Biomedical subjects

S J Santner

Publications and source records attributed to S J Santner.

At least 37 records · Page 2Linked to original sources

PubMed1993

Estrone sulfate promotes human breast cancer cell replication and nuclear uptake of estradiol in MCF-7 cell cultures.

Estradiol levels in breast tumors from post-menopausal women are similar to those in pre-menopausal women even though plasma estrogens are much lower after the menopause. In situ estrogen production by the tumor provides a potential means of maintaining high estradiol levels in post-menopausal breast cancer tissue. The estrone sulfatase pathway has been proposed as the mediator of in situ estrogen production. A number of studies suggest that estrone sulfate may be converted into estradiol in breast tumors via the catalytic activity of estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase. However, these studies used pharmacologic levels of estrogen sulfates and have not shown that physiologic levels can support biologic effects. Accordingly, the present study examined the dose relationship of estrone sulfate to a variety of biologic endpoints in MCF-7 breast cancer cells in culture. These cells converted physiologic concentrations of estrone sulfate to quantities of free estradiol capable of stimulating cell growth. Under these conditions, the nuclear steroids observed were free estrone and estradiol. Increase in cell number after 6 days of exposure to steroid required 100 nM estrone sulfate. However, S-phase, a more sensitive measure of cell proliferation, was stimulated by 0.1 nM estrone sulfate, a clearly physiologic concentration. Stimulation of estrogen-dependent protein markers such as pS2 and progesterone receptor required much higher concentrations of estrone sulfate. These effects were mediated through the estrogen receptor since the pure anti-estrogen, ICI 164384, blocked all effects produced by estrone sulfate. While it has been suggested that anti-estrogens may partly exert their effects by inhibition of sulfatase and 17 beta-hydroxysteroid dehydrogenase, this did not occur under our experimental conditions. These data provide evidence of the relevance of the estrone sulfatase pathway since biologic effects can be demonstrated in response to physiologic concentrations of estrone sulfate.

Biological Transport↗

PubMed1993

Inhibition of estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase by antiestrogens.

Circulating estrone sulfate levels are 10-fold higher than the free estrone and estradiol levels in postmenopausal women and could form a reservoir from which the free estrogens could be synthesized in situ in breast cancer tissues. The enzymes catalyzing conversion of estrone sulfate to free estrone and estradiol are estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase, respectively. Selective blockade of these two enzymes may provide a means of reducing tumor estrogen levels and promoting tumor regression. The present study characterized the kinetics of several potential inhibitors of estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase in vitro in rat breast tumors and compared these effects to those in human tissues. The antiestrogen ICI 164384 as well as tamoxifen and its metabolites inhibit estrone sulfatase via noncompetitive mechanisms at Kis ranging from 11-1130 microM in rat breast tumors. The steroid sulfates (pregnenolone sulfate and dehydroepiandrosterone sulfate) on the other hand, act as competitive inhibitors with Kis ranging from 4 to 6 microM. ICI 164384 and the tamoxifen metabolite 4-hydroxytamoxifen also blocked 17 beta-hydroxysteroid dehydrogenase at concentrations of 470 and 275 microM, respectively. In human breast tumors, 4-hydroxytamoxifen and desmethyltamoxifen blocked estrone sulfatase and 17 beta-hydroxysteroid dehydrogenase but at higher concentrations than in the rat (i.e. IC50s of 1000-2000 microM). The inhibition caused by the antiestrogens requires concentrations at least 100-fold higher than those necessary for antiestrogenic effects. Although blockade of enzyme action is significant in vitro, and could also be in vivo, the effects of antiestrogens on enzyme inhibition are likely to be outweighed by their ability to block estrogen receptor-mediated effects in patients.

17-Hydroxysteroid Dehydrogenases↗

PubMed1993

Effect of androstenedione on growth of untransfected and aromatase-transfected MCF-7 cells in culture.

