Growth hormone-releasing hormone antagonists counteract interferon-γ - induced barrier dysfunction in bovine and human endothelial cells.

GHRH regulates the secretion of GH from the anterior pituitary gland. An emerging body of evidence suggests that the activities of that neuropeptide are not limited to the GH/IGF-I axis, but they expand towards the mediation of inflammatory processes. GHRHAnt were developed to oppose the activities of GHRH in malignancies, and have been associated with strong anti-inflammatory and anti-oxidative effects in a diverse variety of tissues, including the lungs. In the present study we report that GHRHAnt oppose interferon-γ - induced paracellular hyperpermeability and reactive oxygen species generation in bovine and human pulmonary endothelial cells; and suppress interferon-γ - triggered STAT3, cofilin and ERK1/2 activation. Our observations substantiate previous findings on the protective effects of GHRHAnt in endothelial inflammation and barrier break-down.

The influence of a synthetic growth hormone-releasing hormone analogue G11 and opioid peptide biphalin on selected fibroblasts parameters relevant to wound healing.

The treatment of hard-to-heal chronic wounds is still a major medical problem and an economic and social burden. In this work, we examine the proregenerative potential of two peptides, G11 (a trypsin-resistant analogue of growth hormone-releasing hormone [GHRH]) and biphalin (opioid peptide), and their combination in vitro on human fibroblasts (BJ). G11, biphalin and their combination exhibited no toxicity against BJ cells. On the contrary, these treatments significantly stimulated proliferation and migration of fibroblasts. Under inflammatory conditions (LPS-induced BJ cells), we noticed that the tested peptides decreased the levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and interleukin 1ß (IL-1ß). This was correlated with diminished phosphorylation levels of p38 kinase, but not those of ERK1/2. We found also that G11, biphalin and their combination activated the ERK1/2 signalling pathway, which has been previously implicated in promigratory activity of some regeneration enhancers, including opioids or GHRH analogues. Potential application of their combination requires further work, in particular in vivo experiments, in which the organism-level relevance of the discussed cell-level effects would be proven and, additionally, analgesic action of the opioid ingredient could be quantified.

Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in Primary Human Endometrial Carcinomas: Novel Therapeutic Approaches.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various tumors, including endometrial carcinomas (EC). However, tumoral receptors that mediate the antiproliferative effects of GHRH antagonists in human ECs have not been fully characterized. In this study, we investigated the expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors (GHRH-R) in 39 human ECs and in 7 normal endometrial tissue samples using RT-PCR. Primers designed for the PCR amplification of mRNA for the full length GHRH-R and SVs were utilized. The PCR products were sequenced, and their specificity was confirmed. Nine ECs cancers (23%) expressed mRNA for SV1, three (7.7%) showed SV2 and eight (20.5%) revealed mRNA for SV4. The presence of SVs for GHRH-Rs could not be detected in any of the normal endometrial tissue specimens. The presence of specific, high affinity GHRH-Rs was also demonstrated in EC specimens using radioligand binding studies. Twenty-four of the investigated thirty-nine tumor samples (61.5%) and three of the seven corresponding normal endometrial tissues (42.9%) expressed mRNA for GHRH ligand. Our findings suggest the possible existence of an autocrine loop in EC based on GHRH and its tumoral SV receptors. The antiproliferative effects of GHRH antagonists on EC are likely to be exerted in part by the local SVs and GHRH system.

Suppression of reactive oxygen species in endothelial cells by an antagonist of growth hormone-releasing hormone.

