Cyclin D3 maintains growth-inhibitory activity of C/EBPalpha by stabilizing C/EBPalpha-cdk2 and C/EBPalpha-Brm complexes

C/EBPα arrests proliferation of young livers by inhibition of cdk2. In old mice, C/EBPα inhibits growth by repression of E2F-dependent promoters through the C/EBPα-Brm complex. In this paper, we show that cyclin D3-cdk4/cdk6 supports the ability of C/EBPα to inhibit liver proliferation in both age groups. Although cyclin D3-cdk4/cdk6 kinases are involved in the promotion of growth, they are expressed in terminally differentiated cells, suggesting that they have additional functions in these settings. We demonstrate that C/EBPα represents a target for phosphorylation by cyclin D3-cdk4/cdk6 complexes in differentiated liver cells and in differentiated adipocytes. Cyclin D3-cdk4/cdk6 specifically phosphorylate C/EBPα at Ser193 in vitro and in the liver and support growth-inhibitory C/EBPα-cdk2 and C/EBPα-Brm complexes. We found that cyclin D3 is increased in old livers and activates cdk4/cdk6, resulting in stabilization of the C/EBPα-Brm complex. Old livers fail to reduce the activity of cyclin D3-cdk4/cdk6 after partial hepatectomy, leading to high levels of C/EBPα-Brm complexes after partial hepatectomy, which correlate with weak proliferation. We examined the role of cyclin D3 in the stabilization of C/EBPα-cdk2 and C/EBPα-Brm by using 3T3-L1 differentiated cells. In these cells, cyclin D3 is increased during differentiation and phosphorylates C/EBPα at Ser193, leading to the formation of growth-inhibitory C/EBPα-cdk2 and C/EBPα-Brm complexes. The inhibition of cyclin D3 blocks the formation of these complexes. Thus, these studies provide a new function of cyclin D3, which is to support the growth-inhibitory activity of C/EBPα.     

Species Used for Drug Testing Reveal Different Inhibition Susceptibility for 17beta-Hydroxysteroid Dehydrogenase Type 1

Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17β-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17β-HSD types 1, 2, 4, 5 and 7 but also against 17β-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17β-HSDs analyzed were observed. Especially, the rodent 17β-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17β-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution.     

Structure–function relationships in 3α-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms

3α-Hydroxysteroid dehydrogenases (3α-HSDs) inactivate steroid hormones in the liver, regulate 5α-dihydrotestosterone (5α-DHT) levels in the prostate, and form the neurosteroid, allopregnanolone in the CNS. Four human 3α-HSD isoforms exist and correspond to AKR1C1–AKR1C4 of the aldo-keto reductase (AKR) superfamily. Unlike the related rat 3α-HSD (AKR1C9) which is positional and stereospecific, the human enzymes display varying ratios of 3-, 17-, and 20-ketosteroid reductase activity as well as 3α-, 17β-, and 20α-hydroxysteroid oxidase activity. Their k_cat values are 50–100-fold lower than that observed for AKR1C9. Based on their product profiles and discrete tissue localization, the human enzymes may regulate the levels of active androgens, estrogens, and progestins in target tissues. The X-ray crystal structures of AKR1C9 and AKR1C2 (human type 3 3α-HSD, bile acid binding protein and peripheral 3α-HSD) reveal that the AKR1C2 structure can bind steroids backwards (D-ring in the A-ring position) and upside down (β-face inverted) relative to the position of a 3-ketosteroid in AKR1C9 and this may account for its functional plasticity. Stopped-flow studies on both enzymes indicate that the conformational changes associated with binding cofactor (the first ligand) are slow; they are similar in both enzymes but are not rate-determining. Instead the low k_cat seen in AKR1C2 (50-fold less than AKR1C9) may be due to substrate “wobble” at the plastic active site.     

