Kate Groot

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.

In vivo and in vitro release of ACTH by synthetic CRF

The 41-residue corticotropin releasing factor (CRF) was synthesized by the solid phase method. The synthetic CRF and arginine vasopressin (AVP) were examined for ACTH releasing activity and effects on the release of 5 other pituitary hormones in vivo and in vitro. Injection of the CRF into pharmacologically blocked rats increased plasma corticosterone levels in a dose-related manner. The minimum effective dose was 1.6 x 10(-12) mol/100 g body weight. CRF also significantly stimulated release of ACTH-like immunoreactivity in a dose-related manner from rat pituitary quarters beginning at a concentration of 10(-9) M. AVP, a peptide known to have CRF activity, exhibited slightly lower corticotropin releasing activity than the CRF at equimolar dose levels. Secretion of other pituitary hormones was not appreciably altered by either the CRF or AVP.

Inhibition of in vivo proliferation of androgen-independent prostate cancers by an antagonist of growth hormone-releasing hormone.

Tumour-inhibitory effects of a new antagonist of growth hormone-releasing hormone (GH-RH), MZ-4-71, were evaluated in nude mice bearing androgen-independent human prostate cancer cell lines DU-145 and PC-3 and in Copenhagen rats implanted with Dunning R-3327 AT-1 prostatic adenocarcinoma. After 6 weeks of therapy, the tumour volume in nude mice with DU-145 prostate cancers treated with 40 microg day(-1) MZ-4-71 was significantly decreased to 37 +/- 13 mm3 (P < 0.01) compared with controls that measured 194 +/- 35 mm3. A similar inhibition of tumour growth was obtained in nude mice bearing PC-3 cancers, in which the treatment with MZ-4-71 for 4 weeks diminished the tumour volume to 119 +/- 35 mm3 compared with 397 +/- 115 mm3 for control animals. Therapy with MZ-4-71 also significantly decreased weights of PC-3 and DU-145 tumours and increased tumour doubling time. Serum levels of GH and IGF-I were significantly decreased in animals treated with GH-RH antagonist. In PC-3 tumour tissue, the levels of IGF-I and IGF-II were reduced to non-detectable values after therapy with MZ-4-71. The growth of Dunning R-3327 AT-1 tumours in rats was also significantly inhibited after 3 weeks of treatment with 100 microg of MZ-4-71 day(-1) i.p. as shown by a reduction in tumour volume and weight (both P-values < 0.05). Specific high-affinity binding sites for IGF-I were found on the membranes of DU-145, PC-3 and Dunning R-3327 AT-1 tumours. Our results indicate that GH-RH antagonist MZ-4-71 suppresses growth of PC-3, DU-145 and Dunning AT-1 androgen-independent prostate cancers, through diminution of GH release and the resulting decrease in the secretion of hepatic IGF-I, or through mechanisms involving a lowering of tumour IGF-I levels and possibly an inhibition of tumour IGF-I and IGF-II production. GH-RH antagonists could be considered for further development for the therapy of prostate cancer, especially after the 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.

Potentiation of the inhibitory effect of growth hormone-releasing hormone antagonists on PC-3 human prostate cancer by bombesin antagonists indicative of interference with both IGF and EGF pathways

In view of the involvement of various neuropeptides and growth factors in the progression of androgen‐independent prostate cancer, we investigated the effects of antagonists of growth hormone‐releasing hormone (GHRH) alone or in combination with an antagonist of bombesin/gastrin‐releasing peptide (BN/GRP) on PC‐3 human prostate cancers.

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.

in vivo inhibition of PC-3 human androgen-independent prostate cancer by a targeted cytotoxic bombesin analogue, AN-215

The effectiveness of chemotherapy targeted to bombesin (BN) receptors was evaluated in nude mice bearing PC‐3 human androgen‐independent prostate cancers. Cytotoxic BN analogue AN‐215, consisting of 2‐pyrrolinodoxorubicin (AN‐201) linked to BN‐like carrier peptide RC‐3094, was injected i.v. at 150 nmol/kg on days 1, 11 and 21. After treatment with AN‐215, tumor volume was 69% (p < 0.01) smaller than that in controls and tumor doubling time was extended from 8.5 ± 0.7 days to 20.3 ± 3.5 days (p < 0.05). Cytotoxic radical AN‐201, carrier RC‐3094 and their unconjugated mixture administered at the same dosage were ineffective. The mortality rate was 12.5% in the AN‐201 group and 16.7% in the group treated with the mixture, but no deaths occurred in mice receiving AN‐215. Because the ester bond linking AN‐201 to the carrier molecule is hydrolyzed much faster in mouse serum than in human serum, in the second experiment we investigated the tolerance to AN‐215 and its effect in nude mice bearing PC‐3 tumors after pharmacological inhibition of serum carboxylesterases. Two applications of AN‐201 at 200 nmol/kg were lethal, whereas no mortality was observed after 4 injections of AN‐215 at the same dose. Administration of 200 nmol/kg AN‐215 on days 1, 7, 17 and 26 again produced 69% tumor inhibition. BN receptors on membranes of PC‐3 tumors were detected by 125I‐[Tyr4]BN binding, and expression of mRNA for BRS‐3 and GRP‐R subtypes was also found. AN‐215 showed a high affinity to PC‐3 tumors, displacing the radioligand at an IC50 of 12.95 ± 0.35 nM. Because BN receptors are present on primary and metastatic prostate cancer, targeted chemotherapy with AN‐215 might benefit patients with advanced prostatic carcinoma who relapsed androgen ablation. Int. J. Cancer 88:652–657, 2000.

Inhibition of growth and metastases of Mda-mb-435 human estrogen-independent breast cancers by an antagonist of growth hormone-releasing hormone

Antagonists of growth hormone-releasing hormone (GH-RH) inhibit the growth of various cancers by mechanism(s) that include the suppression of the insulin-like growth factors (IGF)-I and/or -II. In this study, nude mice bearing orthotopic implants of MDA-MB-435 human estrogen-independent breast carcinoma received 39 days of therapy with GH-RH antagonist JV-1-36 (20 μg/day). The treatment significantly inhibited tumor growth by 71.1% (p<0.01) and nullified the metastatic potential of MDA-MB-435 cells. Four of eight control mice (50%) developed metastases in the lymph nodes and one (12.5%) in the lung, but none of the animals receiving JV-1-36 showed metastatic spread. GH-RH antagonist JV-1-36 inhibited the growth of MDA-MB-435 cells in vitro, while IGF-I stimulated it. However, mRNA for IGF-I or -II was not detected in MDA-MB-435 cells, indicating that the suppression of autocrine IGFs may not be involved in the antiproliferative mechanism. Using ligand competition assays with 125I-labeled GH-RH antagonist JV-1-42, specific high-affinity binding sites for GH-RH were found on tumor membranes. Reverse transcription-polymerase chain reaction revealed the expression of mRNA for GH-RH receptor splice variant-1 in MDA-MB-435 tumors. Our results suggest that the antitumorigenic action of GH-RH antagonists on MDA-MB-435 breast cancer could be direct and mediated by tumoral GH-RH receptors.

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.