Growth hormone-releasing hormone (GHRH) is a hypothalamic hormone, which regulates the secretion of growth hormone (GH) from the anterior pituitary gland. The effects of GHRH extend beyond the GH-insulin-like growth factor I axis, and that neuropeptide has been involved in the potentiation of several malignancies and other inflammatory disorders. The development of GHRH antagonists (GHRHAnt) delivers an exciting possibility to counteract the pathogenesis of the GHRH-related effects in human pathophysiology, especially when considered that GHRHAnt support endothelial barrier integrity. Those GHRHAnt-mediated effects are exerted at least in part due to the suppression of major inflammatory pathways, and the modulation of major cytoskeletal components. In the present study, we measured the production of reactive oxygen species (ROS) in bovine pulmonary artery endothelial cells, human cerebral microvascular endothelial cells, and human lung microvascular endothelial cells exposed to GHRH or a commercially available GHRHAnt. Our findings reveal the antioxidative effects of GHRHAnt in all three cell lines, which express GHRH receptors. The redox status of NIH/3T3 cells, which do not produce GHRH receptors, was not significantly affected by GHRH or GHRHAnt. Hence, the application of GHRHAnt in pathologies related to increased ROS production should be further investigated.

Agonists of growth hormone-releasing hormone (GHRH) inhibit human experimental cancers in vivo by down-regulating receptors for GHRH.

The effects of the growth hormone-releasing hormone (GHRH) agonist MR409 on various human cancer cells were investigated. In H446 small cell lung cancer (SCLC) and HCC827 and H460 (non-SCLC) cells, MR409 promoted cell viability, reduced cell apoptosis, and induced the production of cellular cAMP in vitro. Western blot analyses showed that treatment of cancer cells with MR409 up-regulated the expression of cyclins D1 and D2 and cyclin-dependent kinases 4 and 6, down-regulated p27kip1, and significantly increased the expression of the pituitary-type GHRH receptor (pGHRH-R) and its splice-variant (SV1). Hence, in vitro MR409 exerts agonistic action on lung cancer cells in contrast to GHRH antagonists. However, in vivo, MR409 inhibited growth of lung cancers xenografted into nude mice. MR409 given s.c. at 5 µg/day for 4 to 8 weeks significantly suppressed growth of HCC827, H460, and H446 tumors by 48.2%, 48.7%, and 65.6%, respectively. This inhibition of tumor growth by MR409 was accompanied by the down-regulation of the expression of pGHRH-R and SV1 in the pituitary gland and tumors. Tumor inhibitory effects of MR409 in vivo were also observed in other human cancers, including gastric, pancreatic, urothelial, prostatic, mammary, and colorectal. This inhibition of tumor growth parallel to the down-regulation of GHRH-Rs is similar and comparable to the suppression of sex hormone-dependent cancers after the down-regulation of receptors for luteinizing hormone-releasing hormone (LHRH) by LHRH agonists. Further oncological investigations with GHRH agonists are needed to elucidate the underlying mechanisms.

Growth hormone-releasing hormone promotes therapeutic effects of peripheral blood endothelial progenitor cells in ischemic repair.

PURPOSE: In peripheral artery disease, blockage of the blood supply to the limbs leads to blood flow attenuation and tissue ischemia. We investigated whether growth hormone-releasing hormone (GHRH) could enhance the biological functions and therapeutic effects of endothelial progenitor cells (EPCs) derived from adult human peripheral blood (PB). METHODS: EPCs were isolated from human PB (PB-EPCs) and cord blood and expanded in vitro. PB-EPCs incubated with or without GHRH were evaluated for proliferation, migration, and angiogenesis capacity and apoptosis rates under oxidative stress conditions. Activation of STAT3 and Akt pathways was evaluated using Western blot. A hind-limb ischemia (HLI) mouse model was used to study the efficacy of GHRH in improving EPC therapy in vivo. RESULTS: GHRH enhanced the proliferation, migration, and angiogenesis capacity of PB-EPCs and reduced apoptosis under H2O2 stimulation. These beneficial effects were GHRH receptor-dependent and were paralleled by increased phosphorylation of STAT3 and Akt. Transplantation of GHRH-preconditioned EPCs into HLI model mice enhanced blood flow recovery by increasing vascular formation density and enhanced tissue regeneration at the lesion site. CONCLUSION: Our studies demonstrate a novel role for GHRH in dramatically improving therapeutic angiogenesis in HLI by enhancing the biological functions of EPCs. These findings support additional studies to explore the full potential of GHRH in augmenting cell therapy for the management of ischemia.

Antagonists of growth hormone-releasing hormone inhibit proliferation induced by inflammation in prostatic epithelial cells.

