Preclinical efficacy of growth hormone-releasing hormone antagonists for androgen-dependent and castration-resistant human prostate cancer.

Advanced hormone-sensitive prostate cancer responds to androgen-deprivation therapy (ADT); however, therapeutic options for recurrent castration-resistant disease are limited. Because growth hormone-releasing hormone (GHRH) and GHRH receptor (GHRH-R) are regulated in an autocrine fashion in prostate cancer, inhibition of GHRH-R represents a compelling approach to treatment. We investigated the effects of the latest series of improved, highly potent GHRH antagonists--MIA-602, MIA-606, and MIA-690--on the growth of androgen-dependent as well as castration-resistant prostate cancer (CRPC) cells in vitro and in vivo. GHRH-R and its splice variant, SV1, were present in 22Rv1, LNCaP, and VCaP human prostate cancer cell lines. Androgen-dependent LNCaP and VCaP cells expressed higher levels of GHRH-R protein compared with castration-resistant 22Rv1 cells; however, 22Rv1 expressed higher levels of SV1. In vitro, MIA-602 decreased cell proliferation of 22Rv1, LNCaP, and VCaP prostate cancer cell lines by 70%, 61%, and 20%, respectively (all P < 0.05), indicating direct effects of MIA-602. In vivo, MIA-602 was more effective than MIA-606 and MIA-690 and decreased 22Rv1 xenograft tumor volumes in mice by 63% after 3 wk (P < 0.05). No noticeable untoward effects or changes in body weight occurred. In vitro, the VCaP cell line was minimally inhibited by MIA-602, but in vivo, this line showed a substantial reduction in growth of xenografts in response to MIA-602, indicating both direct and systemic inhibitory effects. MIA-602 also further inhibited VCaP xenografts when combined with ADT. This study demonstrates the preclinical efficacy of the GHRH antagonist MIA-602 for treatment of both androgen-dependent and CRPC.

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.

Antiamnesic properties of analogs and mimetics of the tripeptide human urocortin 3.

Amnesia is a deficit in memory caused by brain damage, disease, or trauma. Until now, there are no successful medications on the drug market available to treat amnesia. Short analogs and mimetics of human urocortin 3 (Ucn 3) tripeptide were synthetized and tested for their action against amnesia induced by eletroconvulsion in mice. Among the 16 investigated derivatives of Ucn 3 tripeptide, eight compounds displayed antiamnesic effect. Our results proved that the configuration of chiral center of glutamine does not affect the antiamnesic properties. Alkyl amide or isoleucyl amide at the C-terminus may lead to antiamnesic compounds. As concerned the N-terminus, acetyl, Boc, and alkyl ureido moieties were found among the active analogs, but the free amino function at the N-terminus usually led to an inactive derivatives. These observations may lead to the design and synthesis of small peptidomimetics and amino acid derivatives as antiamnesic drug candidates, although the elucidation of the mechanism of the action requires further investigations.

Antagonists of growth hormone-releasing hormone inhibit growth of androgen-independent prostate cancer through inactivation of ERK and Akt kinases.

The management of castration-resistant prostate cancer (CRPC) presents a clinical challenge because of limitations in efficacy of current therapies. Novel therapeutic strategies for the treatment of CRPC are needed. Antagonists of hypothalamic growth hormone-releasing hormone (GHRH) inhibit growth of various malignancies, including androgen-dependent and independent prostate cancer, by suppressing diverse tumoral growth factors, especially GHRH itself, which acts as a potent autocrine/paracrine growth factor in many tumors. We evaluated the effects of the GHRH antagonist, JMR-132, on PC-3 human androgen-independent prostate cancer cells in vitro and in vivo. JMR-132 suppressed the proliferation of PC-3 cells in vitro in a dose-dependent manner and significantly inhibited growth of PC-3 tumors by 61% (P < 0.05). The expression of GHRH, GHRH receptors, and their main splice variant, SV1, in PC-3 cells and tumor xenografts was demonstrated by RT-PCR and Western blot. The content of GHRH protein in PC-3 xenografts was lowered markedly, by 66.3% (P < 0.01), after treatment with JMR-132. GHRH induced a significant increase in levels of ERK, but JMR-132 abolished this outcome. Our findings indicate that inhibition of PC-3 prostate cancer by JMR-132 involves inactivation of Akt and ERK. The inhibitory effect produced by GHRH antagonist can result in part from inactivation of the PI3K/Akt/mammalian target of rapamycin and Raf/MEK/ERK pathways and from the reduction in GHRH produced by cancer cells. Our findings support the role of GHRH as an autocrine growth factor in prostate cancer and suggest that antagonists of GHRH should be considered for further development as therapy for CRPC.

