Gonadotropin-releasing hormone (GnRH) receptors in tumors: a new rationale for the therapeutical application of GnRH analogs in cancer patients?

Gonadotropin-Releasing Hormone (GnRH) is the hypothalamic decapeptide which plays a key role in the control of reproductive functions. By binding to specific receptors present on the pituitary gonadotropes, GnRH regulates gonadotropin release and, consequently, steroid hormone secretion from the gonads. When given continuously and at high doses, GnRH agonists suppress the pituitary gonadal axis through the down-regulation and desensitization of its own receptors. Based on this rationale, pituitary GnRH receptors represent the target for the successful utilization of GnRH agonists (that are more stable than the native peptide) for the treatment of hormone-dependent tumors (e.g., prostate, breast, endometrial, ovarian cancers). The observation that GnRH receptors are expressed in steroid-dependent tumors, and that their activation reduces cell proliferation and metastatic behavior of cancer cell lines, both in vitro and in vivo (when inoculated into nude mice), indicates a possible additional and more direct antitumor activity for these compounds. Interestingly, GnRH receptors have been shown to be expressed also in androgen-independent prostate carcinoma, as well as in tumors that are not classically considered hormone-related (e.g., melanoma), suggesting a clinical utility of the administration of GnRH analogs also in these tumors. More recently, GnRH agonists have been proposed as useful carriers to target cytotoxic drugs or toxins to cancer cells displaying the specific GnRH receptors. A second form of GnRH (designated GnRH-II) has been discovered in most vertebrates, including humans. GnRH-II has been suggested to act through a 'putative' cognate type II GnRH receptor, which is distributed in different tissues, both normal and tumoral. In humans, a full-length functional type II GnRH receptor has not been found. Therefore, its functions as well as its possible utility as a molecular target for a GnRH-II based therapy in oncology still has to be clarified. This review will focus on the role of GnRH receptors in the control of tumor growth, progression and dissemination. It will also be discussed whether the presence of these receptors might represent an additional rationale for the clinical utility of GnRH analogs as anticancer drugs.

Inhibitory activity of luteinizing hormone-releasing hormone on tumor growth and progression.

Luteinizing hormone-releasing hormone (LHRH) is the key hormone in the control of reproductive functions. In recent years, it has become evident that LHRH might act as a growth modulatory factor in tumors of the reproductive system. We have shown that in prostate cancer cells LHRH is expressed, together with its receptors, to negatively regulate cell proliferation. In these cells, LHRH acts as an antimitogenic factor through the activation of the Gi-cAMP intracellular signaling pathway. More recently, we investigated whether an LHRH-based autocrine system might also be expressed in tumors that are not classically related to the reproductive tract, such as melanoma. Malignant melanoma is known to be characterized not only by a high proliferation rate, but also by an aggressive metastatic behavior. We have demonstrated that both LHRH and LHRH receptors are expressed in human melanoma cells (BLM and Me15392). Activation of LHRH receptors by means of a potent LHRH agonist (Zoladex) significantly inhibited cell proliferation. The LHRH agonist also reduced the ability of melanoma cells to invade a reconstituted basement membrane (Matrigel) and to migrate in response to a chemotactic stimulus. These data indicate that: (a) in prostate cancer cells the LHRH receptor is coupled to a Gi-cAMP signal transduction pathway; (b) LHRH and LHRH receptors are also expressed in tumors that are not classically related to the reproductive system, such as melanoma; in melanoma cells, LHRH might act as an inhibitory factor on both cell proliferation and metastatic behavior. It is suggested that, in melanoma, LHRH receptors might represent a diagnostic marker and a possible molecular target for new therapeutic approaches for this pathology.

Oncostatic activity of a thiazolidinedione derivative on human androgen-dependent prostate cancer cells.

