Hiroji Okada

Steroid Receptor Levels and Histology of Endometriosis and Adenomyosis

Steroid receptors in endometriosis and adenomyosis were investigated to clarify their clinical significance. The receptor levels were determined by Scatchard plot analysis (4 degrees C, by dextran-coated charcoal). In the cytosols of both tissues, the 17 beta-estradiol-estrogen receptor (ER) complex demonstrated a dissociation constant (Kd) of 4.5 x 10(-10) M; the Kd of the progesterone-progesterone receptor (PR) complex was 1.5 x 10(-9) M; and the Kd of the dihydrotestosterone-androgen receptor (AR) complex was 4.0 x 10(-10) M. Seven cases of ovarian endometriosis were studied. The ER and PR levels in endometriosis seemed to be lower than those in the corresponding normal endometrium. AR was also present. There was a suggestion that most endometriosis is least responsive to progestogens. Ten cases of adenomyosis were studied. Histologic dating revealed a delay in the most aberrant endometrial tissue in adenomyosis, as compared with dating of corresponding normal endometrial tissue. ER and AR were detected in all cases. PR was not detected in some cases and, when detected, the content seemed to be lower, possibly suggesting the delayed dating.

Evidence for estrogen synthesis in adenomyotic tissues

OBJECTIVE To investigate steroidogenesis in eutopic and ectopic endometrial tissues in adenomyosis. STUDY DESIGN Aromatase and estrone sulfatase activities were determined by anion-exchange resin column chromatography, thin-layer chromatography, and cocrystallization. The effects of danazol on the activity of these enzymes were also examined. Moreover, localization of aromatase in eutopic and ectopic endometrial tissues was immunohistochemically examined with antihuman placental aromatase cytochrome P-450 antibody. RESULTS Aromatase and estrone sulfatase activities were detected in ectopic endometrium. The activity of these enzymes was significantly suppressed by danazol. In addition, aromatase was immunohistochemically detected in glandular cells of eutopic and ectopic endometrial tissues. CONCLUSIONS The results suggest that estrogen is synthesized in the eutopic and ectopic endometrial tissues of women with uterine adenomyosis and that it may affect the growth of adenomyosis. Danazol suppressed synthesis of estrogen in vitro.

Estrogen as a growth factor to central nervous cells. Estrogen treatment promotes development of acetylcholinesterase-positive basal forebrain neurons transplanted in the anterior eye chamber.

In a previous report, we demonstrated in vivo ameliorating effects of conjugated estrogen in women suffering from senile dementia-Alzheimers type. To investigate the effects of estrogen on the growth of cholinergic neurons, the present study was performed using rat cholinergic tissue implanted into the anterior chamber of the eye. Fetal diagonal band tissue containing cholinergic neurons was grafted into the anterior eye chamber of adult female rats that had either been treated or not with 2 mg estradiol valerate injected every 3 days after oophorectomy. Two and four weeks after transplantation, the axonal and/or dendritic growth of cholinergic neurons in the graft was studied using acetylcholinesterase histochemistry. At both times, acetylcholinesterase positive processes were densely distributed in the grafts of estradiol valerate treated rats, while in rats without estradiol valerate treatment acetylcholinesterase positive reaction was essentially localized only on the cell bodies. These findings were more obvious at 2 weeks after transplantation than at 4 weeks. These results suggest that estrogen acts on cholinergic neurons as a growth factor.

Estrogen productivity of endometrium and endometrial cancer tissue; influence of aromatase on proliferation of endometrial cancer cells.

Aromatase, estrone (E1) sulfatase and E1 sulfotransferase activities were examined in endometrium and endometrial cancer tissue preparations. Aromatase and E1 sulfatase activities in endometrial cancer tissues were found to be significantly higher than in normal endometrial tissues. However, E1 sulfotransferase activity did not differ between benign and malignant tissue. We also examined the effect of testosterone (T) on aromatase activity and tritiated thymidine uptake (DNA synthesis) in various cultured cervical or corpus endometrial cancer cell lines (OMC-4, HHUA, Ishikawa, HEC-59). The results demonstrated that only the HEC-59 cell line had high aromatase activity and increased its DNA synthesis in response to T. This increase of DNA synthesis by T was not suppressed by simultaneous addition of cyproterone acetate, but was by tamoxifen. These data suggest that in situ estrogen production in endometrial cancer tissue is biologically important and that aromatase in cancer cells may contribute partially to cell proliferation if androgen substrate is provided.

The Behavior of Endometrial Hyperplasia: A Prospective Study

Objective: To clarify the behavior of endometrial hyperplasia in a prospective study.

Possible mechanism of steroid action of the plant herb extracts glycyrrhizin, glycyrrhetinic acid, and paeoniflorin: Inhibition by plant herb extracts of steroid protein binding in the rabbit

To assess the action of some components of herbal medicine, glycyrrhizin, glycyrrhetinic acid, and paeoniflorin, on steroids, their binding to several classes of intracellular and serum steroid-binding proteins were studied in the rabbit. The affinity (inhibitor constant) for binding of dihydrotestosterone-sex hormone binding globulin in the serum (dissociation constant of 2.0 nmol/L for dihydrotestosterone) was approximately 520 nmol/L, and that for the binding of cortisol-corticosteroid-binding globulin in the serum (dissociation constant of 2.0 nmol/L for cortisol) was approximately 10 mumol/L. In the uterine cytosol, the inhibitor constant value for estradiol receptor binding (dissociation constant of 1.0 nmol/L) was 0.9 mumol/L, and these compounds did not influence progestin receptor binding (dissociation constant of 1.4 nmol/L). The inhibitor constant values for glucocorticoid receptor binding (dissociation constant of 1.0 nmol/L) in the liver cytosol were 3.0 nmol/L for paeoniflorin, 2.0 nmol/L for glycyrrhizin, and 1.7 nmol/L for glycyrrhetinic acid, and those for mineralocorticoid receptor binding (dissociation constant = 1.1 nmol/L) in the kidney cytosol were 3.5 nmol/L for paeoniflorin and glycyrrhetinic acid and 3.0 nmol/L for glycyrrhizin. These results suggest that herbal extracts such as the above compounds influence steroid effects by glucocorticoid and mineralocorticoid receptors and to a lesser extent by estrogen receptors or serum sex hormone-binding globulin and corticosteroid-binding globulin.

