Ontogenesis of Oxidative Reaction of 17β-hydroxysteroid Dehydrogenase and 11β-hydroxysteroid Dehydrogenase in Rat Leydig Cells, a Histochemical Study

The enzyme 17β-hydroxysteroid dehydrogenase is required for the synthesis and 11β-hydroxysteroid dehydrogenase for the regulation of androgens in rat Leydig cells. This histochemical study describes ontogenetic changes in distribution and intensity of these enzymes in Leydig cells from postnatal day (pnd) 1–90. Using NAD or NADP as the cofactor, 17β-hydroxysteroid dehydrogenase (substrate: 5-androstene-3β, 17β-diol) peaks were observed on pnd 16 for fetal Leydig cells and on pnd 19 and 37 for adult Leydig cells. Between pnd 13 and 25 the fetal cells showed a higher intensity for the 17β-enzyme than the adult cells; more fetal Leydig cells were stained with NADP, whereas more adult cells were positive with NAD on pnd 13 and 16. A nearly identical distribution of 11β-hydroxysteroid dehydrogenase (substrate: corticosterone) was observed with NAD or NADP as the cofactor; the reaction was present from pnd 31 onwards, first in a few adult Leydig cells and later in almost all these cells homogeneously. The ontogenetic curves of the two enzymes show an inverse relationship. To conclude: (1) Generally, a stronger reaction for 17β-hydroxysteroid dehydrogenase is shown with NAD as cofactor than with NADP; using NADP, fetal Leydig cells show a stronger staining than adult Leydig cells. (2) The data possibly support the notion of a new isoform of 11β -hydroxysteroid dehydrogenase in addition to types 1 and 2.     

Green Tea and One of Its Constituents,Epigallocatechine-3-gallate,Are Potent Inhibitors of Human 11β-hydroxysteroid Dehydrogenase Type 1

The microsomal enzyme 11β-hydroxysteroid deydrogenase type 1 (11β-HSD1) catalyzes the interconversion of glucocorticoid receptor-inert cortisone to receptor- active cortisol, thereby acting as an intracellular switch for regulating the access of glucocorticoid hormones to the glucocorticoid receptor. There is strong evidence for an important aetiological role of 11β-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and obesity. Hence, modulation of 11β-HSD1 activity with selective inhibitors is being pursued as a new therapeutic approach for the treatment of the metabolic syndrome. Since tea has been associated with health benefits for thousands of years, we sought to elucidate the active principle in tea with regard to diabetes type 2 prevention. Several teas and tea specific polyphenolic compounds were tested for their possible inhibition of cortisone reduction with human liver microsomes and purified human 11β-HSD1. Indeed we found that tea extracts inhibited 11β-HSD1 mediated cortisone reduction, where green tea exhibited the highest inhibitory potency with an IC50 value of 3.749 mg dried tea leaves per ml. Consequently, major polyphenolic compounds from green tea, in particular catechins were tested with the same systems. (−)-Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11β-HSD1 activity (reduction: IC50 = 57.99 µM; oxidation: IC50 = 131.2 µM). Detailed kinetic studies indicate a direct competition mode of EGCG, with substrate and/or cofactor binding. Inhibition constants of EGCG on cortisone reduction were Ki = 22.68 µM for microsomes and Ki = 18.74 µM for purified 11β-HSD1. In silicio docking studies support the view that EGCG binds directly to the active site of 11β-HSD1 by forming a hydrogen bond with Lys187 of the catalytic triade. Our study is the first to provide evidence that the health benefits of green tea and its polyphenolic compounds may be attributed to an inhibition of the cortisol producing enzyme 11β-HSD1.     

Coumarins as novel 17β-hydroxysteroid dehydrogenase type 3 inhibitors for potential treatment of prostate cancer

The synthesis and SAR studies of 3- and 4-substituted 7-hydroxycoumarins as novel 17β-HSD3 inhibitors are discussed. The most potent compounds from this series exhibited low nanomolar inhibitory activity with acceptable selectivity versus other 17β-HSD isoenzymes and nuclear receptors.     