Aromatase is present in human breast tumors and in breast cancer cell lines suggesting the possibility of in-situ estrogen production via the androstenedione to estrone and estradiol pathway. However, proof of the biologic relevance of aromatase in breast cancer tissue requires the demonstration that this enzyme mediates biologic effects on cell proliferation. Accordingly, we studied the effects of the aromatase substrate, androstenedione, on the rate of proliferation of wild-type and aromatase-transfected MCF-7 breast cancer cells. Androstenedione did not increase cell growth in wild-type MCF-7 cells which contained relatively low aromatase activity and produced 4-fold more estrone than estradiol. In contrast, aromatase-transfected cells contained higher amounts of aromatase, produced predominantly estradiol, and responded to androstenedione with enhanced growth. An aromatase inhibitor fadrozole hydrochloride, blocked the proliferative effects of androstenedione providing evidence for the role of aromatase in this process. As further evidence of the requirement for aromatase, cells transfected with the neomycin resistance expression plasmid but lacking the aromatase cDNA did not respond to androstenedione. These studies provide evidence that aromatase may have a biologic role for in-situ synthesis of estrogens in breast cancer tissue.

Analysis of Variance↗

PubMed1993

Pseudoaromatase in circulating lymphocytes.

A variety of data suggesting a relationship between estrogens and the immune system prompted a study of aromatase activity in blood lymphocytes. A tritiated water aromatase assay detected activity of 1.9 to 25.1 pmol/g protein/h in 14 samples of human lymphocytes. To confirm these results, additional tritiated water and product isolation assays were performed on a large pool of lymphocytes obtained from 4 U of blood. An assay using [1 beta-3H]androstenedione generated high apparent aromatase activity, 943 pmol/g protein/h, but this activity could not be blocked by the aromatase inhibitor, CGS 16949A. More direct methods of evaluation yielded the following results: (1) PCR demonstrated no aromatase mRNA production in lymphocytes; (2) direct product isolation using [1,2,6,7-3H]androstenedione yielded insignificant production of estrone and estradiol; (3) immunostaining of fixed lymphocyte smears with a polyclonal antibody to aromatase yielded equivocal results. These data suggest the presence of pseudoaromatase in blood lymphocytes. Since circulating lymphocyte pseudoaromatase levels can be correlated with various factors in patients, such as age, menopausal status, and glucose ingestion, further studies of this activity are warranted.

Androstenedione↗

PubMed1992

Prognostic value of breast cancer aromatase.

The estrogen receptor, progesterone receptor, and intratumoral aromatase content of 127 breast carcinomas were determined. Patients whose tumor contained either estrogen or progesterone receptors had a longer disease-free interval, but no difference in survival was observed. Measurable aromatase activity was detected in 78 of 113 (69%) tumors. There was no relationship between aromatase activity and disease-free interval or survival.

Aromatase↗

PubMed1990

Estrone sulfate stimulates growth of nitrosomethylurea-induced breast carcinoma in vivo in the rat.

Concentrations of estradiol in human breast tumors from pre- and post-menopausal women are similar whereas plasma levels are 5- to 60-fold lower in post-menopausal women. The mechanism for maintaining high tumor tissue estrogen levels in post-menopausal women is unknown but could be related to the ability of plasma estrone sulfate to serve as a precursor for estradiol synthesis in tumor tissue. Estrone sulfate plasma levels are 30-fold higher than free estradiol levels in post-menopausal women and estrone sulfatase is present in many tissues, including breast tumors, supporting this hypothesis. In this study, we examined the ability of exogenously administered estrone sulfate to stimulate growth of a carcinogen-induced, hormone-dependent rat mammary tumor and measured the rate of conversion of estrone sulfate to free estrone and estradiol. Castrate rats bearing nitrosomethylurea-induced mammary tumors were infused with estradiol as a control or estrone sulfate over a 14-day period. Estradiol at low doses significantly increased tumor volume whereas higher amounts paradoxically inhibited growth. By comparison, estrone sulfate infusions significantly increased tumor volume over that observed in castrate animals on both days 7 and 14 of infusion. To determine whether estrone sulfate was converted to free estrone and estradiol during this protocol, 3H-estrone sulfate was substituted for unlabelled steroid and castrate animals were again infused for 14 days. At 7, 10 and 14 days of infusion, 18-26% of estrone sulfate was converted to free estrone and 9-16% to free estradiol. There were no significant differences between the 2 doses used and the rates of conversion were stable over the infusion period. Conversion of estrone sulfate to free estradiol was also demonstrated by radioimmunoassay of free estradiol in plasma during estrone sulfate infusions. These data demonstrate that exogenously administered estrone sulfate can stimulate mammary tumor growth in castrate animals and support the possibility that estrone sulfate may serve as an important source of tumor tissue estradiol.

