Studies on steroid metabolism in human endometrial tissue

1. A computerized technique is described for the quantitative determination of radiometabolites from incubation studies. 2. Seven steroid substrates have been incubated with human endometrial tissue. The principal radiometabolites were identified and determined after 2hr. incubation without the addition of cofactors and after 4hr. incubation with cofactors. 3. The main products from progesterone were 20alpha-dihydroprogesterone and 5alpha-pregnanedione with lower yields of 5beta-pregnanedione and 20beta-dihydroprogesterone. There was no evidence for 17alpha-hydroxylase activity. 4. 17alpha-Hydroxyprogesterone was transformed into small yields of 17alpha,20alpha- and 17alpha,20beta-dihydroxypregn-4-en-3-one. In one incubation there was evidence for conversion into androstenedione. 5. Dehydroepiandrosterone was transformed into small amounts of androstenedione, 5alpha-androstanedione and androsterone. 6. Androstenedione and testosterone were interconvertible, the reaction favouring the formation of androstenedione. 5alpha-Androstanedione and androsterone were formed from both substrates. There was no evidence for the formation of phenolic steroids. 7. Oestrone and oestradiol-17beta were interconvertible, the reaction favouring the formation of oestrone.     

Sex steroid hormone metabolism and prostate cancer

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.     

Sex steroid hormone metabolism takes place in human ocular cells

Steroids are potentially important mediators in the pathophysiology of ocular diseases. In this study, we report on the gene expression in the human eye of a group of enzymes, the 17beta-hydroxysteroid dehydrogenases (17HSDs), involved in the biosynthesis and inactivation of sex steroid hormones. In the eye, the ciliary epithelium, a neuroendocrine secretory epithelium, co-expresses the highest levels of 17HSD2 and 5 mRNAs, and in lesser level 17HSD7 mRNA. The regulation of gene expression of these enzymes was investigated in vitro in cell lines, ODM-C4 and chronic open glaucoma (GCE), used as cell models of the human ciliary epithelium. The estrogen, 17beta-estradiol (10(-7) M) and androgen agonist, R1881 (10(-8) M) elicited in ODM-C4 and GCE cells over a 24 h time course a robust up-regulation of 17HSD7 mRNA expression. 17HSD2 was up-regulated by estradiol in ODM-C4 cells, but not in GCE cells. Under steady-state conditions, ODM-C4 cells exhibited a predominant 17HSD2 oxidative enzymatic activity. In contrast, 17HSD2 activity was low or absent in GCE cells. Our collective data suggest that cultured human ciliary epithelial cells are able to metabolize estrogen, androgen and progesterone, and that 17HSD2 and 7 in these cells are sex steroid hormone-responsive genes and 17HSD7 is responsible to keep on intra/paracrine estrogenic milieu.     

Molecular epidemiology of breast cancer: genetic variation in steroid hormone metabolism

The age-specific incidence rate of breast cancer in women rises until menopause, levels off and then rises again at a much lower rate indicating a possible hormonal influence on the disease risk. A large amount of evidence has implicated hormones and other compounds with oestrogen activity in the pathogenesis of certain endocrine cancers, particularly breast cancer. Widely dispersed hormone-like chemicals, capable of disrupting the endocrine system and interfering with proliferation, have been described. Compounds such as dioxins, some polychlorinated biphenyls and the plastic ingredient bisphenol-A have been shown to interfere with human reproduction and hormonal regulation. The levels of these foreign compounds as well as the levels of endogenous oestradiol may influence the risk of breast cancer. Endogenous oestradiol is synthesised in the ovarian theca cells of premenopausal women or in the stromal adipose cells of the breast of postmenopausal women and minor quantities in peripheral tissue. These cells, as well as breast cancer tissue, express all the necessary enzymes for this synthesis: CYP17, CYP11a, CYP19, hydroxysteroid hydrogenase, steroid sulphatase as well as enzymes further hydroxylating oestradiol such as CYP1A1, CYP3A4, CYP1B1. Polymorphisms in these enzymes may have a possible role in the link between environmental estrogens and hormone-like substances and the interindividual risk of breast cancer.     