Expression of 17beta-hydroxysteroid dehydrogenase type 2 and type 5 in breast cancer and adjacent non-malignant tissue: a correlation to clinicopathological parameters

Estrogens play an important role in the development and progression of breast cancer. 17β-Hydroxysteroid dehydrogenase (17β-HSD) type 2 and type 5 are involved in sex steroid metabolism. 17β-HSD type 2 converts estradiol to estrone while 17β-HSD type 5 converts androstenedione to testosterone. Using immunocytochemistry, we have studied the expression of 17β-HSD type 2 and type 5 in 50 specimens of breast carcinoma and adjacent non-malignant tissues. The results were correlated with the estrogen receptor α (ERα) and β (ERβ), progesterone receptor A (PRA) and B (PRB), androgen receptor and CDC47 and with the tumor stage, tumor size, nodal status and menopausal status. 17β-HSD type 2 was expressed in 20% and 17β-HSD type 5 in 56% of breast cancer specimens. In adjacent normal tissues, both enzymes were highly expressed in almost all the patients. No significant association could be found between the expression of 17β-HSD type 2 and 17β-HSD type 5 and between the expression of each enzyme and the clinicopathological parameters studied. The decrease in 17β-HSD type 2 and 17β-HSD type 5 expressions in breast cancer may play a predominant role in the development and/or progression of the cancer by modifying the intratumoral levels of estrogens and androgens.     

Role of 17β-hydroxysteroid dehydrogenase type 1 in endocrine and intracrine estradiol biosynthesis

Enzymes with 17β-hydroxysteroid dehydrogenase (17β-HSD) activity catalyse reactions between the low-active female sex steroid, estrone, and the more potent estradiol, for example. 17β-HSD activity is essential for glandular (endocrine) sex hormone biosynthesis, but it is also present in several extra-gonadal tissues. Hence, 17β-HSD enzymes also take part in local (intracrine) estradiol production in the target tissues of estrogen action. Four distinct 17β-HSD isozymes have been characterized so far, and the data strongly suggests that different 17β-HSD isozymes have distinct roles in endocrine and intracrine metabolism of sex steroids. Current data suggest that 17β-HSD type 1 is the principal isoenzyme involved in glandular estradiol production both in humans and rodents. During ovarian follicular development and luteinization, rat 17β-HSD type 1 is regulated by gonadotropins, and the effects of gonadotropins are modulated by steroid hormones and paracrine growth factors. Human 17β-HSD type 1 favors the reduction reaction, thereby converting estrone to estradiol both in vitro and in cultured cells. Hence, the enzymatic properties of the enzyme are also in line with its suggested role in estradiol biosynthesis. Interestingly, 17β-HSD type 1 is also expressed in certain target tissues of estrogen action such as normal and malignant human breast and endometrium. Hence, 17β-HSD type 1 could be one of the factors leading to a relatively high tissue/plasma ratio of estradiol in breast cancer tissues of postmenopausal women. We conclude that 17β-HSD type 1 has a central role in regulating the circulating estradiol concentration as well as its local production in estrogen target cells.     

Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines

Smad3, a component of the TGFβ signaling pathway, contributes to G₁ arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild-type (WT) Smad3 or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1) or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers.Key words: Smad3, breast cancer, cyclin E, CDK2, TGFβ     

Analysis of 17β-hydroxysteroid dehydrogenase types 5, 7, and 12 genetic sequence variants in breast cancer cases from French Canadian Families with high risk of breast and ovarian cancer

A family history and estrogen exposure are well-known risk factors for breast cancer. Members of the 17β-hydroxysteroid dehydrogenase family are responsible for important steps in the metabolism of androgens and estrogens in peripheral tissues, including the mammary gland. The crucial biological function of 17β-HSDs renders these genes good candidates for being involved in breast cancer etiology. This study screened for mutations in HSD17B7 and HSD17B12 genes, which encode enzymes involved in estradiol biosynthesis and in AKR1C3, which codes for 17β-HSD type 5 enzyme involved in androgen and progesterone metabolism, to assess whether high penetrance allelic variants in these genes could be involved in breast cancer susceptibility. Mutation screening of 50 breast cancer cases from non-BRCA1/2 high-risk French Canadian families failed to identify germline likely high-risk mutations in HSD17B7, HSD17B12 and AKR1C3 genes. However, 107 sequence variants were identified, including seven missense variants. Assessment of the impact of missense variants on enzymatic activity of the corresponding enzymes revealed no difference in catalytic properties between variants of 17β-HSD types 7 and 12 and wild-type enzymes, while variants p.Glu77Gly and p.Lys183Arg in 17β-HSD type 5 showed a slightly decreased activity. Finally, a haplotype-based approach was used to determine tagging SNPs providing valuable information for studies investigating associations of common variants in these genes with breast cancer risk.     