The etiology of benign prostatic hyperplasia (BPH) is multifactorial, and chronic inflammation plays a pivotal role in its pathogenesis. Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide that has been shown to act as paracrine/autocrine factor in various malignancies including prostate cancer. GHRH and its receptors are expressed in experimental models of BPH, in which antagonists of GHRH suppressed the levels of proinflammatory cytokines and altered the expression of genes related to epithelial-to-mesenchymal transition (EMT). We investigated the effects of GHRH antagonist on prostatic enlargement induced by inflammation. Autoimmune prostatitis in Balb/C mice was induced by a homogenate of reproductive tissues of male rats. During the 8-wk induction of chronic prostatitis, we detected a progressive increase in prostatic volume reaching 92% at week 8 compared with control (P < 0.001). Daily treatment for 1 mo with GHRH antagonist MIA-690 caused a 30% reduction in prostate volume (P < 0.05). Conditioned medium derived from macrophages increased the average volume of spheres by 82.7% (P < 0.001) and elevated the expression of mRNA for N-cadherin, Snail, and GHRH GHRH antagonist reduced the average volume of spheres stimulated by inflammation by 75.5% (P < 0.05), and TGF-ß2 by 91.8% (P < 0.01). The proliferation of primary epithelial cells stimulated by IL-17A or TGF-ß2 was also inhibited by 124.1% and 69.9%, respectively. GHRH stimulated the growth of BPH-1 and primary prostate spheres. This study provides evidence that GHRH plays important roles in prostatic inflammation and EMT and suggests the merit of further investigation to elucidate the effects of GHRH antagonists in prostatitis and BPH.

GHRH-mediated GH release is associated with sympathoactivation and baroreflex resetting: a microneurographic study in healthy humans.

Previous research suggested substantial interactions of growth hormone (GH) and sympathetic nervous activity. This cross talk can be presumed both during physiological (e.g., slow-wave sleep) and pathological conditions of GH release. However, microneurographic studies of muscle sympathetic nerve activity (MSNA) and assessment of baroreflex function during acute GH-releasing hormone (GHRH)-mediated GH release were not conducted so far. In a balanced, double-blind crossover design, GHRH or placebo (normal saline) were intravenously administered to 11 healthy male volunteers. MSNA was assessed microneurographically and correlated with blood pressure (BP) and heart rate (HR) at rest before (pre-) and 30-45 (post-I) and 105-120 min (post-II) after respective injections. Additionally, baroreflex function was assessed via graded infusion of vasoactive drugs. GHRH increased GH serum levels as intended. Resting MSNA showed significant net increases of both burst rate and total activity from pre- to post-I and post-II following GHRH injections compared with placebo (ANOVA for treatment and time, burst rate: P = 0.028; total activity: P = 0.045), whereas BP and HR were not altered. ANCOVA revealed that the dependent variable MSNA was not affected by the independent variables mean arterial BP (MAP) or HR (MAP: P = 0.006; HR: P = 0.003). Baroreflex sensitivity at baroreflex challenge was not altered. GHRH-mediated GH release is associated with a significant sympathoactivation at central nervous sites superordinate to the simple baroreflex feedback loop because GH induced a baroreflex resetting without altering baroreflex sensitivity.

Growth hormone/IGF-1 axis longitudinal evaluation in clinically isolated syndrome patients on interferon ß-1b therapy: stimulation tests and correlations with clinical and radiological conversion to multiple sclerosis.

