Magdolna Kovacs

Hypothalamic hormones and cancer.

The use of peptide analogs for the therapy of various cancers is reviewed. Inhibition of the pituitary-gonadal axis forms the basis for oncological applications of luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists, but direct effects on tumors may also play a role. Analogs of somatostatin are likewise used for treatment of various tumors. Radiolabeled somatostatin analogs have been successfully applied for the localization of tumors expressing somatostatin receptors. Studies on the role of tumoral LH-RH, growth hormone-releasing hormone (GH-RH), and bombesin/GRP and their receptors in the proliferation of various tumors are summarized, but the complete elucidation of all the mechanisms involved will require much additional work. Human tumors producing hypothalamic hormones are also discussed. Treatment of many cancers remains a major challenge, but new therapeutic modalities are being developed based on antagonists of GH-RH and bombesin, which inhibit growth factors or their receptors. Other approaches consist of the use of cytotoxic analogs of LH-RH, bombesin, and somatostatin, which can be targeted to receptors for these peptides in various cancers and their metastases. These new classes of peptide analogs should lead to a more effective treatment for various cancers.

Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix down-regulates the mRNA expression of pituitary receptors for LH-RH by counteracting the stimulatory effect of endogenous LH-RH.

The mechanisms through which LH-RH antagonists suppress gonadotroph functions and LH-RH receptor (LH-RH-R) production are incompletely understood. To elucidate these mechanisms, we investigated the effects of Cetrorelix on the mRNA expression of pituitary LH-RH-R and luteinizing hormone (LH) secretion in three experimental systems with different pituitary LH-RH environments. Ovariectomy induced 3.61-fold and 6.34-fold increases in the mRNA expression of pituitary LH-RH-R in rats after 11 and 21 days, respectively. After (5 h) a single injection of 100 microg Cetrorelix, no significant decrease occurred in the mRNA levels of pituitary LH-RH-R in ovariectomized (OVX) rats with high pituitary exposure to LH-RH, but there was a significant 23.2% reduction in cycling rats with normal hypophysial LH-RH environment. Prolonged treatment for 10 days with a Cetrorelix depot formulation releasing 100 microg/day decreased the concentration of mRNA for pituitary LH-RH-R by 72.6% in OVX rats, but only by 32.9% in normal rats. The decline in serum LH was 98.7% in OVX rats and 63.2% in normal rats, resulting in a minimal 0.1--0.2 ng/ml LH concentration in both groups. A continuous exposure of pituitary cells to 100 nM Cetrorelix in the superfusion system, which is devoid of LH-RH, did not cause any significant changes in LH-RH-R mRNA level. These studies demonstrate that prolonged exposure to Cetrorelix in vivo, but not in vitro, down-regulates the mRNA expression of the pituitary receptors for LH-RH. Our findings indicate that LH-RH antagonists exert their inhibitory effects on the gene expression of pituitary LH-RH-R by counteracting the stimulatory effect of endogenous LH-RH.

Lamprey Gonadotropin Hormone-Releasing Hormone-III has No Selective Follicle-Stimulating Hormone-Releasing Effect In Rats

Lamprey gonadotropin releasing‐hormone (LGnRH)‐III, a hypothalamic neurohormone recently isolated from sea lamprey, was reported to have a selective stimulatory effect on follicle‐stimulating hormone (FSH) release in rats and suggested to be the mammalian FSH‐releasing factor. In this study, we determined the relative luteinizing hormone (LH)‐ and FSH‐releasing potency of LGnRH‐III compared to mammalian gonadotropin‐releasing hormone (LHRH) in normal female rats, ovariectomized (OVX) and oestrogen/progesterone substituted rats and the superfused rat‐pituitary cell system. The specificity of LGnRH‐III for the mammalian LHRH receptor was investigated by blocking the receptor with an LHRH antagonist, MI‐1544. In vitro, LGnRH‐III dose‐dependently stimulated both LH and FSH secretion from rat pituitary cells at 10−7 to 10−5 M concentrations, while LHRH stimulated gonadotropin secretion at a 1000‐fold lower doses (10−10 to 10−8 M). The difference between its LH‐ and FSH‐releasing potency was similar to that of LHRH. LGnRH‐III bound to high affinity binding sites on rat pituitary cells with a Kd of 6.7 nm, Bmax=113±27 fmol/mg protein. In vivo, LGnRH‐III also stimulated both LH and FSH secretion in a dose‐dependent manner and, similar to LHRH, induced a greater rise in the serum LH than the FSH level. In normal cycling rats, it showed 180–650‐fold weaker potency than LHRH in stimulating LH secretion and 70–80‐fold weaker effect in stimulating FSH secretion. In OVX rats, LGnRH‐III demonstrated a similarly weak effect on both gonadotropins. It was found to be 40–210‐fold less potent than LHRH regarding LH release and 50–160‐fold weaker regarding FSH release. LHRH‐receptor antagonist MI‐1544 prevented both the LH‐ and the FSH‐releasing effect of LGnRH‐III both in vitro and in vivo. These results do not support the hypothesis that LGnRH‐III might be the mammalian FSH‐releasing factor but demonstrate that it is a weak agonist for the pituitary LHRH receptor and stimulates both gonadotropins in a dose‐dependent fashion.

