Previous studies showed that antagonists of bombesin (BN)/ ch gastrin-releasing peptide (GRP) inhibit the growth of various pr cancers by interfering with the growth-stimulatory effects of ca BN-like peptides and down-regulating epidermal growth factor see receptors on tumors. Because the overexpression of the human hu epidermal growth factor receptor-2 (ErbB-2/HER-2/neu) oncogene an plays a role in the progression of many breast cancers, we inves- an tigated whether BN/GRP antagonists can affect HER-2 in mammary tumors. Female nude mice bearing orthotopic xenografts of MDA- ge MB-435 human estrogen-independent breast cancers were treated rel daily with BN/GRP antagonists RC-3095 (20 fg) or RC-3940-11 (10 re: uig) for 6 weeks. The expression of BN/GRP receptors on tumors syl was analyzed by reverse transcription-PCR and immunoblotting. ex We also evaluated whether the mRNA expression for the c-jun and (S c-fos oncogenes is affected by the therapy. Both BN/GRP antago- 11 nists significantly inhibited growth of MDA-MB-435 cancers; RC- sig 3095 reduced tumor volume by 40% and RC-3940-11 by 65%. The pr GRP receptors (subtype 1) were detected in MDA-MB-435 tumors, ni, showing that they mediate the inhibitory effect of the antagonists. M Tumor inhibition was associated with a substantial reduction in the H( expression of mRNA and protein levels of the ErbB/HER receptor ca family as well as with a decrease in the expression of c-jun and c-fos oncogenes. BN/GRP antagonists RC-3940-11 and RC-3095 could be M considered for endocrine therapy of estrogen-independent breast mi cancers that express members of the ErbB/HER receptor family and tu the c-jun and c-fos oncogenes. an M

433 Background: Urinary bladder cancer is the fifth most frequent cancer diagnosed and among the most expensive cancers to treat in the United States. The management of muscle-invasive tumors presents a clinical challenge because of the toxicity and limitations in efficacy and durability of current therapeutic modalities. Novel therapeutic strategies for this disease are of paramount importance. Growth hormone-releasing hormone (GHRH) receptors and its splice variant were detected in a series of urothelial malignancies and GHRH has been shown to influence the growth of these tumors. Herein we evaluated the effect of GHRH antagonists on the growth of various experimental human urinary bladder cancers in vitro and in vivo in nude mice. Methods: We investigated the effects of several GHRH antagonists MIA 602, MIA 606 and MIA 690 on growth of urothelial HT-1376, J82, and RT-4 tumors xenografted into nude mice. The presence of GHRH receptors was validated by Western blotting. Results: The receptors for GHRH and their main splice variant, SV1, were detected in tumor samples of all 3 human bladder cancer models. In the HT-1376 tumors, the GHRH antagonists caused a 30-60% reduction in volume and 52-70% decrease in tumor weights (p < 0.05). All three antagonists strongly inhibited growth of J82 cancers as shown by a 62-75% reduction in tumor volume and 54-66% decrease in tumor weights (p < 0.05). Treatment with MIA-602 and MIA-606 resulted in a similar marked inhibition of growth of RT-4 cancers; tumor volume and weights were reduced by about 51-71% in the treated groups (p < 0.05). The mice tolerated this therapy well; body and organ weights were not significantly changed by the treatments. No toxicity from the GHRH antagonists was noted. Conclusions: We demonstrated the efficacy of potent GHRH antagonists and their lack of toxicity in inhibiting the growth of experimental models of bladder cancer in vivo. The expression of GHRH receptors was detected in all 3 models of human primary urothelial bladder carcinomas. Our findings warrant further development of GHRH antagonists for clinical therapy of bladder cancer alone or in combination with current chemotherapeutic agents and Exploration of their mechanism of action.

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.

Many bladder cancers progress to invasion with poor prognosis; new therapeutic methods are needed. We developed a cytotoxic LH-RH analog, AN-152 (AEZS-108) containing doxorubicin (DOX), for targeted therapy of cancers expressing LHRH receptors. We investigated the expression of LH-RH receptors in clinical bladder cancers and in HT-1376, J82, RT-4 and HT-1197 human bladder cancer lines. The effect of analog, AN-152, on growth of these tumor lines xenografted into nude mice was analyzed. Using molecular and functional assays, we also evaluated the differences between the effects of AN-152, and DOX alone. We demonstrated the expression of LH-RH receptors on 18 clinical bladder cancers by immunohistochemistry and on four human urinary bladder cancer lines HT-1376, J82, RT-4 and HT-1197 by Western blotting and binding assays. AN-152 powerfully inhibited growth of these bladder cancers in nude mice. AN-152 exerted greater effects than DOX and was less toxic. DOX activated strong multidrug resistance mechanisms in RT-4 and HT-1197 cancers, while AN-152 had no or less such effect. PCR assays and in vitro studies revealed differences in the action of AN-152 and DOX on the expression of genes involved in apoptosis. These results suggest that targeted cytotoxic LH-RH analog, AN-152 (AEZS-108), should be examined for treatment of patients with LH-RH receptor positive invasive bladder cancers.

