Ren Zhi Cai

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–β-catenin complex and integrins, and decreased the release of humoral regulators of glial growth such as FGF, PDGFβ, and TGFβ, 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.

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.

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

Significance Growth hormone-releasing hormone (GHRH) and its agonistic analogs, besides augmenting the release of growth hormone from the pituitary, can exert direct stimulatory effects on various extrapituitary tissues. Present oncologic evaluation of growth of a glioblastoma cell line revealed that the combination of GHRH agonist, JI-34, with doxorubicin (DOX) produced greater inhibition in vivo than either drug alone. In vitro, JI-34 also potentiated the effects of DOX, decreased the expression of the neuroectodermal stem-cell antigen, nestin, and up-regulated the glial maturation marker, GFAP. These findings indicate that GHRH agonists can induce differentiation of cancer cells, increasing the response to DOX. These observations expand the known spectrum of activities of GHRH and its analogs and have potential clinical implications for therapy. 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 TGFβ. 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.

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.

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.