The radioimmunoassay of steroid glucuronides. The oestrogen C-3 glucuronides as haptens.
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ABSTRACT:
SUBMITTER: Samarajeewa P
PROVIDER: S-EPMC1172367 | biostudies-other | 1975 Nov
REPOSITORIES: biostudies-other
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Project description:Radioimmunoassays for five oestrogen metabolites in urine are described; they are oestrone 3-glucuronide, oestradiol 3-glucuronide, oestradiol 17 beta-glucuronide, oestriol 3-glucuronide and oestriol 16 alpha-glucuronide. These assays have proved accurate and reliable and can be performed rapidly; they have been carried out directly in diluted menstrual cycle urine and pregnancy urine. No sample pretreatment was required. Preliminary results suggest that clinically useful information can be obtained by performing these assays on random urine specimens.
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Project description:BackgroundSteroid sulphatase (STS) is one of the steroid-metabolising enzymes involved in desulphating inactive steroid sulphates and oestrogen sulphotransferase (EST) sulphates active oestrogen. The roles of both STS and EST have not been examined in oestrogen-dependent non-small-cell lung cancer (NSCLC).MethodsWe evaluated the immunoreactivity of STS and EST in NSCLC cases using immunohistochemistry. The function of STS and EST was further demonstrated using NSCLC cell lines.ResultsThe immunoreactivity of STS and EST was detected in 49.5% and 27.8% of NSCLC cases, respectively. The immunoreactivity of STS was significantly higher in female adenocarcinoma cases. The STS-positive NSCLCs were also significantly correlated in an inversed manner with tumour size and cell proliferation and tended to be associated with better clinical outcome. However, the immunoreactivity of EST was significantly correlated with intracellular oestradiol concentration. Results of in vitro analysis demonstrated that oestrone sulphate (E1-S) induced and pregnenolone sulphate (Preg-S) inhibited the proliferation in STS-expressing cell lines. The inhibition by Preg-S was reversed by a specific progesterone receptor blocker. Simultaneous addition of E1-S and Preg-S significantly suppressed the proliferation.ConclusionIn NSCLC patients, STS is considered a good prognostic factor. Results of our present study also indicated the benefits of potential progesterone therapy for NSCLC patients.
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Project description:The properties of the UDP-glucuronyltransferase(s) of guinea-pig liver that catalyse the synthesis of steroid glucuronides were examined. There are many similarities between apparently different substrate-specific forms of these enzymes in that all are activated by bivalent metal ions, and all contain at least 2 thiol groups important for enzyme activity. On the other hand, there are significant differences between the enzymes conjugating steroids and those conjugating non-steroids. Only the latter are activated by UDP-N-acetylglucosamine, which enhances their relatively poor affinity for UDP-glucuronic acid. The steroid-conjugating forms of UDP-glucuronyltransferase are not activated by UDP-N-acetylglucosamine and have relatively high apparent affinities for UDP-glucuronic acid. The rate of glucuronidation of testosterone was inhibited by treatment with phospholipase A. Treatment with cholate or Triton X-100 did not enhance the rates of glucuronidation of any steroid tested. The data indicate several similarities between different forms of UDP-glucuronyltransferase, suggesting that there is a large family of related proteins. At the same time there are important differences in the parameters that modulate the rates of different glucuronidation reactions.
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Project description:A gram scale synthesis of the glucuronide metabolites of curcumin were completed in four steps. The newly synthesized curcumin glucuronide compounds 2 and 3 along with curcumin 1 were tested and their anti-proliferative effects against KBM-5, Jurkat cell, U266, and A549 cell lines were reported. Biological data revealed that as much as 1 ?M curcumin 1 exhibited anticancer activity and almost 100% cell kill was noted at 10 ?M on two out of four cell lines; while curcumin mono-glucuronide 2 as well as di-glucuronide 3 displayed no suppression of cell proliferation.