Conversion of human steroid 5β-reductase (AKR1D1) into 3β-hydroxysteroid dehydrogenase by single point mutation E120H: example of perfect enzyme engineering

Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ(4)-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His(120) (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ(4)-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ(4)-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.     

Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/β-HSDs and 5α/β-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17β-HSD type 2 (HSD17B2). Also, 3α-HSD and 3β-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5β-reductase (AKR1D1) are responsible for the formation of 5α- and 5β-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/β-HSD activities of AKR1C1, AKR1C2 and AKR1C3.     

Molecular characterization of mouse 17β-hydroxysteroid dehydrogenase IV

17β-hydroxysteroid dehydrogenases (17β-HSD) catalyze the conversion of estrogens and androgens at the C17 position. The 17β-HSD type I, II, III and IV share less than 25% amino acid similarity. The human and porcine 17β-HSD IV reveal a three-domain structure unknown among other dehydrogenases. The N-terminal domains resemble the short chain alcohol dehydrogenase family while the central parts are related to the C-terminal parts of enzymes involved in peroxisomal β-oxidation of fatty acids and the C-terminal domains are similar to sterol carrier protein 2. We describe the cloning of the mouse 17β-HSD IV cDNA and the expression of its mRNA. A probe derived from the human 17β-HSD IV was used to isolate a 2.5 kb mouse cDNA encoding for a protein of 735 amino acids showing 85 and 81% similarity with human and porcine 17β-HSD IV, respectively. The calculated molecular mass of the mouse enzyme amounts to 79,524 Da. The mRNA for 17β-HSD IV is a single species of about 3 kb, present in a multitude of tissues and expressed at high levels in liver and kidney, and at low levels in brain and spleen. The cloning and molecular characterization of murine, human and porcine 17β-HSD IV adds to the complexity of steroid synthesis and metabolism. The multitude of enzymes acting at C17 might be necessary for a precise control of hormone levels.     

Hydroxybenzothiazoles as new nonsteroidal inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1)

17β-estradiol (E2), the most potent estrogen in humans, known to be involved in the development and progession of estrogen-dependent diseases (EDD) like breast cancer and endometriosis. 17β-HSD1, which catalyses the reduction of the weak estrogen estrone (E1) to E2, is often overexpressed in breast cancer and endometriotic tissues. An inhibition of 17β-HSD1 could selectively reduce the local E2-level thus allowing for a novel, targeted approach in the treatment of EDD. Continuing our search for new nonsteroidal 17β-HSD1 inhibitors, a novel pharmacophore model was derived from crystallographic data and used for the virtual screening of a small library of compounds. Subsequent experimental verification of the virtual hits led to the identification of the moderately active compound 5. Rigidification and further structure modifications resulted in the discovery of a novel class of 17β-HSD1 inhibitors bearing a benzothiazole-scaffold linked to a phenyl ring via keto- or amide-bridge. Their putative binding modes were investigated by correlating their biological data with features of the pharmacophore model. The most active keto-derivative 6 shows IC₅₀-values in the nanomolar range for the transformation of E1 to E2 by 17β-HSD1, reasonable selectivity against 17β-HSD2 but pronounced affinity to the estrogen receptors (ERs). On the other hand, the best amide-derivative 21 shows only medium 17β-HSD1 inhibitory activity at the target enzyme as well as fair selectivity against 17β-HSD2 and ERs. The compounds 6 and 21 can be regarded as first benzothiazole-type 17β-HSD1 inhibitors for the development of potential therapeutics.     

