exposed to 1 microM B[a]P-7,8-diol revealed that cytotoxicity, as measured by lactate dehydrogenase (LDH) release, occurred over the time course of o-quinone formation leading to 77% of the cellular LDH being released by 16 h. AKR1C9 inhibitors blocked the B[a]P-7,8-diol dependent cytotoxicity indicating that it was mediated by the enzymatically formed BPQ. These data indicate that high stable constitutive expression of AKR1C9 will result in B[a]P-7,8-diol mediated cytotoxicity due to the formation of unconjugated BPQ.