Inhibitory effects of antagonistic analogs of GHRH on GH3 pituitary cells overexpressing the human GHRH receptor.

GH3 rat pituitary tumor cells produce GH and prolactin (PRL), but lack the GHRH receptor (GHRH-R). We expressed human GHRH-R (hGHRH-R) in GH3 cells using recombinant adenoviral vectors and studied the effects of GHRH antagonists. The mRNA expression of the GHRH-R gene in the cells was demonstrated by RT-PCR. An exposure of the GH3 cells infected with hGHRH-R to 10(-10), 10(-9) and 10(-8) m hGHRH for 1 or 2 h in culture caused a dose-dependent elevation of the intracellular cAMP concentration and the cAMP efflux. Exposure to hGHRH also elicited dose-dependent increases in GH and PRL secretion from these cells. Neither the uninfected nor the antisense hGHRH-R-infected control cells exhibited cAMP, GH and PRL responses to GHRH stimulation. GHRH antagonists JV-1-38 and jv-1-36 applied at 3x10(-8) m for 3 h, together with 10(-9) m GHRH, significantly inhibited the GHRH-stimulated cAMP efflux from the hGHRH-R-infected cells by 36 and 80% respectively. The more potent antagonist JV-1-36 also decreased the intracellular cAMP levels in these cells by 55%. Exposure to JV-1-36 for 1 h nullified the stimulatory effect of GHRH on GH secretion and significantly inhibited it by 64 and 77% after 2 and 3 h respectively. In a superfusion system, GHRH at 10(-10), 10(-9) and 10(-8) m concentrations induced prompt and dose-related high cAMP responses and smaller increases in the spontaneous GH secretion of the hGHRH-R-infected cells. Antagonists JV-1-36 and JV-1-38 applied at 3x10(-8) m for 15 min, together with 10(-9) m GHRH, inhibited the GHRH-stimulated cAMP response by 59 and 35% respectively. This work demonstrates that GHRH antagonists can effectively inhibit the actions of GHRH on the hGHRH-R. Our results support the view that this class of compounds would be active clinically.

Expression of mRNA for growth hormone-releasing hormone and splice variants of GHRH receptors in human malignant bone tumors.

Splice variants (SV) of receptors for growth hormone-releasing hormone (GHRH) have been found in several human cancer cell lines. GHRH antagonists inhibit growth of various human cancers, including osteosarcomas and Ewing's sarcoma, xenografted into nude mice or cultured in vitro and their antiproliferative action could be mediated, in part, through these SV of GHRH receptors. In this study, we found mRNA for the SV(1) isoform of GHRH receptors in human osteosarcoma line MNNG/HOS and SK-ES-1 Ewing's sarcoma line. We also detected mRNA for GHRH, which is apparently translated into the GHRH peptide and secreted by the cells, as shown by the presence of GHRH-like immunoreactivity in the conditioned media of cell cultures. In proliferation studies in vitro, the growth of SK-ES-1 and MNNG/HOS cells was dose-dependently inhibited by GHRH antagonist JV-1-38 and an antiserum against human GHRH. Our study indicates the presence of an autocrine stimulatory loop based on GHRH and SV(1) of GHRH receptors in human sarcomas. The direct antiproliferative effects of GHRH antagonists on malignant bone tumors appear to be exerted through the SV(1) of GHRH receptors on tumoral cells.

Inhibition of growth and reduction in tumorigenicity of UCI-107 ovarian cancer by antagonists of growth hormone-releasing hormone and vasoactive intestinal peptide.

PURPOSE: To evaluate the tumor inhibitory activities of antagonists of growth hormone-releasing hormone (GH-RH) and vasoactive intestinal peptide (VIP) in UCI-107 human ovarian cancer model, and to investigate the role of the insulin-like growth factor (IGF) system in the response. METHODS: In the present study we investigated the effects of GH-RH antagonist JV-1-36 and VIP antagonist JV-1-52, on the growth and tumorigenicity of UCI-107 ovarian cell carcinoma xenografted into nude mice. Studies on the effects of hGH-RH(1-29)NH2, IGF-I, IGF-II, JV-1-36, and JV-1-52 on the proliferation of UCI-107 cells cultured in vitro were also performed. RESULTS: After 22 days of therapy with JV-1-36 or JV-1-52 at the dose of 20 microg/day, the final volume of UCI-107 tumors was significantly (P<0.05) decreased by 50.5% and 56%, respectively, compared to controls. The concentration of IGF-II in tumors was reduced by 66% in the JV-1-36-treated group and by 62% in the group given JV-1-52 (both P < 0.05). Exposure in vitro to 1 microM concentrations of JV-1-36 or JV-1-52 for 24 h decreased the tumorigenicity of UCI-107 cells in nude mice. All ten mice injected with cells treated with medium alone developed tumors within 23 days after cell inoculation, while only eight of ten and four of ten mice injected with cells exposed to JV-1-36 or JV-1-52, respectively, had tumors. In vitro exposure of UCI-107 cells to 5-35 ng/ml IGF-II produced a significant suppression in the rate of cell proliferation (P < 0.01). CONCLUSION: Our results suggest that GH-RH and VIP antagonists inhibit the growth of UCI-107 ovarian cell carcinoma by mechanisms that appear to involve direct effects on the cancer cells.

