Karoly Szepeshazi

Hypothalamic hormones and cancer.

The use of peptide analogs for the therapy of various cancers is reviewed. Inhibition of the pituitary-gonadal axis forms the basis for oncological applications of luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists, but direct effects on tumors may also play a role. Analogs of somatostatin are likewise used for treatment of various tumors. Radiolabeled somatostatin analogs have been successfully applied for the localization of tumors expressing somatostatin receptors. Studies on the role of tumoral LH-RH, growth hormone-releasing hormone (GH-RH), and bombesin/GRP and their receptors in the proliferation of various tumors are summarized, but the complete elucidation of all the mechanisms involved will require much additional work. Human tumors producing hypothalamic hormones are also discussed. Treatment of many cancers remains a major challenge, but new therapeutic modalities are being developed based on antagonists of GH-RH and bombesin, which inhibit growth factors or their receptors. Other approaches consist of the use of cytotoxic analogs of LH-RH, bombesin, and somatostatin, which can be targeted to receptors for these peptides in various cancers and their metastases. These new classes of peptide analogs should lead to a more effective treatment for various cancers.

Inhibition of growth of androgen-independent DU-145 prostate cancer in vivo by luteinising hormone-releasing hormone antagonist cetrorelix and bombesin antagonists RC-3940-II and RC-3950-II

The aim of this study was to test the antagonist of LH-RH (Cetrorelix), agonist [D-Trp6]LH-RH (triptorelin) and new bombesin antagonists RC-3940-II and RC-3950-II for their effect on the growth of an androgen-independent prostate cancer cell line, DU-145, xenografted into nude mice. Xenografts were grown in male nude mice, and after 4 weeks, the animals were treated either with saline (control) or with one of the analogues. One group of mice was given a combination of Cetrorelix and RC-3950-II. Treatment was given for 4 weeks. Tumour and body weights, and tumour volumes were measured. At sacrifice, tumours were dissected for histological examination and receptor studies. Serum was collected for measurement of hormone levels. The final tumour volume in control animals injected with saline was 577 +/- 155 mm3 and that of animals treated with Cetrorelix only 121.4 +/- 45 mm3 (P < 0.01). Bombesin antagonists RC-3940-II and RC-3950-II also significantly reduced DU-145 tumour volume in nude mice to 84.9 +/- 19.9 and 96.8 +/- 28 mm3, respectively. Agonist [D-Trp6]LH-RH did not significantly inhibit tumour growth. Serum levels of LH were decreased to 0.08 +/- 0.02 ng/ml (P < 0.05) in the Cetrorelix treated group as compared to 1.02 +/- 0.1 ng/ml for the controls, and testosterone levels were reduced to castration levels (0.01 +/- 0.01 ng/ml). Specific receptors for EGF and LH-RH in DU-145 tumours were significantly downregulated after treatment with Cetrorelix, RC-3940-II and RC-3950-II. Although LH-RH could be a local regulator of growth of prostate cancer, the fall in LH-RH receptors is not fully understood and the inhibitory effects of Cetrorelix and bombesin antagonists on DU-145 tumour growth might be attributed at least in part to a downregulation of EHF receptors. Since Cetrorelix and bombesin antagonists inhibit growth of androgen-independent DU-145 prostate cancers, these compounds could be considered for the therapy of advanced prostate cancer in men, especially after relapse.

Luteinizing hormone‐releasing hormone antagonist Cetrorelix (SB‐75) and bombesin antagonist RC‐3940‐II inhibit the growth of androgen‐independent PC‐3 prostate cancer in nude mice

Hormones like bombesin (BN)/gastrin‐releasing peptide (GRP) and luteinizing hormone‐releasing hormone (LH‐RH) and growth factors such as epidermal growth factor (EGF) might be involved in the relapse of prostate cancer under androgen ablation therapy. Interference with receptors for BN/GRP, LH‐RH, or EGF might provide a therapeutic approach to inhibit tumor growth of androgen‐independent prostate cancer.

