Growth Hormone-releasing Hormone Antagonists Research Articles

Activation of mitogen-activated protein kinases by a splice variant of GHRH receptor

Hypothalamic GHRH controls the release of GH from the pituitary gland and also acts as a growth factor in a variety of cancers. The mitogenetic activity of GHRH is exerted through the binding to the pituitary type receptor (pGHRH-R) and its splice variants, mainly SV1. The intracellular signaling pathways that are activated upon the binding of GHRH to the SV1 receptor have not been elucidated. HeLa cervical cancer cells do not express GHRH or GHRH receptors (GHRHRs) and thus do not respond to GHRH or GHRH antagonists. In order to elucidate the mechanism of action of SV1 receptor, we transfected HeLa cells with plasmids for pcDNA3-GHRHR or pcDNA3-SV1. The transfected cells responded to both GHRH (1-29)NH(2) and GHRH antagonist MZ-5-156, as shown by an increase or decrease respectively in the proliferation rate in vitro and the expression of proliferative cell nuclear antigen. We also demonstrated that when the cells transfected with SV1 plasmid are stimulated with GHRH (1-29)NH(2), SV1 receptor activates the mitogen-activated protein kinases pathway (MAPKs), as shown previously for the cells that express pGHRH-R. Our results show, for the first time, the activation of the MAPKs cascade by the SV1 receptor. Since SV1 receptor is found in various tumors and mediates the responses to GHRH and synthetic antagonists, our findings shed light on the mechanism of action of SV1 receptor in cancer cells.

Growth hormone-releasing hormone activates sleep regulatory neurons of the rat preoptic hypothalamus

We examined whether growth hormone-releasing hormone (GHRH) may promote non-rapid eye movement (NREM) sleep via activation of GABAergic neurons in the preoptic area. Male Sprague-Dawley rats were implanted with EEG, EMG electrodes and a unilateral intracerebroventricular cannula. Groups of rats received injections (3 microl icv) with gonadotropin-releasing hormone (GHRH) (0.1 nmol/100 g body wt) or equal volume of physiological saline at the onset of the dark period and were permitted spontaneous sleep for 90 min. Separate groups of rats were sleep deprived by gentle handling for 90 min, beginning at the time of GHRH or saline injection, at the onset of the dark period. Other groups of rats received intracerebroventricular octreotide (somatostatin analog OCT) injections, intracerebroventricular injection of one of two doses of competitive GHRH antagonist, or intracerebroventricular saline injection at light onset and were then permitted 90 min spontaneous sleep-waking. Rats were killed immediately after the 90-min sleep/wake monitoring period. Brain tissue was processed for immunohistochemistry for c-Fos protein and glutamic acid decarboxylase (GAD). Single c-Fos and dual Fos-GAD cell counts were determined in the median preoptic nucleus (MnPN), and in the core and the extended parts of the ventrolateral preoptic nucleus (cVLPO and exVLPO). Intracerebroventricular GHRH elicited a significant increase in NREM sleep amount. Double-labeled Fos+GAD cell counts were significantly elevated after GHRH injection in the MnPN and VLPO in both undisturbed and sleep-deprived groups. OCT and GHRH antagonist significantly decreased NREM sleep amount compared with control rats. OCT injection increased single c-Fos-labeled cell counts in the MnPN, but not in the VLPO. Double-labeled cell counts were significantly reduced after OCT and the high dose of GHRH antagonist injection in all areas examined. These findings identify GABAergic neurons in the MnPN and VLPO as potential targets of the sleep-regulatory actions of GHRH.

GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10μM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (∆Ψm). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21Waf1/Cip1 was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p

The growth hormone–releasing hormone (GHRH) antagonist JV-1-36 inhibits proliferation and survival of human ectopic endometriotic stromal cells (ESCs) and the T HESC cell line