Antagonists of growth hormone-releasing hormone (GHRH) reduce prostate size in experimental benign prostatic hyperplasia.

Growth hormone-releasing hormone (GHRH), a hypothalamic polypeptide, acts as a potent autocrine/paracrine growth factor in many cancers. Benign prostatic hyperplasia (BPH) is a pathologic proliferation of prostatic glandular and stromal tissues; a variety of growth factors and inflammatory processes are inculpated in its pathogenesis. Previously we showed that potent synthetic antagonists of GHRH strongly inhibit the growth of diverse experimental human tumors including prostate cancer by suppressing various tumoral growth factors. The influence of GHRH antagonists on animal models of BPH has not been investigated. We evaluated the effects of the GHRH antagonists JMR-132 given at doses of 40 µg/d, MIA-313 at 20 µg/d, and MIA-459 at 20 µg/d in testosterone-induced BPH in Wistar rats. Reduction of prostate weights was observed after 6 wk of treatment with GHRH antagonists: a 17.8% decrease with JMR-132 treatment; a 17.0% decline with MIA-313 treatment; and a 21.4% reduction with MIA-459 treatment (P < 0.05 for all). We quantified transcript levels of genes related to growth factors, inflammatory cytokines, and signal transduction and identified significant changes in the expression of more than 80 genes (P < 0.05). Significant reductions in protein levels of IL-1ß, NF-κß/p65, and cyclooxygenase-2 (COX-2) also were observed after treatment with a GHRH antagonist. We conclude that GHRH antagonists can lower prostate weight in experimental BPH. This reduction is caused by the direct inhibitory effects of GHRH antagonists exerted through prostatic GHRH receptors. This study sheds light on the mechanism of action of GHRH antagonists in BPH and suggests that GHRH antagonists should be considered for further development as therapy for BPH.

Inhibitory effects of antagonists of growth hormone releasing hormone on experimental prostate cancers are associated with upregulation of wild-type p53 and decrease in p21 and mutant p53 proteins.

BACKGROUND: The tumor suppressor gene p53 is implicated in cell cycle control and apoptosis. Antagonists of growth hormone-releasing hormone (GHRH) have been shown to inhibit human experimental prostate cancers. METHODS: We investigated the involvement of p53 apoptotic pathways in this effect. Nude mice bearing xenografted PC-3, DU-145, and MDA-PCa-2b human prostate cancer lines were treated with a new potent GHRH antagonist MZ-J-7-138. To determine whether tumor inhibition by MZ-J-7-138 involves apoptotic mechanisms such as p53 and p21, we evaluated by Western Blot the expression of mutant mt-p53 in PC-3 and DU-145 and of wild type (wt-p53) in MDA-PCa-2b prostate cancers as well as p21. RESULTS: MZ-J-7-138 significantly inhibited the growth of PC-3, DU-145, and MDA-PCa-2b xenografts in nude mice. Androgen deprivation with the LHRH antagonist Cetrorelix enhanced the anti-proliferative effect of GHRH antagonist MZ-J-7-138 on MDA-PCa-2b tumors. The expression of mutant (mt-p53) and p21 protein in PC-3 and DU-145 tumors was significantly decreased by treatment with MZ-J-7-138, whereas wild type wt-p53 expression in MDA-PCA-2b tumors was up regulated by treatment with Cetrorelix. All three models investigated expressed specific, high affinity GHRH receptors. CONCLUSIONS: Our findings indicate that the anti-proliferative effects of GHRH antagonist MZ-J-7-138 and LHRH antagonist Cetrorelix on prostate cancers involve p53 and p21 signaling.

Novel antagonists of growth hormone-releasing hormone inhibit growth and vascularization of human experimental ovarian cancers.

