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.

Shrinkage of experimental benign prostatic hyperplasia and reduction of prostatic cell volume by a gastrin-releasing peptide antagonist.

Gastrin releasing-peptide (GRP) is a potent growth factor in many malignancies. Benign prostatic hyperplasia (BPH) is a progressive age-related proliferation of glandular and stromal tissues; various growth factors and inflammatory processes are involved in its pathogenesis. We have demonstrated that potent antagonists of GRP inhibit growth of experimental human tumors including prostate cancer, but their effect on models of BPH has not been studied. Here, we evaluated the effects of GRP antagonist RC-3940-II on viability and cell volume of BPH-1 human prostate epithelial cells and WPMY-1 prostate stromal cells in vitro, and in testosterone-induced BPH in Wistar rats in vivo. RC-3940-II inhibited the proliferation of BPH-1 and WPMY-1 cells in a dose-dependent manner and reduced prostatic cell volume in vitro. Shrinkage of prostates was observed after 6 wk of treatment with RC-3940-II: a 15.9% decline with 25 µg/d; and a 18.4% reduction with 50 µg/d (P < 0.05 for all). Significant reduction in levels of proliferating cell nuclear antigen, NF-κß/p50, cyclooxygenase-2, and androgen receptor was also seen. Analysis of transcript levels of genes related to growth, inflammatory processes, and signal transduction showed significant changes in the expression of more than 90 genes (P < 0.05). In conclusion, GRP antagonists reduce volume of human prostatic cells and lower prostate weight in experimental BPH through direct inhibitory effects on prostatic GRP receptors. GRP antagonists should be considered for further development as therapy for BPH.

Powerful inhibition of in-vivo growth of experimental hepatic cancers by bombesin/gastrin-releasing peptide antagonist RC-3940-II.

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.

Receptor-targeted therapy of human experimental urinary bladder cancers with cytotoxic LH-RH analog AN-152 [AEZS- 108].

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.

Antagonists of growth hormone-releasing hormone inhibit growth of androgen-independent prostate cancer through inactivation of ERK and Akt kinases.

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.

Combining growth hormone-releasing hormone antagonist with luteinizing hormone-releasing hormone antagonist greatly augments benign prostatic hyperplasia shrinkage.

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 µg 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-1ß, nuclear factor-κß 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.

Antagonists of growth hormone-releasing hormone (GHRH) reduce prostate size in experimental benign prostatic hyperplasia.

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-1ß, NF-κß/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.

The use of peptide analogs for the treatment of gastrointestinal, pancreatic, liver and urinary bladder cancers.

Peptide hormones can influence the development and growth of many cancers which are not considered classical hormone-dependent 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.

Preclinical evaluation of properties of a new targeted cytotoxic somatostatin analog, AN-162 (AEZS-124), and its effects on tumor growth inhibition.

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.

Inhibition of human non-small cell lung cancers with a targeted cytotoxic somatostatin analog, AN-162.

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.

Therapy of experimental hepatic cancers with cytotoxic peptide analogs targeted to receptors for luteinizing hormone-releasing hormone, somatostatin or bombesin.

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.

LH-RH receptors in human colorectal cancers: unexpected molecular targets for experimental therapy.

Since the efficacy of chemotherapy can be enhanced by targeting to specific receptors on tumors, we investigated the expression of LH-RH receptors in 5 human colon cancer lines and the effects of cytotoxic LH-RH analogs on these tumors. Nude mice bearing HT-29, HCT-116, HCT-15, LoVo and Colo-320DM cancers were treated with cytotoxic LH-RH analogs AN-152 and AN-207 or their respective cytotoxic radicals doxorubicin (DOX) and 2-pyrrolino-DOX (AN-201). The reduction in tumor growth was evaluated, and cell proliferation characteristics as well as apoptosis were analyzed by histological methods. LH-RH receptors on the tumors were investigated by radioligand binding assays and their mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). All 5 colorectal cancer lines expressed high affinity binding sites for LH-RH, and mRNA for the LH-RH receptors. Both cytotoxic LH-RH analogs AN-152 and AN-207 powerfully inhibited growth of all colon cancers. AN-207 had the strongest effect on HT-29 and HCT-116 tumors, and AN-152 was the most effective on Colo-320DM cancers. Cytotoxic radicals AN-201 and DOX were less effective on these 3 tumors, but had effects similar to AN-152 and AN-207 on HCT-15 and LoVo carcinomas. The four cytotoxic compounds also differently affected apoptosis and proliferation rate of the various tumor lines. Our findings suggest that cytotoxic LH-RH analogs should be considered for the therapy of patients with advanced colorectal carcinoma.

