A new approach to the treatment of acute myeloid leukaemia targeting the receptor for growth hormone-releasing hormone.

Growth hormone-releasing hormone (GHRH) is secreted by the hypothalamus and acts on the pituitary gland to stimulate the release of growth hormone (GH). GHRH can also be produced by human cancers, in which it functions as an autocrine/paracrine growth factor. We have previously shown that synthetic antagonistic analogues of GHRH are able to successfully suppress the growth of 60 different human cancer cell lines representing over 20 cancers. Nevertheless, the expression of GHRH and its receptors in leukaemias has never been examined. Our study demonstrates the presence of GHRH receptor (GHRH-R) on 3 of 4 human acute myeloid leukaemia (AML) cell lines-K-562, THP-1, and KG-1a-and significant inhibition of proliferation of these three cell lines in vitro following incubation with the GHRH antagonist MIA-602. We further show that this inhibition of proliferation is associated with the upregulation of pro-apoptotic genes and inhibition of Akt signalling in leukaemic cells. Treatment with MIA-602 of mice bearing xenografts of these human AML cell lines drastically reduced tumour growth. The expression of GHRH-R was further confirmed in 9 of 9 samples from patients with AML. These findings offer a new therapeutic approach to this malignancy and suggest a possible role of GHRH-R signalling in the pathology of AML.

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.

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.

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.

Carotid-Femoral Pulse Transit Time Variability Predicted Mortality and Improved Risk Stratification in the Elderly.

[Figure: see text].

Association of birth defects with the mode of assisted reproductive technology in a Chinese data-linkage cohort.

OBJECTIVE: To evaluate the impact of assisted reproductive technology (ART) on the offspring of Chinese population. DESIGN: Retrospective, data-linkage cohort. SETTING: Not applicable. PATIENT(S): Live births resulting from ART or natural conception. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Birth defects coded according to ICD-10. RESULT(S): Births after ART were more likely to be female and multiple births, especially after intracytoplasmic sperm injection (ICSI). ART was associated with a significantly increased risk of birth defects, especially, among singleton births, a significantly increased risk in fresh-embryo cycles after in vitro fertilization (IVF) and frozen-embryo cycles after ICSI. Associations between ART and multiple defects, between ART and gastrointestinal malformation, genital organs malformation, and musculoskeletal malformation among singleton births, and between ART and cardiac septa malformation among multiple births were observed. CONCLUSION(S): This study suggests that ART increases the risk of birth defects. Subgroup analyses indicate higher risk for both fresh and frozen embryos, although nonsignificantly for frozen embryos after IVF and for fresh embryos were presented with low power. Larger sample size research is needed to clarify effects from fresh- or frozen-embryo cycles after IVF and ICSI.

Effects of an Antagonistic Analog of Growth Hormone-Releasing Hormone on Endometriosis in a Mouse Model and In Vitro.

Endometriosis is a benign gynecologic disorder causing dysmenorrhea, pelvic pain, and subfertility. Receptors for the growth hormone-releasing hormone (GHRH) were found in endometriotic tissues. Antagonists of GHRH have been used to inhibit the growth of endometriotic endometrial stromal cells. In this study, the GHRH receptor splice variant (SV) 1 was detected in human endometrial tissue samples by Western blots and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The highest messenger RNA (mRNA) and protein levels of SV1 were found in eutopic endometrium from patients with endometriosis compared to ectopic endometriotic tissues and endometrium from normal patients. The highest expression for GHRH mRNA was found by qRT-PCR in ectopic endometriosis lesions. In an in vivo mouse model with human endometrial explants from patients with endometriosis, 10 µg MIA-602 per day resulted in significantly smaller human endometrial xenotransplants after 4 weeks compared to mice treated with vehicle. The endometrial tissues expressed SV1 before and after xenotransplantation. The proliferation of endometrial stromal cells as well as the endometriosis cell lines 12-Z and 49-Z was decreased by exposure to 1 µM MIA-602 after 72 hours. The protein levels of epithelial growth factor receptors in 12-Z and 49-Z cell lines were reduced 48 and 72 hours after the administration of 1 µM MIA-602. MIA-602 decreased the activation of the MAP-kinases ERK-1/2. Our study demonstrates the presence of SV1 receptor as a target for treatment with GHRH antagonist in endometriosis. Endometrial tissues respond to MIA-602 with inhibition of proliferation in vitro and in vivo. The use of MIA-602 could be an effective supplement to the treatment strategies in endometriosis.