BACKGROUND: Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancer cell lines and experimental tumors by mechanisms that include direct action on GHRH receptors in cancer cells. METHODS: In this study, the effects of newly synthesized GHRH antagonists, MIA-313, MIA-602, MIA-604, and MIA-610, were investigated in 2 human ovarian epithelial adenocarcinoma cell lines, OVCAR-3 and SKOV-3, in vitro and in vivo. The expression of receptors for GHRH was demonstrated by Western blot analysis and ligand competition methods in the OVCAR-3 and SKOV-3 cell lines and in tumors from those cells grown in athymic nude mice. The effects of GHRH antagonists on the secretion of vascular endothelial growth factor (VEGF) by OVCAR-3 cells and on the vascularization of OVCAR-3 xenografts also were evaluated. RESULTS: Both the pituitary and the splice variant type 1 (SV1) GHRH receptors were detected in the 2 cell lines and in tumor xenografts, and SV1 was expressed at higher levels. Cell viability assays revealed the antiproliferative effect of all GHRH antagonists that were. Maximal tumor growth inhibition was approximately 75% in both models. MIA-313 and MIA-602 decreased VEGF secretion of OVCAR-3 cells, as measured by enzyme-linked immunosorbent assay, and reduced tumor vascularization in a Matrigel plug assay, but caused no change in the expression of VEGF or VEGF receptor in the terminal ileum of mice with OVCAR-3 tumors. CONCLUSIONS: Results from the current study indicated that a he novel approach based on GHRH antagonists may offer more effective therapeutic alternatives for patients with advanced ovarian cancer and who do not tolerate conventional anti-VEGF therapy.

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-1ß, nuclear factor-κß 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.

Inhibitory effects of GHRH antagonists on human GH-secreting adenoma tissue.

Experimental data indicate that antagonists of growth hormone-releasing hormone (GHRH) could be used clinically in disorders characterized by excessive GHRH/growth hormone (GH) secretion, but direct evidence for the effectiveness of GHRH antagonists on human pituitary tissue is still lacking. In this study, we investigated the inhibitory effect of our GHRH antagonists MZ-4-71 and JV-1-36 and the somatostatin (SST) analog RC-160 on superfused pituitary cells obtained from a human GH-secreting adenoma. Using Western blot analysis and immunohistochemistry, we demonstrated profuse expression of the GHRH receptor and its major splice variant SV1 and an increase in the expression of Gsa protein in the adenoma tissue. Exposure of the tumor cells to exogenous pulses of GHRH induced definite GH responses, causing a 3- to 5-fold elevation of the basal GH level. The antagonists MZ-4-71 and JV-1-36 did not alter basal GH secretion, indicating that the adenoma cells did not secrete GHRH in an autocrine manner. However, both antagonists prevented the stimulatory effect of exogenous GHRH. Similarly to the GHRH antagonists, neither SST-14 nor the SST analog RC-160 had an effect on the basal GH secretion of the tumor cells, but both peptides inhibited the stimulatory effect of exogenous GHRH, with RC-160 being more potent than SST. Our study provides direct evidence for the effectiveness of potent GHRH antagonists such as MZ-4-71 and JV-1-36 on human pituitary GH-secreting adenoma tissue and strongly suggests that these drugs could be used for therapy of GHRH-associated forms of acromegaly, particularly for those patients in whom surgery fails or is not an option.

The effect of GHRH antagonists on human glioblastomas and their mechanism of action.

The effects of new growth hormone-releasing hormone (GHRH) antagonists JMR-132 and MIA-602 and their mechanism of action were investigated on 2 human glioblastoma cell lines, DBTRG-05 and U-87MG, in vitro and in vivo. GHRH receptors and their main splice variant, SV1 were found on both cell lines. After treatment with JMR-132 or MIA-602, the cell viability decreased significantly. A major decrease in the levels of phospho-Akt, phospho-GSK3ß and phosho-ERK 1/2 was detected at 5 and 10 min following treatment with the GHRH antagonists, whereas elevated levels of phospho-p38 were observed at 24 hr. The expression of caspase-3 and poly(ADP-ribose) (PARP), as the downstream executioners of apoptosis were found to be significantly elevated after treatment. Following treatment of the glioblastoma cells with GHRH antagonists, nuclear translocation of apoptosis inducing factor (AIF) and Endonuclease G (Endo G) and the mitochondrial release of cytochrome c (cyt c) were detected, indicating that the cells were undergoing apoptosis. In cells treated with GHRH antagonists, the collapse of the mitochondrial membrane potential was shown with fluorescence microscopy and JC-1 membrane potential sensitive dye. There were no significant differences between results obtained in DBTRG-05 or U-87MG cell lines. After treatment with MIA-602 and JMR-132, the reduction rate in the growth of DBTRG-05 glioblastoma, xenografted into nude mice, was significant and tumor doubling time was also significantly extended when compared with controls. Our study demonstrates that GHRH antagonists induce apoptosis through key proapoptotic pathways and shows the efficacy of MIA-602 for experimental treatment of glioblastoma.