Synthesis and In Vitro Evaluation of New Potent Antagonists of Growth Hormone-Releasing Hormone (GH-RH)

In the search for more potent antagonists of hGH-RH, 20 new analogs were synthesized, purified and tested in vitro. All the analogs were based on the N-terminal sequence of 28 or 29 amino acid residues of hGH-RH, but contained D-Arg2 and Nle27 modifications. Most analogs had Phe (pCl)6 and Agm29 substituents. The effect of other substitutions such as Abu8 and/or Abu15 and Ala15 and various hydrophobic and hydrophilic D or L amino acids at position 8 were also investigated. All the peptides were acylated at the N-terminus in an attempt to increase the antagonistic activity. In the superfused rat pituitary cell system, most analogs inhibited more powerfully the GH release induced by GH-RH than the standard antagonist [Ac-Tyr1, D-Arg2]hGH-RH (1-29)-NH2. Some antagonists were long acting. Among the peptides synthesized, antagonist PhAc[D-Arg2, Phe(pCl)6, Abu15, Nle27]hGH-RH (1-28) Agm (MZ-5-156) appeared to be the most potent and inhibited GH release in vitro 63-200 times more powerfully than the standard antagonist. MZ-5-156 and other antagonists showed high binding affinities to membrane receptors for GH-RH. Some of these hGH-RH antagonists could be further developed for possible onocological applications.

New derivatives of GnRH as potential anticancer therapeutic agents

GnRH (gonadotropin-releasing hormone), a decapeptide produced by the hypothalamus, plays an important role in the reproduction by regulating the pituitary-gonadal axis. Continuous high doses of GnRH or its superactive agonists result in desensitization of the pituitary gonadotropes and a suppression of sex steroid production by the gonads (chemical castration). Based on these effects, the treatment with GnRH agonists has become a widely used hormonal therapy of the sex-steroid dependent tumors. It was also demonstrated that most tumor cells contain GnRH receptors, and the direct antiproliferative effect of GnRH analogs on cancer cells might be mediated by these receptors. Development of new GnRH derivatives is focused on the decrease of their hormonal potency resulting in higher selectivity of the antitumor activity. One of the most promising natural GnRH analogs, lamprey (l) lGnRH-III, was isolated from see lamprey. This variant of GnRH binds to GnRH receptors and inhibits proliferation of various cancer cells. However, its endocrine effect is insignificant in mammals. lGnRH-III dimers and conjugates were prepared and were shown to have increased antiproliferative effects on various cancer cells, while their hormonal activity was lower than that of the native hormone. lGnRH-III was applied as targeting moiety to deliver anticancer agents to tumor cells. Research data concerning lGnRH-III and its analogs represent a new outlook for research trends of the application of GnRH compounds in cancer chemotherapy. Studies on the effects of lGnRH-III derivatives including antiproliferative effects, cytotoxicity, hormonal actions, and enzymatic stability are reviewed in this article.