To determine the effect of the GHRH antagonist JV-1-36 on proliferation and survival of primary ectopic human endometriotic stromal cells (ESCs) and the T HESC cell line. Prospective laboratory study. University hospital. 22 women with endometriosis (aged 34.8+/-5.7 years) undergoing therapeutic laparoscopy. Eutopic (n=10) and ectopic (n=22) endometrial tissues were collected from women who underwent therapeutic laparoscopic surgery for endometriosis (stage III/IV). Expression of GHRH, GHRH receptor (GHRH-R) and GHRH-R splice variant (SV) 1 mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR). The ESC proliferation was assessed by 5-bromo-2-deoxyuridine incorporation, cell survival by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Trypan blue assay. The T HESC survival was evaluated by MTT, cyclic adenosine monophosphate (cAMP) levels by ELISA, extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation by Western blot, and insulin-like growth factor (IGF)-2 mRNA by real-time PCR. The ESCs and T HESCs, but not normal endometrial tissues, expressed GHRH-R mRNA; SV1 mRNA was determined in normal endometrial tissues, ESCs, and T HESCs; GHRH mRNAwas found in T HESCs; JV-1-36 inhibited ESC proliferation and ESC and T HESC survival. In T HESCs, JV-1-36 reduced cAMP production and ERK1/2 phosphorylation but had no effect on IGF-2 mRNA expression. The GHRH antagonist JV-1-36 inhibits endometriotic cell proliferation and survival, suggesting that GHRH antagonist may represent promising tools for treatment of endometriosis.

Combination of a novel growth hormone releasing hormone Antagonist JMR 132 and Tamoxifen enhances the inhibitory effects on growth of ZR 75 and T47D estrogen dependent human breast cancer cells in vitro and in vivo

Background: Tamoxifen is the oldest selective estrogen receptor modulator (SERM) developed to antagonize the estrogen receptor function on human breast cancer cells. JMR 132 is a novel GHRH Antagonist proven to be potent inhibitor of tumor growth in estrogen receptor negative human breast cancer cell lines. A combination of both substances could induce a more potent growth inhibition, as compared to the single drugs. Methods: To determine inhibitory effects of the GHRH antagonist JMR 132 and Tamoxifen alone and in combination, estrogen receptor positive cell lines ZR 75 and T 47D were incubated with substances at concentrations of 2,5µM, 5µM and 10µM. The cell viability after a period of 72h was measured by MTT assay. To assess if the treatment arrested cell growth at a specific phase of the cell cycle or simply caused cell death, we performed a flow cytometric DNA cell cycle analysis on ZR 75 cells exposed to JMR 132, Tamoxifen and their combination. In an in vivo model, the cell line ZR 75 was xenografted subcutaneously into nude mice. Treatment groups were control, JMR 132at a dose of 10µM, Tamoxifen at a dose of 100µgr per mouse q3 per week and the combination of both substances. All given for a period of 4 weeks. Results: In vitro 1o µM JMR 132 and Tamoxifen significantly decreased the proliferation of the ZR 75 and T47D cell lines, as compared to controls (p<0,05). The combination of 1o µM inhibited the growth of ZR 75 was significantly by 14%, as compared to JMR 132 alone. The combination also significantly suppressed the growth of T47D by 14% and 22% respectively, as compared to Tamoxifen and JMR 132. In in vivo treatment with the combination of JMR 132 and Tamoxifen induced a significant inhibition of tumor growth of ZR 75 xenografts after 14 days compared to the single agents groups and inhibition remained significant until the end of the study on day 28. The flow cytometric DNA cell cycle analysis revealed a cell cycle arrest in the S Phase which led to apoptosis of the cells. Conclusion: Treatment of ZR 75 and T47D estrogen receptor positive human breast cancer lines with GHRH antagonist JMR 132 and its combination with Tamoxifen induced potent inhibitory effects in vitro and in vivo and led to subsequent cell death. Further studies are being conducted to elucidate the mechanisms of action of these effects

Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia–reperfusion injury in rat heart

