Bianca Marchetti

Modulation of hippocampal LHRH receptors by sex steroids in the rat

The modulation of brain LHRH receptors by sex steroids was assessed in the female rat hippocampus using in vitro autoradiography and iodinated [D-Ser (TBU)6, des-Gly-NH2(10)]LHRH ethylamide as the radioligand. As evaluated by optical densitometry, the density of hippocampal LHRH receptors was increased by castration. In castrated animals, estradiol administration produced a small decrease in receptor concentration, whereas the concomitant administration of progesterone and estradiol as well as the injection of dihydrotestosterone induced a marked decrease in receptor density. Progesterone administration did not produce any significant change in receptor concentration. These results indicate that brain LHRH receptors can be modulated by sex steroids. Interestingly, this modulation of hippocampal LHRH receptors is similar to that previously observed in pituitary gland.

Adrenal steroids stimulate growth and progesterone receptor levels in rat uterus and DMBA-induced mammary tumors

We have studied the effect of treatment with the adrenal steroids androst-5-ene-3β,17β-diol (Δ5-diol) and dehydroepiandrosterone (DHEA) on the growth and progesterone receptor levels of dimethylbenz-(a)anthracene (DMBA)-induced mammary tumors in the rat.While the total number of tumors in ovariectomized animals was 0.60 ± 0.19 tumor per rat after 24 days, it increased to 2.54 ± 0.50 (p<0.01) and 1.42 ± 0.26 (p<0.01) in the Δ5-diol and DHEA (2 mg, twice daily) treated animals, respectively. While very few new tumors developed during a 24-day period in ovariectomized animals (0.07 ± 0.07/rat), an average of 0.47 ± 0.19 (p<0.05) new tumor per animal appeared in intact rats. In ovariectomized animals treated with Δ5-diol or DHEA, the numbers of new tumors were 0.77 ± 0.26 (p<0.05) and 0.42 ± 0.15 (p<0.05), respectively. An even more striking effect was observed on average total tumor area, which decreased from 4.70 ± 0.95 cm2 in intact animals to 0.75 ± 0.27 cm2 (p<0.01) following ovariectomy. Values of 9.79 ± 2.25 (p<0.01) and 3.93 ± 0.86 cm2 (p<0.01) were found in the Δ5-diol- and DHEA-treated ovariectomized animals, respectively.Treatment of ovariectomized animals with Δ5-diol and DHEA caused a marked increase (p<0.01) in progesterone receptor levels in both the uteri and DMBA-induced mammary tumors. Uterine weight was also stimulated (p<0.01) by treatment with the two adrenal steroids.The present data show that two adrenal C19-Δ5 steroids, Δ5-diol and DHEA, possess stimulatory effects analogous to those of estrogens on DMBA-induced mammary tumor growth and progesterone receptor levels in the rat, thus supporting the suggestion of an important role of these adrenal steroids in breast cancer and other estrogen-sensitive diseases in the human.

Characteristics of flutamide action on prostatic and testicular functions in the rat

The effect of daily treatment with the pure antiandrogen Flutamide has been studied either alone or in combination with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide (LHRH-A), on testicular and prostatic functions in adult male rats. Treatment for 10 days with Flutamide (5 mg/rat, twice daily) caused a marked stimulation of plasma testosterone (T) associated with a significant increase in plasma gonadotropin concentrations and inhibited plasma PRL levels. Testicular weight is not changed following antiandrogen administration but testicular LH/hCG receptor levels are markedly decreased with no change in FSH receptor levels. Moreover, Flutamide treatment alone produces an important inhibition of ventral prostate and seminal vesicle weights associated with a significant decrease in prostatic beta-adrenergic receptor levels but no change is observed in specific ornithine decarboxylase (ODC) activity. Daily LHRH-A treatment at the dose of 1 microgram/day for 10 days decreases plasma T to levels comparable to those found in orchiectomized men (0.30 +/- 0.5 ng/ml). This effect is associated with an almost complete loss of testicular LH/hCG receptors, a decrease in testicular weight, a significant increase in plasma gonadotropins and a marked inhibition of plasma PRL concentration. A relatively smaller inhibition of ventral prostate and seminal vesicle weights follows treatment with the LHRH agonist alone, this effect being accompanied by a significant reduction in beta-adrenergic receptor concentration but no change in prostatic ODC activity. Combination of the two drugs, however, caused a potent inhibitory effect on both ventral prostate and seminal vesicle weight to values similar to those found in castrated rats. The prostatic weight loss is accompanied by a marked fall in ODC activity and in the concentration of beta-adrenergic receptors. The present data clearly show that combined treatment with an LHRH agonist and a pure antiandrogen is highly effective in inhibiting, not only prostatic growth, but also two androgen-sensitive parameters of prostatic activity.