Synergistic inhibition of growth of lung carcinomas by antagonists of growth hormone-releasing hormone in combination with docetaxel.

We investigated the effect of antagonists of growth hormone-releasing hormone (GHRH) MZ-J-7-138 and JV-1-92 on H460 human non-small cell lung carcinoma (NSCLC) xenografted orthotopically into nude mice. Treatment with MZ-J-7-138 or JV-1-92 inhibited orthotopic growth of H460 NSCLC by 52-65% (P < 0.001) and was associated with a significant decrease in protein expression of K-Ras, cyclooxygenase-2 (Cox-2) and phospho-Akt (pAkt). In other experiments, treatment with MZ-J-7-138 or docetaxel reduced tumor volume of s.c. xenografted H460 human NSCLC by 30-36% (P < 0.01). The combination of MZ-J-7-138 and docetaxel resulted in a synergistic growth inhibition of H460 NSCLC xenografts of 63%. MZ-J-7-138 alone or in combination with docetaxel significantly reduced protein levels of K-Ras, Cox-2, and pAkt by 56-63%. Docetaxel given singly diminished the protein levels only of Cox-2 and did not affect K-Ras and pAkt. High-affinity binding sites, mRNA, and protein expression of pituitary GHRH receptors and its splice variant (SV) 1 were found in H460. H460 NSCLC cells contained GHRH peptide, and its growth was significantly inhibited in vitro by 10 microM MZ-J-7-138 (P < 0.001). Serum insulin-like growth factor 1 (IGF1) was not reduced by either GHRH antagonists. These findings suggest that antiproliferative effects of GHRH antagonists in H460 NSCLC are associated with down-regulation of K-Ras, Cox-2, and pAkt. In conclusion, GHRH antagonists in combination with docetaxel synergistically inhibit growth of H460 NSCLC and the expression of K-ras, Cox-2, and pAkt, which might abrogate the signal transduction pathways for cell growth stimulation and therapeutic resistance.

Targeted chemotherapy with cytotoxic bombesin analogue AN-215 inhibits growth of experimental human prostate cancers.

We developed a powerful cytotoxic analogue of bombesin AN-215, in which the bombesin (BN)-like carrier peptide is conjugated to 2-pyrrolino doxorubicin (AN-201). Human prostate cancers express high levels of receptors for BN/gastrin releasing peptide (GRP) that can be used for targeted chemotherapy. The effects of targeted chemotherapy with cytotoxic BN analogue AN-215 were evaluated in nude mice bearing subcutaneous xenografts of DU-145, LuCaP-35, MDA-PCa-2b and intraosseous implants of C4-2 human prostate cancers. Intraosseous growth of C4-2 tumors was monitored by serum PSA. BN/GRP receptors were evaluated by 125I-[Tyr4]BN binding assays and RT-PCR. The effects of AN-215 on apoptosis and cell proliferation were followed by histology, and the expression of Bcl-2 and Bax protein was determined by Western blot analysis. Targeted analog AN-215 significantly inhibited growth of subcutaneously implanted DU-145, LuCaP-35 and MDA-PCa-2b prostate cancers by 81% to 91% compared to controls, while cytotoxic radical AN-201 was less effective and more toxic. Serum PSA levels of mice bearing intraosseous C4-2 prostate tumors were significantly reduced. In LuCaP-35 tumors administration of BN antagonist RC-3095 prior to AN-215 blocked the receptors for BN/GRP and inhibited the effects of AN-215. High affinity receptors for BN/GRP and their m-RNA were detected on membranes of all 4 tumor models. Therapy with AN-215, but not with AN-201, decreased the ratio of Bcl-2/Bax in DU-145 and the expression of antiapoptotic Bcl-2 in LuCaP-35 tumors. The presence of BN/GRP receptors on primary and metastatic prostate cancers makes possible targeted chemotherapy with AN-215 for the treatment of this malignancy.

