New therapies for relapsed castration-resistant prostate cancer based on peptide analogs of hypothalamic hormones

It is a pleasure to contribute our presentation at the International Prostate Forum of the Annual Meeting of the American Urological Association (AUA) to this special issue of the Asian Journal of Andrology

Targeted cytotoxic analog of luteinizing hormone-releasing hormone (LHRH), AEZS-108 (AN-152), inhibits the growth of DU-145 human castration-resistant prostate cancer in vivo and in vitro through elevating p21 and ROS levels

Management of castration-resistant prostate cancer (CRPC) is challenging due to lack of efficacious therapy. Luteinizing hormone-releasing hormone (LHRH) analogs appear to act directly on cells based on the LHRH receptors on human prostate adenocarcinoma cells. We explored anticancer activity of a cytotoxic analog of LHRH, AEZS-108, consisting of LHRH agonist linked to doxorubicin. Nude mice bearing DU-145 tumors were used to compare antitumor effects of AEZS-108 with its individual constituents or their unconjugated combination. The tumor growth inhibition of conjugate was greatest among treatment groups (90.5% inhibition vs. 41% by [D-Lys(6)]LHRH+DOX). The presence of LHRH receptors on DU-145 cells was confirmed by immunocytochemistry. In vitro, AEZS-108 significantly inhibited cell proliferation (61.2% inhibition) and elevated apoptosis rates (by 46%). By the detection of the inherent doxorubicin fluorescence, unconjugated doxorubicin was seen in the nucleus; the conjugate was perinuclear and at cell membrane. Autophagy, visualized by GFP-tagged p62 reporter, was increased by AEZS-108 (7.9-fold vs. 5.3-fold by DOX+[D-Lys(6)]LHRH. AEZS-108 more effectively increased reactive oxygen species (ROS, 2-fold vs. 1.4-fold by DOX+[D-Lys(6)]LHRH) and levels of the apoptotic regulator p21 in vivo and in vitro. We demonstrate robust inhibitory effects of the targeted cytotoxic LHRH analog, AEZS-108, on LHRHR positive castration-resistant prostate cancer cells

Growth hormone-releasing hormone agonist attenuates vascular calcification in diabetic db/db mice

IntroductionVascular calcification (VC) is an independent risk factor for cardiovascular diseases. VC increases mortality of all-causes. VC is one of most common cardiovascular complications in type II diabetes. So far, no therapy has been proven to be effective in treatment of clinical VC. The present study investigated the therapeutic effects of MR409, an agonistic analog of growth hormone-releasing hormone (GHRH-A), on VC in diabetic db/db mice.Method and resultDiabetic mice were injected with MR409 subcutaneously every day for 8 weeks. Long-term treatment with MR409 improved serum lipid profile and endothelium-dependent relaxation to acetylcholine, and reduced vascular structural injury in diabetic mice without affecting serum growth hormone level. Echocardiography showed that calcium plaques present in heart valve of diabetic mice disappeared in diabetic mice after treatment with MR409. MR409 inhibited vascular calcium deposition associated with a marked reduction in the expressions of osteogenic-regulated alkaline phosphatase (ALP) and transcription osteogenic marker gene Runx2 in diabetic mice. MR409 also inhibited vascular reactive oxygen species (ROS) generation and upregulated the expressions of anti-calcifying protein Klotho in diabetic mice.DiscussionOur results demonstrate that GHRH-A MR409 can effectively attenuate VC and heart valve calcification, and protect against endothelial dysfunction and vascular injury in diabetic mice without significantly affecting pituitary-growth hormone axis. The mechanisms may involve upregulation of anti-calcifying protein Klotho and reduction in vascular ROS and the expression of redox sensitive osteogenic genes Runx2 and ALP. GHRH-A may represent a new pharmacological strategy for treatment of VC and diabetics associated cardiovascular complications

Actions and Potential Therapeutic Applications of Growth Hormone-Releasing Hormone Agonists.

Abstract In this article, we briefly review the identification of GHRH, provide an abridged overview of GHRH antagonists, and focus on studies with GHRH agonists. Potent GHRH agonists of JI and MR class were synthesized and evaluated biologically. Besides the induction of the release of pituitary GH, GHRH analogs promote cell proliferation and exert stimulatory effects on various tissues, which express GHRH receptors (GHRH-Rs). A large body of work shows that GHRH agonists, such as MR-409, improve pancreatic β-cell proliferation and metabolic functions and facilitate engraftment of islets after transplantation in rodents. Accordingly, GHRH agonists offer a new therapeutic approach to treating diabetes. Various studies demonstrate that GHRH agonists promote repair of cardiac tissue, producing improvement of ejection fraction and reduction of infarct size in rats, reduction of infarct scar in swine, and attenuation of cardiac hypertrophy in mice, suggesting clinical applications. The presence of GHRH-Rs in ocular tissues and neuroprotective effects of GHRH analogs in experimental diabetic retinopathy indicates their possible therapeutic applications for eye diseases. Other effects of GHRH agonists, include acceleration of wound healing, activation of immune cells, and action on the central nervous system. As GHRH might function as a growth factor, we examined effects of GHRH agonists on tumors. In vitro, GHRH agonists stimulate growth of human cancer cells and upregulate GHRH-Rs. However, in vivo, GHRH agonists inhibit growth of human cancers xenografted into nude mice and downregulate pituitary and tumoral GHRH-Rs. Therapeutic applications of GHRH analogs are discussed. The development of GHRH analogs should lead to their clinical use