The hypothalamic neuropeptide growth hormone-releasing hormone (GHRH) stimulates GH synthesis and release in the pituitary. GHRH also exerts proliferative effects in extrapituitary cells, whereas GHRH antagonists have been shown to suppress cancer cell proliferation. We investigated GHRH effects on cardiac myocyte cell survival and the underlying signalling mechanisms. Reverse transcriptase-polymerase chain reaction analysis showed GHRH receptor (GHRH-R) mRNA in adult rat ventricular myocytes (ARVMs) and in rat heart H9c2 cells. In ARVMs, GHRH prevented cell death and caspase-3 activation induced by serum starvation and by the beta-adrenergic receptor agonist isoproterenol. The GHRH-R antagonist JV-1-36 abolished GHRH survival action under both experimental conditions. GHRH-induced cardiac cell protection required extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide-3 kinase (PI3K)/Akt activation and adenylyl cyclase/cAMP/protein kinase A signalling. Isoproterenol strongly upregulated the mRNA and protein of the pro-apoptotic inducible cAMP early repressor, whereas GHRH completely blocked this effect. Similar to ARVMs, in H9c2 cardiac cells, GHRH inhibited serum starvation- and isoproterenol-induced cell death and apoptosis through the same signalling pathways. Finally, GHRH improved left ventricular recovery during reperfusion and reduced infarct size in Langendorff-perfused rat hearts, subjected to ischaemia-reperfusion (I/R) injury. These effects involved PI3K/Akt signalling and were inhibited by JV-1-36. Our findings suggest that GHRH promotes cardiac myocyte survival through multiple signalling mechanisms and protects against I/R injury in isolated rat heart, indicating a novel cardioprotective role of this hormone.

0141 Combination of antagonists of growth hormone-releasing hormone with docetaxel as potential therapy in breast cancer in a preclinical study

0141 Combination of antagonists of growth hormone-releasing hormone with docetaxel as potential therapy in breast cancer in a preclinical study

Antioxidant activity of growth hormone-releasing hormone antagonists in LNCaP human prostate cancer line

Hypothalamic growth hormone-releasing hormone (GHRH) controls the release of growth hormone and acts as a growth factor in various tumors. Potent antagonistic analogues of GHRH have been synthesized that strongly suppress the growth of diverse cancers through several mechanisms. However, the influence of GHRH antagonists on the redox (reduction/oxidation) status of cancers has not been investigated. Cellular generation of reactive oxygen species (ROS) is central to redox signaling and is implicated in the initiation, development, and progression of cancer. In this study, we evaluated by Western blot the effects in vitro of GHRH and its antagonist JMR-132 on proliferating cell nuclear antigen, tumor suppressor protein p53, transcription factor NF-kappaB p50 and its phosphorylated form, caspase 3, and cleaved caspase 3 in the LNCaP human prostate cancer cell line. GHRH stimulated and GHRH antagonist inhibited the expression of the major antioxidant enzymes, as well as the expression of COX 2 and cytochrome c oxidase IV, which are enzymes involved in the generation of ROS. GHRH augmented and GHRH antagonist suppressed lipid and protein oxidative stress markers, as well as the intracellular generation of ROS. In all these tests, GHRH antagonists exerted strong antioxidant activity. Because the metabolism of ROS and oxidative stress have been associated with initiation and progression of not only prostate tumors but also other malignancies, our findings reinforce previous experimental evidence that GHRH antagonists could be useful for cancer therapy.

Essential role of p21/waf1 in the mediation of the anti-proliferative effects of GHRH antagonist JMR-132

GHRH, besides its neuroendocrine action in controlling the release of GH from the pituitary, stimulates the growth of various cancers in vivo and in vitro by direct mechanism(s). However, the molecular mechanism that mediates these proliferative effects of GHRH in extrapituitary tissues remains poorly characterized. In the present study, we investigated whether the tumor suppressor p21/waf1 is involved in the mediation of the proliferative effects of GHRH in A549 human lung cancer epithelial cells. Exposure of A549 cells to the GHRH antagonist JMR-132 caused a significant inhibition in the rate of cell proliferation. In A549 cells, GHRH suppressed while JMR-132 increased the levels of p21 expression in a dose-dependent manner. This suggests that GHRH could regulate p21 levels. We then evaluated whether p21 is required in A549 cells for the regulation of cell proliferation by GHRH. To this end, we knocked-down p21 expression in A549 cells by siRNA and assessed the effects of antagonist JMR-132 on cell proliferation. We found that the loss of p21 expression abolished the anti-proliferative effects of JMR-132. Suppression of p21 expression by siRNA in human HT29 colon cancer cells and non-transformed mouse osteoblasts KS483 also blocked the anti-proliferative effects of JMR-132 suggesting that the regulation of cell proliferation by GHRH is p21 dependent. These results shed light on the molecular mechanism of action of GHRH antagonists in tumor tissues and suggest that the antineoplastic activity of GHRH antagonists could be considered for the treatment of cancers expressing p21.