Inhibitors of type 1 17β-hydroxysteroid dehydrogenase with reduced estrogenic activity: Modifications of the positions 3 and 6 of estradiol

Breast cancer is the second most frequent cancer affecting women. Among all endocrine therapies for the treatment of breast cancer, inhibition of estrogen biosynthesis is becoming an interesting complementary approach to the use of antiestrogens. The enzyme type 1 17β-hydroxysteroid dehydrogenase (17β-HSD) plays a critical role in the biosynthesis of estradiol catalyzing preferentially the reduction of estrone into estradiol, the most active estrogen. Consequently, this enzyme is an interesting biological target for designing drugs for the treatment of estrogen-sensitive diseases such as breast cancer. Our group has reported the synthesis and the biological evaluation of N-methyl, N-butyl 6β-(thiaheptamamide)estradiol as a potent reversible inhibitor of type 1 17β-HSD. Unfortunately, this inhibitor has shown an estrogen effect, thus reducing its possible therapeutic interest. Herein three strategies to modify the biological profile (estrogenicity and inhibitory potency) of the initial lead compound were reported. In a first approach, the thioether bond was replaced with a more stable ether bond. Secondly, the hydroxyl group at position 3, which is responsible for a tight binding with the estrogen receptor, was removed. Finally, the amide group of the side-chain was changed to a methyl group. Moreover, the relationship between the inhibitory potency and the configuration of the side-chain at position 6 was investigated. The present study confirmed that the 6β-configuration of the side chain led to a much better inhibition than the 6α-configuration. The replacement of the 3-OH by a hydrogen atom as well as that of the amide group by a methyl was clearly unfavorable for the inhibition of type 1 17β-HSD. Changing the thioether for an ether bond decreased by 10-fold the estrogenic profile of the lead compound while the inhibitory potency on type 1 17β-HSD was only decreased by 5-fold. This study contributes to the knowledge required for the development of compounds with the desired profile, that is, a potent inhibitor of type 1 l7β-HSD without estrogen-like effects.     

Effects of phthalates on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities in human and rat testes

The 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. Human and rat testis microsomes were used to investigate the inhibitory potencies on 3β-HSD and 17β-HSD3 activities of 14 different phthalates with various carbon numbers in the ethanol moiety. The results demonstrated that the half-maximal inhibitory concentrations (IC(50)s) of dipropyl (DPrP), dibutyl (DBP), dipentyl (DPP), bis(2-butoxyethyl) (BBOP) and dicyclohexyl (DCHP) phthalate were 123.0, 24.1, 25.5, 50.3 and 25.5μM for human 3β-HSD activity, and 62.7, 30.3, 33.8, 82.6 and 24.7μM for rat 3β-HSD activity, respectively. However, only BBOP and DCHP potently inhibited human (IC(50)s, 23.3 and 8.2μM) and rat (IC(50)s, 30.24 and 9.1μM) 17β-HSD3 activity. Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effects on both enzyme activities even at concentrations up to 1mM. The mode of action of DCHP on 3β-HSD activity was competitive with the substrate pregnenolone but noncompetitive with the cofactor NAD+. The mode of action of DCHP on 17β-HSD3 activity was competitive with the substrate androstenedione but noncompetitive with the cofactor NADPH. In summary, our results showed that there are clear structure-activity responses for phthalates in the inhibition of both 3β-HSD and 17β-HSD3 activities. The length of carbon chains in the ethanol moieties of phthalates may determine the potency to inhibit these two enzymes.     

Androsterone Derivatives Substituted at Position 16: Chemical Synthesis,Inhibition of Type 3 17β-hydroxysteroid Dehydrogenase,Binding Affinity for Steroid Receptors and Proliferative/antiproliferative Activity on Shionogi (AR +) Cells

A series of androsterone (ADT) derivatives substituted at position 16 were efficiently synthesized in short reaction sequences; the ether analogues were also synthesized in the case of the methyl and allyl derivatives. The aim of this study was to develop inhibitors of the steroidogenic enzyme type 3 17 β -hydroxysteroid dehydrogenase and then evaluate their ability to inhibit this activity in transfected HEK-293 cells. For each compound we measured the percentage of inhibition of the transformation of 4-androstene-3,17-dione, the natural substrate of this steroidogenic enzyme, into the active androgen testosterone. The synthesized compounds proved to be weak inhibitors of this enzyme, but interestingly, these ADT derivatives do not bind to androgen, estrogen, glucocorticoid, and progestin receptors, suggesting no unsuitable receptor-mediated effects. One exception, 16 α -(3′-bromopropyl)-5 α -androstane-3 α,17 β -diol (8), the only compound bearing a hydroxy group at position 17 β instead of a ketone, showed a strong binding affinity for the androgen receptor (70% at 1 μM) and also exhibited an antiproliferative activity on Shionogi (AR +) cells (86% at 1 μM), which was comparable to that of hydroxyflutamide, a pure antiandrogen (100% at 1 μM).     

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.     

