Different conversion metabolic rates of testosterone are associated to hormone-sensitive status and -response of human prostate cancer cells

The main goal of the present work was to compare the ability of human prostate cancer (PCa) cells to metabolize testosterone (T) in living conditions. To this end we studied three different human PCa cell lines (LNCaP, DU145 and PC3) having different hormone-sensitive status and capability of response to androgens. We used an original approach which allows the evaluation of conversion metabolic rates in growing cells after administration of labeled steroid precursor (presently T), at physiological concentrations (1–10 nM). Analysis of both precursor degradation and formation of several products was carried out using reverse phase-high performance liquid chromatography (RP-HPLC) and “on line” radioactive detection. Comparison of the three human PCa cells revealed that their metabolic aptitude differed in many respects: (i) rates of precursor degradation, (ii) different products' formation, and (iii) extent of conjugate production. In detail, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione (A-4-ene-Ad); both DU145 and LNCaP cells mostly retained high levels of unconverted T, with a limited production of A-4-ene-Ad and its 17-keto derivatives (if any). Either LNCaP or DU145 cells generated a relatively high amount of dihydrotestosterone (DHT). In contrast, neither DHT nor its main metabolites were detected in PC3 cells at both short and longer incubation times. As expected, T degradation and A-4-ene-Ad production were highly correlated (r = 0.97; P < 0.03); similarly, A-4-ene-Ad and DHT formation showed a negative, significant correlation. Negligible production of conjugates was noted in both PC3 and DU145 cells, whilst it was remarkable in LNCaP cells (ranging from 43 to 57%). Overall, our data indicate that human PCa cells degrade T quite differently, favoring alternatively reductive or oxidative patterns of androgen metabolism.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

From methyl -glucopyranoside to methyl -allopyranoside via the Mitsunobu reaction

Methyl β--glucopyranoside reacted with a 4-molar excess of the Mitsunobu reagents (triphenylphosphine–diethyl azodicarboxylate–benzoic acid) under Weinges et al. [Carbohydr. Res., 164 (1987) 453–458] conditions to furnish four differently benzoylated methyl β--allopyranosides in a very good overall yield. The same reaction applied to methyl α--glucopyranoside yielded allosides in a low yield and nine other sugar products. These results give an insight into the course of the Mitsunobu esterification–inversion reaction.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

A novel approach to predicting protein structural classes in a (20–1)-D amino acid composition space

The development of prediction methods based on statistical theory generally consists of two parts: one is focused on the exploration of new algorithms, and the other on the improvement of a training database. The current study is devoted to improving the prediction of protein structural classes from both of the two aspects. To explore a new algorithm, a method has been developed that makes allowance for taking into account the coupling effect among different amino acid components of a protein by a covariance matrix. To improve the training database, the selection of proteins is carried out so that they have (1) as many non-homologous structures as possible, and (2) a good quality of structure. Thus, 129 representative proteins are selected. They are classified into 30 α, 30 β, 30 α + β, 30 α/β, and 9 ζ (irregular) proteins according to a new criterion that better reflects the feature of the structural classes concerned. The average accuracy of prediction by the current method for the 4 × 30 regular proteins is 99.2%, and that for 64 independent testing proteins not included in the training database is 95.3%. To further validate its efficiency, a jackknife analysis has been performed for the current method as well as the previous ones, and the results are also much in favor of the current method. To complete the mathematical basis, a theorem is presented and proved in Appendix A that is instructive for understanding the novel method at a deeper level. © 1995 Wiley-Liss, Inc.     

Effects of endocrine disrupters on sex steroid synthesis and metabolism pathways in fish

The interactions of estrogenic (nonylphenol, dicofol, atrazine), androgenic (organotins, phthalates, fenarimol) and anti-androgenic compounds (vinclozolin, diuron, p,p'-DDE) with key enzymatic activities involved in both synthesis and metabolism of sex hormones was investigated. Carp testicular microsomes incubated in the presence of androstenedione and different xenobiotics evidenced higher sensitivity of 5alpha-reductase activity than 17beta-hydroxysteroid dehydrogenase activity towards those chemicals. Dicofol, organotins and phthalates were among the most effective inhibitors. In contrast, ovarian synthesis of maturation-inducing hormones (20alpha- and 20beta-hydroxysteroid dehydrogenase activities) were enhanced by nonylphenol, dicofol, fenarimol and p,p'-DDE. Metabolic clearance pathways of hormones were also affected. Fenarimol, nonylphenol and triphenyltin inhibited the glucuronidation of testosterone and estradiol at concentrations as low as 10, 50 and 100 microM, respectively. Triphenyltin, tributyltin and nonylphenol were also inhibitors of estradiol sulfation with IC(50) values of 17, 18 and 41 microM. Overall, the data indicates the interaction of selected chemicals with key enzymatic pathways involved in both synthesis and metabolism of sex hormones. This interference might be one of the underlying mechanisms for the reported hormonal disrupting properties of the tested compounds, and might finally affect physiological processes such as gamete growth and maturation.     

Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.     

Regulation of the rat interstitial collagenase promoter by IL-1β, c-Jun,and ras-dependent signaling in growth plate chondrocytes

In an attempt to better define molecular influences on rat interstitial collagenase gene expression in cartilage, the promoter function was characterized using transient transfection assay, electrophoresis mobility shift assay, and genetic analysis in isolated growth plate chondrocytes. Data from 5′-flanking deletion and selected mutations suggest that multiple cis elements in both the proximal and distal regions of the promoter were important in the regulation of promoter activity. A proximal tumor response element (TRE) was shown to be necessary for basal and interleukin (IL)-1β–inducible reporter gene activity. Cells stimulated by IL-1β (1 ng/ml; 18 h) had elevated TRE binding activity, and one of the factors involved was identified as the nuclear protein, c-Jun. Indeed, c-Jun directed antisense oligonucleotides reduced rat interstitial collagenase mRNA. A sense oligonucleotide was ineffective. Regulation of promoter activity was susceptible to Ras-dependent signaling as expression of dominant negative mutant of Ras kinase (pZIP-RasN17) reduced reporter gene activity. In a comparison of proximal promoter reporter plasmid activity between proliferative and hypertrophic cells, inhibition of Ras-dependent signaling was less effective in the later cell type. This study suggests that the activation of nuclear binding proteins that bind TRE may be a common event with IL-1β regulation. Moreover, these data suggest that the regulation of rat interstitial collagenase gene expression is a combinatorial process and multiple cis-acting regulatory sites may interact to exert different effects dependent on the stage of chondrocyte differentiation. J. Cell. Biochem. 67:92–102, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.