Thiazolidinedione derivatives with potent antiarthritic activity, such as CGP 52608, have been suggested to exert their biological effects through the activation of the orphan nuclear receptor RORalpha. Since response elements for this receptor are present in the promoter region of cell cycle-related genes (i.e., p21(WAF1/CIP1) and cyclin A), we reasoned that CGP 52608 might affect cell proliferation, cell cycle progression and the expression of cell cycle-related genes. This hypothesis has been verified in the human androgen-dependent prostate cancer cell line LNCaP. We found that the treatment of LNCaP cells with CGP 52608 brings about a significant and dose-dependent decrease of cell proliferation. Thiazolidinedione affected cell cycle distribution, inducing an accumulation of the cells in the G0/G1 phase and a decrease in the S phase. This effect was accompanied by an increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and a decreased expression of cyclin A. These data indicate that, in human androgen-dependent LNCaP prostate cancer cells, the thiazolidinedione derivative CGP 52608 exerts a strong cytostatic activity, by reducing cell proliferation and by affecting cell cycle distribution through the modulation of the expression of cell cycle-related genes. These biological actions of CGP 52608 might be mediated by the activation of the orphan nuclear RORalpha receptor, which is expressed in LNCaP cells.

LHRH analogues as anticancer agents: pituitary and extrapituitary sites of action.

Two classes of luteinising hormone-releasing hormone (LHRH) analogues have been developed so far to be used for oncological therapies: LHRH agonists and antagonists. LHRH agonists are widely and successfully used for the management of steroid-dependent malignancies. Chronic administrations of these compounds result in downregulation and desensitisation of pituitary LHRH receptors and, therefore, in a complete suppression of gonadal function. LHRH agonist administration is effective, safe and reversible, suffering only from the 'flare-up' phenomenon at the beginning of treatment. LHRH antagonists suppress the pituitary-gonadal function by competing with native LHRH for binding to its pituitary receptor but without giving rise to the intracellular cascade of events evoked by the natural hormone or LHRH agonists. Synthetic peptides belonging to the last generations of LHRH antagonists have already been successful in clinical trials. They are completely devoid of the 'flare-up' phenomenon and seem to be free of side effects, such as histamine release. Recently, the expression of LHRH and LHRH receptors has been reported in a number of hormone-responsive tumours. In contrast with the pituitary LHRH receptor which is coupled to the Gq/11-PLC intracellular system of events, stimulation of the tumour LHRH receptor by LHRH is followed by the activation of a Gi protein and a decrease in cAMP levels. This intracellular pathway mediates the inhibitory action of the autocrine/paracrine LHRH system on tumour cell proliferation. The activation of LHRH receptors at tumour level may then represent an additional and more direct mechanism of action for the antitumoural activity of LHRH agonists. Surprisingly, LHRH antagonists also exert a marked antimitogenic activity on a number of hormone-responsive cancer cell lines, indicating that these compounds might behave as antagonists at pituitary level and as agonists at the level of the tumour. The observation that the inhibitory LHRH autocrine system is also present in some steroid-unresponsive cancer cell lines might suggest a possible clinical utility of LHRH analogues also for those tumours that have escaped the initial phase of hormone dependency.

Activation of the orphan nuclear receptor RORalpha induces growth arrest in androgen-independent DU 145 prostate cancer cells.

BACKGROUND: RORalpha is a transcription factor which belongs to the family of orphan nuclear receptors. The regulatory functions of this receptor are still poorly understood. However, response elements for RORalpha are present on the promoter of cell cycle-related genes suggesting that it might be involved in the control of cell proliferation. In this study, we investigated the expression and the possible function of RORalpha in a human androgen-independent prostate cancer cell line (DU 145). The thiazolidinedione-derivative CGP 52608 has been utilized as the specific ligand and activator of RORalpha. METHODS: The effects of CGP 52608 on DU 145 cell proliferation and cell cycle distribution were analyzed by hemocytometer and by FACS analysis, respectively. The expression of RORalpha as well as the effects of RORalpha activation on the expression of cell cycle-related genes were evaluated by RT-PCR. To clarify whether RORalpha activation might affect the proliferation of prostate cancer cells also in vivo, nude mice bearing DU 145 tumor xenografts were treated with CGP 52608 at different doses and the growth of the tumors was followed by caliper measurement. RESULTS: RORalpha is expressed in DU 145 cells and the treatment of the cells with the thiazolidinedione-derivative CGP 52608 brought about a dose-dependent and significant decrease of cell proliferation. Ligand-induced activation of RORalpha affected cell cycle distribution, inducing an accumulation in the G(0)/G(1) phase and a decrease in the S phase. This effect was accompanied by an increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and a decreased expression of cyclin A. The growth of DU 145 tumors in nude mice was significantly reduced by treatment with CGP 52608. CONCLUSIONS: These data indicate that, in androgen-independent DU 145 prostate cancer cells, activation of the orphan nuclear receptor RORalpha inhibits cell growth, both in vitro and in vivo. RORalpha also induces cell cycle arrest, possibly through the modulation of the expression of cell cycle-related genes.