Animals↗

PubMed1990

Potency and specificity of CGS-16949A as an aromatase inhibitor.

Inhibitors of aromatase are useful for treatment of estrogen-dependent breast cancer and as probes for study of normal physiology. Inhibitors of this enzyme with more favorable properties are necessary since the most extensively utilized inhibitor, aminoglutethimide, lacks specificity and causes frequent side effects. The present study compared the potency and specificity of a new aromatase inhibitor, CGS-16949A, with that of aminoglutethimide in a variety of in vitro enzyme preparations. CGS-16949A blocked aromatase by 50% in human breast cancer homogenates, live breast cancer cells, human placental microsomes and porcine ovarian microsomes at concentrations of 0.008 to 0.02 microM. In contrast, concentrations of 10-25 microM of aminoglutethimide were required to inhibit aromatase similarly in these tissues. For human placental microsomes, the Ki for CGS 16949A was 0.17 nM and for aminoglutethimide, 0.54 microM. Preincubation studies indicated that CGS-16949A acts by a competitive inhibitory mechanism and not by "suicide inhibitory" properties. With respect to specificity, CGS-16949A had no effect on cholesterol side-chain cleavage activity in rat testicular mitochondria. A 54% reduction in enzyme activity was observed in adrenal mitochondria but only at concentrations five orders of magnitude higher (i.e. 100 microM) than required to inhibit aromatase. In contrast, 10 microM concentrations of aminoglutethimide blocked cholesterol side-chain cleavage activity in rat testicular and adrenal mitochondria by 67 and 91%, respectively. These data suggest that CGS-16949A has favorable properties as a specific and potent aromatase inhibitor.

Animals↗

PubMed1988

Correlation of aromatase activity with histological differentiation of breast cancer--a morphometric analysis.

Differentiation is a term that indicates the degree to which a tumor resembles histologically the tissue or cell of origin. A system to quantitate the proportion of breast cancer cells participating in glandular differentiation or remaining within ducts was employed. The degree of tumor differentiation of 58 primary breast cancers was correlated with estrogen (ER) and progesterone receptors (PR) and tumor aromatase activity. There was a significant association between tumor differentiation (greater than or equal to 2% cancer cells exhibiting glandular differentiation) and the presence of ER or PR in tumors. Conversely, there was no correlation between tumor differentiation and measurable tumor aromatase activity.

Aromatase↗

PubMed1988

Marked heterogeneity of aromatase activity in human malignant melanoma tissue.

The prognosis from human malignant melanoma varies according to sex and to multiple histologic, biologic and cell kinetic parameters. Thus melanomas exhibit a major degree of heterogeneity in their biologic properties and further characterization of their biochemical heterogeneity should yield important information. The present study sought to demonstrate the activity of a biochemical marker of estrogen synthesis, the aromatase enzyme, in melanoma tissue and to determine its range of activity. Initially, we validated a highly sensitive radiometric assay for aromatase by comparing it with a direct product isolation method. We detected production of 417 pmol/g protein/h of estrone and 37.3 pmol/g protein/h of estradiol by direct product isolation in a human melanoma and 398 pmol estrone/g protein/h by the radiometric assay. The activity present was blocked by similar amounts of the aromatase inhibitor, aminoglutethimide, as were necessary to block placental, breast cancer, and rat brain aromatase activity. We then assayed aromatase radiometrically in 19 human melanomas and found measurable activity ranging from 9 to 398 pmol estrone/g protein/h in 15 tissues. No relationship with the patient's age or sex was demonstrated. The activity exceeded by 2-fold that previously detected in 49/61 human breast cancers. This study identified a marker enzyme in melanoma tissue which varied by 40-fold among human tumors. Correlation of aromatase activity with prognosis and response to various types of therapy is now necessary to establish the biologic relevance of this finding.

