Spironolactone-related inhibitors of type II 17beta-hydroxysteroid dehydrogenase: chemical synthesis, receptor binding affinities, and proliferative/antiproliferative activities.

The family of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyzes the formation and inactivation of testosterone (T), dihydrotestosterone (DHT), and estradiol (E2), thus playing a crucial role in the regulation of active steroid hormones in target tissues. Among the five known 17beta-HSD enzymes, type II catalyzes the oxidation of E2 into estrone (E1), T into androstenedione, DHT into androstanedione, and 20alpha-dihydroprogesterone into progesterone. Specific inhibitors are thus an interesting means to study the regulation and to probe the structure of type II 17beta-HSD. In this context, we have efficiently synthesized a series of 7alpha-thioalkyl and 7alpha-thioaryl derivatives of spironolactone that inhibit type II 17beta-HSD. These new C19-steroidal inhibitors possess two important pharmacophores, namely 17-spiro-gamma-lactone and a bulky side-chain at the 7alpha-position. It was found that a para-substituted benzylthio group at the 7alpha-position enhances the inhibitory potency of spironolactone derivatives on type II 17beta-HSD. In fact, the compound with a para-hydroxy-benzylthio group showed an IC50 value of 0.5 microM against type II 17beta-HSD, whereas the compound with a para-[2-(1-piperidinyl)-ethoxy]-benzylthio group inhibited this enzyme with an IC50 value of 0.7 microM. The latter inhibitor is more selective than the former because it did not show any inhibitory potency against P450 aromatase as well as any affinity towards four steroid receptors (AR, PR, GR, ER). As a result, this inhibitor did not show any proliferative effect on androgen-sensitive Shionogi cells and estrogen-sensitive ZR-75-1 cells. These findings contribute to a better knowledge of the structure of type II 17beta-HSD and offer an interesting tool to study the regulation of this enzyme in several biological systems.     

Detection of Synenkephalin, the Amino-Terminal Portion of Proenkephalin, by Antisera Directed Against Its Carboxyl Terminus

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.     

Phytochemical composition of Cymbopogon citratus and Eucalyptus citriodora essential oils and their anti-inflammatory and analgesic properties on Wistar rats

Cymbopogon citratus and Eucalyptus citriodora are widely used herbs/plants as a source of ethnomedicines in tropical regions of the world. In this work, we studied the anti-inflammatory and gastroprotective effects of C. citratus and E. citriodora essential oils on formol-induced edema, and acetic acid induced abdominal cramps in Wistar rats. To fully understand the chemically induced anti-inflammatory properties of these plants, we first analyzed the chemical composition of the essential oils. A total of 16 chemical constituents accounting for 93.69 % of the oil, were identified in C. citratus among which, Geranial (27.04 %), neral (19.93 %) and myrcene (27.04 %) were the major constituents. For E. citriodora, 19 compounds representing 97.2 % of the extracted oil were identified. The dominant compound of E. citriodora essential oil was citronellal (83.50 %). In vivo analysis and histological assay showed that the two essential oils displayed significant dose dependent edema inhibition effect over time. They displayed strong analgesic and antipyretic properties similar to that induced by 50 mg/kg of acetylsalicylate of lysine. However, the E. citriodora essential oil was more effective than that of C. citratus. We identified significant numbers of aldehyde molecules in both essential oils mediating antioxidant activity that may contribute to the anti-inflammatory effects observed on the rats. Altogether, this work demonstrates the anti-inflammatory property of C. citratus and E. citriodora suggesting their potential role as adjuvant therapeutic alternatives in dealing with inflammatory-related diseases.     

P2Y2 receptor promotes intestinal microtubule stabilization and mucosal re‐epithelization in experimental colitis

P2Y₂ receptor expression is increased in intestinal epithelial cells (IECs) during inflammatory bowel diseases (IBDs). In this context, P2Y₂ stimulates PGE₂ release by IECs, suggesting a role in wound healing. For this study, we have used the non‐cancerous IEC‐6 cell line. IEC‐6 cell migration was determined using Boyden chambers and the single‐edged razor blade model of wounding. The receptor was activated using ATP, UTP, or 2‐thioUTP. Pharmacological inhibitors, a blocking peptide, a neutralizing antibody and interfering RNAs were used to characterize the signaling events. Focal adhesions and microtubule (MT) dynamics were determined by immunofluorescence using anti‐vinculin and anti‐acetylated‐α‐tubulin antibodies, respectively. In vivo, the dextran sodium sulfate mouse model of colitis was used to characterize the effects of P2Y₂ agonist 2‐thioUTP on remission. We showed that P2Y₂ increased cell migration and wound closure by recruiting Go protein with the cooperation of integrin α_v. Following P2Y₂ activation, we demonstrated that GSK3β activity was inhibited in response to Akt activation. This leads to MT stabilization and increased number of focal adhesions. In vivo, P2Y₂ activation stimulates remission, as illustrated by a reduction in the disease activity index values and histological scores as compared to control mice. These findings highlight a novel function for this receptor in IECs. They also illustrate that P2Y receptors could be targeted for the development of innovative therapies for the treatment of IBDs. J. Cell. Physiol. 228: 99–109, 2013. © 2012 Wiley Periodicals, Inc.