Beta-adrenergic receptors in DMBA-induced rat mammary tumors: Correlation with progesterone receptor and tumor growth

SummaryIn order to gain further knowledge about the potential role of catecholamines in mammary carcinoma, we have used the potent β-adrenergic antagonist cyanopindolol (CYP) as iodinated ligand to characterize β-adrenergic receptors in membranes prepared from mammary tumors induced by dimethylbenz(a)anthraene (DMBA) administration in the rat. The binding of [125I]CYP to membrane preparations of DMBA-induced rat mammary tumors is rapid at room temperature, reaching half maximal specific binding at 30 min of incubation. Scatchard analysis of the data indicates that [125I]CYP binds to a single class of high affinity sites (114 ± 2.1 fmoles/mg protein) at an apparent KD value of 38.0 ± 0.3 pM. The order of potency of a series of agonists to compete for [125I]CYP binding is consistent with interaction with a β2-subtype receptor: zinterol > (−)isoproterenol > (−)epinephrine» (−)norepinephrine. In addition, the potency of a series of specific β1, and β2 synthetic compounds to displace [125I]CYP in mammary tumors is similar to their potency in typical β2-adrenergic tissues. The binding of [125I]CYP to DMBA-induced rat mammary tumors shows a marked stereoselectivity, the (−)isomers of isoproterenol and propranolol being 150 and 80 times more potent, respectively, than their respective enantiomers. The autoradiographic localization of [125I]CYP performed on frozen sections revealed the presence of specific β-adrenergic receptors in all the malignant cells. Spontaneous mammary tumors of aging (18–22 months) female rats have high levels of β-adrenergic receptors. Castration decreased the concentration of [125I]CYP binding sites in DMBA-induced mammary tumors. A close correlation was observed between progressing, static, and regressing tumors after ovariectomy and β-adrenergic receptor concentration. The presence of β-adrenergic receptors in mammary tumors as well as the modulation of their level by ovarian hormones provides a mechanism for catecholaminergic influence in mammary cancer tissue.

Effects of the aromatase inhibitor 4-hydroxyandrostenedione and the antiandrogen flutamide on growth and steroid levels in DMBA-induced rat mammary tumors

SummaryUsing dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in the rat as model, comparison was made of the effect of treatment for 20 days with the aromatase inhibitor 4-hydroxyandrostenedione (4-OH-A) (7.5 mg, twice daily) or the antiandragen flutamide (5 mg, twice daily) on tumor growth as well as on plasma and tumor content of estrogens, androgens, and their precursors and metabolites. Tumor number and size were markedly decreased following treatment with either drug, the effect of treatment being more important on size than number, and on new tumors which developed during treatment than on tumors already present at start of treatment. Treatment with the aromatase inhibitor 4-OH-A caused a parallel decrease in plasma and tumor levels of pregnenolone (Preg), progesterone (P), and 17-OH P, while there was a marked increase in dehydroepiandrosterone (DHEA), androst-5-ene-3β,17β-diol (Δ5-diol), androstenedione (Δ4-dione), testosterone (T), androstane-3α, 17β-diol (3α-diol), and androstane-3β, 17β-diol (3β-diol), with no significant change in dihydrotestosterone (DHT) and 17β-estradiol levels. The marked increase in tissue T content coupled to a decrease in P levels could well contribute to the inhibition of tumor growth induced by 4-OH-A. Flutamide, on the other hand, caused a marked fall in plasma and tissue levels of Preg, 17-OH Preg, P, and 17-OH P, with no significant change in the concentration of the other steroids, thus suggesting a possible role of the fall in tissue P levels in the inhibition of tumor growth. Since both drugs are potent inhibitors of DMBA-induced tumor growth in intact animals, better knowledge of their mechanism of action should add to our understanding of the multiple endocrine factors controlling the growth of these tumors.

Specificity of the direct effect of an LHRH agonist on testicular 17-hydroxylase but not on 5α-reductase activity in hypophysectomized adult rats

The direct effect of treatment with a potent LHRH agonist on testicular steroidogenesis was studied by incubation of radioactive steroids with a testicular homogenate or with a suspension of interstitial cells obtained following 7 days of treatment of adult hypophysectomized male rats. The animals received [D-Ser(tBu)6,des-Gly-NH2(10)]LHRH ethylamide (25 micrograms) administered 3 times a day, hCG (5 or 25 IU) once daily or a combination of both drugs. The metabolism of tritiated progesterone into delta 4-metabolites by a suspension of interstitial cells was markedly reduced by treatment with the LHRH agonist (LHRH-A) alone or following combined treatment with hCG and LHRH-A. No formation of 5 alpha-reduced steroids was detected in the medium following incubation with testicular homogenate or interstitial cells. Similar findings were obtained by measurement of testicular steroid content. The present data demonstrate that the direct effect of the LHRH agonist is limited to the Leydig cells on 17-hydroxylase activity. This inhibitory effect is reflected by an accumulation of testicular pregnenolone and progesterone content and a marked inhibition of progesterone metabolism into delta 4-androgens. However, no stimulation of 5 alpha-reductase, an enzyme localized in seminiferous tubules, could be detected. Such data show clear differences between the direct and the pituitary-mediated effects of treatment with LHRH agonists on testicular steroidogenesis. While the LHRH agonist administered at high doses in the rat can directly inhibit 17-hydroxylase activity, the stimulatory effect on 5 alpha-reductase activity is regulated by another mechanism.

Combination Therapy with the Antiandrogen Flutamide and the LHRH Agonist [D-Trp6, des-Gly-NH2 10]LHRH Ethylamide in Prostate Cancer: Rationale and 5-Year Clinical Experience

Man is unique among species in having adrenals which secrete large amounts of precursor steroids which are converted into active androgens in the prostate and other peripheral tissues (Acevedo and Goldzieher, 1965; Harper et al., 1974; Cameron et al., 1966; Voigt and Bartsch, 1986).