Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Thesis (M.D. with Distinction in Research) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Partial embargo. Vita. Bibliography: 44-46

Abstract Prostate cancer is the most frequently diagnosed cancer in men in industrialized countries. Despite the substantial clinical importance of the disease, the mechanisms underlying the development and progression of prostate cancer are poorly understood. In the present study, fragment analysis of chromosome arm 16q was carried out with the aim of searching for sites of consistent chromosomal deletion, possibly uncovering the location of target genes that become inactivated in prostate carcinogenesis. The highest percentage of loss of heterozygosity (LOH) was found at chromosomal region 16q24.1-q24.2, including the gene for 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase (17HSD/KSR) type 2, HSD17B2. The data further indicated an association between loss of the most commonly deleted region and clinically aggressive features of the disease. A fragment analysis performed using sequential primary and locally recurrent prostate cancer specimens suggested the location of the genes related to prostate cancer progression to be at 16q24.3 and, further, gave rise to a hypothesis of the potential role of locus HSD17B2 as a prognostic marker for prostate cancer progression. Quantitative real-time polymerase chain reaction (PCR) revealed a decreased HSD17B2 gene copy number in prostate cancer specimens compared to their normal counterparts. A diminished HSD17B2 gene copy number was significantly associated with poor differentiation of the tumor. The progression of prostate cancer during androgen deprivation is a serious clinical problem, the molecular mechanisms of which largely remain to be clarified. The present data of enzyme activity measurements performed using high-performance liquid chromatography (HPLC) provided evidence of a substantial decrease in oxidative and an increase in reductive 17HSD/KSR activity during the transition of prostate cancer LNCaP cells into an androgen-independent state. Further, the changes detected in the activities largely coincided with the changes in the relative expression levels of genes for the potential 17HSD/KSR isoenzymes; 17HSD/KSR types 2, 5, and 7, as evidenced by relative quantitative reverse transcription PCR (RT-PCR). The data on the expression analysis of mRNA for 17HSD/KSR types 5 and 7 in prostate tissue specimens performed using in situ hybridization showed a moderately low but constitutive level for 17HSD/KSR7 mRNA in tissues of cancerous as well as hyperplastic origin. The expression of mRNA for 17HSD/KSR type 5, instead, varied considerably between different specimens, the highest expressions being strongly associated with aggressive and metastatic prostate cancer. Interestingly, furthermore, the intense expression of 17HSD/KSR5 was significantly associated with the androgen deprivation achieved either surgically or medically. Since 17HSD/KSRs critically contribute to the control of the bioavailability of active sex steroid hormones locally in the prostate, the variation in intraprostatic 17HSD/KSR activity might be crucially involved in the regulation of the growth and function of the organ.

Abstract The biosynthesis and metabolism of sex steroids are controlled by a series of steroidogenic enzymes. In the placenta and the breast, 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) is essential for the synthesis of all steroid hormones by catalyzing pregnenolone to progesterone (P) or dehydroepiandrosterone (DHEA) to androstenedione (A-dione). P450 aromatase (P450arom) converts androgens to estrogens and is therefore critical for estrogen production. 17β-hydroxysteroid dehydrogenases (17HSDs) are a group of enzymes responsible for the interconversion between low-activity 17-ketosteroids and high-activity 17β-hydroxysteroids, thus acting as key enzymes modulating the biosynthesis and metabolism of both estrogens and androgens. In situ hybridization assays showed that 3β-HSD1, P450arom and 17HSD1, 2, 5 and 7 are expressed in early and mid-gestation placentas. Abundant expressions of 3β-HSD1, P450arom and 17HSD1 were seen in syncytiotrophoblast (ST) cells. Signals of these three enzymes were also detected in some column cytotrophoblast (CCT) cells. 17HSD2 and 5 were located in intravillous stromal (IS) cells, whereas 17HSD7 mRNA was present in all types of placental cells. This suggests that the human placenta produces not only P and estrogens, but also androgens. Moreover, the placenta possesses a function, by the action of 17HSD2, to protect the fetus and the maternal body from excessive sex steroid influence. In tubal pregnancy, P450arom and 17HSD1 were found in ST cells, implying an estrogen biosynthesis mechanism similar to that in normal intrauterine pregnancy. In both JEG-3 choriocarcinoma cell line and cultured normal human cytotrophoblast (CTB) cells, retinoic acids were shown to promote the enzyme activity as well as mRNA expression of P450arom and 17HSD1, and hence their action on the biosynthesis of E2. The mRNA expressions of 17HSD1, 2 and 5 in 794 breast carcinoma specimens were analyzed and correlated with ERα, ERβ, PR, Ki67, c-erbB2 and clinical parameters. 17HSD1, 2 and 5 were detected in epithelial cells in normal and malignant breast tissues. In breast cancer specimens, the positive cases for 17HSD1, 2 and 5 were 16%, 25% and 65%, respectively. 17HSD1 was found to be an independent prognostic marker of the progression of breast cancer.