Antagonistic analogs of growth hormone-releasing hormone increase the efficacy of treatment of triple negative breast cancer in nude mice with doxorubicin; A preclinical study

Introduction This study evaluated the effects of an antagonistic analog of growth hormone-releasing hormone, MIA-602, on tumor growth, response to doxorubicin, expression of drug resistance genes, and efflux pump function in human triple negative breast cancers. Methods HCC1806 (doxorubicin-sensitive) and MX-1 (doxorubicin-resistant), cell lines were xenografted into nude mice and treated with MIA-602, doxorubicin, or their combination. Tumors were evaluated for changes in volume and the expression of the drug resistance genes MDR1 and NANOG. In-vitro cell culture assays were used to analyze the effect of MIA-602 on efflux pump function. Results Therapy with MIA-602 significantly reduced tumor growth and enhanced the efficacy of doxorubicin in both cell lines. Control HCC1806 tumors grew by 435%, while the volume of tumors treated with MIA-602 enlarged by 172.2% and with doxorubicin by 201.6%. Treatment with the combination of MIA-602 and doxorubicin resulted in an increase in volume of only 76.2%. Control MX-1 tumors grew by 907%, while tumors treated with MIA-602 enlarged by 434.8% and with doxorubicin by 815%. The combination of MIA-602 and doxorubicin reduced the increase in tumor volume to 256%. Treatment with MIA-602 lowered the level of growth hormone-releasing hormone and growth hormone-releasing hormone receptors and significantly reduced the expression of multidrug resistance (MDR1) gene and the drug resistance regulator NANOG. MIA-602 also suppressed efflux pump function in both cell lines. Conclusions We conclude that treatment of triple negative breast cancers with growth hormone-releasing hormone antagonists reduces tumor growth and potentiates the effects of cytotoxic therapy by nullifying drug resistance

Powerful Inhibition of Experimental Human Pancreatic Cancers by Receptor Targeted Cytotoxic LH-RH analog AEZS-108

Pancreatic carcinoma is one of the cancers with the worse prognosis, thus any therapeutic improvement is imperative. Cytotoxic LH-RH analog, AN-152 (proprietary designation, AEZS-108), consisting of doxorubicin (DOX) conjugated to D-Lys6LH-RH, is now in clinical trials for targeted therapy of several sex hormone-dependent tumors that express LH-RH receptors. We investigated LH-RH receptors in human pancreatic carcinoma and the effects of AN-152 (AEZS-108) on experimental pancreatic cancers. We determined LH-RH receptor presence in human pancreatic cancer samples by immunohistochemistry and, in three human pancreatic cancer lines (SW-1990, Panc-1 and CFPAC-1), by binding assays and Western blotting. The effects of the cytotoxic LH-RH analog were investigated on growth of these same cancer lines xenografted into nude mice. We also analyzed differences between the antitumor effects of the cytotoxic analog and its cytotoxic radical alone, doxorubicin (DOX), on the expression of cancer-related genes by PCR arrays. LH-RH receptors were expressed in two randomly selected surgically removed human pancreatic cancer samples and in all three cancer lines. Cytotoxic LH-RH analogs powerfully inhibited growth of all three tumor lines in nude mice; AN-152 was significantly stronger than DOX on Panc-1 and CFPAC-1 cancers. PCR array showed that cytotoxic LH-RH analog AN-152 affected the expression of genes associated with cellular migration, invasion, metastasis and angiogenesis more favorably than DOX, however the changes in gene expression varied considerably among the three cancer lines. Cytotoxic LH-RH analog, AEZS-108, may be a useful agent for the treatment of LH-RH receptor positive advanced pancreatic carcinoma

Growth hormone-releasing hormone antagonist MIA-602 inhibits inflammation induced by SARS-CoV-2 spike protein and bacterial lipopolysaccharide synergism in macrophages and human peripheral blood mononuclear cells

