Tea Lanišnik Rižner

AKR1C1 and AKR1C3 may determine progesterone and estrogen ratios in endometrial cancer

Endometrial cancer is the most common malignancy of the female genital tract. Its incidence correlates with prolonged estrogen stimulation unopposed by progesterone or synthetic progestins. Estrogen and progestin action is regulated at the pre-receptor level, by interconversion of active hormones (estradiol (E2), progesterone (P)) with their inactive counterparts (estrone (E1), 20alpha-hydroxyprogesterone (20alpha-OHP)) in target tissues. Expression of enzymes that control the ratio of E2 and P may thus play role in the disease process. We first confirmed that AKR1C1 (human 20alpha-hydroxysteroid dehydrogenase) in a cellular context inactivates P by forming 20alpha-OHP but does not catalyze the reverse reaction. We next examined the expression of AKR1C1 and AKR1C3 (type 5 17beta-hydroxysteroid dehydrogenase) in 16 paired specimens of endometrial cancer and adjacent normal endometrium. Quantification by isoform specific real-time PCR revealed higher expression of AKR1C1 in nine specimens and higher expression of AKR1C3 in four specimens of endometrial cancer. Importantly, upregulation of both enzymes in the same specimen was observed. Since AKR1C1 inactivates P its elevated expression in diseased endometrium may contribute to diminished protection by P, while elevated expression of AKR1C3 which forms E2 in vivo, may contribute to the enhanced estrogen action. It is suggested that the expression of AKR1C1 and AKR1C3 in endometrial cancer will govern the ratio of P:E2.

Expression analysis of the genes involved in estradiol and progesterone action in human ovarian endometriosis

Endometriosis is defined as the presence of endometrial glands and stroma within extrauterine sites, and it is well known that endometriosis is an estrogen-dependent disease. The defective formation and metabolism of steroid hormones is responsible for the promotion and development of endometriosis. In the present study we examined the mRNA levels of six enzymes that are involved in the metabolism of estrogen and progesterone – aromatase, 17β-hydroxysteroid dehydrogenase (17β-HSD) types 1, 2 and 7, sulfatase and sulfotransferase – and of the steroid receptors – estrogen receptors α and β (ERα, ERβ) and progesterone receptors A and B (PRAB) – implicated in human ovarian endometriosis. We analyzed 16 samples of ovarian endometriosis and 9 of normal endometrium. The real-time polymerase chain reaction analyses revealed that six of the nine genes investigated are differentially regulated. Aromatase, 17β-HSD types 1 and 7, sulfatase and ERβ were statistically significantly upregulated, while ERα was significantly downregulated, in the endometriosis group compared with the control group. There were no significant differences in 17β-HSD type 2, sulfotransferase and PRAB gene expression. Our results indicate that, in addition to the previously reported upregulation of aromatase, upregulation of 17β-HSD types 1 and 7 and sulfatase can also increase the local estradiol concentration. This could thus be responsible for the estrogen-dependent growth of endometriotic tissue. Surprisingly ERα was downregulated.

A novel 17beta-hydroxysteroid dehydrogenase in the fungus Cochliobolus lunatus: new insights into the evolution of steroid-hormone signalling.

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) catalyses the reduction of steroids and of several o- and p-quinones. After purification of the enzyme, its partial amino acid sequence was determined. A PCR fragment amplified with primers derived from peptide sequences was generated for screening the Coch. lunatus cDNA library. Three independent full-length cDNA clones were isolated and sequenced, revealing an 810-bp open reading frame encoding a 270-amino-acid protein. After expression in Escherichia coli and purification to homogeneity, the enzyme was found to be active towards androstenedione and menadione, and was able to form dimers of Mr 60000. The amino acid sequence of the novel 17beta-HSD demonstrated high homology with fungal carbonyl reductases, such as versicolorin reductase from Emericella nidulans (Aspergillus nidulans; VerA) and Asp. parasiticus (Ver1), polyhydroxynaphthalene reductase from Magnaporthe grisea, the product of the Brn1 gene from Coch. heterostrophus and a reductase from Colletotrichum lagenarium, which are all members of the short-chain dehydrogenase/reductase superfamily. 17beta-HSDcl is the first discovered fungal 17beta-hydroxysteroid dehydrogenase belonging to this family. The primary structure of this enzyme may therefore help to elucidate the evolutionary history of steroid dehydrogenases.

Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism.

Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ(4)-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency.

Steroid-transforming enzymes in fungi.

Fungal species are a very important source of many different enzymes, and the ability of fungi to transform steroids has been used for several decades in the production of compounds with a sterane skeleton. Here, we review the characterised and/or purified enzymes for steroid transformations, dividing them into two groups: (i) enzymes of the ergosterol biosynthetic pathway, including data for, e.g. ERG11 (14α-demethylase), ERG6 (C-24 methyltransferase), ERG5 (C-22 desaturase) and ERG4 (C-24 reductase); and (ii) the other steroid-transforming enzymes, including different hydroxylases (7α-, 11α-, 11β-, 14α-hydroxylase), oxidoreductases (5α-reductase, 3β-hydroxysteroid dehydrogenase/isomerase, 17β-hydroxysteroid dehydrogenase, C-1/C-2 dehydrogenase) and C-17-C-20 lyase. The substrate specificities of these enzymes, their cellular localisation, their association with protein super-families, and their potential applications are discussed. Article from a special issue on steroids and microorganisms.