Comparison of mechanisms of action of luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix and LHRH agonist triptorelin on the gene expression of pituitary LHRH receptors in rats

The mechanisms through which luteinizing hormone (LH)-releasing hormone (LHRH) antagonists suppress pituitary gonadotroph functions and LHRH-receptor (LHRH-R) expression are incompletely understood. Consequently, we investigated the direct effect of LHRH antagonist cetrorelix in vitro on the expression of the pituitary LHRH-R gene and its ability to counteract the exogenous LHRH and the agonist triptorelin in the regulation of this gene. We also compared the effects of chronic administration of cetrorelix and triptorelin on the LHRH-R mRNA level and gonadotropin secretion in ovariectomized (OVX) and normal female rats. The exposure of pituitary cells in vitro to 3-min pulses of 1 nM LHRH or 0.1 nM triptorelin for 5 h increased the LHRH-R mRNA level by 77–88%. Continuous perfusion of the cells with 50 nM cetrorelix did not cause any significant changes, but prevented the stimulatory effect of LHRH pulses on the receptor mRNA expression. In OVX rats, 10 days after administration of a depot formulation of cetrorelix, releasing 100 μg of peptide daily, the elevated LHRH-R mRNA level was decreased by 73%, whereas daily injection of 100 μg of triptorelin caused a 41% suppression. In normal female rats, cetrorelix treatment suppressed the LHRH-R mRNA level by 33%, but triptorelin increased it by 150%. The highly elevated serum LH levels in OVX rats and the normal LH concentration of cycling rats were rapidly and completely suppressed by cetrorelix. Triptorelin decreased the serum LH in OVX rats to the precastration level, but had no effect on basal LH in normal rats. Our results confirm that LHRH antagonists, such as cetrorelix, inhibit the gene expression of pituitary LHRH-R indirectly, by counteracting the stimulatory effect of LHRH. A rapid suppression of serum LH by LHRH antagonists would be advantageous in the treatment of sex hormone-dependent tumors and other conditions.

Effects of long-term treatment with the luteinizing hormone-releasing hormone (LHRH) agonist Decapeptyl and the LHRH antagonist Cetrorelix on the levels of pituitary LHRH receptors and their mRNA expression in rats

The effects of depot formulations of the luteinizing hormone-releasing hormone (LHRH) agonist Decapeptyl (25 μg/day) for 30 days or LHRH antagonist Cetrorelix pamoate (100 μg/day) for 30 days and daily injections of 100 μg of Decapeptyl for 10 days on the expression of mRNA for pituitary LHRH receptor (LHRH-R) and the levels of LHRH-R protein were evaluated in rats. Serum sex steroid concentrations and the weights of the reproductive organs were greatly reduced in all groups treated with analogs, demonstrating an efficient blockade of the pituitary–gonadal axis. Decapeptyl microcapsules elevated serum LH in female rats, but decreased it in male rats. LHRH-R mRNA expression in female pituitaries was reduced to 41% and 56–65% on days 10 and 30, respectively, whereas LHRH-R protein was 64% of control on day 10 and returned to pretreatment levels on day 30. Decapeptyl microcapsules reduced LHRH-R mRNA expression in male pituitaries to 58% on day 30 but not LHRH-R protein. Daily injections of Decapeptyl caused a desensitization of LH responses in female rats, while raising LHRH-R mRNA expression in female rats by 23% and LHRH-R protein levels by 119%. Cetrorelix pamoate reduced serum LH in female rats and diminished LHRH-R mRNA to 30% and 26% and LHRH-R protein to 57% and 48% on days 10 and 30, respectively. Elevated LHRH-R protein levels of ovariectomized rats were reduced after 10-day treatment with Cetrorelix or 100 μg/day Decapeptyl. Thus, changes in the mRNA expression after treatment with Cetrorelix, but not always Decapeptyl, paralleled those of LHRH-R protein. The inhibitory effect of Cetrorelix on serum LH, pituitary LHRH-R mRNA, and LHRH-R protein was greater than that of Decapeptyl.

Combining Growth Hormone-Releasing Hormone Antagonist With Luteinizing Hormone-Releasing Hormone Antagonist Greatly Augments Benign Prostatic Hyperplasia Shrinkage

