Five new C21 steroidal glycosides from Cynanchum komarovii Al.Iljinski

Five new C21 steroidal glycosides, namely, komarosides D (1), E (2), F (3), G (4), and H (5), along with two known C21 steroidal glycosides cynatratoside E (6) and hancoside A (7), were isolated from the ethanol extract of the roots of Cynanchum komarovii Al.Iljinski (Asclepiadaceae). Their structures were determined by physiochemical and spectroscopic analysis. Among these glycosides, five had an aberrant 13,14:14,15-disecopregnane-type skeleton, and the other two had normal four-ring C21 steroid skeletons. The existence of more than one type of C21 steroid skeleton in one species is rare in the plants of the family Asclepiadaceae, and this has chemotaxonomic significance for this species.     

Syntheses of acetylated steroid glycosides and selective cleavage of O-acetyl groups in sugar moiety

Acetylated 3β-O-β-glycosyl steroid derivatives were synthesized by the reaction of a new polyhydroxysteroid 3β,5α,6β-trihydroxypregn-16-en-20-one (2) with the peracetylated 1-bromo derivatives of d-glucose and d-galactose, respectively. Subsequent protection by excess acetic anhydride in pyridine selectively gave the 6β-O-acetylated steroid glycosides. Deprotection of the acetylated steroid glycosides separately with moderate catalysis dibutyltin oxide in methanol selectively removed all acetyl groups of sugar moiety, whereas the acetyl group of the steroid part was retained. The structures of the steroid glycosides were confirmed by mass spectrometry, NMR and IR. The complete protocol was shown to be non-destructive at all stages to the sugar moieties and the steroid nucleus. These regioselective reactions open a route to the synthesis of a series of closely related isomers of 2 and other widespread polyhydroxysteroids and steroid glycosides in marine organisms and some terrestrial species.     

Tibolone: a steroid with a tissue-specific mode of action

In postmenopausal women tibolone has proved to prevent bone-loss and relieve climacteric symptoms as effectively as estrogens, but it does not stimulate the endometrium and the breast. This clinical profile strongly suggests that tibolone is a compound with tissue-specific action. Tibolone is quickly metabolized into its main active metabolites, 3 and 3β-OH, which are also present in an inactive, sulphated, form. In addition a Δ4-metabolite is found in circulation. The 3-OH-metabolites bind only to the estrogen receptor while the Δ4-isomer shows affinity only to the progesterone and androgen receptors. Tibolone prevents bone loss in a similar way to estrogens. Studies on bone mass using anti-estrogen, antiprogestin and anti-androgen in combination with tibolone, confirmed the sole involvement of the estradiol receptor. Increases in skin temperature as well as vaginal atrophy can be prevented by tibolone in a similar way to estrogens. Breast safety studies showed that tibolone clearly inhibited the growth of tumors in a DMBA model. In breast cell lines, tibolone profoundly inhibited sulphatase activity and an increase in apoptosis and decrease in cell proliferation was found. The stimulation of the endometrium is prevented by the local formation of the Δ4-isomer from tibolone or the 3β-OH-metabolite. We conclude that tibolone acts as a tissue-specific compound by mediating its effects via steroid receptors and enzymatic pathways. This dual effect of tibolone explains it's positive clinical effects on bone, vagina and brain, and avoids stimulation of the endometrium and breast tissue.     

Genomics of steroid hormones: in silico analysis of nucleotide sequence variants (polymorphisms) of the enzymes involved in the biosynthesis and metabolism of steroid hormones

Alterations of steroid hormone biosynthesis and metabolism are suspected to be involved in the pathogenesis of several diseases. Several polymorphisms of the enzymes involved in these processes have already been described and some could be associated with certain diseases. We attempted to examine the sequence variants of these genes in order to find novel variants by an in silico analysis. We analyzed the known human nucleotide sequences of the enzymes p450 side-chain cleavage enzyme, steroid 17-alpha-hydroxylase/17,20-lyase, 3-beta-hydroxysteroid dehydrogenase types 1 and 2, 21-hydroxylase, 11-beta-hydroxylase, aldosterone synthase, aromatase, 11-beta-hydroxysteroid dehydrogenase types 1 and 2, steroid 5-alpha-reductase types 1 and 2, steroid 5-beta-reductase, dehydroepiandrosterone sulfotransferase, 17-beta-hydroxysteroid dehydrogenase types 1–3. The analysis was performed using the National Center for Biotechnology Information Database by the search tool blastn. We found numerous sequence variants in both coding and non-coding sequences. The majority of these sequence variants have already been described, nevertheless, some appear as novel variants. Some of these may also have functional significance. We hypothesize over the possible significance of these findings and briefly review the available literature.     

A cell surface protein controls endocrine ring gland morphogenesis and steroid production

Identification of signals for systemic adaption of hormonal regulation would help to understand the crosstalk between cells and environmental cues contributing to growth, metabolic homeostasis and development. Physiological states are controlled by precise pulsatile hormonal release, including endocrine steroids in human and ecdysteroids in insects. We show in Drosophila that regulation of genes that control biosynthesis and signaling of the steroid hormone ecdysone, a central regulator of developmental progress, depends on the extracellular matrix protein Obstructor-A (Obst-A). Ecdysone is produced by the prothoracic gland (PG), where sensory neurons projecting axons from the brain integrate stimuli for endocrine control. By defining the extracellular surface, Obst-A promotes morphogenesis and axonal growth in the PG. This process requires Obst-A-matrix reorganization by Clathrin/Wurst-mediated endocytosis. Our data identifies the extracellular matrix as essential for endocrine ring gland function, which coordinates physiology, axon morphogenesis, and developmental programs. As Obst-A and Wurst homologs are found among all arthropods, we propose that this mechanism is evolutionary conserved.     