Inhibition of human experimental prostate cancers by a targeted cytotoxic luteinizing hormone-releasing hormone analog AN-207.

BACKGROUND: Receptors for luteinizing hormone-releasing hormone (LHRH) on human prostate cancers can be used for targeted chemotherapy with cytotoxic analogs of LHRH, such as AN-207, which consists of superactive doxorubicin derivative 2-pyrrolino doxorubicin (AN-201) linked to carrier [D-Lys6] LHRH. METHODS: The effects of AN-207 and AN-201 were investigated in DU-145 androgen independent and LuCaP-35 androgen sensitive human prostate cancers xenografted into nude mice. Toxicity was evaluated by survival rates, changes in body weights, and leukocyte counts. LHRH receptors on DU-145 and LuCaP-35 tumors were evaluated by radioreceptor assays and RT-PCR. The effects on apoptosis and cell proliferation were investigated by histology and evaluation of apoptotic oncogenes Bcl-2 and Bax by Western Blot analysis. RESULTS: AN-207 inhibited growth of DU-145 tumors significantly by 75% (P < 0.01) and LuCaP-35 human prostate cancers by 80% (P < 0.01), and was less toxic than AN-201. Receptors for LHRH were expressed on DU-145 and LuCaP-35 tumors. Blockade of LHRH receptors with LHRH agonist Triptorelin nullified the effects of AN-207. Treatment with AN-207, but not with AN-201, decreased Bcl-2/Bax ratio in DU-145 tumors and Bcl-2 in LuCaP-35 tumors indicating an increase in apoptotic activity. AN-207, but not AN-201, decreased cell proliferation in both models. CONCLUSIONS: Targeted chemotherapy with AN-207 could be considered for treatment of advanced prostate cancer.

The expression of the pituitary growth hormone-releasing hormone receptor and its splice variants in normal and neoplastic human tissues.

Various attempts to detect human pituitary growth hormone-releasing hormone receptor (pGHRH-R) in neoplastic extrapituitary tissues have thus far failed. Recently, four splice variants (SVs) of GHRH-R have been described, of which SV1 has the highest structural homology to pGHRH-R and likely plays a role in tumor growth. The aim of this study was to reinvestigate whether human tumors and normal human extrapituitary tissues express the pGHRH-R and to corroborate our previous findings on its SVs. Thus, we developed a real-time PCR method for the detection of the mRNA for the pGHRH-R, its SVs, and the GHRH peptide. Using real-time PCR, Western blotting, and radioligand-binding assays, we detected the mRNA for pGHRH-R and pGHRH-R protein in various human cancer cell lines grown in nude mice and in surgical specimens of human lung cancers. The expression of mRNA for SVs of pGHRH-R and GHRH was likewise found in xenografts of human non-Hodgkin's lymphomas, pancreatic cancer, glioblastoma, small-cell lung carcinomas, and in human nonmalignant prostate, liver, lung, kidney, and pituitary. Western blots showed that these normal and malignant human tissues contain SV1 protein and immunoreactive GHRH. Our results demonstrate that some normal human tissues and tumors express mRNA and protein for the pGHRH-R and its splice variants. These findings confirm and extend the concept that GHRH and its receptors play an important role in the pathophysiology of human cancers.

Inhibition of growth of experimental human and hamster pancreatic cancers in vivo by a targeted cytotoxic bombesin analog.