Growth-inhibitory activity of melatonin on human androgen-independent DU 145 prostate cancer cells.

BACKGROUND: The pineal hormone melatonin has been shown to exert a direct oncostatic activity on neoplastic cells, particularly from breast cancer. In the present study, we evaluated the effects of melatonin on the proliferation and on the cell cycle distribution of human androgen-independent DU 145 prostate cancer cells. Experiments were also performed to gain insights into the possible mechanism of action of the hormone. METHODS: The effects of melatonin on DU 145 cell proliferation was analyzed by counting the cells by hemocytometer at the end of treatment. The effects of the pineal hormone on cell cycle distribution were evaluated by FACS analysis. RT-PCR studies were performed to detect Mel(1a) and Mel(1b) expression in DU 145 cells. The cellular localization of (125)I-melatonin binding sites was investigated by radioreceptor assay. A commercially available binding-protein assay kit was utilized to evaluate intracellular cAMP levels. RESULTS: Melatonin, in physiological doses, significantly inhibited DU 145 cell proliferation and induced cell cycle withdrawal by accumulating cells in G0/G1 phase. The mRNA for Mel(1a) receptors was found to be expressed in DU 145 cells; however, by radioreceptor assay, no binding sites for (125)I-melatonin could be detected in membrane preparations, suggesting that, in these cells, the level of translation of this mRNA is too low to possibly mediate the antiproliferative action of the hormone. In agreement with this hypothesis, melatonin did not affect forskolin-induced intracellular cAMP accumulation. Binding sites for (125)I-melatonin were found in nuclear extracts of DU 145 cells. CONCLUSIONS: Melatonin exerts a direct oncostatic activity on human androgen-independent prostate cancer cells, by affecting cell cycle progression. This activity seems to be mediated by nuclear, but not by membrane, receptors.

Antiproliferative action of melatonin on human prostate cancer LNCaP cells.

Recent experimental evidence suggests that melatonin, the major pineal hormone, might possess oncostatic properties. The present experiments were performed to verify whether melatonin might modulate the growth of androgen-dependent prostate cancer cells (LNCaP) and to obtain information on its possible mechanism of action. We have shown that melatonin, when given in the nanomolar range, significantly inhibits the proliferation of LNCaP cells; moreover, the pineal gland hormone affects cell cycle distribution by inducing an accumulation of the cells in G0/G1 and a decrease in S phase. To investigate the mechanism of action of melatonin, by RT-PCR analysis we were able to demonstrate the expression, in prostate cancer cells, of a mRNA coding for the membrane Mel1a melatonin receptor. However, by radioreceptor assay, no detectable binding of 2-[125I]iodomelatonin could be observed in membrane preparations from these cells, suggesting that the levels of translation of the mRNA for Mel1a are possibly too low to mediate the antiproliferative action of the hormone. This hypothesis is further supported by the following observations: i) melatonin analogs, specifically acting through membrane receptors (i.e., 2-bromomelatonin), were completely ineffective in modulating prostate cancer cell proliferation; ii) melatonin failed to prevent forskolin-induced cAMP accumulation. These results indicate that melatonin, at nanomolar concentrations, exerts a direct antiproliferative action on androgen-dependent prostate cancer cells, significantly affecting their distribution throughout the cell cycle. Membrane receptors do not seem to be involved in the oncostatic action of the pineal gland hormone.

The luteinizing hormone-releasing hormone receptor in human prostate cancer cells: messenger ribonucleic acid expression, molecular size, and signal transduction pathway.