Identification of an 80kD Protein Associated with the α3β1 Integrin as a Proteolytic Fragment of the α3 Subunit: Studies with Human Keratinocytes

We have characterised a protein of approximately 80kD previously observed to co-immunoprecipitate with the α₃β₁ integrin in lysates of surface labelled human epiderrnalkerati-nocytes. The 80kD protein only appeared when keratinocytes were harvested with trypsin/EDTA prior to lysis and a protein of similar molecular mass could be immunoprecipitated from human dermal fibroblasts following treatment of the cells with trypsin/EDTA. N terminal sequencing established that the 80kD protein had homology with the as integrin subunit. Peptide-mass fingerprinting was used to confirm that the protein comprised the amino terminus of α₃ and established that the site of cleavage was after amino acid 629. The 80kD fragment could be coimmunoprecipitated with α₃β₁ using an antibody to the cytoplasmic domain of the α₃ subunit, showing that the fragment remained complexed with intact α₃β₁. When antibodies to the cytoplasmic and extracellular domains of α₃ were used to label human epidermis by immunofluorescence, the staining patterns were indistinguishable and there is therefore no evidence that proteolysis of α₃ plays a role in keratinocyte detachment from the basement membrane during terminal differentiation. Whether the 80kD fragment has any effects, positive or negative, on α₃β₁-mediated adhesion remains to be determined.     

Further studies on the bile salt induction of 7α- and 7β-hydroxysteroid dehydrogenases in Clostridium absonum

Optimal induction of 7α- and 7β-hydroxysteroid dehydrogenase in 100-ml cultures grown to stationary phase was achieved by the addition of metabolizable bile salt inducers: chenodeoxycholate, 7-ketolithocholate or cholate at 2.5–3 h after inoculation. Bile salt addition prior to or after this period markedly reduced the enzyme levels induced. However, when the non-metabolizable inducers deoxycholate and 12-ketolithocholate were similarly added, no significant differences in enzyme levels were observed between addition at 2.5–3 h or at earlier times. The ability of both metabolizable and non-metabolizable bile salts to induce the enzymes fell markedly when additions were made later than approximately 3.5 h. Kinetic studies using 1-l cultures suggest that in a larger culture a somewhat earlier inducer addition period is optimal. When ranked according to the level of enzymes induced the order in decreasing induction power was: chenodeoxycholate, 7-ketolithocholate, deoxycholate, 12-ketolithocholate and cholate. Mixtures of cholate and suboptimal concentrations of deoxycholate induced the culture better than the sum of the two concentrations individually. The end product, ursodeoxycholate, was very effective in blocking the induction by chenodeoxycholate or deoxycholate. Ursocholate (3α,7β,12α-trihydroxy-5β-cholanoate) was less effective. Cultures when grown for 3 h with various bile salts or none, then centrifuged and recultured for a further 3 h in fresh medium containing chenodeoxycholate, all yielded identical enzyme levels within experimental error. We conclude that exposure of the organism to bile salt inducer in the last 3 h of culture was important, while the history of the culture prior to this time was unimportant in the induction process.     

Studies on the Chemical Synthesis of Potential Antimetabolites. 32.1 Synthesis of β-D-Pentofuranosyldeazaadenines as Candidate Inhibitors for S-Adenosylhomocysteinases and Methyltransferases

Abstract

9-β-D-Arabinofuranosyldeazaadenines [1-deaza-araA (4a) and 3-deaza-araA (4b)] were prepared from 6-chloro-β-D-ribofuranosyl-1- (6a) and -3-deazapurine (6b), respectively. Synthesis of 2′-deoxy-1-deaza-adenosine (5a) from 1-deazaadenosine (6c) is also described.     

Studies on the Phosphonate Isostere of Nucleoside 3′- and 2′-Phosphates as Precursors of the Related Oligonucleotides

Abstract

Synthesis of 2′,3′-dideoxy-3′-C-(dihydroxyphosphinylmethyl)-adenosine and -thymidine 5, as well as of 2′-deoxy-2′-C-(dihydroxyphosphinylmethyl)-adenosine and -thymidine 9 was accomplished with the use of the universal carbohydrate precursor 3-deoxy-1,2;5,6-di-O-isopropylidene-3-C-(mesyloxymethyl)-α-D-allofuranose (1).     

Upregulation of PPARβ/δ Is Associated with Structural and Functional Changes in the Type I Diabetes Rat Diaphragm

Background

Diabetes mellitus is associated with alterations in peripheral striated muscles and cardiomyopathy. We examined diaphragmatic function and fiber composition and identified the role of peroxisome proliferator-activated receptors (PPAR α and β/δ) as a factor involved in diaphragm muscle plasticity in response to type I diabetes.