Adult↗

PubMed1987

Aromatase activity in primary and metastatic human breast cancer.

Aromatase activity was measured in 104 primary and 24 metastatic breast cancer patients. The assay employed quantitates the production of 3H water release from 1 beta-[3H]androstenedione after aromatization. Of 104 human primary breast tumors studied, 64 contained measurable aromatase activity, ranging from 5-70.5 pmol estrone formed/g protein/hour. In primary breast cancers there was no difference in levels of aromatase activity when analyzed by menstrual status or age by decade. Aromatase activity was similar in small and large primary tumors. The median aromatase activity of primary breast tumors (8.6 pmol/g/h) was similar to that found in metastatic breast cancer deposits (12.0 pmol/g/h). Aromatase activity did not correlate with either estrogen (ER) or progesterone (PR) receptor concentration in the tissues assayed. In this regard there were 33 ER- PR- tumor biopsies. Twelve of these 33 tumors contained aromatase activity greater than 10 pmol/g/hour.

Adult↗

PubMed1987

Role of polyamines in the synthesis of prolactin-regulated growth factors by experimental breast cancer in culture.

The present experiments were designed to test whether polyamines play an essential role in the synthesis of growth factors induced by ovine prolactin (oPRL), using the N-nitrosomethylurea (NMU)-induced rat mammary tumor cultured in the soft agar clonogenic assay. Conditioned media (CM) obtained from tumors treated with oPRL and the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) (1 mM) no longer exerted the colony-stimulating effect which was observed with oPRL-CM. Such growth-promoting activity was restored with conditioned media obtained from tumors treated with oPRL, DFMO, and increasing concentrations of spermidine from 1 to 500 microM. The colony-stimulating effect of the CM employed could not be accounted for by the contaminating presence in the media of oPRL, DFMO, and polyamines. These results indicate that in our system polyamines play an important role in the synthesis of oPRL-regulated growth factors.

Animals↗

PubMed1987

Inhibition of aromatase as treatment of breast carcinoma in postmenopausal women.

Recent treatment strategies have been directed toward blockade of estrogen action or inhibition of estrogen biosynthesis as a means of inducing regression of hormone-dependent breast cancer. The major source of estrogen in postmenopausal women is the peripheral conversion of androstenedione to estrone through the enzyme aromatase. It is known that aromatase activity increases proportionately with degree of obesity in women. To test the importance of this modulatory factor, we correlated body weight with estrogen excretion in our population of patients with breast cancer and found significant relationships. In situ production of estradiol from plasma precursors within breast cancer tissue may provide another source of estrogen. Major enzymes mediating estrogen biosynthesis were found to be present in tumor biopsy specimens. Aromatase activity was found to be present in 48/61 human tumors, sulfatase in 35/35, and 17 beta -hydroxysteroid dehydrogenase in 41/41. One inhibitor of aromatase, aminoglutethimide, has been extensively studied in patients with breast cancer. The additional effects of this drug on cholesterol side-chain cleavage and on 11-hydroxylase activity require coadministration of replacement glucocorticoid in treatment regimens. In pilot trials, 37% of patients experienced objective tumor regression with a combination of 1000 mg aminoglutethimide and 40 mg hydrocortisone daily. In randomized clinical trials with this regimen, aromatase inhibition with aminoglutethimide produced tumor regression with similar frequency as did surgical hypophysectomy, surgical adrenalectomy, or tamoxifen administration. The side effects of aminoglutethimide, including lethargy, skin rash, and ataxia complicate its use even though these problems are generally transient. Regimens of low-dose aminoglutethimide are being developed to reduce these side effects. Low-dose aminoglutethimide appears to block aromatase effectively and to have limited side effects, and is undergoing extensive clinical trial. A more specific aromatase inhibitor, 4-hydroxyandrostenedione, is now also being tested clinically, whereas MDL 18962, another new selective inhibitor, is undergoing study in animals.

Aminoglutethimide↗

PubMed1986

Estrone sulfate: a potential source of estradiol in human breast cancer tissues.