Expression and regulation of aromatase and 17β-hydroxysteroid dehydrogenase type 4 in human THP 1 leukemia cells

Estradiol is active in proliferation and differentiation of sex-related tissues like ovary and breast. Glandular steroid metabolism was for a long time believed to dominate the estrogenic milieu around any cell of the organism. Recent reports verified the expression of estrogen receptors in “non-target” tissues as well as the extraglandular expression of steroid metabolizing enzymes. Extraglandular steroid metabolism proved to be important in the brain, skin and in stromal cells of hormone responsive tumors. Aromatase converts testosterone into estradiol and androstenedione into estrone, thereby activating estrogen precursors. The group of 17β-hydroxysteroid dehydrogenases catalyzes the oxidation and/or reduction of the forementioned compounds, e.g. estradiol/estrone, thereby either activating or inactivating estradiol. Aromatase is expressed and regulated in the human THP 1 myeloid leukemia cell line after vitamin D/GMCSF-propagated differentiation. Aromatase expression is stimulated by dexamethasone, phorbolesters and granulocyte/macrophage stimulating factor (GMCSF). Exons I.2 and I.4 are expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. Vitamin D-differentiated THP 1 cells produce a net excess of estradiol in culture supernatants, if testosterone is given as aromatase substrate. In contrast, the 17β-hydroxysteroid dehydrogenase type 4 (17β-HSD 4) is abundantly expressed in unstimulated THP 1 cells and is further stimulated by glucocorticoids (2-fold). The expression is unchanged after vitamin D/GMCSF-propagated differentiation. 17β-HSD 4 expression is not altered by phorbolester treatment in undifferentiated cells but is abolished after vitamin D-propagated differentiation along with downregulation of β-action. Protein kinase C activation therefore appears to dissociate the expression of aromatase and 17β-HSD 4 in this differentiation stage along the monocyte/phagocyte pathway of THP 1 myeloid cells. The expression of steroid metabolizing enzymes in myeloid cells is able to create a microenvironment which is uncoupled from dominating systemic estrogens. These findings may be relevant in the autocrine, paracrine or iuxtacrine cellular crosstalk of myeloid cells in their respective states of terminal differentiation, e.g. in bone metabolism and inflammation.     

Seasonal changes in the expression of PACAP,VPAC1, VPAC2, PAC1 and testicular activity in the testis of the muskrat (Ondatra zibethicus)

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the steroidogenesis and spermatogenesis in the testis through its receptors PAC1, VPAC1 and VPAC2. In this study, we investigated the seasonal expressions of PACAP, PAC1, VPAC1, VPAC2, luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD) and CYP17A1 in the testis of male muskrat during the breeding season and non-breeding season, respectively. Histologically, we observed the presence of Leydig cells, Sertoli cells and various types of germ cells in the testis during the breeding season, yet only Leydig cells, Sertoli cells, spermatogonia and primary spermatocyte during the non-breeding season. In addition, the immunohistochemical localizations of PACAP and VPAC1 were identified in the Leydig cells, spermatogonia and spermatozoa during the breeding season, while only in the Leydig cells and spermatogonia during the non-breeding season, and PAC1 and VPAC2 were localized in the Leydig cells in both seasons, among which LHR, StAR, 3β-HSD and CYP17A1 were also expressed. Meanwhile, the protein and mRNA expression levels of PACAP, PAC1, VPAC1, VPAC2, LHR, FSHR, StAR, 3β-HSD and CYP17A1 in the testis during the breeding season were significantly higher than those during the non-breeding season. These results suggested that PACAP is involved in the regulation of steroidogenesis and spermatogenesis via an endocrine, autocrine or paracrine manner in the testis of muskrat.Key words: Pituitary adenylate cyclase-activating peptide (PACAP), PACAP receptors, steroidogenesis, testis, Ondatra zibethicus.     

Progesterone inhibits estrogen-induced cyclin D1 and cdk4 nuclear translocation, cyclin E- and cyclin A-cdk2 kinase activation, and cell proliferation in uterine epithelial cells in mice