Dose‐dependent growth inhibition in vivo of PC‐3 prostate cancer with a reduction in tumoral growth factors after therapy with GHRH antagonist MZ‐J‐7‐138

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers and affect tumoral growth factors. We investigated the effect of a new GHRH antagonist MZ-J-7-138 at doses of 1.25, 2.5, 5 and 10 microg/day s.c. on the growth of PC-3 human androgen independent prostate cancers xenografted s.c. into nude mice. Binding assays were used to investigate GHRH receptors. The levels of IGF-II and VEGF in tumors were measured by radioimmunoassays. Treatment with 2.5, 5, and 10 microg/day MZ-J-7-138 caused a significant dose-dependent growth reduction of PC-3 tumors. The greatest inhibition of 78% was obtained with 10 microg/day. The suppression of IGF-II protein levels in tumors was seen at all doses of MZ-J-7-138, but only 10 microg dose induced a significant inhibition. MZ-J-7-138 also reduced VEGF protein levels, the inhibition being significant at doses of 5 and 10 microg. Specific high affinity binding sites for GHRH were found on PC-3 tumors using (125)I-labeled GHRH antagonist JV-1-42. MZ-J-7-138 displaced radiolabeled JV-1-42 with an IC(50) of 0.32 nM indicating its high affinity to GHRH receptors. Real-time PCR analyses detected splice variant 1 (SV1) of GHRH receptor (GHRH-R) as well as pituitary type of GHRH-R and GHRH ligand. Our results demonstrate the efficacy of GHRH antagonist MZ-J-7-138 in suppressing growth of PC-3 prostate cancer at doses lower than previous antagonists. The reduction of levels of growth factors such as VEGF and IGF-II in tumors by GHRH antagonist was correlated with the suppression of tumor growth.

Triple-negative breast cancers express receptors for growth hormone-releasing hormone (GHRH) and respond to GHRH antagonists with growth inhibition

Triple-negative breast cancers do not express receptors for estrogen or progesterone and do not overexpress HER2. These tumors have an unfavorable prognosis and at present chemotherapy is the only treatment option. Because the antagonists of growth hormone-releasing hormone (GHRH) have been shown to inhibit growth of a variety of cancers by endocrine and paracrine/autocrine mechanisms, we evaluated the expression of GHRH receptors in human specimens of triple-negative breast cancers and the response to GHRH by in vitro models. In samples of triple-negative breast cancers we found mRNA expression for the GHRH receptor and its functional splice variant SV1 in 25 and 70% of the cases, respectively and for GHRH in 80% of the samples. Immunoreaction of SV1 was detected in the human triple-negative breast cancer cell line HCC1806 while HCC1937 was negative. The growth of HCC1806 was stimulated by GHRH(1-44)NH(2) and inhibited by GHRH antagonist MZ-J-7-118. In addition, in HCC1806 MAP-kinases ERK-1/2 were activated by GHRH. Our findings suggest the existence of an autocrine loop consisting of GHRH and GHRH receptors in triple-negative breast cancers. Our in vitro studies demonstrate that targeting the GHRH receptor may be a therapeutic option which should be evaluated in studies in vivo.

Differential expression of GHRH receptor and its splice variant 1 in human normal and malignant mucosa of the oesophagus and colon

Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as a growth factor for gastrointestinal (GI) tumours. The tumourigenic effects of GHRH appear to be mediated by the splice variant 1 (SV-1) of GHRH receptor as well as the full length pituitary type receptor for GHRH (GHRH-R). We examined the protein and mRNA expression of GHRH-R and SV-1 in normal human tissues and tumours of the gastrointestinal (GI-) tract by immunohistochemical staining and reverse transcriptase (RT)-PCR. Squamous cells and squamous cell carcinoma of the oesophagus were negative for GHRH-R and SV-1, while Barrett's mucosa and adenocarcinomas of the oesophagus showed a strong expression of both receptors. The expression of GHRH-R was absent in normal colonic mucosa other than neuroendocrine cells (NE) and lining epithelium (LE) but strong in tubular adenomas of the colon, while the staining for SV-1 was absent in cells other than NE. However, the expression of both receptors was significantly increased in tubulovillous adenomas and colorectal cancers. No differences were seen in protein levels for both receptors between normal and neoplastic tissues of the stomach, pancreas and liver. Because of low mRNA levels for both receptors in all samples tested, only a qualitative assessment could be made. However, mRNA for GHRH-R and SV-1 showed a near-perfect correlation with the assessment of receptor proteins by immunostaining. Our study shows that in contrast to normal mucosa, transformed mucosa of the oesophagus and the colon expresses GHRH-R and SV-1. This aberrant expression of GHRH-R and SV-1 in oesophageal and colorectal malignancies may provide a molecular target for a therapeutic approach based on GHRH antagonists.

Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines

Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines in vitro. However, the replacement of the knocked down GHRH expression by exogenous GHRH (1–29)NH2 re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1–29)NH2 and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor.

Inhibition of proliferation, VEGF secretion of human neuroendocrine tumor cell line NCI-H727 by an antagonist of growth hormone-releasing hormone (GH-RH) in vitro

Growth hormone-releasing hormone (GH-RH) can stimulate not only growth hormone (GH) secretion by anterior pituitary gland but also proliferation of many cancer cell lines in vitro and in xenografts tumor models in vivo. Several antagonists of GH-RH have been shown to inhibit several cancer growths, but the role of GH-RH antagonists in the regulation of neuroendocrine cancers cell proliferation and tumor progression remains obscure. The aim of the study was to evaluate the influence of JV-1-36 (synthetic GH-RH antagonist) on proliferation and VEGF secretion by human neuroendocrine lung non-small cell carcinoma (NCI-H727) using cell culture model. The in vitro effect of JV-1-36 on the proliferation of NCI-H727 cells was assessed by the measurement of BrdU incorporation by colorimetric immunoassay. The presence of VEGF and membrane GH-RH receptors on the surface of H727 cells were visualized by immunocytochemistry using specific anti-GH-RH receptor antibody directed to the carboxy-terminal region. VEGF secretion to the cell cultures supernatants was assessed by ELISA methods. Immunoreactive cell membrane GH-RH receptors and VEGF–immunopositive cytoplasmatic granules were clearly confined on the surface of nearly all cancer cells. JV-1-36 at the concentration of 10 −6–10 −10 M significantly inhibited growth of H727 cells, compared with untreated controls. In H727 cells, the antiproliferative JV-1-36 effect was associated with a dose-dependent reduction of VEGF secretion. In conclusion, our findings demonstrate the strong evidence for the antiproliferative action of GH-RH antagonist JV-1-36 for the NCI-H727 cells. In addition the suppression of VEGF secretion by H727 cells might contribute, at least in part, to the antitumor action of GH-RH antagonists.

New Approaches to the Therapy of Various Tumors Based on Peptide Analogues

The discovery of hypothalamic hormones was briefly reviewed. The development of new hormonal methods for the therapy of various cancers based on analogues of hypothalmic hormones is then presented. My group isolated luteininzing hormone-releasing hormone (LH-RH), also known as Gn-RH, from pig hypothalmi, elucidated its amino acid sequence, and synthesized it in 1971. The interest in medical applications of LH-RH led to the synthesis of LH-RH analogues by various groups. LH-RH agonists substituted in positions 6 or 10 including Decapeptyl, Leuprolide and Zoladex are much more active than LH-RH and on continuous administration produce inhibition of pituitary and gonads. Chronic administration of LH-RH agonists is being utilized for the treatment of prostate and breast cancer. Octapeptide analogues of somatostatin have various applications in Oncology. In 1980 we developed a new endocrine therapy for advanced prostate cancer based on agonists of LH-RH, which is now preferred by 70-90% of prostate cancer patients for primary treatment. LH-RH antagonists such as Cetrorelix can be used for therapy of BPH. On the basis of the presence of specific receptors for hypothalamic peptides on human cancers, we developed targeted cytotoxic analogues of LH-RH, somatostatin, and bombesin/GRP linked to doxorubicin or 2-pyrrolinodoxorubicin. These analogues inhibit the growth of experimental human prostate, breast, ovarian and endometrial cancer, renal cell carcinoma, pancreatic, colorectal and gastric cancers, small cell lung carcinoma (SCLC) and non-SCLC, brain tumors, melanomas, and lymphomas. Cytotoxic LH-RH analogues are now in clinical trials. Recently we demonstrated that growth hormone-releasing hormone (GH-RH) also serves as an autocrine growth factor in many cancers. Antagonistic analogues of GH-RH synthesized in our laboratory inhibit the growth of diverse tumors. The discovery of LH-RH and somatostatin has led to clinical use of their analogues in the field of cancer treatment and GH-RH antagonists also show a great promise.