Evidence has accumulated indicating that LHRH might behave as an autocrine/paracrine growth inhibitory factor in some peripheral tumors. However, LHRH receptors in tumor cells have not been fully characterized, so far. The present experiments were performed to analyze: 1) the messenger RNA expression; 2) the molecular size; and 3) the signal transduction pathway of LHRH receptors in prostate cancer. For these studies, the human androgen-dependent LNCaP and androgen-independent DU 145 prostate cancer cell lines were used. 1) By RT-PCR, a complementary DNA product, which hybridized with a 32P-labeled oligonucleotide probe specific for the pituitary LHRH receptor complementary DNA, was found both in LNCaP and in DU 145 cells. 2) Western blot analysis, using a monoclonal antibody raised against the human pituitary LHRH receptor, revealed the presence of a protein band of approximately 64 kDa (corresponding to the molecular mass of the pituitary receptor) in both cell lines. 3) In LNCaP and DU 145 cells, pertussis toxin completely abrogated the antiproliferative action of a LHRH agonist (LHRH-A). Moreover, LHRH-A substantially antagonized the pertussis toxin-catalyzed ADP-ribosylation of a Galpha(i) protein. Finally, LHRH-A significantly counteracted the forskolin-induced increase of intracellular cAMP levels in both cell lines. These data demonstrate that the LHRH receptor, which is present in prostate cancer cells, independently of whether they are androgen-dependent or not, corresponds to the pituitary receptor, in terms of messenger RNA expression and protein molecular size. However, at variance with the receptor of the gonadotrophs, prostate cancer LHRH receptor seems to be coupled to the Galpha(i) protein-cAMP signal transduction pathway, rather than to the Galpha(q/11)-phospholipase C signaling system. This might be responsible for the different actions of LHRH in anterior pituitary and in prostate cancer.

Luteinizing hormone-releasing hormone agonists interfere with the mitogenic activity of the insulin-like growth factor system in androgen-independent prostate cancer cells.

We have previously shown that LHRH agonists exert a direct and specific inhibitory action on the proliferation of the androgen-independent DU 145 prostate cancer cell line; however, the cellular mechanisms mediating this antiproliferative action are not well defined. It is well known that the insulin-like growth factor (IGF) system plays a crucial role in the local regulation of the growth of androgen-independent prostate cancer. The present experiments were performed to evaluate whether LHRH agonists might exert their antimitogenic effect by interfering with the activity of the locally expressed IGF system. To this purpose, the effects of the LHRH agonist Zoladex (LHRH-A) on 1) the mitogenic action of IGF-I, 2) the tyrosine phosphorylation of type 1 IGF-I receptor (IGF-IR), 3) the concentration of IGF-IR, and 4) the secretion of IGF-binding protein-3 were studied. The results obtained show that in DU 145 cells, LHRH-A 1) counteracts the mitogenic action of IGF-I in a dose-dependent manner, 2) prevents the IGF-I-induced tyrosine phosphorylation of the IGF-IR, 3) reduces the concentration of IGF-IR without affecting its Kd value, and 4) does not affect the secretion of IGF-binding protein-3 in the conditioned medium from these cells. These data suggest that LHRH agonists may inhibit the proliferation of human androgen-independent prostate tumor cells by interfering with some of the cellular mechanisms mediating the stimulatory action of the IGF system.

Mechanism of action of interleukin-1 in modulating gonadotropin secretion. In vivo and in vitro studies.

To obtain further information on the mode of action of interleukin (IL)-1 in modulating gonadotropin secretion, a series of in vivo and in vitro studies has been performed with the beta-isoform of IL-1. IL-1 beta injected in a lateral ventricle of 3-week-castrated female rats resulted in the expected decrease in serum levels of gonadotropins luteinizing hormone (LH), and follicle-stimulating hormone (FSH), accompanied by a decrease in the number of LH-releasing hormone (LHRH) receptors. These results may indicate that the inhibition of gonadotropin release may result from a decrease in the number of LHRH pituitary receptors either through a direct effect on the pituitary or by modulating the release of LHRH from hypothalamic neurons able to induce a reduction in pituitary LHRH receptors. In vitro studies using the GT1-1 cell line, which specifically produces and secretes LHRH, demonstrated that IL-beta stimulates LHRH release but does not influence intracellular levels of LHRH mRNA. These results seem to indicate that IL-1 beta may act at several levels of the nervous machinery leading to gonadotropin secretion, with a series of effects more complex than previously anticipated.

Growth-inhibitory effects of luteinizing hormone-releasing hormone (LHRH) agonists on xenografts of the DU 145 human androgen-independent prostate cancer cell line in nude mice.

