Anti-AKR1C4 polyclonal antibody

Recent studies have shown that an adrenal steroid 11 beta-hydroxy-4-androstene-3,17-dione serves as the precursor to androgens, 11-ketotestosterone and 11-ketodihydrotestosterone (11KDHT). The biosynthetic pathways include the reduction of 3- and 17-keto groups of the androgen precursors 11-keto-C-19-steroids, which has been reported to be mediated by three human enzymes; aldo-keto reductase (AKR)1C2, AKR1C3 and 17 beta-hydroxysteroid dehydrogenase (HSD) type-3. To explore the contribution of the enzymes in the reductive metabolism, we kinetically compared the substrate specificity for 11-keto-C-19-steroids among purified recombinant preparations of four AKRs (1C1, 1C2,1C3 and 1C4) and DHRS11, which shows 17 beta-HSD activity. Although AKR1C1 did not reduce the 11-keto-C-19-steroids, AKR1C3 and DHRS11 reduced 17-keto groups of 11-keto-4-androstene-3,17-dione, 11-keto-5 alpha-androstane-3,17-dione (11K-Adione) and 11-ketoandrosterone with K-m values of 5-28 mu M. The 3-keto groups of 11KDHT and 11K-Adione were reduced by AKR1C4 (K-m 1 mu M) more efficiently than by AKR1C2 (K-m 5 and 8 mu M, respectively). GC/MS analysis of the products showed that DHRS11 acts as 17 beta-HSD, and that AKR1C2 and AKR1C4 are predominantly 3 alpha-HSDs, but formed a minor 3 beta-metabolite from 11KDHT. Since DHRS11 was thus newly identified as 11-keto-C-19-steroid reductase, we also investigated its substrate-binding mode by molecular docking and site-directed mutagenesis of Thr163 and Val200, and found the following structural features: 1). There is a space that accommodates the 11-keto group of the 11-keto-C-19-steroids in the substrate-binding site. 2) Val200 is a critical determinant for exhibiting the strict 17 beta-HSD activity of the enzyme, because the Val200Leu mutation resulted in both significant impairment of the 17 beta-HSD activity and emergence of 3 beta-HSD activity towards 5 alpha-androstanes including 11KDHT.

Aldo-keto reductases (AKRs) oxidize structurally diverse PAH trans-dihydrodiols to yield reactive and redox active o-quinones. This study examined the ability of AKR1C2 and AKR1C4 to oxidize PAH trans-dihydrodiols of the benz[a]anthracene series. The enzymes oxidized 100% of the racemic trans-dihydrodiols and the highest utilization ratios were observed for the more potent proximate carcinogens 7,12-dimethylbenz[a]anthracene-3,4-diol (DMBA-3,4-diol) and 7-methylbenz[a]anthracene-3,4-diol (7-MBA-3,4-diol). Human multiple tissue expression array analysis revealed high expression of AKR1C isoforms in the human lung carcinoma cell line A549. Both Western blot analysis using AKR1C9 antisera and enzymatic assays using 1-acenapthanol as substrate confirmed the presence of active AKR1C enzymes in A549 cells. To determine the importance of AKR1C-mediated trans-dihydrodiol oxidation in A549 cells, DMBA-3,4-diol was incubated with cell lysates in the presence of 2-mercaptoethanol. Liquid chromatography/mass spectrometric analysis identified peaks that corresponded to the synthetically prepared mono- and bis-thioether conjugates of DMBA-3,4-dione confirming the ability of these cells to oxidize DMBA-3,4-diol to the corresponding o-quinone. Together, these studies demonstrate the importance of human AKR1Cs in PAH activation and their possible role in lung cancer.