Cellular mechanisms of growth inhibition of human endometrial cancer cell line by an antagonist of growth hormone-releasing hormone

The expression of growth hormone-releasing hormone (GHRH) and its receptors has been demonstrated in peripheral tissues as well as CNS. Recently, the functional splice variant SV1 of GHRH receptor was identified in various human cancers and cancer cell lines. Although antineoplastic activity of GHRH antagonists has been clearly demonstrated, the mechanism of action is incompletely understood. The objective of this study was the investigation of direct anti-proliferative effect of GHRH antagonist MZ-5-156 on HEC-1A human endometrial cancer cell line and the elucidation of underlying mechanisms. RT-PCR revealed the expression of mRNA for GHRH and SV1 of GHRH receptor in HEC-1A cells. MZ-5-156, at concentrations between 10(-7) and 10(-5) M, had a dose-dependent antiproliferative effect on HEC-1A cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, (MTS) assay. Hoechst 33342 staining and flow cytometric analysis indicated that MZ-5-156, at 10(-6) M, induced apoptosis in HEC-1A cells after 48 h of treatment. Western blot analysis of apoptosis-related proteins demonstrated that treatment with MZ-5-156 (10(-6) M) for 48 h significantly increased the protein levels of Fas, phospho-p53 (Ser46), p53AIP1 (p53-regulated Apoptosis-Inducing Protein 1), and caspase-8, -9, and -3, and decreased the protein level of Bcl-2. These results demonstrate that MZ-5-156 can directly inhibit the proliferation of human endometrial cancer cells, which express mRNA for GHRH and SV1 of GHRH receptor, presumably through the induction of p53-dependent apoptosis coupled with the up-regulation of Fas, phospho-p53 (Ser46), p53AIP1, and caspase-8, -9, and -3, and the down-regulation of Bcl-2.

Endocrine and Antineoplastic Actions of Growth Hormone-Releasing Hormone Antagonists

Potent antagonists of growth hormone-releasing hormone (GHRH) have been developed for the treatment of disorders caused by excessive GHRH or growth hormone (GH) production and for therapy of cancers. GHRH antagonists suppressed the release of GH and insulin-like growth factor (IGF)-I in transgenic mice overexpressing human (h) GHRH gene, an animal model of human acromegaly. It was also shown in GH3 rat pituitary tumor cells overexpressing the human pituitary GHRH receptor (pGHRH-R) that GHRH antagonists can inhibit c-AMP production and GH secretion through the human receptor. These observations indicate that GHRH antagonists could be used clinically in disorders characterized by excessive GHRH/GH secretion. Many recent studies demonstrate that GHRH antagonists can inhibit tumor growth by several mechanisms. By indirect action through pGHRH-Rs these antagonists suppress circulating GH/IGF-I level, which results in the inhibition of cancers that depend on GH and/or IGF-I as growth factors. However, GHRH antagonists are also effective inhibitors of tumor IGF-II production, which is a potent mitogen but independent of GH. GHRH antagonists can inhibit tumor cell proliferation by direct action on tumor cell receptors, suppressing the IGF-II and other growth factor production of tumor cells. In addition, various human tumors and tumor cell lines secrete GHRH peptide and respond to GHRH with proliferation. This finding suggests that GHRH functions as an autocrine growth factor and that GHRH antagonists can block its effects on tumor growth. Recently, we demonstrated the expression of hGHRH-R and its splice variants in various human cancers. Antiproliferative action of GHRH antagonists on these cancers indicates that the direct inhibitory effects of GHRH antagonists are mediated by tumoral GHRH receptors.

