Petra Popovics

Hypothalamo-pituitary and immune-dependent adrenal regulation during systemic inflammation.

Significance In several critically ill patients the homeostatic regulation of adrenocortical hormone secretion is impaired. Toll-like receptors (TLR) play a substantial role in HPA axis activation in the course of systemic inflammation. Here, using mice with conditional deletion of a crucial TLR adapter protein, MyD88, we investigated the role of systemic and local adrenal TLR signaling in the activation of adrenal glucocorticoid responses to stress and regulation of immune-adrenal crosstalk during systemic inflammatory response syndrome. Our results suggest that immune cells, rather than adrenal cells, are major regulators of the systemic and intra-adrenal inflammatory response to LPS. However, the full HPA axis activation in SIRS is not entirely dependent on systemic TLR signaling. Inflammation-related dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis is central to the course of systemic inflammatory response syndrome or sepsis. The underlying mechanisms, however, are not well understood. Initial activation of adrenocortical hormone production during early sepsis depends on the stimulation of hypothalamus and pituitary mediated by cytokines; in late sepsis, there is a shift from neuroendocrine to local immune–adrenal regulation of glucocorticoid production. Therefore, the modulation of the local immune–adrenal cross talk, and not of the neuroendocrine circuits involved in adrenocorticotropic hormone production, may be more promising in the prevention of the adrenal insufficiency associated with prolonged sepsis. In the present work, we investigated the function of the crucial Toll-like receptor (TLR) adaptor protein myeloid differentiation factor 88 (MyD88) in systemic and local activation of adrenal gland inflammation and glucocorticoid production mediated by lipopolysachharides (LPSs). To this end, we used mice with a conditional MyD88 allele. These mice either were interbred with Mx1 Cre mice, resulting in systemic MyD88 deletion, predominantly in the liver and hematopoietic system, or were crossed with Akr1b7 Cre transgenic mice, resulting thereby in deletion of MyD88, which was adrenocortical-specific. Although reduced adrenal inflammation and HPA-axis activation mediated by LPS were found in Mx1Cre+-MyD88fl/fl mice, adrenocortical-specific MyD88 deletion did not alter the adrenal inflammation or HPA-axis activity under systemic inflammatory response syndrome conditions. Thus, our data suggest an important role of immune cell rather than adrenocortical MyD88 for adrenal inflammation and HPA-axis activation mediated by LPS.

Targeting the 5′-AMP-activated protein kinase and related metabolic pathways for the treatment of prostate cancer

Introduction: Increasing evidence suggests that prostate cancer cells undergo unique metabolic reprogramming during transformation. A master regulator of cellular homeostasis, 5′-AMP-activated protein kinase (AMPK), directs metabolic adaptation that supports the growth demands of rapidly dividing cancer cells. The utilization of AMPK as a therapeutic target may therefore provide an effective strategy in the treatment of prostate cancer. Areas covered: Our review describes the regulation of AMPK by androgens and upstream kinases including the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in prostate cancer. Oncogenic, AMPK-regulated pathways that direct various metabolic processes are also addressed. Furthermore, we discuss the role of AMPK in growth arrest and autophagy as a potential survival pathway for cancer cells. In addition, by regulating non-metabolic pathways, AMPK may stimulate migration and mitosis. Finally, this review summarizes efforts to treat prostate cancer with pharmacological agents capable of modulating AMPK signaling. Expert opinion: Current research is primarily focused on developing drugs that activate AMPK as a treatment for prostate cancer. However, oncogenic aspects of AMPK signaling calls for caution about employing such therapies. We think that inhibitors of CaMKK2 or AMPK, or perhaps the modulation of downstream targets of AMPK, will gain importance in the clinical management of prostate cancer.

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.

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

Significance The current therapeutic approaches to the treatment of benign prostatic hyperplasia (BPH) do not take into consideration that inflammation is an important factor in the pathogenesis of this disease. We previously demonstrated that growth hormone-releasing hormone (GHRH) antagonists reduce prostatic weights and decrease the level of inflammatory cytokines in a testosterone-induced BPH model. This study sheds light on the paracrine roles of GHRH in prostatic inflammation and demonstrates that GHRH stimulates the growth of BPH-1 and primary prostate epithelial spheres and that GHRH antagonists reduce prostate volume in an experimental model of prostatic inflammation. 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-β2 by 91.8% (P < 0.01). The proliferation of primary epithelial cells stimulated by IL-17A or TGF-β2 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.