The response of the uterine epithelium to female sex steroid hormones provides an excellent model to study cell proliferation in vivo since both stimulation and inhibition of cell proliferation can be studied. Thus, when administered to ovariectomized adult mice 17beta-estradiol (E2) stimulates a synchronized wave of DNA synthesis and cell division in the epithelial cells, while pretreatment with progesterone (P4) completely inhibits this E2-induced cell proliferation. Using a simple method to isolate the uterine epithelium with high purity, we have shown that E2 treatment induces a relocalization of cyclin D1 and, to a lesser extent, cdk4 from the cytoplasm into the nucleus and results in the orderly activation of cyclin E- and cyclin A-cdk2 kinases and hyperphosphorylation of pRb and p107. P4 pretreatment did not alter overall levels of cyclin D1, cdk4, or cdk6 nor their associated kinase activities but instead inhibited the E2-induced nuclear localization of cyclin D1 to below the control level and, to a lesser extent, nuclear cdk4 levels, with a consequent inhibition of pRb and p107 phosphorylation. In addition, it abrogated E2-induced cyclin E-cdk2 activation by dephosphorylation of cdk2, followed by inhibition of cyclin A expression and consequently of cyclin A-cdk2 kinase activity and further inhibition of phosphorylation of pRb and p107. P4 is used therapeutically to oppose the effect of E2 during hormone replacement therapy and in the treatment of uterine adenocarcinoma. This study showing a novel mechanism of cell cycle inhibition by P4 may provide the basis for the development of new antiestrogens.     

Inhibition of Human Steroid 5��-Reductase (AKR1D1) by Finasteride and Structure of the Enzyme-Inhibitor Complex

The Δ⁴-3-ketosteroid functionality is present in nearly all steroid hormones apart from estrogens. The first step in functionalization of the A-ring is mediated in humans by steroid 5α- or 5β-reductase. Finasteride is a mechanism-based inactivator of 5α-reductase type 2 with subnanomolar affinity and is widely used as a therapeutic for the treatment of benign prostatic hyperplasia. It is also used for androgen deprivation in hormone-de pend ent prostate carcinoma, and it has been examined as a chemopreventive agent in prostate cancer. The effect of finasteride on steroid 5β-reductase (AKR1D1) has not been previously reported. We show that finasteride competitively inhibits AKR1D1 with low micromolar affinity but does not act as a mechanism-based inactivator. The structure of the AKR1D1·NADP⁺·finasteride complex determined at 1.7 Å resolution shows that it is not possible for NADPH to reduce the Δ^1-2-ene of finasteride because the cofactor and steroid are not proximal to each other. The C3-ketone of finasteride accepts hydrogen bonds from the catalytic residues Tyr-58 and Glu-120 in the active site of AKR1D1, providing an explanation for the competitive inhibition observed. This is the first reported structure of finasteride bound to an enzyme involved in steroid hormone metabolism.The Δ⁴-3-ketosteroid functionality is present in many important steroid hormones, e.g. testosterone, cortisone, and progesterone. An initial step in steroid hormone metabolism is the reduction of the Δ⁴-ene, which in humans is mediated by steroid 5α-reductases (SRD5A1, SRD5A2) or steroid 5β-reductase (AKR1D1)³ to yield the corresponding 5α- or 5β-dihydrosteroids, respectively (1, 2). The products of these reactions are not always inactive. 5α-Reductase is responsible for the conversion of testosterone to 5α-dihydrotestosterone (5α-DHT), which is the most potent natural ligand for the androgen receptor. By contrast, in addition to being involved in bile acid biosynthesis, 5β-reductase is responsible for generating 5β-pregnanes, which are natural ligands for the pregnane-X receptor (PXR) in the liver (3, 4). PXR is involved in the induction of CYP3A4, which is responsible for the metabolism of a large proportion of drugs (5, 6). Thus both 5α-reductase and 5β-reductase are involved in the formation of potent ligands for nuclear receptors.Finasteride is a selective 5α-reductase type 2 inhibitor that reduces plasma 5α-dihydrotestosterone levels and shrinks the size of the prostate (7). It is a widely used therapeutic agent in the treatment of benign prostatic hyperplasia (8, 9), it is used in androgen deprivation therapy to treat prostate cancer (10), and it has been examined as a chemopreventive agent for hormone-dependent prostate cancer (11). Finasteride was originally thought to act as a competitive inhibitor with nanomolar affinity for 5α-reductase type 2 (12). More recently, it was found that finasteride acts as a mechanism-based inactivator of this enzyme (13). Subsequent to inhibitor binding, there is hydride transfer from the NADPH cofactor to the Δ^1-2-ene double bond of finasteride. The intermediate enolate tautomerizes at the enzyme active site to form a bisubstrate analogue in which dihydrofinasteride is covalently bound to NADP⁺ (13). The bisubstrate analogue has subnanomolar affinity for 5α-reductase type 2 (Fig. 1). No structural information exists for 5α-reductase type 1 or type 2; therefore, it is not possible to determine how finasteride would bind to the active site of a human steroid double bond reductase in the absence of an experimentally determined crystal structure.Open in a separate windowFIGURE 1.Mechanism-based inactivation of 5α-reductase type 2 by finasteride. Adapted from Bull et al. (13). R = −C(=O)-NH₂; PADPR = 2′-phosphoadenosine-5″-diphosphoribose.Human steroid 5β-reductase is a member of the aldo-keto reductase (AKR) superfamily and is formally designated (AKR1D1) (14). The AKRs are soluble NADP(H)-dependent oxidoreductases with monomeric molecular masses of 37 kDa. These enzymes are amenable to x-ray crystallography, and during the last year, we and others have reported crystal structures of ternary complexes of AKR1D1 (15–17). The ternary complexes containing steroid substrates include: AKR1D1·NADP⁺·testosterone (PDB: 3BUR), AKR1D1·NADP⁺·progesterone (PDB: 3COT), AKR1D1·NADP⁺·cortisone (PDB: 3CMF), and AKR1D1·NADP⁺·Δ⁴-androstene-3,17-dione (PDB: 3CAS) (17). In addition, ternary complexes containing the products 5β-dihydroprogesterone (PDB: 3CAV) and 5β-dihydrotestosterone (PDB: 3DOP) have also been described (16, 18).As part of an ongoing inhibitor screen of AKR1D1, we now report that finasteride acts as a competitive inhibitor with low micromolar affinity. Additionally, we report the x-ray crystal structure of the AKR1D1·NADP⁺·finasteride complex.     