Experiments have been performed to clarify whether LHRH agonists might decrease growth of hormone-unresponsive prostate cancer in vivo. Male nude mice were injected s.c. with the human androgen-independent prostate tumor DU 145 cells; osmotic minipumps releasing the LHRH agonist Zoladex (LHRH-A) for 14 days were simultaneously implanted under the skin. Treatment with LHRH-A induced a significant decrease in tumor growth up to the end of the treatment. In subsequent experiment, minipumps releasing LHRH-A were implanted in nude mice either 7 or 14 days after cell inoculation. When the treatment was started 7 days after inoculation of the cells, tumor growth was significantly decreased up to 28 days; thereafter, tumor volume remained lower than in controls, although not significantly. When LHRH-A was administered beginning 14 days after cell inoculation, tumor growth was not significantly affected at any time interval considered. LHRH-A did not appear to induce apoptosis in DU 145 cells, at least on the basis of the apoptotic index and immunohistochemical staining of the p53 protein. On the other hand, treatment with LHRH-A was accompanied by a significant decrease of the concentration of epidermal growth factor receptors in DU 145 prostate cancer specimens. Our results show that the LHRH agonist used significantly inhibits the growth of DU 145 androgen-independent prostate tumor xenografts in nude mice.

Mechanism of action of interleukin-1 in modulating gonadotropin secretion. In vivo and in vitro studies

To obtain further information on the mode of action of interleukin (IL)-1 in modulating gonadotropin secretion, a series of in vivo and in vitro studies has been performed with the beta-isoform of IL-1. IL-1 beta injected in a lateral ventricle of 3-week-castrated female rats resulted in the expected decrease in serum levels of gonadotropins luteinizing hormone (LH), and follicle-stimulating hormone (FSH), accompanied by a decrease in the number of LH-releasing hormone (LHRH) receptors. These results may indicate that the inhibition of gonadotropin release may result from a decrease in the number of LHRH pituitary receptors either through a direct effect on the pituitary or by modulating the release of LHRH from hypothalamic neurons able to induce a reduction in pituitary LHRH receptors. In vitro studies using the GT1-1 cell line, which specifically produces and secretes LHRH, demonstrated that IL-beta stimulates LHRH release but does not influence intracellular levels of LHRH mRNA. These results seem to indicate that IL-1 beta may act at several levels of the nervous machinery leading to gonadotropin secretion, with a series of effects more complex than previously anticipated.

Effects of LHRH agonists on the growth of human prostatic tumor cells: "in vitro" and "in vivo" studies.

Luteinizing Hormone Releasing Hormone (LHRH) agonists exert both "in vitro" and "in vivo" a direct inhibitory action on the growth of both androgen-dependent (LNCaP) and androgen-independent (DU 145) human prostatic cancer cell lines. The present experiments have been performed to investigate the mechanisms involved in this direct antiproliferative action of LHRH agonists. In particular, the aim was to study whether these compounds might exert their antiproliferative effect by interfering with the stimulatory action of epidermal growth factor (EGF) both "in vitro" and "in vivo". To this purpose, the effects of LHRH agonist, Zoladex (LHRH-A), on the mitogenic action of EGF, on EGF-activated intracellular signaling mechanisms (tyrosine phosphorylation of EGF receptor and c-fos proto-oncogene expression), and on the concentration of EGF receptors have been evaluated in both LNCaP and DU 145 cells. The results of these "in vitro" studies show that in LNCaP cells LHRH-A counteracts the mitogenic action of EGF, abrogates the EGF-induced c-fos expression and reduces the concentration of EGF-binding sites, without modifying the EGF induced tyrosine phosphorylation. In DU 145 cells, LHRH-A antagonizes the proliferative action of EGF, inhibits tyrosine phosphorylation of EGF receptor induced by EGF and significantly reduces the number of EGF binding sites, without altering the stimulation of c-fos expression induced by EGF. For the "in vivo" experiments, male nude mice were s.c. injected in the flank with DU 145 cells and treated for 14 days with LHRH-A (100 micrograms/days). At the end of the treatment, the concentration of EGF receptors on membrane preparations as well as on tumor volume were found to be significantly lower in LHRH-A treated animals than in control mice. The mitotic index and the expression of the proliferation-associated antigen Ki67 were found similar in control as well as in treated animals. In addition no modification of apoptotic index (expression of p53) was observed. These data suggest that LHRH agonists may inhibit the proliferation of the tumor cells by interfering with the stimulatory actions of EGF.