The modulation of choline phosphoglyceride metabolism in human colon cancer

Colorectal cancer has a high incidence of morbidity and mortality in the North American population. Elevated levels of plasmalogens have been reported in some neoplastic tissues including colon tumors, but the mechanism for this increase has not been defined. Since changes in plasmalogen level are usually associated with changes in the other phospholipid subclasses, a general increase in all phospholipid subclasses may also be found in colonic neoplasms. In this study, the levels of the major phospholipids, including their plasmalogen and diacylphospholipid subclasses, were found to be elevated in human malignant colonic tissues. Since phosphatidylcholine is the most prominent type of phospholipid found in both malignant and control tissues, the mechanism for its accumulation during malignancy was investigated. Decreases in phospholipase C and D activities were observed in tumor samples, but an enhancement of the CTP: phosphocholine cytidylyltransferase activity was also detected. Immunoblotting analysis revealed that the elevated cytidylyltransferase activity was caused by a three-fold increase in the level of enzyme protein during tumor development. Based on these enzyme studies, we conclude that the high level of phosphatidylcholine in colon tumors resulted from a decrease in its turnover and an increase in its expression.     

Alkaline phosphatases and steroid receptors in human breast cancer

Estrogen receptors (ER), progesterone receptors (PR) and alkaline phosphatases (AP) were measured in 150 tumors from patients who underwent mastectomy for primary breast cancer. The percentage of ER positive samples was inversely related to the AP activity ranging from 88.9% in low activity samples (less than 30 U/mg prot.) down to 30.6% in the high activity ones (greater than 400 U/mg prot.). When considering only ER positive samples, the ER content was inversely related to the AP activity. This could not be demonstrated for PR. Therefore, the authors suggest the hypothesis that in human breast cancer, the AP may play a role in the dephosphorylation of the ER molecule and in the consequent modulation of its binding capability.     

Role of human adipose tissue in the production and metabolism of steroid hormones

Radioimmunological methods have been employed for the simultaneous determination of dehydroepiandrosterone, androstenedione, testosterone, oestrogens (oestradiol + oestrone), progesterone, 17 alpha-hydroxyprogesterone and cortisol in human adipose tissue and peripheral blood to compare the hormone pool of adipose tissue with that in the general circulation. Extremely high steroid concentrations in the adipose tissue and hormone pool in the fat of obese subjects were observed. For adipose tissue/serum steroid ratios, the highest values were obtained for dehydroepiandrosterone and the lowest ones for cortisol. A preliminary study showed a great accumulation of steroids in adjacent adipose tissue of breast tumors. Striking differences were observed in the adipose tissue steroid concentrations between benign and malignant mammary tumors. The present findings revealed that blood hormone determinations may be insufficient to consider the steroid hormone availability in various endocrinopathies or steroid responsive tumors, especially when the endocrine state of extremely obese subjects is observed.     

Altered sphingolipid metabolism in human endometrial cancer

There is a growing body of evidence indicating that bioactive sphingolipids play a key role in cancer development, progression and metastasis. However, sphingolipid metabolism in malignant tumors is poorly investigated. Therefore, the aim of the present study was to examine the content of selected intermediates of ceramide metabolism and the activity of key enzymes of ceramide de novo synthesis and sphingosine-1-phosphate (S1P) production in the endometrial cancer. The specimens of cancer tissue and healthy endometrium were obtained from women undergoing surgery because of the cancer (n = 23) and because of myomas (n = 18), respectively. The content of sphinganine, dihydroceramide, ceramide, sphingosine and S1P was measured using high pressure liquid chromatography. The activity of the enzymes was determined using radioactive substrates. It has been found that the content of each examined sphingolipid was markedly elevated in the cancer tissue compared with the healthy endometrium. Namely, sphinganine, sphingosine and dihydroceramide by 3–4.6-fold, ceramide and S1P by 1.9- and 1.6-fold, respectively. Interestingly, the ratio of S1P to ceramide remained stable. The activity of serine palmitoyltransferase and sphingosine kinase 1 was increased by 2.3- and 2.6-fold, respectively. We conclude that endometrial carcinoma is characterized by profound changes in sphingolipid metabolism that likely contribute to its progression and chemoresistance.     