Antiproliferative effect of growth hormone-releasing hormone (GHRH) antagonist on ovarian cancer cells through the EGFR-Akt pathway.

BACKGROUND: Antagonists of growth hormone-releasing hormone (GHRH) are being developed for the treatment of various human cancers. METHODS: MTT assay was used to test the proliferation of SKOV3 and CaOV3. The splice variant expression of GHRH receptors was examined by RT-PCR. The expression of protein in signal pathway was examined by Western blotting. siRNA was used to block the effect of EGFR. RESULTS: In this study, we investigated the effects of a new GHRH antagonist JMR-132, in ovarian cancer cell lines SKOV3 and CaOV3 expressing splice variant (SV)1 of GHRH receptors. MTT assay showed that JMR-132 had strong antiproliferative effects on SKOV3 and CaOV3 cells in both a time-dependent and dose-dependent fashion. JMR-132 also induced the activation and increased cleaved caspase3 in a time- and dose-dependent manner in both cell lines. In addition, JMR-132 treatments decreased significantly the epidermal growth factor receptor (EGFR) level and the phosphorylation of Akt (p-Akt), suggesting that JMR-132 inhibits the EGFR-Akt pathway in ovarian cancer cells. More importantly, treatment of SKOV3 and CaOV3 cells with 100 nM JMR-132 attenuated proliferation and the antiapoptotic effect induced by EGF in both cell lines. After the knockdown of the expression of EGFR by siRNA, the antiproliferative effect of JMR-132 was abolished in SKOV3 and CaOV3 cells. CONCLUSIONS: The present study demonstrates that the inhibitory effect of the GHRH antagonist JMR-132 on proliferation is due, in part, to an interference with the EGFR-Akt pathway in ovarian cancer cells.

The immunohistochemical expression of growth hormone-releasing hormone receptor splice variant 1 is a favorable prognostic marker in colorectal cancer.

Hypothalamic growth hormone (GH)-releasing hormone (GHRH) regulates the release of GH from the pituitary gland. The receptors for GHRH (GHRH-R) are expressed predominantly in the pituitary. Recent evidence demonstrates that splice variants of the GHRH receptor are also expressed in several nonpituitary tissues, both normal and tumoral, as well as in cancer cell lines. The aim of this study was to investigate the expression of the splice variant 1 (SV-1) of GHRH-R in colorectal cancer (CRC). Seventy patients who underwent partial colectomy for CRC were enrolled in the study. Immunohistochemical expression of SV-1 was studied in paraffin-embedded sections of patient tumor tissue. A cytoplasmic supranuclear expression of SV-1 was observed in CRC as well as in the normal colon mucosa. Tumor grade and pathological stage were negatively correlated with expression of SV-1 (P = 0.012 and P = 0.013, respectively). CRCs metastatic to the liver showed a lower expression of SV-1 than did primary tumors, but this difference was not statistically significant. Kaplan-Meier and Cox univariate survival analyses indicated an improved survival time in patients with high SV-1 compared with those with low GHRH-R expression, but this difference was not statistically significant. The immunohistochemical expression of SV-1 seems to be a favorable prognostic factor in CRC.

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.

BACKGROUND: Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers and affect tumoral growth factors. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

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.

Differences between normal and alpha-synuclein overexpressing SH-SY5Y neuroblastoma cells after Abeta(1-42) and NAC treatment.