Luteinizing hormone-releasing hormone agonists interfere with the stimulatory actions of epidermal growth factor in human prostatic cancer cell lines, LNCaP and DU 145.

LH-releasing hormone (LHRH) agonists exert a direct inhibitory action on the growth of both androgen-dependent (LNCaP) and androgen-independent (DU 145) human prostatic cancer cell lines. The present studies were aimed at clarifying whether these compounds might exert their antiproliferative action by interfering with the stimulatory action of epidermal growth factor (EGF). To this purpose, the effects of a LHRH agonist (Zoladex, LHRH-A) on the mitogenic action of EGF, on some of the EGF-activated intracellular signaling mechanisms (tyrosine phosphorylation of the 170-kDa EGF receptor, and c-fos protooncogene expression), as well as on the concentration of EGF receptors have been evaluated. These studies have been performed in both LNCaP and DU 145 cells. The results obtained show that in LNCaP cells, LHRH-A counteracts the mitogenic action of EGF, completely abrogates EGF-induced c-fos expression, and significantly reduces the concentration of EGF-binding sites. The EGF-activated tyrosine phosphorylation of the EGF receptor is not affected by LHRH-A in LNCaP cells. In DU 145 cells, LHRH-A antagonizes the proliferative action of EGF, inhibits the tyrosine phosphorylation of the EGF receptor induced by EGF, and significantly reduces the number of EGF-binding sites. In these cells, LHRH-A is not able to modify the increased expression of c-fos that follows the treatment with EGF. These data suggest that LHRH agonists may inhibit the proliferation of human prostatic tumor cells by interfering with the stimulatory actions of EGF. The intracellular mechanism of action of these compounds appears to differ in androgen-dependent LNCaP and androgen-independent DU 145 cells.

Growth factors in steroid-responsive prostatic tumor cells.

Androgens stimulate the growth of prostatic carcinoma, possibly by modulating the activity of locally expressed growth factors. Recently, we have shown that an LHRH (or LHRH-like) system exerting an inhibitory action on cell proliferation is present in the human androgen-dependent prostatic tumor cell lines LNCaP. The following experiments have been performed in LNCaP cells to clarify whether LHRH might inhibit cell proliferation by interfering with the two major mitogenic factors for these cells: (a) testosterone (T), the major exogenous stimulating factor, and (b) epidermal growth factor (EGF), one of the locally produced growth factors. (a) It has been shown that an LHRH agonist (LHRH-A, Zoladex) counteracts the proliferative action of T in a dose-dependent way. To clarify whether LHRH might interfere with the activity of T in prostate tumors, LNCaP cells were treated with LHRH agonist over different time intervals, and the effects of treatment evaluated in terms of expression of androgen receptor mRNA. The data obtained indicate that LHRH-A does not affect androgen receptor expression at any time interval examined. (b) LHRH-A inhibits the mitogenic action of EGF on LNCaP cells and significantly reduces the concentration of EGF receptors in these cells. Experiments have been performed to explore whether LHRH-A might alter intracellular signaling mechanisms mediating the activity of EGF. In LNCaP cells LHRH-A blocks EGF-induced expression of the c-fos proto-oncogene but does not modify EGF-induced tyrosine phosphorylation of the EGF receptor. These data suggest that, in androgen-dependent prostate tumors, LHRH might inhibit cell proliferation by interfering with some but not all of the mechanisms mediating the mitogenic action of EGF. Possible interactions between LHRH and T-activated events still remain to be elucidated.

Role of growth factors, steroid and peptide hormones in the regulation of human prostatic tumor growth.