Beneficial effects of growth hormone-releasing hormone agonists on rat INS-1 cells and on streptozotocin-induced NOD/SCID mice

Significance Improved therapeutic strategies for transplantation of pancreatic islet cells to selected patients with type-1 diabetes are urgently needed. Growth hormone-releasing hormone (GHRH) agonists to influence growth, function, and engraftment of islet cells were previously reported by us. This study demonstrates greater stimulation by improved GHRH agonists, on proliferation, gene expressions, and the signaling pathways in pancreatic β cells. Agonist MR-409 in vivo reduced the severity of streptozotocin-induced diabetes in nonobese diabetic severe combined immunodeficiency mice. Transplantation of rat islets preconditioned in vitro with MR-409 and its administration in vivo promoted growth, function, and engraftment of exogenous islets, supporting the use of GHRH agonists in type-1 diabetes. The beneficial effects of GHRH agonists on the functions of β cells may also provide approaches to their application in type-2 diabetes. Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote growth, function, and engraftment of islet cells following transplantation. Here we evaluated recently synthesized GHRH agonists on the proliferation and biological functions of rat pancreatic β-cell line (INS-1) and islets. In vitro treatment of INS-1 cells with GHRH agonists increased cell proliferation, the expression of cellular insulin, insulin-like growth factor-1 (IGF1), and GHRH receptor, and also stimulated insulin secretion in response to glucose challenge. Exposure of INS-1 cells to GHRH agonists, MR-356 and MR-409, induced activation of ERK and AKT pathways. Agonist MR-409 also significantly increased the levels of cellular cAMP and the phosphorylation of cAMP response element binding protein (CREB) in INS-1 cells. Treatment of rat islets with agonist, MR-409 significantly increased cell proliferation, islet size, and the expression of insulin. In vivo daily s.c. administration of 10 μg MR-409 for 3 wk dramatically reduced the severity of streptozotocin (STZ)-induced diabetes in nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice. The maximal therapeutic benefits with respect to the efficiency of engraftment, ability to reach normoglycemia, gain in body weight, response to high glucose challenge, and induction of higher levels of serum insulin and IGF1 were observed when diabetic mice were transplanted with rat islets preconditioned with GHRH agonist, MR-409, and received additional treatment with MR-409 posttransplantation. This study provides an improved approach to the therapeutic use of GHRH agonists in the treatment of diabetes mellitus.

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

HighlightsWe report synthesis of new GHRH analogs with increased anticancer activity.Incorporation of pentafluoro Phe and Orn12,21 into GHRH analogs augments antitumor activity.ω‐amino acids at N‐and C‐terminus produce an increased inhibition of tumor growth.GHRH analogs inhibit growth of prostatic and other cancers in vitro and in vivo.GHRH analogs target G protein‐coupled GHRH receptors. Abstract 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 &bgr; cell lymphoma, and ACHN renal cell carcinoma cell lines were xenografted into nude mice and treated subcutaneously with GHRH antagonists at doses of 1–5 &mgr;g/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 5 &mgr;M 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 &bgr; 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.

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.

Growth hormone-releasing hormone antagonists reduce prostatic enlargement and inflammation in carrageenan-induced chronic prostatitis

Inflammation plays a key role in the etiology of benign prostatic hyperplasia (BPH) through multiple pathways involving the stimulation of proliferation by cytokines and growth factors as well as the induction of the focal occurrence of epithelial‐to‐mesenchymal transition (EMT). We have previously reported that GHRH acts as a prostatic growth factor in experimental BPH and in autoimmune prostatitis models and its blockade with GHRH antagonists offer therapeutic approaches for these conditions. Our current study was aimed at the investigation of the beneficial effects of GHRH antagonists in λ‐carrageenan‐induced chronic prostatitis and at probing the downstream molecular pathways that are implicated in GHRH signaling.

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.

MP17-03 INFLAMMATION-INDUCED PROSTATIC ENLARGEMENT AND PROLIFERATION OF PROSTATE EPITHELIAL CELLS IS REDUCED BY GROWTH HORMONE-RELEASING HORMONE (GHRH) ANTAGONISTS THROUGH THE INHIBITION OF EPITHELIAL-TO-MESENCHYMAL TRANSITION

(red arrowhead in Fig.1D) IL-18 levels were elevated in banked urine specimens of BPH patients. CONCLUSIONS: Co-localized immunoreactivity of NLRP1, and its downstream product IL-18 supports the assembly and activation of inflammasome in BPH specimens positive for infiltration of inflammatory cells. Recapitulation of findings from the animal model demonstrate that inflammasome plays a major role in the prostatic inflammation associated with BPH and therefore inflammasome targeted therapies can be an option for BPH management.