Inhibitory Effects of Antagonists of Growth Hormone-Releasing Hormone (GHRH) in Thyroid Cancer.

Growth hormone-releasing hormone (GHRH) is a peptide hormone secreted by the hypothalamus that regulates the synthesis and secretion of growth hormone (GH) in the pituitary. The extra-hypothalamic GHRH and its cognate receptors (GHRHR and splice variants) play a mitogenic role by stimulating cell proliferation and preventing apoptotic cell death. It is well established that GHRH antagonists inhibit the growth, tumorigenicity, and metastasis of various human malignancies. In this work, we studied the effect of two new GHRH antagonists, MIA602 and MIA690, on thyroid cancer. We studied the effect of MIA602 and MIA690 on thyroid cancer in vitro, using human thyroid cancer cell lines, and in vivo, using chicken embryo chorioallantoic membrane (CAM) assays. We found that mRNA for GHRH and GHRH receptor is expressed in thyroid cell lines and in samples of thyroid tumors. Immunohistochemistry confirmed the expression of GHRHR protein in specimens of thyroid tumor. We observed that GHRH antagonists inhibited the growth and increased apoptosis of thyroid cancer cells. In vivo, the antagonists inhibited growth and angiogenesis of engrafted thyroid tumors. Our results suggest that GHRH expression may play a role in growth of thyroid cancer and that GHRH antagonists can be a therapeutic option for thyroid cancer patients.

Inhibition of human androgen-independent PC-3 and DU-145 prostate cancers by antagonists of bombesin and growth hormone releasing hormone is linked to PKC, MAPK and c-jun intracellular signalling.

Bombesin/gastrin-releasing peptide (BN/GRP) antagonists RC-3940-II and RC-3940-Et, and growth hormone-releasing hormone (GHRH) antagonists MZ-J-7-118 and RC-J-29-18 inhibit the growth of human androgen-independent PC-3 and DU-145 prostate cancers in nude mice. Additive inhibitory effects were observed after treatment with both classes of analogs. In the present study, we investigated the effects of these antagonists on intracellular signalling pathways of protein kinase C (PKC), mitogen activated protein kinases (MAPK) and c-fos and c-jun oncogenes that are involved in tumour cell proliferation. In PC-3 tumours, antagonists of BN/GRP and GHRH decreased significantly the expression of PKC isoforms alpha (alpha), eta (eta) and zeta (zeta) and increased that of delta (delta) PKC protein. MAPK was not detectable. In DU-145 tumours, which constitutively express MAPK, all treatments strongly decreased the levels of p42/44 MAPK. Treatment with the antagonists tended to reduce m-RNA for c-jun in both tumour models. In proliferation assays in vitro, inhibitors of PKC and MAPK diminished growth of DU-145 and PC-3 cells. These findings suggest that antagonists of BN/GRP and GHRH inhibit the growth of androgen-independent prostate cancer by affecting intracellular signalling mechanisms of PKC, MAPK and c-jun.

New therapeutic approach to heart failure due to myocardial infarction based on targeting growth hormone-releasing hormone receptor.

BACKGROUND: We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair. METHODS AND RESULTS: H9c2 cells were cultured in serum-free medium, mimicking nutritional deprivation. GHRH agonists decreased calcium influx and significantly improved cell survival. Rats with cardiac infarction were treated with GHRH agonists or placebo for four weeks. MI size was reduced by selected GHRH agonists (JI-38, MR-356, MR-409); this accompanied an increased number of cardiac c-kit+ cells, cellular mitotic divisions, and vascular density. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-10 and TNF-α compared to placebo. Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and pro-fibrotic systems, and by elevation of bone morphogenetic proteins. CONCLUSIONS: Treatment with GHRH agonists appears to reduce the inflammatory responses post-MI and may consequently improve mechanisms of healing and cardiac remodeling by regulating pathways involved in fibrosis, apoptosis and cardiac repair. Patients with cardiac dysfunction could benefit from treatment with novel GHRH agonists.

Growth hormone-releasing hormone agonists reduce myocardial infarct scar in swine with subacute ischemic cardiomyopathy.