PURPOSE Benign prostatic hyperplasia often affects aging men. Antagonists of the neuropeptide growth hormone-releasing hormone reduced prostate weight in an androgen induced benign prostatic hyperplasia model in rats. Luteinizing hormone-releasing hormone antagonists also produce marked, protracted improvement in lower urinary tract symptoms, reduced prostate volume and an increased urinary peak flow rate in men with benign prostatic hyperplasia. We investigated the influence of a combination of antagonists of growth hormone-releasing hormone and luteinizing hormone-releasing hormone on animal models of benign prostatic hyperplasia. MATERIALS AND METHODS We evaluated the effects of the growth hormone-releasing hormone antagonist JMR-132, given at a dose of 40 μg daily, the luteinizing hormone-releasing hormone antagonist cetrorelix, given at a dose of 0.625 mg/kg, and their combination on testosterone induced benign prostatic hyperplasia in adult male Wistar rats in vivo. Prostate tissue was examined biochemically and histologically. Serum levels of growth hormone, luteinizing hormone, insulin-like growth factor-1, dihydrotestosterone and prostate specific antigen were determined. RESULTS Marked shrinkage of the rat prostate (30.3%) occurred in response to the combination of growth hormone-releasing hormone and luteinizing hormone-releasing hormone antagonists (p<0.01). The combination strongly decreased prostatic prostate specific antigen, 6-transmembrane epithelial antigen of the prostate, interleukin-1β, nuclear factor-κβ and cyclooxygenase-2, and decreased serum prostate specific antigen. CONCLUSIONS A combination of growth hormone-releasing hormone antagonist with luteinizing hormone-releasing hormone antagonist potentiated a reduction in prostate weight in an experimental benign prostatic hyperplasia model. Results suggest that this shrinkage in prostate volume was induced by the direct inhibitory effects of growth hormone-releasing hormone and luteinizing hormone-releasing hormone antagonists exerted through their respective prostatic receptors. These findings suggest that growth hormone-releasing hormone antagonists and/or their combination with luteinizing hormone-releasing hormone antagonists should be considered for further development as therapy for benign prostatic hyperplasia.

A correlation of endocrine and anticancer effects of some antagonists of GHRH.

GHRH receptor antagonists inhibit growth and metastasis of a large number of experimental tumors expressing the pituitary GHRH receptor (pGHRH-R) and its major splice variant SV1. In this study, using Western blot, we demonstrated that DBTRG-05 and U-87MG human glioblastoma cell lines express pGHRH-R at levels 6-15 times higher than SV1. To reveal a correlation between the anticancer activity and the endocrine potency on inhibition of GH release, we compared the antitumor effect of GHRH antagonists JV-1-63 and MZJ-7-138 on growth of DBTRG-05 human glioblastomas grafted into athymic nude mice with their inhibitory potency on GH release. JV-1-63 strongly suppressed the stimulated GH secretion induced by clonidine in rats and inhibited the exogenous GHRH-induced GH surge by 88-99% in vivo and in vitro. MZJ-7-138 decreased the stimulated GH secretion by 58% in vitro and showed only a tendency to inhibit GH secretion in vivo. The strong inhibitor of GH release JV-1-63 reduced tumor growth of DBTRG-05 glioblastomas in nude mice by 46%, while the weak GH release suppressor MZJ-7-138 did not have an effect. Exposure of DBTRG-05 cells to the GHRH antagonists in vitro caused an upregulation of mRNA expression for pGHRH-R and a downregulation of SV1 expression, with JV-1-63 having significantly greater effects than MZJ-7-138. Our results demonstrate that a positive correlation exists between the endocrine potency and the antiproliferative efficacy of GHRH antagonists in tumors strongly expressing pGHRH-R.

Structure–activity study on the LH- and FSH-releasing and anticancer effects of gonadotropin-releasing hormone (GnRH)-III analogs

UNLABELLED GnRH-III was reported to have selective FSH-releasing activity in rats and significant anticancer potency on human breast cancer cells. To improve either of these effects, 14 analogs were synthesized and investigated for FSH/LH stimulation and breast cancer inhibition. Analogs with single amino acid changes in positions 5-7 or 10 showed small or no difference in the FSH- or LH-releasing activity compared with GnRH-III but their anticancer potency decreased significantly. Modification of the terminal amino acids, side chain cyclization at the 6-8 regions, or combined amino acid changes at positions 4, 6 and/or 8 resulted in the decrease of both effects. Gonadotropin-releasing activity of Arg(8)-GnRH-III was improved 3-11-fold. A copolymer conjugate of GnRH-III showed 2-3-fold anticancer activity while losing endocrine potency. CONCLUSION The activation of GnRH-receptors on pituitary and breast cancer cells requires a specific structure and/or conformation that makes possible to improve the anticancer selectivity of GnRH analogs.