Estrogen metabolism in human colorectal cancer cells

Epidemiological and "in vitro" studies support a direct role of estrogens in the pathogenesis and/or progression of colorectal cancer (CRC). Recent observations suggest a local synthesis of 17beta-estradiol (E(2)). In the present study, the CRC estrogen receptor beta (ERbeta) positive HCT8, HCT116, DLD-1 and LoVo cell lines were evaluated for expression of functional 17beta-hydroxysteroid dehydrogenase (17betaHSD) types 1, 2, 3, and 4. RT-PCR analysis revealed that while 17betaHSD1 and 17betaHSD4 were expressed in all the four cell lines, 17betaHSD2 and 17betaHSD3 were expressed in a cell-specific manner. The interconversion of tritiated estrone (E(1)) or E(2) evaluated by thin layer chromatography of conditioned media revealed that in HCT8, HCT116, and DLD-1 cells both reductive and oxidative activities were present, the latter showing K(m) values (approximately 10 microM) 40-fold higher than the former (approximately 250 nM). On the contrary, in LoVo cells, estrogens were almost (approximately 90%) completely metabolized to hydrophile compounds. Charcoal-dextrane (DC) stripped fetal calf serum (FCS) (10%), E(2) (10nM), Vitamin D(3) (100nM) and the combined E(2) and Vitamin D(3) treatment were evaluated for modulation of 17betaHSD isoenzymes gene expression and activity. Gene expression and activity of 17betaHSD reductive and oxidative isoenzymes were respectively inhibited and enhanced by Vitamin D(3) in HCT8 and LoVo cells. Surprisingly, DC-FCS induced a marked increase of estrogen metabolism toward hydrophile metabolites in all four cell lines. In conclusion, our results clearly show that metabolism of estrogens by 17betaHSD isoenzymes is functional and modulated by external stimuli in continuous neoplastic colonic epithelial cell lines.     

Anabolic steroid metabolism in skeletal muscle

Three hundred and sixty male albino rats weighing 180 to 200 g were used to determine the effect of anabolic steroid hormones on adaptive changes in the synthesis of ribosomal RNA both in sedentary animals and in animals involved in a training programme. One injection of Retabolil (0.1 mg/100 g body weight) increased the α-amanitin insensitive RNA polymerase activity of nuclei from skeletal muscles. Fourteen h after this hormone injection the enzyme activity was 45% higher than in control animals and it remained at this level for 4 days. Under these conditions a selective binding of 19-nortestosterone with cytoplasmic proteins of skeletal muscle was found. Physical training increased the RNA polymerase activity by 50% (P < 0.05). It was found that the testosterone binding capacity of a cytoplasmic extract from trained animals was 70% greater than that of the control animals (P < 0.05). Four injections of Retabolil during training resulted in an additional increase of RNA polymerase activity of 40% (P < 0.05) but reduced the testosterone binding capacity of the cytoplasmic proteins that occurred with training by 21%. These results demonstrate the effect of anabolic hormones in the regulations of RNA synthesis in skeletal muscle nuclei in the process of their adaptation to systematic physical training.     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

Pituitary control of hepatic steroid metabolism

Hypophysectomy of male animals has little effect on the hepatic 4-androstene-3,17-dione (androstenedione) metabolism except at the kinetic level where changes in the apparent Km of the 16 alpha- and 7 alpha-hydroxylases are seen. On the other hand, hypophysectomy of female animals leads to a "masculinization" of hepatic androstenedione metabolism, following the changes seen in Vmax of the respective enzymes probably due to the removal of the source of "feminizing factor" thought to maintain the female-type metabolism in the liver. There seems to be a temporal dissociation of the effects on the various enzymes indicating different cellular control mechanisms for these enzymes. Oestrogen treatment of male rats causes "feminization" of the hepatic androstenedione metabolism. The time study shows an initial increase in 17-hydroxysteroid oxidoreductase, 6 beta- and 16 alpha-hydroxylase activities followed by a decrease to the levels of females. This biphasic effect is possibly due to an initial direct effect of the oestrogen on the liver followed by an indirect effect via the hypothalamo-pituitary system. The changes in enzyme activity noted are related to changes in Vmax of the respective enzymes although changes in apparent Km are also seen.