Corticosterone in foetal tissues after injection of the mother with ¹⁴C-corticosterone was determined by acetylation. with ³H-acetic anhydride and crystallization to constant specific activity. The corticosterone content of whole foetal tissue varied between gestational days 13 and 17 from 641 to 300 ng/g respectively. The specific activity of foetal hormone recovered remained essentially constant; after a 15-minute pulse this was as much as one-fourth that of maternal hormone. However, placenta, head and liver showed distinctly different patterns of metabolism, which changed greatly during this time in head and liver, with a decrease in the conversion of corticosterone to 11-dehydrocorticosterone and a rise in foetal liver 113-hydroxysteroid:NADP oxidoreductase activity. This mitochondrial enzyme, Km=33yM, pH optimum 6, which reduces the 11-dehydro metabolite to the biologically active 116-OH compound, increased sharply, raising the relative amount of the latter in foetal tissues from 15 to 91% during this period. One day after removal of maternal adrenals, foetal corticosterone was normal and maternal levels close to normal, indicating ability of foetal adrenals to function. Maternal hormone, however, crossed to the foetus readily and it was considered most likely that, normally, the maternal source predominates. Regardless of origin, foetal or maternal, however, the hormone is maintained in different foetal tissues in a distinct and different manner. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21719号 / 薬科博第110号 / 新制||薬科||12(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 教授 竹島 浩, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Intra-adipose cortisol is derived from the systemic circulation via the hypothalamic-pituitaryadrenal axis (HPAA) and generated locally through conversion of inactive cortisone to cortisol by the intra-cellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1). This thesis addresses the relative contributions of the HPAA and adipose tissue 11βHSD1 to the adipose tissue glucocorticoid pool and describes development and validation of a novel stable isotope tracer, 1,2 [2H]2-cortisone (d2-cortisone), to measure 11βHSD1- dehydrogenase activity in adipose tissue and skeletal muscle in vivo. In otherwise healthy females (n=6) undergoing hysterectomy for a benign indication, an intravenous infusion of d4-cortisol was administered and subcutaneous and omental adipose tissue biopsies were obtained along with concomitant peripheral venous blood, to measure the rate of exchange of cortisol between plasma and adipose tissue for comparison with rates of intra-cellular cortisol generation by 11βHSD1. Cortisol concentrations and enrichment with d4-cortisol were lower in adipose tissue than in plasma. The rate of accumulation of d4-cortisol in adipose tissue depots was ~0.5nmol/kg/h despite the infusion contributing 1.9μmol/h d4-cortisol into the circulation, and the proportion of the intra-adipose cortisol pool replaced each hour was ~10%. The contribution of 11βHSD1 to this turnover could not be quantified since very little substrate d3-cortisone accumulated in adipose during infusion. Method development for d2-cortisone included optimising LC-MS/MS conditions, confirming that d2-cortisone was a substrate for human 11βHSD1 and that no significant primary isotope effect existed. The pharmacokinetics of d2-cortisone were assessed in vivo in healthy male volunteers (n=3). The method was validated by measuring whole body cortisone production in healthy volunteers (n=3) before and after eating liquorice which resulted in a ~50% fall in cortisone production. 11βHSD1-dehydrogenase activity was measured in adipose tissue and skeletal muscle in healthy volunteers (n=6) using d2- cortisone and substantial 11β-dehydrogenase activity was present in both tissues (~1.5-fold higher 11β-dehydrogenase activity than 11β-reductase activity in adipose tissue and approximately equal 11β-reductase and 11β-dehydrogenase activity in skeletal muscle). 