Evidence for 1,8‐dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and non‐saline conditions

The ascomycetous black yeasts Hortaea werneckii, Phaeotheca triangularis, and Trimmatostroma salinum are halophilic fungi that inhabit hypersaline water of solar salterns. They are characterized by slow, meristematic growth and very thick, darkly pigmented cell walls. The dark pigment, generally thought to be melanin, is consistently present in their cell walls when they grow under saline and non-saline conditions. We used the inhibitor tricyclazole to test the fungi in this study for the presence of 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, since fungal melanins reportedly are derived either from DHN, tyrosine via 3,4-dihydroxyphenylalanine, gamma-glutaminyl-3,4-dihydroxybenzene, or catechol. Tricyclazole-treated cultures of the fungi were reddish-brown in color and contained typical intermediates of the DHN-melanin pathway, as demonstrated by high-performance liquid chromatography. This investigation showed that the three fungi synthesized DHN-melanin under saline and non-saline growth conditions.

Estrogen biosynthesis, phase I and phase II metabolism, and action in endometrial cancer

Endometrial cancer is the most common gynecological malignancy in the developed World. Based on their histopathology, clinical manifestation, and epidemiology, the majority of endometrial cancer cases can be divided into two groups: the more prevalent type 1 which is associated with unopposed estrogen exposure; and the less common type 2, which is usually not associated with hyper-estrogenic factors. This manuscript overviews the published data on the expression of genes encoding the estrogen biosynthetic enzymes, the phase I and phase II estrogen metabolic enzymes, and the estrogen receptors in endometrial cancer, at the mRNA, protein and enzyme activity levels. The potential role of altered expression of these enzymes and receptors in cancerous versus control endometrial tissue, and the implication of estrogens in tumor initiation and promotion, are discussed. Finally, based on the published data, a model of estrogen metabolism and actions is proposed for pre-cancerous and cancerous endometrial tissue, and the role of the estrogens in the progression of endometrial cancer from endometrial hyperplasia is suggested.

Disturbed expression of phase I and phase II estrogen-metabolizing enzymes in endometrial cancer: Lower levels of CYP1B1 and increased expression of S-COMT

Expression levels of genes encoding phase I and phase II estrogen-metabolizing enzymes: CYP1A1, CYP1A2, CYP1B1, CYP3A5, CYP3A7, SULT1A1, SULT1E1, SULT2B1, COMT, UGT2B7, and GSTP1 were studied by real-time PCR in 38 samples of cancerous and adjacent control endometrium. We found significantly lower levels of CYP1B1 and CYP3A7, higher levels of SULT2B1, UGT2B7 and GSTP1, and no differences in expression of COMT, CYP1A1, CYP3A5, SULT1E1 and SULT2A1 in the endometrial cancers. The CYP1B1 and COMT proteins were also examined by Western blotting and immunohistochemical staining, supporting the real-time PCR analysis. Lower levels of CYP1B1 detected in cancerous endometrium suggest its important role in control, precancerous tissue. Additionally, we showed for the first time higher protein levels of soluble COMT in cancerous endometrium, and higher levels of membrane-bound COMT in control, precancerous endometrium. The importance of the changed ratio between soluble and membrane-bound COMT still needs to be evaluated in further studies.

Phytoestrogens as inhibitors of the human progesterone metabolizing enzyme AKR1C1.

Phytoestrogens are plant-derived, non-steroidal constituents of our diets. They can act as agonists or antagonists of estrogen receptors, and they can modulate the activities of the key enzymes in estrogen biosynthesis. Much less is known about their actions on the androgen and progesterone metabolizing enzymes. We have examined the inhibitory action of phytoestrogens on the key human progesterone-metabolizing enzyme, 20alpha-hydroxysteroid dehydrogenase (AKR1C1). This enzyme inactivates progesterone and the neuroactive 3alpha,5alpha-tetrahydroprogesterone, to form their less active counterparts, 20alpha-hydroxyprogesterone and 5alpha-pregnane-3alpha,20alpha-diol, respectively. We overexpressed recombinant human AKR1C1 in Escherichia coli, purified it to homogeneity, and examined the selected phytoestrogens as inhibitors of NADPH-dependent reduction of a common AKR substrate, 9,10-phenantrenequinone, and progesterone. The most potent inhibitors were 7-hydroxyflavone, 3,7-dihydroxyflavone and flavanone naringenin with IC(50) values in the low microM range. Docking of the flavones in the active site of AKR1C1 revealed their possible binding modes, in which they are sandwiched between the Leu308 and Trp227 of AKR1C1.

Expression analysis of estrogen-metabolizing enzymes in human endometrial cancer.

Estrogen-dependent endometrial cancer is related to unopposed and prolonged estrogen stimulation. We examined the expression of estrogen-metabolizing enzymes in correlation with the ERalpha and ERbeta estrogen receptors in human endometrial Ishikawa adenocarcinoma cells and in endometrial cancer specimens and adjacent normal endometrium from the same patients. Real-time PCR analysis revealed that both estrogen receptors and selected estrogen-metabolizing enzymes were expressed in the Ishikawa cells and in endometrial tissue. We detected higher expression of ERalpha than ERbeta, higher expression of sulfatase than sulfotransferase and low expression of aromatase in the Ishikawa cells and the tissue, as well as higher levels of type 2 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in normal and diseased tissue than in the Ishikawa cells. When we compared the expression in endometrial cancer samples and in the adjacent normal endometrium, ERalpha and ERbeta, sulfatase and sulfotransferase were seen to be downregulated in the majority of the cancerous tissue specimens.