OBJECTIVES: Targeting anticancer agents to receptors for peptide hormones such as bombesin/gastrin-releasing peptide (GRP) on tumor cells increases the efficacy and lowers the toxicity of cancer therapy. We studied the expression of bombesin/GRP receptors in 6 experimental pancreatic cancers and evaluated tumor inhibition in vivo produced by targeted chemotherapy with the cytotoxic bombesin analog AN-215. METHODS: Nude mice with xenografts of Panc-1, CFPAC-1, Capan-1, Capan-2, MiaPaCa-2, and SW-1990 human ductal pancreatic cancers, as well as hamsters with nitrosamine-induced pancreatic cancers, were treated with AN-215 or its cytotoxic radical 2-pyrrolinodoxorubicin (AN-201) for 7 to 12 weeks. Tumor growth reduction and survival were analyzed, and cell proliferation rate and apoptosis were examined by histologic methods. Bombesin/GRP receptors on the tumors were studied by ligand-binding assays and their mRNA expression was studied by reverse transcriptase-polymerase chain reaction. RESULTS: All tumors expressed mRNA for subtype 1 bombesin/GRP receptor, but MiaPaCa-2, and in one experiment, SW-1990 tumors did not show binding sites for bombesin. AN-215 powerfully inhibited the growth of all pancreatic cancers that expressed functional receptors for bombesin/GRP. AN-201 was less effective on most tumors and somewhat more toxic than AN-215. CONCLUSIONS: Bombesin/GRP receptors are expressed on most ductal pancreatic carcinoma cell lines and can be used for targeted chemotherapy with the cytotoxic bombesin analog AN-215.

Inhibitory effect of antagonists of bombesin and growth hormone-releasing hormone on orthotopic and intraosseous growth and invasiveness of PC-3 human prostate cancer in nude mice.

PURPOSE: To determine whether antagonists of growth hormone-releasing hormone (GHRH) and bombesin/gastrin-releasing peptide (BN/GRP) can inhibit the orthotopic and metastatic growth of PC-3 human androgen-independent prostate cancers. EXPERIMENTAL DESIGN: The effects of administration of GHRH antagonist MZ-J-7-118, BN/GRP antagonist RC-3940-II, and their combination on the growth and metastatic spread of PC-3 tumors implanted orthotopically into nude mice were evaluated. The efficacy of this treatment on PC-3 tumors implanted intratibially and s.c. was also determined. RESULTS: Treatment with MZ-J-7-118, RC-3940-II, or their combination significantly inhibited the growth of PC-3 tumors implanted orthotopically, intraosseously, and s.c. The combination of the two antagonists had the greatest effect, inhibiting orthotopic tumor growth by 77%, intratibially implanted tumors by 86%, and s.c. tumors by 86%. The therapy with BN/GRP and GHRH antagonists, especially in combination, also reduced the local tumor spread and distant metastases in animals bearing orthotopic tumors. Combination therapy was likewise the most effective in reducing the incidence and severity of tibial osteolytic lesions and pathologic fractures in intraosseously implanted tumors. High-affinity binding sites for BN/GRP and GHRH were found in s.c. and orthotopic PC-3 tumor samples. MZ-J-7-118, RC-3940-II, and the combination of both compounds inhibited in vitro growth of PC-3 cells. CONCLUSIONS: Our findings show the efficacy of BN/GRP antagonists and GHRH antagonists for the treatment of advanced prostate cancer in preclinical metastatic models. As BN/GRP antagonists are already in clinical trials and GHRH antagonists are effective in androgen-independent prostate cancer models, these analogues could be considered for the management of advanced prostate carcinoma.

Effective treatment of experimental androgen sensitive and androgen independent intraosseous prostate cancer with targeted cytotoxic somatostatin analogue AN-238.