BACKGROUND AND PURPOSE: Growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis abnormalities in multiple sclerosis (MS) suggest their role in its pathogenesis. Interferon ß (IFN-ß) efficacy could be mediated also by an increase of IGF-1 levels. A 2-year longitudinal study was performed to estimate the prevalence of GH and/or IGF-1 deficiency in clinically isolated syndrome (CIS) patients and their correlation with conversion to MS in IFN treated patients. METHODS: Clinical and demographic features of CIS patients were collected before the start of IFN-ß-1b. IGF-1 levels and GH response after arginine and GH releasing hormone + arginine stimulation tests were assessed. Clinical and magnetic resonance imaging evaluations were performed at baseline, 1 year and 2 years. RESULTS: Thirty CIS patients (24 female) were enrolled. At baseline, four patients (13%) showed a hypothalamic GH deficiency (GHD), whilst no one had a pituitary GHD. Baseline demographic, clinical and radiological data were not related to GHD, whilst IGF-1 levels were inversely related to age (P < 0.001) and GH levels (P = 0.03). GH and IGF-1 serum mean levels were not significantly modified after 1 and 2 years of treatment in the whole group, although 3/4 GHD patients experienced a normalization of GH levels, whilst one dropped out. After 2 years of treatment 13/28 (46%) patients converted to MS. The presence of GHD and GH and IGF-1 levels were not predictive of relapses, new T2 lesions or conversion occurrence. CONCLUSIONS: Growth hormone/IGF-1 axis function was found to be frequently altered in CIS patients, but this was not related to MS conversion. Patients experienced an improvement of GHD during IFN therapy. Longer follow-up is necessary to assess its impact on disease progression.

Profound Actions of an Agonist of Growth Hormone-Releasing Hormone on Angiogenic Therapy by Mesenchymal Stem Cells.

OBJECTIVE: The efficiency of cell therapy is limited by poor cell survival and engraftment. Here, we studied the effect of the growth hormone-releasing hormone agonist, JI-34, on mesenchymal stem cell (MSC) survival and angiogenic therapy in a mouse model of critical limb ischemia. APPROACH AND RESULTS: Mouse bone marrow-derived MSCs were incubated with or without 10(-8) mol/L JI-34 for 24 hours. MSCs were then exposed to hypoxia and serum deprivation to detect the effect of preconditioning on cell apoptosis, migration, and tube formation. For in vivo tests, critical limb ischemia was induced by femoral artery ligation. After surgery, mice received 50 µL phosphate-buffered saline or with 1×10(6) MSCs or with 1×10(6) JI-34-reconditioned MSCs. Treatment of MSCs with JI-34 improved MSC viability and mobility and markedly enhanced their capability to promote endothelial tube formation in vitro. These effects were paralleled by an increased phosphorylation and nuclear translocation of signal transducer and activator of transcription 3. In vivo, JI-34 pretreatment enhanced the engraftment of MSCs into ischemic hindlimb muscles and augmented reperfusion and limb salvage compared with untreated MSCs. Significantly more vasculature and proliferating CD31(+) and CD34(+) cells were detected in ischemic muscles that received MSCs treated with JI-34. CONCLUSIONS: Our studies demonstrate a novel role for JI-34 to markedly improve therapeutic angiogenesis in hindlimb ischemia by increasing the viability and mobility of MSCs. These findings support additional studies to explore the full potential of growth hormone-releasing hormone agonists to augment cell therapy in the management of ischemia.

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists.

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFß. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.

Agonists of growth hormone-releasing hormone stimulate self-renewal of cardiac stem cells and promote their survival.

The beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R) in heart failure models are associated with an increase in the number of ckit(+) cardiac stem cells (CSCs). The goal of the present study was to determine the presence of GHRH-R in CSCs, the effect of GHRH-R agonists on their proliferation and survival, and the mechanisms involved. We investigated the expression of GHRH-R in CSCs of different species and the effect of GHRH-R agonists on their cell proliferation and survival. GHRH-R is expressed in ckit(+) CSCs isolated from mouse, rat, and pig. Treatment of porcine CSCs with the GHRH-R agonist JI-38 significantly increased the rate of cell division. Similar results were observed with other GHRH-R agonists, MR-356 and MR-409. JI-38 exerted a protective effect on survival of porcine CSCs under conditions of oxidative stress induced by exposure to hydrogen peroxide. Treatment with JI-38 before exposure to peroxide significantly reduced cell death. A similar effect was observed with MR-356. Addition of GHRH-R agonists to porcine CSCs induced activation of ERK and AKT pathways as determined by increased expression of phospho-ERK and phospho-AKT. Inhibitors of ERK and AKT pathways completely reversed the effect of GHRH-R agonists on CSC proliferation. Our findings extend the observations of the expression of GHRH-R by CSCs and demonstrate that GHRH-R agonists have a direct effect on proliferation and survival of CSCs. These results support the therapeutic use of GHRH-R agonists for stimulating endogenous mechanisms for myocardial repair or for preconditioning of stem cells before transplantation.