Aldo-keto reductase 1C3 expression in MCF-7 cells reveals roles in steroid hormone and prostaglandin metabolism that may explain its over-expression in breast cancer

Aldo-keto reductase (AKR) 1C3 (type 5 17β-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD₂ to 9α,11β-PGF₂, Δ⁴-androstenedione to testosterone, progesterone to 20α-hydroxyprogesterone, and to a lesser extent, estrone to 17β-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1 μM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17β-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor α induced proliferation. MCF-7-AKR1C3 cells also reduced PGD₂, limiting its dehydration to form PGJ₂ products. The AKR1C3 product was confirmed as 9α,11β-PGF₂ and quantified with a stereospecific stable isotope dilution liquid chromatography–mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD₂ on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor γ (PPARγ) signaling by reducing formation of 15-deoxy-Δ^12,14-PGJ₂ (15dPGJ₂).     

Therapeutic Potential of Date Palm Pollen for Testicular Dysfunction Induced by Thyroid Disorders in Male Rats

Hyper- or hypothyroidism can impair testicular function leading to infertility. The present study was designed to examine the protective effect of date palm pollen (DPP) extract on thyroid disorder-induced testicular dysfunction. Rats were divided into six groups. Group I was normal control. Group II received oral DPP extract (150 mg kg^-1), group III (hyperthyroid group) received intraperitoneal injection of L-thyroxine (L-T4, 300μg kg^-1; i.p.), group IV received L-T4 plus DPP extract, group V (hypothyroid group) received propylthiouracil (PTU, 10 mg kg^-1; i.p.) and group VI received PTU plus DPP extract. All treatments were given every day for 56 days. L-T4 or PTU lowered genital sex organs weight, sperm count and motility, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone (T), testicular function markers and activities of testicular 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD). Moreover, L-T4 or PTU increased estradiol (E2) serum level, testicular oxidative stress, DNA damage and apoptotic markers. Morphometric and histopathologic studies backed these observations. Treatment with DPP extract prevented LT4- or PTU induced changes. In addition, supplementation of DPP extract to normal rats augmented sperm count and motility, serum levels of LH, T and E2 paralleled with increased activities of 3β-HSD and 17β-HSD as well as testicular antioxidant status. These results provide evidence that DPP extract may have potential protective effects on testicular dysfunction induced by altered thyroid hormones.     