THE EXPRESSION OF GROWTH HORMONE-RELEASING HORMONE (GHRH) AND SPLICE VARIANTS OF ITS RECEPTOR IN HUMAN GASTROENTEROPANCREATIC CARCINOMAS

Splice variants (SVs) of receptors for growth hormone-releasing hormone (GHRH) have been found in primary human prostate cancers and diverse human cancer cell lines. GHRH antagonists inhibit growth of various experimental human cancers, including pancreatic and colorectal, xenografted into nude mice or cultured in vitro, and their antiproliferative action could be mediated in part through SVs of GHRH receptors. In this study we examined the expression of mRNA for GHRH and for SVs of its receptors in tumors of human pancreatic, colorectal, and gastric cancer cell lines grown in nude mice. mRNA for both GHRH and SV1 isoform of GHRH receptors was expressed in tumors of pancreatic (SW1990, PANC-1, MIA PaCa-2, Capan-1, Capan-2, and CFPAC1), colonic (COLO 320DM and HT-29), and gastric (NCI-N87, HS746T, and AGS) cancer cell lines; mRNA for SV2 was also present in Capan-1, Capan-2, CFPAC1, HT-29, and NCI-N87 tumors. In proliferation studies in vitro, the growth of pancreatic, colonic, and gastric cancer cells was stimulated by GHRH(1–29)NH2 and inhibited by GHRH antagonist JV-1–38. The stimulation of some gastroenteropancreatic cancer cells by GHRH was followed by an increase in cAMP production, and GHRH antagonist JV-1–38 competitively inhibited this effect. Our study indicates the presence of an autocrine/paracrine stimulatory loop based on GHRH and SV1 of GHRH receptors in human pancreatic, colorectal, and gastric cancers. The finding of SV1 receptor in human cancers provides an approach to an antitumor therapy based on the blockade of this receptor by specific GHRH antagonists.

New approaches to therapy of cancers of the stomach, colon and pancreas based on peptide analogs

Cancers of the stomach, colon and exocrine pancreas are major international health problems and result in more than a million deaths worldwide each year. The therapies for these malignancies must be improved. The effects of gastrointestinal (GI) hormonal peptides and endogenous growth factors on these cancers were reviewed. Some GI peptides, including gastrin and gastrin-releasing peptide (GRP) (mammalian bombesin), appear to be involved in the growth of neoplasms of the GI tract. Certain growth factors such as insulin-like growth factor (IGF)-I, IGF-II and epidermal growth factor and their receptors that regulate cell proliferation are also implicated in the development and progression of GI cancers. Experimental investigations on gastric, colorectal and pancreatic cancers with analogs of somatostatin, antagonists of bombesin/GRP, antagonists of growth hormone-releasing hormone as well as cytotoxic peptides that can be targeted to peptide receptors on tumors were summarized. Clinical trials on peptide analogs in patients with gastric, colorectal and pancreatic cancers were reviewed and analyzed. It may be possible to develop new approaches to hormonal therapy of GI malignancies based on various peptide analogs.

Antagonists of growth hormone-releasing hormone (GH-RH) inhibit IGF-II production and growth of HT-29 human colon cancers

Insulin-like growth factors (IGFs) I and II are implicated in progression of various tumours including colorectal carcinomas. To interfere with the production of IGFs, we treated male nude mice bearing xenografts of HT-29 human colon cancer with various potent growth hormone-releasing hormone (GH-RH) antagonists. Twice daily injections of antagonist MZ-4-71, 10 μg intraperitoneally or 5 μg subcutaneously (s.c.) resulted in a significant 43–45% inhibition of tumour growth. Longer acting GH-RH antagonists, MZ-5-156 and JV-1-36 given once daily at doses of 20 μg s.c. produced a 43–58% decrease in volume and weight of cancers. Histological analyses of HT-29 cancers demonstrated that both a decreased cell proliferation and an increased apoptosis contributed to tumour inhibition. GH-RH antagonists did not change serum IGF-I or IGF-II levels, but significantly decreased IGF-II concentration and reduced mRNA expression for IGF-II in tumours. In vitro studies showed that HT-29 cells produced and secreted IGF-II into the medium, and addition of MZ-5-156 dose-dependently decreased IGF-II production by about 40% as well as proliferation of HT-29 cells. Our studies demonstrate that GH-RH antagonists inhibit growth of HT-29 human colon cancers in vivo and in vitro. The effect of GH-RH antagonists may be mediated through a reduced production and secretion of IGF-II by cancer cells.

Antagonists of growth hormone-releasing hormone (GH-RH) inhibit in vivo proliferation of experimental pancreatic cancers and decrease IGF-II levels in tumours.