Soil restoration under pasture after lignite mining: Management effects on soil biochemical properties and their relationships with herbage yields

The recovery of soil biochemical properties under grazed, grass-clover pasture, after simulated lignite mining, was studied over a 5-year period in a mesic Typic Dystrochrept soil at Waimumu, Southland, New Zealand. The restoration procedures involved four replacement treatments, after A, B, and C horizon materials had been separately removed, from all except the control, and stockpiled for 2–3 weeks. In each replacement treatment, the effects of ripping to 1.8 m depth, mole drainage, and the use of fertilizer nitrogen were also investigated.Replacement treatment markedly influenced the recovery of herbage production and soil organic C and total N contents, N mineralization, microbial biomass (as indicated by mineral-N flush) and invertase and sulphatase activities. The effectiveness of replacement treatments decreased in the order: 1. control (no stripping or replacement). 2 A, B, and C horizon materials replaced in the same order. 3. A, B, and C horizon materials each mixed with an equal amount of siltstone overburden and replaced in order, 4. A and B horizon materials mixed before replacing over C horizon material.Ripping increased herbage production, net N mineralization, and to some extent microbial biomass. Drainage had little, if any, effect.Fertilizer N also stimulated herbage production, but depressed clover growth. Over 2.5 years, it had little detectable effect on the soil properties.Increases in soil invertase and, to a lesser extent, sulphatase activity during the trial were closely related to changes in herbage production. Microbial biomass increased more rapidly than did soil organic C in the early stages of the trial.Rates of net N mineralization strongly suggest that N availability would have limited pasture growth, especially in the treatments with mixed soil materials.     

Regulation of steroid sulphatase expression and activity in breast cancer

Steroid sulphatase (STS) catalyzes the conversion of oestrone sulphate (E1S) to oestrone (E1) and its action in breast tumours makes a major contribution to in situ oestrogen production in this tissue. Although expression of STS mRNA and STS activity are increased in malignant breast tissues compared with that in non-malignant tissues, little is known about the regulation of its expression or activity. In the present study we have used a RT-PCR technique to investigate the regulation of STS mRNA expression in cultured breast tissue fibroblasts and MCF-7 cells. STS mRNA expression was readily detectable in fibroblasts derived from breast tissue proximal to tumours, breast tumour tissue and reduction mammoplasty tissue. For two pre-menopausal subjects, STS mRNA expression was similar in proximal and tumour fibroblasts whereas for a third, post-menopausal subject, expression in breast tumour fibroblasts was 2.4-fold that in proximal fibroblasts. The cytokine tumour necrosis factor alpha (TNFalpha) or the STS inhibitor, 2-methoxyoestrone-3-O-sulphamate, had no effect on STS mRNA expression in fibroblasts. STS mRNA was detectable in MCF-7 cells but neither TNFalpha nor interleukin 6 (IL-6) affected its expression. Transient transfection of COS-1 and MCF-7 cells with a STS cDNA lacking STS 5' and 3' sequences increased activity 17-fold and 2-fold, respectively. TNFalpha plus IL-6 increased STS activity in mock transfected MCF-7 cells and further increased STS activity in transfected MCF-7 cells. This indicates that activation can occur independently of STS promoter and enhancer elements. In conjunction with the lack of regulation of STS mRNA it suggest that TNFalpha and IL-6 may increase STS activity via a post-translational modification of the enzyme or by increasing substrate availability.     

Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors

Steroid sulfatase (STS) regulates the formation of active steroids from systemic precursors, such as estrone sulfate and dehydroepiandrosterone sulfate (DHEAS). In breast tissues, this pathway is a source for local production of estrogens, which support the growth of endocrine-dependent tumours. Therefore, inhibitors of STS could have therapeutic potential. In this study, we report on substituted chromenone sulfamates as a novel class of non-steroidal irreversible inhibitors of STS. The compounds are substantially more potent (6- to 80-fold) than previously described types of non-steroidal inhibitors when tested against purified STS. In MCF-7 breast cancer cells, they inhibit STS activity with IC₅₀ below 100 pM. Importantly, the compounds also potently block estrone sulfate-stimulated growth of MCF-7 cells, again with IC₅₀ below 100 pM. For one compound, we also observed a lack of any estrogenic effect at high concentrations (1 μM). We also demonstrate for the first time that STS inhibitors can block the DHEAS-stimulated growth of MCF-7 cells. Interestingly, this cannot be achieved with specific inhibitors of the aromatase, suggesting that stimulation of MCF-7 cell growth by DHEAS follows an aromatase-independent pathway. This gives further justification to consider steroid sulfatase inhibitors as potential drugs in the therapy of breast cancer.     

Ontogenic variations in the content and distribution of progesterone receptor isoforms in the reproductive tract and brain of chicks

Progesterone participates in the regulation of several functions in chicks such as ovulation, gonadal differentiation, and sexual and nesting behaviors. Many progesterone actions are mediated by specific intracellular receptors (PR) which are ligand-induced transactivators. Two PR isoforms that are functionally distinct in their ability to activate genes and regulate distinct physiological processes have been described in chicks: a full length form PR-B and the N-terminally truncated one PR-A which lacks the amino-terminal 128 amino acids of PR-B. PR isoforms have been detected in several tissues of both the adult and the embryo chick such as brain, ovary and oviduct. PR isoforms expression ratio varies among progesterone target tissues and under different hormonal and environmental conditions such as those presented during avian sexual maturity and the seasons of the year. These data let us to conclude that progesterone actions in brain, ovary, and oviduct highly depend on PR isoforms expression pattern and regulation.