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.

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.

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.

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.

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.