BACKGROUND: Growth hormone-releasing hormone agonists (GHRH-As) stimulate cardiac repair following myocardial infarction (MI) in rats through the activation of the GHRH signaling pathway within the heart. We tested the hypothesis that the administration of GHRH-As prevents ventricular remodeling in a swine subacute MI model. METHODS AND RESULTS: Twelve female Yorkshire swine (25 to 30 kg) underwent transient occlusion of the left anterior descending coronary artery (MI). Two weeks post MI, swine were randomized to receive injections of either 30 µg/kg GHRH-A (MR-409) (GHRH-A group; n=6) or vehicle (placebo group; n=6). Cardiac magnetic resonance imaging and pressure-volume loops were obtained at multiple time points. Infarct, border, and remote (noninfarcted) zones were assessed for GHRH receptor by immunohistochemistry. Four weeks of GHRH-A treatment resulted in reduced scar mass (GHRH-A: -21.9 ± 6.42%; P=0.02; placebo: 10.9 ± 5.88%; P=0.25; 2-way ANOVA; P=0.003), and scar size (percentage of left ventricular mass) (GHRH-A: -38.38 ± 4.63; P=0.0002; placebo: -14.56 ± 6.92; P=0.16; 2-way ANOVA; P=0.02). This was accompanied by improved diastolic strain. Unlike in rats, this reduced infarct size in swine was not accompanied by improved cardiac function as measured by serial hemodynamic pressure-volume analysis. GHRH receptors were abundant in cardiac tissue, with a greater density in the border zone of the GHRH-A group compared with the placebo group. CONCLUSIONS: Daily subcutaneous administration of GHRH-A is feasible and safe in a large animal model of subacute ischemic cardiomyopathy. Furthermore, GHRH-A therapy significantly reduced infarct size and improved diastolic strain, suggesting a local activation of the GHRH pathway leading to the reparative process.

Novel GHRH antagonists suppress the growth of human malignant melanoma by restoring nuclear p27 function.

Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent forms remains a challenge. It has recently been reported that growth hormone-releasing hormone (GHRH) receptor is involved in the pathogenesis of melanoma. Therefore, we investigated the effects of our new GHRH antagonists on a human melanoma cancer cell line. Antiproliferative effects of GHRH antagonists, MIA-602, MIA-606 and MIA-690, on the human melanoma cell line, A-375, were studied in vitro using the MTS assay. The effect of MIA-690 (5 µg/day 28 d) was further evaluated in vivo in nude mice bearing xenografts of A-375. Subcellular localization of p27 was detected with Western blot and immunofluorescent staining. MIA-690 inhibited the proliferation of A-375 cells in a dose-dependent manner (33% at 10 µM, and 19.2% at 5 µM, P < 0 .05 vs. control), and suppressed the growth of xenografted tumors by 70.45% (P < 0.05). Flow cytometric analysis of cell cycle effects following the administration of MIA-690 revealed a decrease in the number of cells in G2/M phase (from 19.7% to 12.9%, P < 0.001). Additionally, Western blot and immunofluorescent studies showed that exposure of A-375 cells to MIA-690 triggered the nuclear accumulation of p27. MIA-690 inhibited tumor growth in vitro and in vivo, and increased the translocation of p27 into the nucleus thus inhibiting progression of the cell cycle. Our findings indicate that patients with malignant melanoma could benefit from treatment regimens, which combine existing chemotherapy agents and novel GHRH-antagonists.

Suppression of the proliferation of human U-87 MG glioblastoma cells by new antagonists of growth hormone-releasing hormone in vivo and in vitro.

Five-year survival of patients afflicted with glioblastoma multiforme (GBM) is rare, making this cancer one of the most feared malignancies. Previously, we reported that growth hormone-releasing hormone (GHRH) is a potent growth factor in cancers. The present work evaluated the effects of two antagonistic analogs of GHRH (MIA-604 and MIA-690) on the proliferation of U-87 MG GBM tumors, in vivo as well as in vitro. Both analogs were administered subcutaneously and dose-dependently inhibited the growth of tumors transplanted into nude mice (127 animals in seven groups). The analogs also inhibited cell proliferation in vitro, decreased cell size, and promoted apoptotic and autophagic processes. Both antagonists stimulated contact inhibition, as indicated by the expression of the E-cadherin-ß-catenin complex and integrins, and decreased the release of humoral regulators of glial growth such as FGF, PDGFß, and TGFß, as revealed by genomic or proteomic detection methods. The GHRH analogs downregulated other tumor markers (Jun-proto-oncogene, mitogen-activated protein kinase-1, and melanoma cell adhesion molecule), upregulated tumor suppressors (p53, metastasis suppressor-1, nexin, TNF receptor 1A, BCL-2-associated agonist of cell death, and ifκBα), and inhibited the expression of the regulators of angiogenesis and invasion (angiopoetin-1, VEGF, matrix metallopeptidase-1, S100 calcium binding protein A4, and synuclein-γ). Our findings indicate that GHRH antagonists inhibit growth of GBMs by multiple mechanisms and decrease both tumor cell size and number.