Local formation of estradiol in human breast tumors could provide a more important source of estrogen than is delivered from plasma. Prior studies have suggested that estrone is primarily synthesized from estrone sulfate. The enzyme 17 beta-hydroxysteroid dehydrogenase (HSD) would be required to convert estrone to estradiol. This study characterized HSD in 1000 X g supernatants from human breast tumors. Estradiol synthesis was linearly related to tissue concentration or time over the range studied. Cofactor requirements varied with estrone concentration. High and low affinity sites were found in 50% of tissues studied, while the remainder contained only low affinity sites. Screen assays showed measurable activity in all 42 samples tested. This activity ranged from 0.73- greater than 100 nmol estrone synthesized/g protein/hr, with a median activity of 5.9 nmol/g/hr. We evaluated the biological relevance of the sulfatase-HSD pathway by testing the ability of estrone sulfate to stimulate colony formation in soft agar cultures of nitrosomethylurea-induced rat mammary tumors. The maximally effective concentration ranged from 10(-7) to 10(-4)M. Significant stimulation of colony formation was observed in 7 of 8 experiments. The estrone sulfate stimulation pattern was similar to that previously observed with estradiol. Of the 3H-estrone sulfate added to the dishes, 20-98% was recovered as estrone and 0.2-6% as estradiol. These studies suggest that the requisite enzymes are present in human breast tumors for conversion of estrone sulfate to estradiol, and that this pathway may be biologically significant.

17-Hydroxysteroid Dehydrogenases↗

PubMed1986

The assessment of diminished testicular function in boys of pubertal age.

Nine boys with anorchia and 9 individuals with diminished testicular function were followed between ages 10 and 18 with serial determinations of urine gonadotrophins and serum testosterone. Ten hCG stimulation tests were performed in eight of these patients. Adult levels of LH (greater than 500 mIU/h) and low measurements of serum testosterone (less than 54 ng/dl) confirmed the anorchic state. Boys with diminished testicular reserve exhibited urinary gonadotrophin changes similar to peripubertal anorchic patients but testosterone levels increased progressively with age (90-715 ng/dl). FSH attained adult castrate levels (greater than 1700 mIU/h) by age 13 in these boys and served to distinguish them from normal controls. The longitudinal assessment of basal hormone levels can separate anorchia from diminished testicular reserve in boys of pubertal age and testing with hCG is not required for evaluation.

Adolescent↗

PubMed1986

Enzymatic control of estrogen production in human breast cancer: relative significance of aromatase versus sulfatase pathways.

One-third of the cases of breast cancer in postmenopausal women are hormone-dependent and the lesions regress upon treatment with antiestrogens or inhibition of estrogen biosynthesis. In these patients, estrogens are synthesized in extraglandular tissues from adrenal precursors and re-enter plasma to produce estrone levels of 52 +/- 6.5 pg/ml (mean +/- SEM) and estradiol concentrations of 13.1 +/- 0.7 pg/ml. However, the fact that the levels of estrogen in breast tumor tissue are an order of magnitude higher than plasma levels suggested the possibility of in situ estrogen production. To address this possibility, we measured several enzymes involved in estradiol biosynthesis in human tumors. Forty-eight of 61 tumors contained aromatase (estrogen synthetase) activity ranging from 5-80 pg/gm protein per hour. By comparison, the levels of estrone sulfatase were 10(6) higher, ranging from 0.8-125 micrograms/gm protein per hour. Because the sulfatase enzyme was of lower affinity (i.e., Km = 27 microM) than that of aromatase (i.e., 0.027 microM), the amount of estrogen formed under conditions of similar substrate concentrations was compared and found to be 10-fold higher via the sulfatase enzyme. In 41 additional tumors, the 17 beta-hydroxysteroid dehydrogenase enzyme, catalyzing the conversion of estrone to estradiol, was uniformly present. To test the biologic relevance of the estrone sulfate to estrone to estradiol pathway, estrogen-dependent nitrosomethylurea rat mammary tumors were grown in soft agar in the presence of estrone sulfate. Concentrations of estrone sulfate of 10(-6) microM significantly (p less than 0.01) stimulated colony formation in this system in which 75.5-98.6% of estrone sulfate was converted to estrone and 0.2 to 6% to estradiol. These data support the hypothesis that mammary carcinomas can synthesize estradiol in situ from circulating estrogen precursor and that local conversion is biologically important. On the basis of comparative data, the estrone sulfate to estrone to estradiol pathway is quantitatively more important than that involving androstenedione to estrone to estradiol.