Protection of neonatal rat cardiac myocytes against radiation-induced damage with agonists of growth hormone-releasing hormone.

Despite the great clinical significance of radiation-induced cardiac damage, experimental investigation of its mechanisms is an unmet need in medicine. Beneficial effects of growth hormone-releasing hormone (GHRH) agonists in regeneration of the heart have been demonstrated. The aim of this study was the evaluation of the potential of modern GHRH agonistic analogs in prevention of radiation damage in an in vitro cardiac myocyte-based model. Cultures of cardiac myocytes isolated from newborn rats (NRVM) were exposed to a radiation dose of 10Gy. The effects of the agonistic analogs, JI-34 and MR-356, of human GHRH on cell viability, proliferation, their mechanism of action and the protein expression of the GHRH/SV1 receptors were studied. JI-34 and MR-356, had no effect on cell viability or proliferation in unirradiated cultures. However, in irradiated cells JI-34 showed protective effects on cell viability at concentrations of 10 and 100nM, and MR-356 at 500nM; but no such protective effect was detected on cell proliferation. Both agonistic analogs decreased radiation-induced ROS level and JI-34 interfered with the activation of SAFE/RISK pathways. Using Western blot analysis, a 52kDa protein isoform of GHRHR was detected in the samples in both irradiated and unirradiated cells. Since GHRH agonistic analogs, JI-34 and MR-356 alleviated radiation-induced damage of cardiac myocytes, they should be tested in vivo as potential protective agents against radiogenic heart damage.

Peripheral activities of growth hormone-releasing hormone.

Growth hormone (GH)-releasing hormone (GHRH) is produced by the hypothalamus and stimulates GH synthesis and release in the anterior pituitary gland. In addition to its endocrine role, GHRH exerts a wide range of extrapituitary effects which include stimulation of cell proliferation, survival and differentiation, and inhibition of apoptosis. Accordingly, expression of GHRH, as well as the receptor GHRH-R and its splice variants, has been demonstrated in different peripheral tissues and cell types. Among the direct peripheral activities, GHRH regulates pancreatic islet and ß-cell survival and function and endometrial cell proliferation, promotes cardioprotection and wound healing, influences the immune and reproductive systems, reduces inflammation, indirectly increases lifespan and adiposity and acts on skeletal muscle cells to inhibit cell death and atrophy. Therefore, it is becoming increasingly clear that GHRH exerts important extrapituitary functions, suggesting potential therapeutic use of the peptide and its analogs in a wide range of medical settings.

Effects of growth hormone-releasing hormone on sleep and brain interstitial fluid amyloid-ß in an APP transgenic mouse model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by impairment of cognitive function, extracellular amyloid plaques, intracellular neurofibrillary tangles, and synaptic and neuronal loss. There is substantial evidence that the aggregation of amyloid ß (Aß) in the brain plays a key role in the pathogenesis of AD and that Aß aggregation is a concentration dependent process. Recently, it was found that Aß levels in the brain interstitial fluid (ISF) are regulated by the sleep-wake cycle in both humans and mice; ISF Aß is higher during wakefulness and lower during sleep. Intracerebroventricular infusion of orexin increased wakefulness and ISF Aß levels, and chronic sleep deprivation significantly increased Aß plaque formation in amyloid precursor protein transgenic (APP) mice. Growth hormone-releasing hormone (GHRH) is a well-documented sleep regulatory substance which promotes non-rapid eye movement sleep. GHRHR(lit/lit) mice that lack functional GHRH receptor have shorter sleep duration and longer wakefulness during light periods. The current study was undertaken to determine whether manipulating sleep by interfering with GHRH signaling affects brain ISF Aß levels in APPswe/PS1ΔE9 (PS1APP) transgenic mice that overexpress mutant forms of APP and PSEN1 that cause autosomal dominant AD. We found that intraperitoneal injection of GHRH at dark onset increased sleep and decreased ISF Aß and that delivery of a GHRH antagonist via reverse-microdialysis suppressed sleep and increased ISF Aß. The diurnal fluctuation of ISF Aß in PS1APP/GHRHR(lit/lit) mice was significantly smaller than that in PS1APP/GHRHR(lit/+) mice. However despite decreased sleep in GHRHR deficient mice, this was not associated with an increase in Aß accumulation later in life. One of several possibilities for the finding is the fact that GHRHR deficient mice have GHRH-dependent but sleep-independent factors which protect against Aß deposition.