Contribution of aromatase to the deoxyribonucleic acid synthesis of MCF-7 human breast cancer cells and its suppression by aromatase inhibitors ☆

We have studied the effects of various steroids on DNA synthesis in MCF-7 human breast carcinoma cells, which have aromatase activity and which exert an oestrogen receptor-mediated growth, to assess the significance of intracellular aromatase on growth stimulation as well as inhibition by aromatase inhibitors. The cells were cultured for 96 h in phenol red-free medium containing 10% charcoal-treated fetal bovine serum and test reagents and pulse-labelled with [3H]thymidine. Physiological concentrations of oestradiol, oestrone, testosterone (T) and androstenedione (AD) stimulated thymidine incorporation. However, oestrone-sulphate and dihydrotestosterone (DHT) only stimulated at concentrations greater than the physiological levels. T and DHT stimulation was blocked by tamoxifen, but not by cyproterone acetate, suggesting that the stimulation was mediated via the oestrogen receptor but not by the androgen receptor. Stimulation by T and AD was reduced by aminoglutethimide and 14 alpha-hydroxy-4-androstene-3,6,17-trione, both of which inhibit aromatase activity, however, stimulation by nonaromatizable DHT was not reduced by the inhibitors, suggesting that androgens were converted by the intracellular aromatase to oestrogens which stimulated the thymidine incorporation. It is suggested that intracellular aromatase significantly contributes to the stimulation of DNA synthesis and that aromatase inhibitors suppress the stimulation.

Estradiol-17β- progesterone and 5α-dihydrotestosterone receptors of uterine myometrium and myoma in the human subject

Steroid receptors in human uterine myomas and the corresponding myometria were characterized and investigated to clarify their relation to tumor growth. The 248,800 g supernatant of tissue homogenates was used, in which steroid receptors were characterized and determined by Scatchard plot analysis using dextran coated charcoal at 4°C. In cytosols of both myoma and moymetrium, the estrogen receptors (ER) were sedimented at approximately the 7S, 5S and 4S regions, and the estradiol-17β(E2)-ER complex had a dissociation constant (KD) = 4.5 × 10−10M. Progesterone receptors (PR) were sedimented at approximately the 7S and 4–5S regions. The KD of the progesterone-PR complex was 1.5 × 10−9M and the KD of the R5020-PR complex was 5.0 × 10−10M. Androgen receptors (AR) were sedimented at approximately the 6–7S and 5S regions and dihydrotestosterone (DHT)-AR complex had a KD = 4.0 × 10−10M. All of steroid-7S binding in the cytosol were easily degraded during 5–20% sucrose gradient centrifugation. Ligand specificity studies characterized the specificity of receptors. There was no significant difference in steroid receptor levels (expressed as fmol/mg protein) between myoma and the corresponding myometrium; however if the receptor level was expressed as fmol/μDNA, the estrogen receptor level in the myometrium was higher than that in the myoma (P < 0.05). There was no significant difference in mitotic index between the two tissues.

Relationship between aromatase activity and steroid receptor levels in ovarian tumors from postmenopausal women

Aromatase activity, as well as steroid receptors, exists in nonfunctional ovarian tumors. Steroid receptor status has been reported to be related to prognosis in ovarian cancer patients. We determined aromatase activity and progesterone receptor (PR) and estrogen receptor (ER) levels in 43 ovarian tumors obtained from postmenopausal women. Aromatase activity was detected in 35 tumors (81%), PR in 21 tumors (49%) and ER in 13 tumors (30%). Eighty-three percent (10/12) of mucinous cystadenoma tissues showed positive PR with high aromatase activity, while 93% (13/14) of malignant tumors showed negative PR and low aromatase activity. Aromatase activity was detected in 95% (20/21) of PR-positive tumors, being greater than in PR-negative tumors (P < 0.002). There was a positive correlation between aromatase activity and PR (rs = 0.49, P < 0.001). However, there was no correlation between aromatase activity and ER. In 17 patients (43%), the serum estradiol level was higher than 30 pg/ml and there was a positive correlation among estradiol, estrone, androstenedione and testosterone. However, serum steroid levels were not correlated with aromatase activity, PR or ER. Aminoglutethimide inhibited aromatase activity of benign and malignant ovarian tumors, uterine myoma, choriocarcinoma cells and purified human placental P-450arom in a similar manner. These results suggest that aromatase activity is correlated with PR in ovarian tumors of postmenopausal women. In addition to steroid receptor status, aromatase activity may be a useful prognostic factor in ovarian cancers.

Danazol binding to steroid receptors in human uterine endometrium

To understand the mechanism of action of danazol, the binding of danazol to multiple classes of intracellular steroid binding proteins was studied in the human uterine endometrium. Danazol bound to endometrial receptors for estrogen, progesterone, and androgen and seemed to bind to endometrial intracellular corticosteroid-binding globulin and sex-hormone-binding globulin. Danazol occupies almost all binding sites of steroids in the steroid target cells in spite of the presence of endogenous steroids. It is speculated that the binding behavior of danazol may be related to its therapeutic effect on endometriosis.