Beneficial effects of novel antagonists of GHRH in different models of Alzheimer's disease.

Alzheimer's disease is the most frequent debilitating disorder of the central nervous system. Neuroendocrine mechanisms appear to play an important role in this insidiously developing disease. In the present study, the effects of a recently developed growth hormone-releasing hormone (GHRH) antagonist (MIA-690) were evaluated in vivo observing the behavior of genetically modified "Alzheimer's" 5XFAD mice in a Morris water maze (MWM). The effects of the antagonist were also evaluated in vitro using HCN2 human cortical cell cultures treated with amyloid-ß1-42. In vivo, the indices of cognitive performance (latency, cumulative index etc.) were followed up for 6 months. In vitro, the formation of reactive oxygen species, markers of inflammatory and neurohormonal signaling were measured by fluorescent detection, PCR, and ELISA. Accumulation of amyloid-ß1-42 rafts and τ filaments in necropsied brain samples was verified with the help of ELISA. In the MWM experiments, MIA-690 decreased escape latency, and, in the brain samples, it inhibited the concentration of amyloid-ß1-42 and τ filaments. In cell cultures, the GHRH analog showed anti-oxidative and neuro-protective properties and inhibited the GHRH-growth hormone-insulin like growth factor axis. Our data strongly suggest the merit of further studies with GHRH analogs in models of Alzheimer's disease and in elementary clinical trials.

Antagonists of growth hormone releasing hormone (GHRH) and of bombesin/gastrin releasing peptide (BN/GRP) suppress the expression of VEGF, bFGF, and receptors of the EGF/HER family in PC-3 and DU-145 human androgen-independent prostate cancers.

BACKGROUND: Antagonists of growth hormone releasing hormone (GHRH) as well as antagonists of bombesin/gastrin releasing peptide (BN/GRP) inhibit the growth of various malignancies (cancers) including prostate cancer. METHODS: We investigated the effects of GHRH antagonists MZ-J-7-118 and RC-J-29-18, BN/GRP antagonists RC-3940-II and RC-3940-Et and the combination of MZ-J-7-118 and RC-3940-II on the growth of PC-3 and DU-145 human androgen independent prostate cancers xenografted s.c. into nude mice. To elucidate the mechanisms of action of these analogs, growth factors like IGF-II (insulin-like growth factor-II), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor receptor/human epidermal growth factor receptor (EGF-R/HER) family were measured in tumors as well as IGF-I in serum. RESULTS: Antagonists of GHRH and BN/GRP alone or in combination significantly inhibited growth of PC-3 and DU-145 tumors, the greatest inhibition of tumor volume being achieved by combination of MZ-J-7-118 (5 microg/day) and RC-3940-II (10 microg/day). BN/GRP and GHRH antagonists and their combination also decreased the expression of VEGF significantly in PC-3 and non-significantly in DU-145, as measured by radioimmunoassay for VEGF protein and RT-PCR for mRNA levels of VEGF. GHRH and BN/GRP antagonists reduced bFGF concentrations and the maximal binding capacity of EGF receptors, and their mRNA levels in PC-3 and DU-145 tumors. mRNA levels for HER-2 and -3 were also diminished in PC-3 tumors by GHRH and BN/GRP antagonists. No changes in HER-4 were found after treatment. Serum IGF-I and tumoral IGF-II levels were not affected by the analogs. CONCLUSIONS: BN/GRP and GHRH antagonists inhibit growth of PC-3 and DU-145 prostate cancers by suppressing the expression of tumoral growth factors such as VEGF and bFGF as well as the receptors for EGF and related HER-2 and -3. Additive effects on tumor inhibition (TI) in vivo, but not on VEGF, bFGF, or members of the EGF/HER receptor family, can be achieved by the joint administration of both classes of analogs.