Activation of growth hormone releasing hormone (GHRH) receptor stimulates cardiac reverse remodeling after myocardial infarction (MI).

Both cardiac myocytes and cardiac stem cells (CSCs) express the receptor of growth hormone releasing hormone (GHRH), activation of which improves injury responses after myocardial infarction (MI). Here we show that a GHRH-agonist (GHRH-A; JI-38) reverses ventricular remodeling and enhances functional recovery in the setting of chronic MI. This response is mediated entirely by activation of GHRH receptor (GHRHR), as demonstrated by the use of a highly selective GHRH antagonist (MIA-602). One month after MI, animals were randomly assigned to receive: placebo, GHRH-A (JI-38), rat recombinant GH, MIA-602, or a combination of GHRH-A and MIA-602, for a 4-wk period. We assessed cardiac performance and hemodynamics by using echocardiography and micromanometry derived pressure-volume loops. Morphometric measurements were carried out to determine MI size and capillary density, and the expression of GHRHR was assessed by immunofluorescence and quantitative RT-PCR. GHRH-A markedly improved cardiac function as shown by echocardiographic and hemodynamic parameters. MI size was substantially reduced, whereas myocyte and nonmyocyte mitosis was markedly increased by GHRH-A. These effects occurred without increases in circulating levels of growth hormone and insulin-like growth factor I and were, at least partially, nullified by GHRH antagonism, confirming a receptor-mediated mechanism. GHRH-A stimulated CSCs proliferation ex vivo, in a manner offset by MIA-602. Collectively, our findings reveal the importance of the GHRH signaling pathway within the heart. Therapy with GHRH-A although initiated 1 mo after MI substantially improved cardiac performance and reduced infarct size, suggesting a regenerative process. Therefore, activation of GHRHR provides a unique therapeutic approach to reverse remodeling after MI.

Growth hormone-releasing hormone antagonists abolish the transactivation of human epidermal growth factor receptors in advanced prostate cancer models.

Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in a variety of cellular phenotypes related with tumorigenesis process. Human epidermal growth factor receptor family members (HER) such as EGFR and HER2 are involved in mitogenic signaling pathways implicated in the progression of prostate cancer. We analyzed the cross-talk between GHRH and EGF receptors in prostate cancer. The effects of GHRH in HER signaling were evaluated on human androgen-independent PC3 prostate cancer cells in vitro and GHRH antagonist in vitro and in nude mice xenografts of PC3 prostate cancer. Time-course studies indicated that GHRH had a stimulatory activity on both the expression of EGFR and HER2. GHRH analogues, JMR-132 and JV-1-38, endowed with antagonistic activity for GHRH receptors, abrogated the response to GHRH in PC3 cells. GHRH stimulated a rapid ligand-independent activation of EGFR and HER2 involving at least cAMP/PKA and Src family signaling pathways. GHRH also stimulated a slow ligand-dependent activation of EGFR and HER2 involving an extracellular pathway with an important role for ADAM. Preliminary results also revealed an increase of mRNA for GHRH and GHRH receptor induced by EGF. The inhibition of tumor growth, in vivo, was associated with a substantial reduction in the expression of mRNA and protein levels of EGFR and HER2 in the tumors. GHRH antagonist JV-1-38, significantly decreased the phosphorylated Src levels. The cross-talk between HER and GHRH-R may be impeded by combining drugs acting upon GHRH receptors and HER family members in human advanced prostate cancer.