Previous work carried out in the authors' laboratory has shown that LHRH agonists directly inhibit the proliferation of hormone-responsive and hormone-independent human prostatic cancer cell lines (respectively LNCaP and DU145). In addition, the hormone-dependent LNCaP cells respond to a challenge with testosterone with an increase in growth rate. The following experiments have been performed to investigate whether the LHRH agonists might act by interfering with the stimulatory actions of either the EGF/TGF alpha system or androgens. The results obtained in LNCaP and DU145 cells show that LHRH agonists counteract the mitogenic action of the EGF/TGF alpha system. This effect is mediated by a decrease in the concentration of EGF receptors. In addition, in the hormone-dependent LNCaP cells, the treatment with LHRH agonists antagonizes the proliferation promoting effect of testosterone, which in turn appears to be mediated by the activation of the locally expressed EGF/TGF alpha system. Finally, the results suggest the presence in LNCaP cells of a soluble peptidase able to degrade LHRH. In conclusion, the present data suggest an intimate interplay among the actions of LHRH agonists, of androgens and of growth factors, thus, supporting the hypothesis that LHRH agonists may interfere with the EGF/TGF alpha stimulatory loop and with androgens in the control of the proliferation of human prostatic tumors.

Effects of steroids on the brain opioid system.

The experiments reported here add further evidence in support of the view that sex steroids may influence the binding characteristics of brain opioid receptors. In particular, it has been shown that: (a) the number of mu-opioid receptors varies in the hypothalamus of regularly cycling female rats according to the different phases of the estrous cycle, which are characterized by fluctuations of circulating levels of sex steroids; (b) the number of mu-opioid receptors decreases in the hypothalamus and in the corpus striatum when ovariectomized rats are submitted to treatments with estradiol and progesterone able to induce a "positive" feedback effect on LH release. A treatment with estrogen alone able to induce a "negative" feedback effect on LH release brings about an increase of the number of mu-opioid receptors in the thalamus and in the hippocampus; (c) in addition to the mu-receptors, receptors of the delta type may also be involved in the control of gonadotropin secretion; recent results here presented indicate that a line of immortalized hypothalamic cells (GT1 cells), which synthesize and secrete LHRH, present delta opioid receptors on their membranes; these are apparently involved in the control of LHRH release from these cells.

Growth of the androgen-dependent tumor of the prostate: role of androgens and of locally expressed growth modulatory factors.

The crucial role played by androgens in the growth of prostatic carcinoma is now well established. However, the mechanisms of this proliferative action are still poorly understood. Experiments have been performed to clarify: (1) the metabolism of androgens in prostatic tumor cells; and (2) the role played by locally produced growth factors in the autocrine regulation of prostatic tumor cell proliferation and the possible regulation exerted by testosterone (T) on the activity of these factors. These studies have been performed by utilizing the human androgen-responsive prostatic cancer LNCaP cell line. (1) By incubating LNCaP cells with different 14C-labeled androgenic precursors, it has been shown that all the major key enzymes involved in the metabolism of androgens (5 alpha-reductase, 17 beta-hydroxysteroid-oxidoreductase, 3 alpha- and 3 beta-hydroxysteroid-oxidoreductases) are present and active in these cells. In particular, the 5 alpha-reductase, which converts T and delta 4 to DHT and 5 alpha-A respectively, seems to be more active when delta 4 is the substrate, suggesting a preference for this precursor. (2) The hypothesis that LNCaP cells might produce LHRH (or a LHRH-like peptide) has been verified by RT-PCR, performed in the presence of a pair of specific oligonucleotide primers. A cDNA band of the expected size (228 bp), which specifically hybridized with a 32P-labeled LHRH oligonucleotide probe, has been obtained in LNCaP cells. To clarify the possible role played by this factor in the regulation of tumor growth, LNCaP cells, cultured in steroid-free conditions, have been treated with a LHRH antagonist; the treatment resulted in a significant increase of cell proliferation. Taken together, these data indicate that a LHRH (or LHRH-like) growth modulatory system is expressed in LNCaP cells and plays an inhibitory role in the regulation of tumor cell proliferation. This system seems to be regulated in a negative way by steroids. Growth factors endowed with stimulatory activity, such as EGF and TGF alpha, have also been shown to be produced by LNCaP cells. The present studies show that the immunoprecipitation of the EGF receptor with a specific monoclonal antibody (Ab225) reveals a protein band of the expected size (170 kDa) which is phosphorylated even in basal conditions. Moreover, the treatment of LNCaP cells, cultured in serum-free conditions, either with a monoclonal antibody against the EGF receptor, or with immunoneutralizing antibodies against EGF and TGF alpha, results in a significant decrease of cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)