11βHSD1-reductase activity was also assessed using a 9,11,12,12 [2H]4-cortisol infusion (d4-cortisol). Skeletal muscle and adipose tissue displayed 11β-reductase activity. In adipose tissue this activity was of a similar magnitude to previous reports. Insulin increased whole body 11β-reductase activity, but did not switch 11βHSD1 direction in muscle or adipose tissue, indicating the predominant effect of insulin may be on hepatic 11βHSD1. Therefore, turnover of the intra-adipose tissue glucocorticoid pool is slow and it is unlikely that rapid acute fluctuations in circulating cortisol are reflected in adipose tissue, although this has not been confirmed under normal physiological conditions. Secondly, 11βHSD1 may be bidirectional in human subcutaneous adipose tissue and skeletal muscle in vivo, and insulin does not regulate the balance of activities. However, in this study blood sampling occurred from blood vessels which express 11βHSD2, and thus some of the measured dehydrogenase activity in this study may reflect endothelial 11βHSD2 activity. Together these findings further our understanding of adipose tissue cortisol physiology in health, suggesting that 11βHSD1 may play a relatively important role in modulating activation of glucocorticoid receptors in adipose tissue, and that dysregulation or inhibition of 11βHSD1 may affect cortisol inactivation as well as regeneration.

Inflammation plays a key role in the underlying pathogenesis of obesity and its associated health risks, with increased markers of inflammation evident in both liver and adipose tissue. In parallel, there is dysregulation of glucocorticoid metabolism in obesity, with increased adipose levels of the glucocorticoid-regenerating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and increased hepatic levels of 5α-reductase type 1 (5αR1), which catalyses the reduction of glucocorticoids. Both the mechanisms and consequences of this glucocorticoid metabolism dysregulation remain unclear, however, there is evidence that it may be related to inflammation. In vitro studies have demonstrated that pro-inflammatory markers upregulate 11βHSD1 expression in adipocytes, potentially explaining increased expression of this enzyme in obesity. Previous work has also demonstrated that the glucocorticoid metabolites produced by 5αR1 lack the metabolic effects of the parent glucocorticoid, but retain its anti-inflammatory properties, indicating that increased expression of hepatic 5αR1 may serve to dampen down inflammation in the liver. The hypotheses addressed in this thesis are that in obesity, inflammation regulates adipose glucocorticoid metabolism through 11βHSD1, and that hepatic glucocorticoid metabolism regulates the inflammatory state of the liver through 5αR1. The role of inflammation in the regulation of 11βHSD1 was assessed in vivo in mice treated with the anti-inflammatory compound sodium salicylate (salicylate). In diet-induced obese mice, salicylate downregulated 11βHSD1 expression and activity selectively in visceral adipose tissue, alongside improved glucose tolerance, reduced plasma non-esterified fatty acids, and changes in adipose lipid metabolism. 11βHSD1-deficient mice fed a high-fat diet were resistant to the insulin sensitising effects of salicylate treatment. These results indicate a novel role for 11βHSD1 down-regulation in mediating the insulin sensitising effect of anti-inflammatory treatment. The mechanisms underpinning the anti-inflammatory properties of 5α-reduced glucocorticoids were explored in vitro and in vivo. In lipopolysaccharide-stimulated murine macrophages, both 5α-reduced glucocorticoid metabolites tested, namely 5α-dihydrocorticosterone (5αDHB) and 5α-tetrahydrocorticosterone (5αTHB), suppressed tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) release, although to a lesser extent than corticosterone (B). Similar to B, both 5αDHB and 5α THB suppressed phosphorylation of intra-cellular inflammatory signalling mitogen-activated protein kinases (MAPK) proteins c-Jun N-terminal kinase (JNK) and p38, as well as increasing