Secretagogue induction of GH release in QNR/D cells: prevention of cell death.

Retinal ganglion cells (RGCs) in the chick embryonic neural retina are extrapituitary sites of growth hormone (GH) synthesis and release. The regulation of GH secretion by these cells is largely unknown, although we recently discovered several of the hypothalamic releasing factors involved in pituitary GH regulation (including GH-releasing hormone (GHRH) and thyrotropin releasing hormone, TRH) to be present in the cytoplasm of immortalized quail RGCs (QNR/D cells). QNR/D cells may therefore provide an experimental model for studies on GH regulation in the chick neural retina. The possibility that GHRH and TRH might stimulate GH secretion in QNR/D cells was therefore investigated. Both peptides acutely depleted the GH content of the QNR/D cells, as demonstrated by immunocytochemistry and ELISA, whilst increasing the GH content in incubation media. Both peptides also increased the immunochemical and ELISA content of the QNR/D cells and the content of GH in the incubation media after long-term incubation. Cell survival, determined by metabolic activity of the QNR/D cells and by TUNEL-labeling, was reduced when the endogenous GH content was reduced by GH immunoneutralization, even in the presence of exogenous GHRH or TRH. Cell survival was also reduced when endogenous GHRH was blocked by GHRH immunoneutralization, although the immunoneutralization of endogenous TRH did not affect QNR/D cell survival. In summary, these results demonstrate secretagogue actions of exogenous GHRH and TRH on the secretion of GH from QNR/D cells. They also suggest that endogenous GHRH, but not endogenous TRH, prevents cell death by increasing endogenous GH secretion in QNR/D cells.

Inhibitory effects of antagonists of growth hormone-releasing hormone on growth and invasiveness of PC3 human prostate cancer.

New approaches are needed to the therapy of advanced prostate cancer. This study determined the effect of growth hormone-releasing hormone (GHRH) antagonists, JMR-132 and JV-1-38 on growth of PC3 tumors as well as on angiogenesis and metastasis through the evaluation of various factors that contribute largely to the progression of prostate cancer. Human PC3 androgen-independent prostate cancer cells were injected subcutaneously into nude mice. The treatment with JMR-132 (10 µg/day) or JV-1-38 (20 µg/day) lasted 41 days. We also evaluated the effects of JMR-132 and JV-1-38 on proliferation, cell adhesion and migration in PC-3 cells in vitro. Several techniques (Western blot, reverse transcription polymerase chain reaction, immunohistochemistry, ELISA and zymography) were used to evaluate the expression levels of GHRH receptors and its splice variants, GHRH, vascular endothelial growth factor (VEGF), hypoxia inducible factor (HIF)-1α, metalloproteinases (MMPs) -2 and -9, ß-catenin and E-cadherin. GHRH antagonists suppressed the proliferation of PC-3 cells in vitro and significantly inhibited growth of PC3 tumors. After treatment with these analogues, we found an increase in expression of GHRH receptor accompanied by a decrease of GHRH levels, a reduction in both VEGF and HIF-1α expression and in active forms of MMP-2 and MMP-9, a significant increase in levels of membrane-associated ß-catenin and a significant decline in E-cadherin. These results support that the blockade of GHRH receptors can modulate elements involved in angiogenesis and metastasis. Consequently, GHRH antagonists could be considered as suitable candidates for therapeutic trials in the management of androgen-independent prostate cancer.

Expression of lymphocyte-derived growth hormone (GH) and GH-releasing hormone receptors in aging rats.

In the present study, we show that higher levels of lymphocyte GH are expressed in spleen cells from aging animals compared to young animals. Further, leukocytes from primary and secondary immune tissues and splenic T and B cells from aging rats all express higher levels of GHRH receptors compared to younger animals. Bone marrow and splenic T cells express the highest levels of GHRH receptor in aging animals. Spleen cells from aging animals showed no significant change in proliferation or GH induction after treatment with GHRH. Taken together, the data for the first time show alterations in GH synthesis and expression of the GHRH receptor on cells of the immune system that may play a role in the immune response in aging.