PURPOSE: The targeted cytotoxic somatostatin analogue AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier octapeptide RC-121, is scheduled for clinical trials. To extend previous findings we tested AN-238 on human androgen sensitive MDA-PCa-2b prostate cancers grown subcutaneously and androgen independent LNCaP derived C4-2 prostate cancers xenografted into the tibiae of nude mice. MATERIALS AND METHODS: Changes in serum prostate specific antigen (PSA) levels were monitored by radioimmunoassay. Somatostatin receptors in tumor samples were characterized. RESULTS: Three intravenous injections of AN-238 at 150 nmol/kg doses inhibited the growth of subcutaneous MDA-PCa-2b tumors by 62% vs controls (p <0.05) and were more effective than equimolar doses of the radical AN-201 (p <0.05). AN-238 also decreased serum PSA levels by 62% vs controls (p <0.01). In nude mice bearing intra-osseous implanted C4-2 prostate cancers AN-238 decreased serum PSA levels by 65% compared with controls after 5 weeks of therapy (p <0.05), while AN-201 was ineffective. All AN-238 treated mice were alive at the termination of the experiment, while only 50% of controls and 60% of animals treated with AN-201 survived (p <0.01). Histological evaluation of intraosseous C4-2 tumors showed that AN-238 induced a significant increase in apoptosis (p <0.05). MDA-PCa-2b and C4-2 tumors showed high affinity binding for somatostatin and the expression of mRNA for somatostatin receptor subtypes 1, 2A and 5. CONCLUSIONS: The current study demonstrates the efficacy of the somatostatin analogue AN-238 for subcutaneous MDA-PCa-2b as well as for intraosseous C4-2 prostate cancers xenografted into nude mice. This targeted cytotoxic analogue could represent a new therapy for patients with advanced metastatic prostate carcinoma.

Antagonists of growth hormone-releasing hormone inhibit the proliferation of experimental non-small cell lung carcinoma.

Recent studies show that antagonists of growth hormone-releasing hormone (GH-RH) inhibit proliferation of various cancers indirectly through blockage of the endocrine GH-insulin-like growth factor (IGF) I axis and directly by an action on tumor cells involving the suppression of autocrine/paracrine IGF-I, IGF-II, or GH-RH. The effectiveness of therapy with GH-RH antagonist JV-1-38 and its mechanisms of action were investigated in NCI-H838 non-small cell lung carcinoma (NSCLC) xenografted s.c. into nude mice and in vitro. Treatment with GH-RH antagonist JV-1-38 significantly (P < 0.05-0.001) inhibited tumor growth as demonstrated by a 58% decrease in final tumor volume, 54% reduction in tumor weight, and the extension of tumor-doubling time from 8.5 +/- 1.38 to 12 +/- 1.07 days as compared with controls. Using ligand competition assays with (125)I-labeled GH-RH antagonist JV-1-42, specific high-affinity binding sites for GH-RH were found on tumor membranes. Reverse transcription-PCR revealed the expression of mRNA for GH-RH and splice variant 1 (SV(1)) of GH-RH receptor in H838 tumors. Reverse transcription-PCR analysis also demonstrated that H838 tumors express IGF-I and IGF-I receptors. Tumoral concentration of IGF-I and its mRNA expression were significantly decreased by 25% (P = 0.05) and 65% (P < 0.001), respectively, in animals receiving JV-1-38, whereas serum IGF-I levels remained unchanged. In vitro studies showed that H838 cells secreted GH-RH and IGF-I into the medium. The growth of tumor cells in vitro was stimulated by IGF-I and inhibited by GH-RH antagonist JV-1-38 and a GH-RH antiserum. Our results extend the findings on the involvement of IGF-I in NSCLC and suggest that GH-RH may be an autocrine growth factor for H838 NSCLC. The antitumorigenic action of GH-RH antagonists could be partly direct and mediated by SV(1) of tumoral GH-RH receptors. The finding of GH-RH and SV(1) of GH-RH receptors in NSCLC provides a new approach to the treatment of this malignancy based on the use of antagonistic analogues of GH-RH.