Insulin-like growth factors (IGF-I and IGF-II) are implicated in the pathogenesis of pancreatic carcinoma. Antagonists of growth hormone-releasing hormone (GH-RH) suppress the GH-RH-GH-IGF-I axis and also act directly on tumours to reduce production of IGF-I or II. The aim of this study was to investigate the effects of two potent GH-RH antagonists in two experimental models of pancreatic cancer. Syrian golden hamsters with nitrosamine-induced pancreatic tumours were treated with 10 micrograms/day of GH-RH antagonist MZ-4-71 for 60 days. The therapy reduced the number of tumorous animals, decreased the weight of tumorous pancreata by 55%, and lowered AgNOR numbers in tumour cells. In two other experiments, GH-RH antagonists MZ-4-71 and MZ-5-156 significantly inhibited growth of SW-1990 human pancreatic cancers xenografted into nude mice, as shown by a reduction in tumour volume and tumour weights, and a decrease in AgNORs in cancer cells. IGF-I levels in serum and in pancreatic cancer tissue remained unchanged after therapy, suggesting that an effect on IGF-I is not involved in tumour inhibition. In contrast, IGF-II concentrations in tumours were significantly reduced by 50-60% after treatment with the GH-RH antagonists as compared with controls. In vitro studies showed that the concentration of IGF-II in the culture medium was increased after seeding of SW-1990 cells, indicating that this pancreatic cancer cell line produced and released IGF-II. This finding was also supported by the expression of IGF-II mRNA in the SW-1990 cells. Addition of 3 x 10(-6) M of GH-RH antagonist MZ-5-156 to the reduced-serum medium decreased cell proliferation, IGF-II mRNA expression in the cells and IGF-II concentration in the medium. Our findings indicate that inhibitory effects of GH-RH antagonists on the growth of experimental pancreatic cancers, may result from a decrease in the production and concentration of IGF-II in the tumours.

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.

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-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.

A targeted cytotoxic somatostatin (SST) analogue, AN-238, inhibits the growth of H-69 small-cell lung carcinoma (SCLC) and H-157 non-SCLC in nude mice

Recently, we developed a cytotoxic analogue of somatostatin (SST), AN-238, in which the SST carrier peptide RC-121 was linked to 2-pyrrolinodoxorubicin (2-pyrrolino-DOX) (AN-201), a potent derivative of doxorubicin. AN-238 can be targeted to SST receptors (SSTRs) on tumours. In the present study, we evaluated the effects of AN-238 on the growth of H-69 small-cell lung carcinoma (SCLC) and H-157 non-SCLC xenografted into nude mice. High affinity binding sites for SST are present in H-69 SCLC and were now detected in H-157 non-SCLC xenografts, but not in H-157 cells. A strong expression of the human SSTR subtype 2 (hSSTR-2) and a weaker expression of subtype 5 (hSSTR-5) was found in H-69 SCLC cells, but not in H-157 non-SCLC cells. However, a strong expression of mRNA for mouse (m)SSTR-2 could be detected in H-157 xenografts. AN-238 effectively inhibited the growth of H-69 SCLC tumours in nude mice. Twenty-six days after a single injection of AN-238 at 200 nmol/kg, the volume of H-69 tumours was decreased by approximately 55% (P<0.05) compared with the controls, while AN-201 at the same dose was highly toxic and produced only a minor tumour inhibition. To evaluate the potency of multiple doses of AN-238, nude mice bearing H-69 SCLC received three injections of AN-238 at 150 nmol/kg on days 1, 12 and 28. In the period of 42 days after the first injection, the growth rate of H-69 tumours was approximately 50% lower than that of controls. In nude mice bearing H-157 non-SCLC tumours, a single i.v. administration of AN-238 at 200 nmol/kg inhibited tumour volume by 91% after 28 days (P<0.01 compared with controls). AN-201 was toxic and ineffective at the same dose. Two injections of AN-238 at 150 nmol/kg given on days 1 and 18 produced 83% inhibition of H-157 tumour growth (P<0.01 versus controls). AN-238 given as a single dose of 200 nmol/kg induced necrosis, while two injections of 150 nmol/kg induced apoptosis in the tumour tissue. Our results indicate that targeted cytotoxic SST analogue AN-238 could be considered for therapy of both SCLC and non-SCLC.