Inhibition of proliferation of small intestinal and bronchopulmonary neuroendocrine cell lines by using peptide analogs targeting receptors

Currently, no consistently effective therapy is available to inhibit cell proliferation or metastasis of neuroendocrine tumor (NET) disease. The effects of 4 novel peptides were analyzed: a targeted cytotoxic analog of luteinizing hormone-releasing hormone (LH-RH) analog (AN-152), a targeted cytotoxic analog of somatostatin (AN-238), and 2 antagonists of growth hormone-releasing hormone (GH-RH) on 3 NET (carcinoid) cell lines that expressed respective peptide receptors. The effects of the compounds were evaluated on cell proliferation in vitro using MTT uptake and Ki67 expression, apoptosis (caspase 3 expression and activity), and cell cycle parameters (DNA distribution). Proliferation of the LH-RH receptor-expressing lung NET, NCI-H720 line, was inhibited 2-fold by AN-152 containing doxorubicin compared with the chemotherapy alone (IC50 of 9.1 nM vs 24 nM). This was associated with a reduction in Ki67 transcript and an increase in both caspase 3 mRNA levels and activity. Proliferation of the GH-RH receptor expressing lung NET, NCI-H727 line, was inhibited by both GH-RH antagonists, the effects being mediated through changes in Ki67 expression, but not in caspase 3-mediated apoptosis. The small intestinal NET, KRJ-I line, was 8x more sensitive to inhibition by AN-238 than to 2-pyrolino-doxorubicin, reflected by increased caspase 3 transcript as well as activity. AN-238-mediated growth inhibition culminated in complete G1 arrest. The data demonstrate GH-RH antagonists or peptide-linked antineoplastic agents such as AN-152 and AN-238 are effective inhibitors of NET proliferation in vitro. Because peptide receptors such as those for GH-RH, LH-RH, and SST subtypes are commonly expressed by NETs, the development of antineoplastic agents targeted to specific tumor receptors may provide a more efficacious strategy than systemic chemotherapeutic agents currently in use.

Expression of growth hormone–releasing hormone receptor splice variant 1 in primary human melanomas

Growth hormone–releasing hormone (GHRH) is secreted by the hypothalamus and upon binding to specific GHRH receptors in the pituitary stimulates growth hormone production and release. In addition to its neuroendocrine action GHRH plays a role in tumorigenesis. Consistently with this latter role, the splice variant 1 (SV1) of GHRH receptor, which is widely expressed in non-pituitary normal tissues and cancers, can mediate the proliferative effects of GHRH and even in the absence of GHRH is capable of eliciting mitogenic signals in the tissues in which it is expressed. The aim of the present study was to investigate the expression of GHRH and its tumoral receptor SV1 in primary human melanomas and dysplastic nevi by immunohistochemistry. None of the specimens tested expressed GHRH. Only 1 of 12 (8%) dysplastic nevi expressed SV1 but 14 of 23 (61%) melanomas showed moderate or strong staining for SV1 (association p<0.005). This is the first report demonstrating the involvement of SV1 in the pathogenesis of melanomas. Our work implies that the progression from a state of dysplasia into malignancy is accompanied by expression of SV1 receptor. Our findings also suggest that treatment with GHRH antagonists should be further explored for the management of malignant melanomas.

Antagonists of growth-hormone-releasing hormone: an emerging new therapy for cancer

This article reviews the potential clinical uses of antagonists of growth-hormone-releasing hormone (GHRH) for tumor therapy. GHRH antagonists suppress the growth of various human cancer lines xenografted into nude mice; such tumors include breast, ovarian, endometrial and prostate cancers, lung cancers (small-cell lung carcinomas and non-small-cell lung carcinomas), renal, pancreatic, gastric and colorectal carcinomas, brain tumors (malignant gliomas), osteogenic sarcomas and non-Hodgkin's lymphomas. The antitumor effects of GHRH antagonists are exerted in part indirectly through the inhibition of the secretion of GH from the pituitary and the resulting reduction in the levels of hepatic insulin-like growth factor I (IGF-I). The main effects of the GHRH antagonists are, however, exerted directly on tumors. GHRH ligand is present in various human cancers and might function as an autocrine and/or paracrine growth factor. Pituitary-type GHRH receptors and their splice variants are also found in many human cancers. The inhibitory effects of GHRH antagonists seem to be due to the blockade of action of tumoral GHRH. Antagonists of GHRH can also suppress cancer growth by blocking production of IGF-I and/or IGF-II by the tumor. Further development of GHRH antagonists that are still-more potent should lead to potential therapeutic agents for various cancers.