Alpha-synuclein (alphaSN) plays a major role in numerous neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Intracellular inclusions containing aggregated alphaSN have been reported in Alzheimer's and Parkinson's affected brains. Moreover, a proteolytic fragment of alphaSN, the so-called non-amyloid component of Alzheimer's disease amyloid (NAC) was found to be an integral part of Alzheimer's dementia related plaques. Despite the extensive research on this topic, the exact toxic mechanism of alphaSN remains elusive. We have taken the advantage of an alphaSN overexpressing SH-SY5Y cell line and investigated the effects of classical apoptotic factors (e.g. H(2)O(2), amphotericin B and ruthenium red) and aggregated disease-related peptides on cell viability compared to wild type neuroblastoma cells. It was found that alphaSN overexpressing cells are more sensitive to aggregated peptides treatment than normal expressing counterparts. In contrast, cells containing elevated amount of alphaSN were less vulnerable to classical apoptotic stressors than wild type cells. In addition, alphaSN overexpression is accompanied by altered phenotype, attenuated proliferation kinetics, increased neurite arborisation and decreased cell motility. Based on these results, the alphaSN overexpressing cell lines may represent a good and effective in vitro model for Alzheimer's and Parkinson's disease.

Inhibition of estrogen receptor positive and negative breast cancer cell lines with a growth hormone-releasing hormone antagonist.

GHRH antagonists have been shown to inhibit growth of various human cancer cell lines xenografted into nude mice including estrogen receptor negative human breast cancers. Previous observations also suggest that GHRH locally produced in diverse neoplasms including breast cancer might directly affect proliferation of tumor cells. In the present study we demonstrate that a novel highly potent GHRH antagonist JMR-132 strongly inhibits the proliferation of both estrogen receptor negative SKBR 3 and estrogen receptor positive ZR 75 human breast cancer cell lines in vitro. The proliferation in vitro of ZR 75 and SKBR 3 was increased after direct stimulation with GHRH(1-29)NH2. The GHRH antagonist JMR-132 had a significant antiproliferative activity in the absence of GHRH and nullified the proliferative effect of GHRH in these cell lines. SKBR 3 and ZR 75 expressed the GHRH ligand as well as the pituitary type of GHRH-receptor, which likely appears to mediate the antiproliferative mechanisms in these cell lines. These in vitro results suggest that JMR-132 is a potent inhibitor of breast cancer growth, independent of the estrogen receptor status. Further investigations on the combination treatment with endocrine agents affecting the estrogen pathway and GRHR antagonists are needed in order to improve the treatment of breast cancer.

Alterations of EGFR/HER, angiogenesis and apoptosis pathways after therapy with antagonists of growth hormone releasing hormone and bombesin in non-small cell lung cancer.

New therapeutic strategies are necessary to improve the treatment of lung cancer. We investigated the effects of bombesin/gastrin-releasing peptide (GRP) antagonist, RC-3940-II, and growth hormone-releasing hormone (GHRH) antagonists, MZ-J-7-114 and MZ-J-7-118, on the expression of epidermal growth factor receptor (EGFR)/HER (-2, -3, and -4) family, angiogenic factors, VEGF-A and VEGF receptors (VEGF-R1 and VEGF-R2), and the apoptotic molecules Bax and Bcl-2, in H-460 and A-549 non-small cell lung carcinomas (NSCLC). Nude mice bearing xenografts of H-460 and A-549 NSCLC were treated daily with these peptide analogues for 4 weeks. The treatment resulted in growth inhibition of H-460 by 22-77% and A-549 NSCLCs by 64-84%. The inhibition of tumor growth was associated with a down-regulation of members of EGFR/HER family. A significant reduction of the levels of expression of EGFR/HER family on both tumors varied from 29-96%: the greatest inhibition being induced by RC-3940-II. Similarly, a significant decrease in the levels of VEGF-A in tumors by 19-60% and VEGF receptors (VEGF-R1, 24-74% and VEGF-R2, 25-50%) was detected after therapy. An up-regulation of Bax by 21-63% and a down-regulation of Bcl-2 by 23-39% was observed only for H-460 NSCLC. Our study demonstrates that human H-460 and A-549 NSCLC, express receptors for GHRH and bombesin/GRP, and respond to the respective antagonists. The antagonists of bombesin/GRP and GHRH could provide a new strategy for treatment of NSCLC through down-regulation of EGFR/HER family and an interference with the angiogenic and apoptotic pathways.