Hepatic carcinoma is a major health problem worldwide. Its incidence is increasing in Western countries and there is currently no effective systemic therapy against it. Targeted treatment modalities developed in the past few years have provided very limited success. Development of new treatment strategies is therefore essential. We investigated the effects of bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC-3940-II on experimental human liver cancers in nude mice. SK-Hep-1 and Hep-G2 cancers transplanted subcutaneously into nude mice were treated daily with 10 or 20 µg of RC-3940-II. Tumor growth was monitored for 50-184 days in five experiments. Tumor gene expression was analyzed with PCR array and protein expression by immunoblotting. Characteristics of BN/GRP receptors in the tumors were analyzed by binding assays. Effects of RC-3940-II on cell proliferation were investigated in vitro. RC-3940-II inhibited the growth of SK-Hep-1 cancers in nude mice by 65-98%, with total regression in 9 of 36 tumors in three experiments. The BN/GRP antagonist inhibited the growth of Hep-G2 cancers as well by 73-82% in two experiments, being effective even on originally large tumors. Gene expression analysis showed an increase in several angiogenesis inhibitors and decrease in proangiogenic genes after RC-3940-II treatment. Receptor assays demonstrated high-affinity binding sites for BN/GRP in both tumor lines. BN/GRP antagonist RC-3940-II powerfully inhibits growth of SK-Hep-1 and Hep-G2 cancers in nude mice. Its effect may be linked to changes in expression of those cancer genes important in angiogenesis, invasion, and metastasis. RC-3940-II may be considered for further investigations in treatment of liver cancers.

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20121066 EFFECTIVE TREATMENT OF URINARY BLADDER CANCERS BY TARGETED CYTOTOXIC LHRH ANALOG AEZS-108 (AN-152) A PRECLINICAL REPORT Ferenc Rick, Luca Szalontay, Gustavo Fernandez-Castro, Norman Block, Gunhild Keller, Karoly Szepeshazi, and Andrew Schally Ferenc RickFerenc Rick Miami, FL More articles by this author , Luca SzalontayLuca Szalontay Miami, FL More articles by this author , Gustavo Fernandez-CastroGustavo Fernandez-Castro Miami, FL More articles by this author , Norman BlockNorman Block Miami, FL More articles by this author , Gunhild KellerGunhild Keller Hamburg, Germany More articles by this author , Karoly SzepeshaziKaroly Szepeshazi Miami, FL More articles by this author , and Andrew SchallyAndrew Schally Miami, FL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1172AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urinary bladder cancer is the fifth most common new cancer diagnosis and among the most expensive cancers to treat in the United States. The management of muscle-invasive tumors presents a clinical challenge because of the toxicity and limitations in durability, and efficacy of current therapeutic modalities. Novel therapeutic strategies for this disease are of paramount importance. Targeted cytotoxic luteinizing hormone-releasing hormone (LHRH or GnRH) analogs powerfully inhibit growth of various experimental cancers; they are more powerful and better tolerated than the cytotoxic radical, doxorubicin (DOX) alone. We evaluated, therefore, the effects of cytotoxic LHRH analog AN-152 on four human bladder cancers xenografted into nude mice. METHODS We investigated the effects of AN-152 on growth of HT-1376, J82, RT-4 and HT-1197 tumors xenografted into nude mice. Surgical specimens of 18 human primary urothelial bladder carcinomas were immunohistochemically stained for LHRH receptor, also. RESULTS In all four in vivo experiments, final tumor volumes (62%, 84%, 85%, and 89% inhibition, respectively; p<0.05) and tumor weights were significantly lower in the groups receiving AN-152; AN-152 had greater effect and was less toxic than DOX. Treatment with DOX alone resulted in significant reduction of tumor volume only in HT-1197 tumors. Positive staining for LHRH receptors was observed in all examined surgical specimens and enhanced staining of the plasma membrane as well as cytoplasmatic staining was detected in these malignant cells and in positive control (human pituitary). CONCLUSIONS We demonstrated the effectiveness of the potent targeted cytotoxic LHRH analog, AN-152, in inhibiting the growth of experimental models of bladder cancer in vivo. The expression of LHRH receptors was detected in all surgical specimens of human primary urothelial bladder carcinomas. Our findings warrant further development of AN-152 for clinical therapy of bladder cancer. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e432-e433 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ferenc Rick Miami, FL More articles by this author Luca Szalontay Miami, FL More articles by this author Gustavo Fernandez-Castro Miami, FL More articles by this author Norman Block Miami, FL More articles by this author Gunhild Keller Hamburg, Germany More articles by this author Karoly Szepeshazi Miami, FL More articles by this author Andrew Schally Miami, FL More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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 mu g 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-1 beta, nuclear factor-kappa beta 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.

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.

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-1β, NF-κβ/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.