Methodology/Principal Findings

Streptozotocin-treated rats were studied after 8 weeks and compared with their controls. Diaphragmatic strips were stimulated in vitro and mechanical and energetic variables were measured, cross bridge kinetics assessed, and the effects of fatigue and hypoxia evaluated. Morphometry, myosin heavy chain isoforms, PPAR α and β/δ gene and protein expression were also assessed. Diabetes induced a decrease in maximum velocity of shortening (−14%, P<0.05) associated with a decrease in myosin ATPase activity (−49%, P<0.05), and an increase in force (+20%, P<0.05) associated with an increase in the number of cross bridges (+14%, P<0.05). These modifications were in agreement with a shift towards slow myosin heavy chain fibers and were associated with an upregulation of PPARβ/δ (+314% increase in gene and +190% increase in protein expression, P<0.05). In addition, greater resistances to fatigue and hypoxia were observed in diabetic rats.

Conclusions/Significance

Type I diabetes induced complex mechanical and energetic changes in the rat diaphragm and was associated with an up-regulation of PPARβ/δ that could improve resistance to fatigue and hypoxia and favour the shift towards slow myosin heavy chain isoforms.     

Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

Summary The localization of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.Supported by grants from the Ministry of Education Science and Culture, Japan     

Rat small-intestinal β-galactosidases. Studies on the fractionation of `acid' β-galactosidase with isoelectric focusing, gel filtration and ion-exchange chromatography

1. Different forms of the rat small-intestinal ;acid' beta-galactosidase were separated by using the isoelectric-focusing technique. The isoelectric points of the different forms were at pH4.2, 4.6, 5.4, 6.1 and approx. 8. 2. The two forms of ;acid' beta-galactosidase isoelectric at pH4.2 and 4.6 were completely excluded from the Sephadex G-200 gel, whereas the form isoelectric at pH8 had K(av.) 0.4. The concentration and pH of the elution buffer influenced the distribution of enzyme activity between different forms. Thus, under certain conditions of ionic strength and pH, the enzyme seems to form high-molecular-weight aggregates with low isoelectric points. These may be homopolymeric aggregates or the result of binding of enzyme to, for example, membrane fragments. The forms isoelectric at pH5.4 and 6.1 are probably aggregates of intermediate size. 3. During ion-exchange chromatography at pH6.0 one fraction of ;acid' beta-galactosidase was not retained on the column and was isoelectric at pH8 and another fraction was eluted when the buffer concentration in the eluate had increased to about 50mm. The main part of enzyme eluted in this second fraction was also isoelectric at pH8, indicating that the elution of this fraction is not a simple ion-exchange procedure but probably also involves a splitting of high-molecular-weight aggregates, originally retained because of their low isoelectric points. The enzyme subunits have a higher isoelectric point, and are therefore no longer bound to the ion-exchange resin.     

Human familial and sporadic breast cancer: analysis of the coding regions of the 17β-hydroxysteroid dehydrogenase 2 gene (EDH17B2) using a single-strand conformation polymorphism assay

17-Hydroxysteroid dehydrogenase (17HSD) is one of the key enzymes in estrogen metabolism, catalyzing the reversible reaction between estradiol and the less active estrogen, estrone. The gene encoding this enzyme, EDH17B2, has been mapped to chromosome 17, region q12–q21, in the vicinity of BRCA1, an as yet unidentified gene that appears to be involved in familial breast cancer and in familial ovarian cancer. The possibility that EDH17B2 gene is the same as BRCA1 was tested by screening for mutations in the coding regions of EDH17B2, using a polymerase chain reaction/single-strand conformation polymorphism method. An AG transition creating a new BstUI site at exon 6 was the only frequent sequence alteration found in the coding region of the gene. This mutation also led to an amino acid substitution of serine to glycine at position 312 (312^S312^G) in the 17HSD protein. Since the nucleotide change was detected both in specimens from patients with familial or sporadic cancer and in control samples, and at similar rates, this mutation appears to be of a polymorphic nature. In addition, a rare polymorphism located at intron 5 was detected. This CT substitution creates a BbvI site and is not thought to have any effect on 17HSD activity. The results indicate that there are no major alterations in the coding areas of EDH17B2 and thus studies testing the hypothesis that EDH17B2 may be the same as BRCA1 should be extended to the promoter and regulatory elements of EDH17B2.