Endomorphin-2, an endogenous tetrapeptide, protects against Abeta1-42 in vitro and in vivo.

The underlying cause of Alzheimer's disease (AD) is thought to be the beta-amyloid aggregates formed mainly by Abeta1-42 peptide. Protective pentapeptides [e.g., Leu-Pro-Phe-Phe-Asp (LPFFD)] have been shown to prevent neuronal toxicity of Abeta1-42 by arresting and reversing fibril formation. Here we report that an endogenous tetrapeptide, endomorphin-2 (End-2, amino acid sequence: YPFF), defends against Abeta1-42 induced neuromodulatory effects at the cellular level. Although End-2 does not interfere with the kinetics of Abeta fibrillogenesis according to transmission electron microscopic studies and quasielastic light scattering measurements, it binds to Abeta1-42 during aggregation, as revealed by tritium-labeled End-2 binding assay and circular dichroism measurements. The tetrapeptide attenuates the inhibitory effect on cellular redox activity of Abeta1-42 in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazolyl-2)-2,-5-diphenyltetrazolium bromide (MTT) assay. In vitro and in vivo electrophysiological experiments show that End-2 also protects against the field excitatory postsynaptic potential attenuating and the NMDA-evoked response-enhancing effect of Abeta1-42. Studies using [D-Ala (2), N-Me-Phe (4), Gly (5)-ol]-enkephalin (DAMGO), a mu-opioid receptor agonist, show that the protective effects of the tetrapeptide are not mu-receptor modulated. The endogenous tetrapeptide End-2 may serve as a lead compound for the drug development in the treatment of AD.

Synthesis and structure-activity studies on novel analogs of human growth hormone releasing hormone (GHRH) with enhanced inhibitory activities on tumor growth.

The syntheses and biological evaluations of new GHRH analogs of Miami (MIA) series with greatly increased anticancer activity are described. In the design and synthesis of these analogs, the following previous substitutions were conserved: D-Arg2, Har9, Abu15, and Nle27. Most new analogs had Ala at position 8. Since replacements of both Lys12 and Lys21 with Orn increased resistance against enzymatic degradation, these modifications were kept. The substitutions of Arg at both positions 11 and 20 by His were also conserved. We kept D-Arg28, Har29 -NH2 at the C-terminus or inserted Agm or 12-amino dodecanoic acid amide at position 30. We incorporated pentafluoro-Phe (Fpa5), instead of Cpa, at position 6 and Tyr(Me) at position 10 and ω-amino acids at N-terminus of some analogs. These GHRH analogs were prepared by solid-phase methodology and purified by HPLC. The evaluation of the activity of the analogs on GH release was carried out in vitro on rat pituitaries and in vivo in male rats. Receptor binding affinities were measured in vitro by the competitive binding analysis. The inhibitory activity of the analogs on tumor proliferation in vitro was tested in several human cancer cell lines such as HEC-1A endometrial adenocarcinoma, HCT-15 colorectal adenocarcinoma, and LNCaP prostatic carcinoma. For in vivo tests, various cell lines including PC-3 prostate cancer, HEC-1A endometrial adenocarcinoma, HT diffuse mixed ß cell lymphoma, and ACHN renal cell carcinoma cell lines were xenografted into nude mice and treated subcutaneously with GHRH antagonists at doses of 1-5µg/day. Analogs MIA-602, MIA-604, MIA-610, and MIA-640 showed the highest binding affinities, 30, 58, 48, and 73 times higher respectively, than GHRH (1-29) NH2. Treatment of LNCaP and HCT-15 cells with 5µM MIA-602 or MIA-690 decreased proliferation by 40%-80%. In accord with previous tests in various human cancer lines, analog MIA-602 showed high inhibitory activity in vivo on growth of PC-3 prostate cancer, HT-mixed ß cell lymphoma, HEC-1A endometrial adenocarcinoma and ACHN renal cell carcinoma. Thus, GHRH analogs of the Miami series powerfully suppress tumor growth, but have only a weak endocrine GH inhibitory activity. The suppression of tumor growth could be induced in part by the downregulation of GHRH receptors levels.