COVID-19 is characterized by an excessive inflammatory response and macrophage hyperactivation, leading, in severe cases, to alveolar epithelial injury and acute respiratory distress syndrome. Recent studies have reported that SARS-CoV-2 spike (S) protein interacts with bacterial lipopolysaccharide (LPS) to boost inflammatory responses in vitro, in macrophages and peripheral blood mononuclear cells (PBMCs), and in vivo. The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting pituitary GH release, exerts many peripheral functions, acting as a growth factor in both malignant and non-malignant cells. GHRH antagonists, in turn, display potent antitumor effects and antinflammatory activities in different cell types, including lung and endothelial cells. However, to date, the antinflammatory role of GHRH antagonists in COVID-19 remains unexplored. Here, we examined the ability of GHRH antagonist MIA-602 to reduce inflammation in human THP-1-derived macrophages and PBMCs stimulated with S protein and LPS combination. Western blot and immunofluorescence analysis revealed the presence of GHRH receptor and its splice variant SV1 in both THP-1 cells and PBMCs. Exposure of THP-1 cells to S protein and LPS combination increased the mRNA levels and protein secretion of TNF-α and IL-1β, as well as IL-8 and MCP-1 gene expression, an effect hampered by MIA-602. Similarly, MIA-602 hindered TNF-α and IL-1β secretion in PBMCs and reduced MCP-1 mRNA levels. Mechanistically, MIA-602 blunted the S protein and LPS-induced activation of inflammatory pathways in THP-1 cells, such as NF-κB, STAT3, MAPK ERK1/2 and JNK. MIA-602 also attenuated oxidative stress in PBMCs, by decreasing ROS production, iNOS and COX-2 protein levels, and MMP9 activity. Finally, MIA-602 prevented the effect of S protein and LPS synergism on NF-кB nuclear translocation and activity. Overall, these findings demonstrate a novel antinflammatory role for GHRH antagonists of MIA class and suggest their potential development for the treatment of inflammatory diseases, such as COVID-19 and related comorbidities

Growth hormone-releasing hormone receptor antagonists modify molecular machinery in the progression of prostate cancer

Background: Therapeutic strategies should be designed to transform aggressive prostate cancer phenotypes to a chronic situation. To evaluate the effects of the new growth hormone-releasing hormone receptor (GHRH-R) antagonists: MIA-602, MIA-606, and MIA-690 on processes associated with cancer progression as cell proliferation, adhesion, migration, and angiogenesis. Methods: We used three human prostate cell lines (RWPE-1, LNCaP, and PC3). We analyzed several molecules such as E-cadherin, ?-catenin, Bcl2, Bax, p53, MMP2, MMP9, PCNA, and VEGF and signaling mechanisms that are involved on effects exerted by GHRH-R antagonists. Results: GHRH-R antagonists decreased cell viability and provoked a reduction in proliferation in LNCaP and PC3 cells. Moreover, GHRH-R antagonists caused a time-dependent increase of cell adhesion in all three cell lines and retarded the wound closure with the highest value with MIA-690 in PC3 cells. GHRH-R antagonists also provoked a large number of cells in SubG0 phase revealing an increase in apoptotic cells in PC3 cell line. Conclusions: Taken all together, GHRH-R antagonists of the MIAMI series appear to be inhibitors of tumor progression in prostate cancer and should be considered for use in future therapeutic strategies on this malignancy.Junta de Comunidades de Castilla-La ManchaUniversidad de Alcal

Tumorigenic transformation of human prostatic epithelial cell line RWPE-1 by growth hormone-releasing hormone (GHRH)

Background: Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in the progression of various tumors. In this study, we analyzed the carcinogenetic potential of exposure to GHRH of a nontumor human prostate epithelial cell line (RWPE-1) as well as its transforming effect in a xenograft model. Methods: We performed cell viability, cell proliferation, adhesion and migration assays. In addition, metalloprotease (MMP)-2 activity by means gelatin zymography, GHRH-R subcellular location using confocal immunofluorescence microscopy and vascular endothelial growth factor (VEGF) levels by enzyme-linked immunoassay were assessed. Besides, we developed an in vivo model in order vivo model to determine the role of GHRH on tumorigenic transformation of RWPE-1 cells. Results: In cell cultures, we observed development of a migratory phenotype consistent with the gelatinolytic activity of MMP-2, expression of VEGF, as well as E-cadherin-mediated cell-cell adhesion and increased cell motility. Treatment with 0.1 µM GHRH for 24 h significantly increased cell viability and cell proliferation. Similar effects of GHRH were seen in RWPE-1 tumors developed by subcutaneous injection of GHRH-treated cells in athymic nude mice, 49 days after inoculation. Conclusions: Thus, GHRH appears to act as a cytokine in the transformation of RWPE-1 cells by mechanisms that likely involve epithelial-mesenchymal transition, thus reinforcing the role of GHRH in tumorigenesis of prostate.Universidad de Alcal