Expression of cyclins and cdks throughout murine carcinogenesis.

The overexpression and/or amplification of cell cycle regulating genes is an important factor in the progression of cancer. Recent attention has been focused on several cyclin and cdks genes whose expression were increased in many types of tumor. In this study, we investigated the expression kinetics of cyclins A, B, D1, E and cdks 1, 2, 4, 6 by RT-PCR coupled with densitometry and correlated to the growth fraction (percentage of S cells). This analysis was performed using an experimental murine leukemic model, generated by in vivo administration of murine clonogenic cells Wehi-3b injected into balb-c mice. Differential expression of cyclins and cdks was observed between normal and tumoral cells with different patterns of expression between G1 and G2M cyclins-cdks. G1 cyclins cdks expression was significantly increased in tumor cells when compared to normal cells. In the same manner, G2M cyclins cdks expression was only observed in tumor cells at a lower level than for G1 cyclins cdks, but not detected in normal cells. These differences correlated with the growth fraction for both the G1 cyclins cdks (r = 0.91, 0.94, 0.85, 0.90 and 0.96 for cyclin D1, cyclin E, cdk2, cdk4 and cdk6, respectively) and the G2M cyclins cdks (r = 0.96, 0.97 and 0.93 for cyclins A, B and cdkl respectively). Analysis of cyclins cdks expression kinetics during tumoral progression shows that cyclins A, B and cdkl were expressed from the 12th day on of disease, increased until the death of the animals and correlated with the growth fraction (r = 0.94, 0.95 and 0.97 for cyclins A, B and cdk1 respectively) (n = 20). Overexpression of other cyclins cdks were observed, from the 6th day on for cyclin D1, the 12th day for cdk2 and cdk4, the 15th day for cdk6 and the 20th day for cyclin E. These increases persisted during tumoral progression and correlated with the growth fraction (r = 0.85, 0.94, 0.93, 0.96, and 0.98 for cyclin D1, cyclin E, cdk2, cdk4 and cdk6, respectively) (n = 20). Our results demonstrated that G1 and G2-M cyclins cdks mRNA levels were increased at approximately the same time of maximal tumor growth. Only cyclin D1 overexpression occured at the initiation of tumoral development, and could therefore be considered as an early marker of cell proliferation.     

Inhibitors of type 5 17β-hydroxysteroid dehydrogenase (AKR1C3): overview and structural insights

There is considerable interest in the development of an inhibitor of aldo-keto reductase (AKR) 1C3 (type 5 17β-hydroxysteroid dehydrogenase and prostaglandin F synthase) as a potential therapeutic for both hormone-dependent and hormone-independent cancers. AKR1C3 catalyzes the reduction of 4-androstene-3,17-dione to testosterone and estrone to 17β-estradiol in target tissues, which will promote the proliferation of hormone dependent prostate and breast cancers, respectively. AKR1C3 also catalyzes the reduction of prostaglandin (PG) H(2) to PGF(2α) and PGD(2) to 9α,11β-PGF(2), which will limit the formation of anti-proliferative prostaglandins, including 15-deoxy-Δ(12,14)-PGJ(2), and contribute to proliferative signaling. AKR1C3 is overexpressed in a wide variety of cancers, including breast and prostate cancer. An inhibitor of AKR1C3 should not inhibit the closely related isoforms AKR1C1 and AKR1C2, as they are involved in other key steroid hormone biotransformations in target tissues. Several structural leads have been explored as inhibitors of AKR1C3, including non-steroidal anti-inflammatory drugs, steroid hormone analogues, flavonoids, cyclopentanes, and benzodiazepines. Inspection of the available crystal structures of AKR1C3 with multiple ligands bound, along with the crystal structures of the other AKR1C isoforms, provides a structural basis for the rational design of isoform specific inhibitors of AKR1C3. We find that there are subpockets involved in ligand binding that are considerably different in AKR1C3 relative to the closely related AKR1C1 or AKR1C2 isoforms. These pockets can be used to further improve the binding affinity and selectivity of the currently available AKR1C3 inhibitors. Article from the special issue on Targeted Inhibitors.