protein expression of MAPK phosphatase-1 (MKP-1). Treatment of phorbol ester-stimulated HEK293 kidney cells with these 5α-metabolites revealed that 5αDHB suppressed nuclear factor κB (NFκB) and activator protein-1 (AP-1) activation to a similar extent to that of B, whilst 5αTHB increased activation of these pro-inflammatory transcription factors, indicating cell-specific effects of 5αTHB. In conclusion, reduced intra-adipose glucocorticoid regeneration by 11βHSD1 mediates the insulin sensitising effects of salicylate, suggesting that altered glucocorticoid metabolism may reflect altered intra-adipose inflammation in obesity. Furthermore, these data support the concept that this enzyme provides a therapeutic target in obesity-related metabolic disorders. 5α-reduced metabolites of glucocorticoids have similar anti-inflammatory properties to the parent glucocorticoid, indicating that the elevated hepatic levels of 5α-reductase in obesity may be a protective mechanism to limit the adverse metabolic effects of glucocorticoids upon the liver, but maintain the beneficial anti-inflammatory properties. These 5α-reduced glucocorticoid metabolites may provide a potential therapeutic treatment as selective glucocorticoid receptor modulators for inflammatory conditions.

Epithelial Ovarian Cancer (EOC) kills more women annually in the United Kingdom than any other gynaecological cancer. Survival rates for women diagnosed with EOC have not improved over the past 30 years, due to the often advanced stage at presentation, where widespread intra-peritoneal dissemination has occurred. The natural history of the disease remains uncertain but the ovarian surface epithelium (OSE) is a strong candidate for the tissue of origin. The OSE undergoes cyclical damage and repair in women of reproductive age following ovulation, which can be considered an acute inflammatory event. Factors that prevent ovulation (pregnancy, breastfeeding and contraceptive pill use) also protect against the development of EOC. Previously published data show that the OSE is able to upregulate the enzyme 11-beta hydroxysteroid dehydrogenase type 1 (11βHSD1) in response to inflammation, the enzyme responsible for converting inactive cortisone to anti-inflammatory cortisol. This thesis hypothesises that 11βHSD isozymes are deregulated in ovarian cancer; that the peritoneal surface epithelium (PSE) is indistinguishable from the OSE in its response to inflammation and should be considered a potential source of some “ovarian cancers”; and finally that the expression of the tumour suppressor gene OPCML (OPioid binding Cell adhesion Molecule-Like) is altered by inflammation. These hypotheses were examined at three levels. Firstly, primary cultures of EOC were established, and glucocorticoid metabolism and the response to inflammation was compared to normal OSE. Results from these investigations reveal that the11βHSD1 response to IL-1α stimulation is impaired in EOC compared to normal OSE at the mRNA level but there is no significant difference when 11βHSD1 enzyme activity is measured in these tissues. When basal levels of 11βHSD1, 11βHSD2 and COX2 are compared amongst untreated samples of EOC and OSE, there was a significant correlation between 11βHSD1 and COX2 mRNA expression (P<0.001). 11βHSD2 mRNA expression was significantly higher in the EOC specimens compared to OSE (P<0.05). Secondly the response to inflammation was compared in primary cultures of human peritoneal surface epithelial (PSE) cells and OSE. The data suggest that the mRNA response to inflammation was similar in OSE and PSE, but that the 11βHSD1 enzyme activity was reduced in PSE (P<0.05), which may result in differences in tissue healing. Finally, the effect of inflammation on the expression of the ovarian cancer associated tumour suppressor gene (TSG), OPCML (OPioid binding Cell adhesion Molecule-Like) and the other members of the IgLON family, was examined in OSE. These results suggest that OPCML mRNA expression can be induced by IL-1α, an effect that is inhibited by cortisol.