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Project description:Our publications demonstrate that physiological concentrations of oestrogen (E2) induce endoplasmic reticulum and oxidative stress which finally result in apoptosis in E2-deprived breast cancer cells, MCF-7:5C. c-Src is involved in the process of E2-induced stress. To mimic the clinical administration of c-Src inhibitors, we treated cells with either E2, a c-Src inhibitor PP2, or the combination for 8 weeks to further explore the apoptotic potential of the c-Src inhibitor and E2 on MCF-7:5C cells.Protein levels of receptors and signalling pathways were examined by immunoblotting. Expression of mRNA was detected through real-time polymerase chain reaction (PCR). Cell cycles were analysed by flow cytometry.Long-term treatment with PP2 alone or E2 alone decreased cell growth. In contrast, a combination of PP2 and E2 blocked apoptosis and the resulting cell line (MCF-7:PF) was unique, as they grew vigorously in culture with physiological levels of E2, which could be blocked by the pure antioestrogen ICI182,780. One major change was that PP2 collaborated with E2 to increase the level of insulin-like growth factor-1 receptor beta (IGF-1R?). Blockade of IGF-1R? completely abolished E2-stimulated growth in MCF-7:PF cells. Furthermore, combination treatment up-regulated transcription factors, Twist1 and Snail, and repressed E-cadherin expression which made MCF-7:PF cells display a characteristic phenotype of epithelial-mesenchymal transition (EMT).These data illustrate the role of the c-Src inhibitor to block E2-induced apoptosis and enhance E2-stimulated growth. Caution must be exercised when considering c-Src inhibitors in clinical trials following the development of acquired resistance to aromatase inhibitors, especially in the presence of the patient's own oestrogen.
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Project description:We reported previously that the tumour suppressor ATBF1 (AT motif-binding factor 1) formed an autoregulatory feedback loop with oestrogen-ER? (oestrogen receptor ?) signalling to regulate oestrogen-dependent cell proliferation in breast cancer cells. In this loop ATBF1 inhibits the function of oestrogen-ER? signalling, whereas ATBF1 protein levels are fine-tuned by oestrogen-induced transcriptional up-regulation as well as UPP (ubiquitin-proteasome pathway)-mediated protein degradation. In the present study we show that EFP (oestrogen-responsive finger protein) is an E3 ubiquitin ligase mediating oestrogen-induced ATBF1 protein degradation. Knockdown of EFP increases ATBF1 protein levels, whereas overexpression of EFP decreases ATBF1 protein levels. EFP interacts with and ubiquitinates ATBF1 protein. Furthermore, we show that EFP is an important factor in oestrogen-induced ATBF1 protein degradation in which some other factors are also involved. In human primary breast tumours the levels of ATBF1 protein are positively correlated with the levels of EFP protein, as both are directly up-regulated ER? target gene products. However, the ratio of ATBF1 protein to EFP protein is negatively correlated with EFP protein levels. Functionally, ATBF1 antagonizes EFP-mediated cell proliferation. These findings not only establish EFP as the E3 ubiquitin ligase for oestrogen-induced ATBF1 protein degradation, but further support the autoregulatory feedback loop between ATBF1 and oestrogen-ER? signalling and thus implicate ATBF1 in oestrogen-dependent breast development and carcinogenesis.
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Project description:Nepeta curviflora Boiss. (Syrian catnip) is native to the Middle East. This medicinal plant is commonly used against nervous disorders, rheumatic pains, and high blood pressure. Herbal infusions prepared from various Nepeta spp. are extensively consumed as functional food. However, limited information has been known about the phenolic constituents of Syrian catnip. In this study, two acylated flavone 7-O-glucuronides, apigenin 7-O-(2?-O-(2?-(E-caffeoyl)-?-glucuronopyranosyl)-?-glucuronopyranoside) (1) and luteolin 7-O-(2?-O-(2?-(E-caffeoyl)-?-glucuronopyranosyl)-?-glucuronopyranoside) (2), along with the known phenolic compounds rosmarinic acid, caffeic acid, apigenin, and apigenin 7-O-?-glucopyranoside were isolated from the aerial parts of N. curviflora. The characterizations of these compounds were based on high-resolution mass spectrometry, UV, and extensive use of multidimensional NMR spectroscopy. The new compounds (1 and 2) were identified in the unmodified state and as dimethylesters.
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Project description:Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that ?-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of ?-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the ?-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular ?-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.
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