17-Hydroxysteroid Dehydrogenases↗

PubMed1986

Inhibition of testosterone production with ketoconazole alone and in combination with a gonadotropin releasing hormone analogue in the rat.

Ketoconazole is a well-tolerated, synthetic, imidazole derivative currently in widespread therapeutic use against mycotic infections. Recent evidence that it depresses testosterone synthesis in humans prompted us to investigate the effects in rats of its administration alone or in combination with the gonadotropin releasing hormone superagonist analogue leuprolide. Plasma luteinizing hormone, testosterone, and ketoconazole levels as well as ventral prostate weight and tumor growth in rats bearing the androgen-dependent Dunning R3327H model of prostate adenocarcinoma were measured. Doses of 30 mg/kg twice daily of ketoconazole alone depressed plasma testosterone levels by approximately 75% to a nadir of 0.47 +/- 0.08 (SE) ng/ml on day 20 (P less than 0.001 versus basal). This effect of ketoconazole was exerted directly at the testicular level since plasma luteinizing hormone levels were not suppressed. In response, ventral prostate weight declined and growth of the Dunning R3327H tumor was retarded to rates observed in castrate controls. Leuprolide alone lowered basal testosterone levels to 0.20 +/- 0.02 ng/ml after 35 days of daily administration but persistent androgen increments after each injection (acute-on-chronic effect) were observed (i.e., to 4.41 +/- 0.62 ng/ml). The addition of ketoconazole to leuprolide inhibited the acute-on-chronic rise in testosterone to 0.33 +/- .07 ng/ml and also lowered basal testosterone levels further to 0.11 +/- 0.01 on day 10 of combined administration. Ketoconazole also blunted the response to the first injection of leuprolide from a 3-h peak level of 8.74 +/- 0.53 to 4.17 +/- 0.80 with the 40 mg/kg dose. These results indicate that combining ketoconazole with leuprolide achieves greater suppression of testosterone than either agent alone. When such protocols are applied to humans with prostate cancer, more extensive effects may be expected because of the greater sensitivity of patients than of the rodent species to these agents.

Animals↗

PubMed1985

Effective inhibition by low dose aminoglutethimide of peripheral aromatization in postmenopausal breast cancer patients.

Aminoglutethimide without glucocorticoid has been shown to be a clinically effective treatment for postmenopausal breast cancer in low dosage (250 mg day-1). The mechanism of action of this approach is thought to be the inhibition of peripheral aromatase, the enzyme which converts androstenedione to oestrone. The activity of this enzyme was measured in vivo by injection with 3H-androstenedione and 14C-oestrone and found to be 0.20% +/- 0.05 in 5 patients on low dose AG therapy. In comparison with previously published data this demonstrates a 92% inhibition of peripheral aromatase activity confirming aromatase inhibition as a viable aim in the endocrine treatment of breast cancer.

Aged↗

PubMed1985

Response of the prepubertal ovary to acute chorionic gonadotropin administration: absence of modulation by growth hormone.

Twenty-five short term hCG stimulation tests were performed in seven prepubertal girls, aged 3-11 yr, who were being evaluated for short stature. Provocative testing revealed GH deficiency in all patients, but reevaluation of one girl at a later date showed normal somatotropin levels. The study protocol lasted 18 months and included testing before, during, and after 1 yr of GH therapy. Delta 4-Androstenedione, testosterone, estrone, and estradiol were determined 0, 24, 48, and 72 h after initiation of a two-injection course of CG. Significant responses (approximately 2-fold over baseline) to the stimulation tests occurred for all steroids except testosterone, though no augmented effects were found in the presence of human GH. The results indicate functional capability of the prepubertal ovary when exposed acutely to a LH-like material, but no role for somatotropin in gonadal steroid production in the prepubertal female.

Androstenedione↗