Peptide hormones can influence the development and growth of many cancers which are not considered classical hormonedependent tumors. Analogs of somatostatin, bombesin/gastrin- releasing peptide (GRP), luteinizing hormone-releasing hormone (LH-RH) and growth hormone-releasing hormone (GH-RH) can interfere with receptors on tumor cells or intracellular pathways that are important in cell proliferation and in this way inhibit tumor growth. The first part of this review explains how these peptide hormones and their analogs affect tumors. The second part of this review describes how various hormone analogs can be used for the treatment of gastric, colorectal, pancreatic, liver and urinary bladder cancers. These tumors are major health problems worldwide and their treatment remains a great challenge. Receptors for somatostatin, bombesin/GRP, LH-RH and GH-RH are present in a large percentage of these cancers. We have developed a series of cytotoxic peptides based on doxorubicin or its derivative 2-pyrrolino-doxorubicin coupled to an analog of LH-RH, somatostatin or bombesin. This new class of targeted analogs might provide a more effective therapy for various cancers that express receptors for these carrier peptides, while producing significantly reduced peripheral toxicity. Under experimental conditions, these peptide hormone analogs strongly inhibited the growth of these tumors. Cytotoxic analogs were particularly effective on tumors that express the specific peptide receptors and acted more powerfully than the carrier peptide or the cytotoxic compound alone. Clinical trials on these peptide analogs are in progress.

In view of findings that various tumors express receptors for somatostatin, a new targeted cytotoxic analog of somatostatin, AN-162 (AEZS-124), consisting of doxorubicin linked through glutaric acid to the somatostatin octapeptide RC-121 was developed in our laboratory. We studied the toxicity in vivo and the effect of AN-162 on growth of the MDA-MB-231 estrogen-independent human breast cancer cell line xenografted into nude mice. AN-162 induced significant tumor growth inhibition compared with the control and the group treated with doxorubicin in equimolar doses. We also evaluated the stability of AN-162 in various sera in vitro, as this conjugate is susceptible to hydrolysis by serum carboxylesterase enzymes in the circulation. This study shows for the first time that AN-162 is a safe and effective compound for the treatment of experimental breast cancer. Our findings support the concept of targeted chemotherapy based on cytotoxic peptide analog AN-162 for the treatment of breast cancers and other cancers expressing somatostatin receptors.

Human non-small cell lung cancers (NSCLCs) express receptors for somatostatin. The cytotoxic analog of somatostatin AN-162 (AEZS-124), consisting of doxorubicin linked to a somatostatin analog RC-121 binds to receptors for somatostatin and is targeted to tumors expressing these receptors. The aim of this study was to investigate the effect of targeted cytotoxic somatostatin analog AN-162 on a panel of human NSCLC cell lines (A549, H460, H838, H1299) in vitro (at 0.5-100 microM concentrations) and in vivo on H460 and H1299 NSCLCs xenografted into nude mice (at the dose of 2.5 micromol/kg, i.v., once a week). The expression of mRNA for somatostatin receptor subtypes was investigated by RT-PCR in cell lines and tumor tissues. Somatostatin receptor proteins were also characterized by ligand competition assay and Western blotting. AN-162 significantly decreased cell proliferation in vitro and tumor growth (p<0.05 vs. all groups) of H460 and H1299 NSCLCs in vivo. Based on real-time PCR array data, AN-162 induced several apoptosis-related genes in vivo in both models. Our results suggest that cytotoxic somatostatin analog AN-162 (AEZS-124) should be considered for the further development of a therapy of patients with NSCLC.

As there is no effective systemic therapy for advanced hepatocellular carcinoma (HCC), we investigated the presence of receptors for somatostatin, bombesin and luteinizing hormone-releasing hormone (LHRH) in SK-Hep-1 human hepatic carcinoma and the effects of cytotoxic analogs of somatostatin (AN-238), bombesin (AN-215) and LHRH (AN-207) on the growth of this tumor. Nude mice bearing SK-Hep-1 HCCs were treated with AN-238, AN-215, AN-207 and their combination, or cytotoxic radical 2-pyrrolinodoxorubicin (AN-201). Tumor growth reduction was determined and cell proliferation characteristics and apoptosis were studied by histologic analysis. The expression of receptors for somatostatin, bombesin and LHRH was investigated by radioreceptor assays and immunohistochemistry. High-affinity binding sites for somatostatin, bombesin and LHRH were detected in SK-Hep-1 cancers. All three cytotoxic peptide analogs inhibited growth of SK-Hep-1 tumors and decreased the cell proliferation rate. Combination therapy with two or three cytotoxic analogs resulted in the strongest tumor inhibition. Receptors for somatostatin, bombesin and LHRH are expressed in SK-Hep-1 human HCC. Cytotoxic peptide analogs targeted to these receptors inhibit growth of this tumor. Targeting to multiple receptors enhances the efficacy of therapy. The results of our study encourage additional experimental investigations to permit the introduction of these cytotoxic analogs into clinical trials.