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 microg/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 (125)I-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 GHRH decrease production of GH and IGF-I in MXT mouse mammary cancers and inhibit tumor growth.

The involvement of IGF-I in mammary carcinogenesis is well established, but the role of GH, as an autocrine growth factor for breast cancers is poorly understood. The goal of our study was to investigate whether antagonists of GHRH can interfere with the effects of GH and IGF-I in MXT mouse mammary cancers. GHRH antagonists JV-1-36 and JV-1-38 inhibited growth of estrogen-independent MXT mouse mammary cancers in vivo, producing about 50% reduction in tumor volume (P < 0.05). This growth inhibition was associated with a decrease in cell proliferation and an increase in apoptosis in MXT cancers. RIA and RT- PCR analyses showed that the concentrations of GH and IGF-I and the levels of mRNA for GH and IGF-I in MXT tumors were reduced by the therapy with GHRH antagonists. Messenger RNA for GH receptors was also decreased. In vitro, the proliferation of MXT cancer cells was strongly stimulated by GH and less effectively by IGF-I, indicating that both GH and IGF-I may act as growth factors for this mammary carcinoma. GHRH antagonist JV-1-38 inhibited the autonomous growth of MXT cells and the proliferation induced by IGF-I or GH and diminished (3)H-thymidine-incorporation stimulated by IGF-I and GH. These findings and a sustained increase in cyclin B2 concentrations in the cells shown by immunoblotting indicate that JV-1-38 causes a block at the end of the G(2) phase of cell cycle. Our results demonstrate that GHRH antagonists decrease the local production of both GH and IGF-I in MXT mouse mammary cancers, the resulting growth inhibition being the consequence of reduced cell proliferation and increased apoptosis.

Antiproliferative actions of growth hormone-releasing hormone antagonists on MiaPaCa-2 human pancreatic cancer cells involve cAMP independent pathways.

We evaluated the effects of GHRH antagonists on the proliferation of MiaPaCa-2 human pancreatic cancer cells and cAMP signaling in vitro. GHRH antagonists inhibited the proliferation of MiaPaCa-2 cells in vitro in a dose-dependent way and caused a significant elevation in cAMP production. In a superfusion system, short-term exposure of the cells to GHRH antagonists evoked an acute, dose-dependent release of cAMP into the medium. Native GHRH, which stimulates cAMP efflux from pituitary at nanomolar doses, did not influence cAMP release from cultured or superfused MiaPaCa-2 cells even at 10-30 microM. VIP, PACAP, secretin and glucagon also did not influence cell proliferation or cAMP production. Adenylate cyclase activator forskolin (FSK) caused a greater cAMP response, but a smaller antiproliferative effect than GHRH antagonists. Combined treatment with FSK and GHRH antagonist JV-1-38 potentiated the cAMP-inducing effect of FSK, but did not produce a greater inhibition of cell proliferation than JV-1-38 alone. A selective accumulation of radiolabeled GHRH antagonist [(125)I]JV-1-42 in vivo in MiaPaCa-2 carcinoma xenografted into nude mice was also observed. In conclusion, second messengers other than cAMP participate in the signal transduction pathways of GHRH analogs mediated by tumoral GHRH receptors.

Antagonists of growth hormone-releasing hormone and somatostatin analog RC-160 inhibit the growth of the OV-1063 human epithelial ovarian cancer cell line xenografted into nude mice.

The effects of antagonists of GHRH and the somatostatin analog RC-160 on the growth of OV-1063 human epithelial ovarian cancer cells xenografted into nude mice were investigated. Treatment with 20 microg/day of the GHRH antagonist JV-1-36 or MZ-5-156 and 60 microg/day of the somatostatin analog RC-160 for 25 days decreased tumor volume by 70.9% (P < 0.01), 58.3% (P < 0.05), and 60.6% (P < 0.01), respectively, vs. the control value. The levels of GH in serum were decreased in all of the treated groups, but only RC-160 significantly reduced serum insulin-like growth factor I (IGF-I). The levels of messenger ribonucleic acid (mRNA) for IGF-I and -II and for their receptors in OV-1063 tumors were investigated by multiplex RT-PCR. No expression of mRNA for IGF-I was detected, but treatment with JV-1-136 caused a 51.8% decrease (P < 0.05) in the level of mRNA for IGF-II in tumors. Exposure of OV-1063 cells cultured in vitro to GHRH, IGF-I, or IGF-II significantly (P < 0.05) stimulated cell growth, but 10(-5) mol/L JV-1-36 nearly completely inhibited (P < 0.001) OV-1063 cell proliferation. OV-1063 tumors expressed mRNA for GHRH receptors and showed the presence of binding sites for GHRH. Our results indicate that antagonistic analogs of GHRH and the somatostatin analog RC-160 inhibit the growth of epithelial ovarian cancers. The effects of RC-160 seem to be exerted more on the pituitary GH-hepatic IGF-I axis, whereas GHRH antagonists appear to reduce IGF-II production and interfere with the autocrine regulatory pathway. The antitumorigenic action of GHRH antagonists appears to be mediated by GHRH receptors found in OV-1063 tumors.

Direct action of growth hormone-releasing hormone agonist JI-38 on normal human fibroblasts: evidence from studies on cell proliferation and c-myc proto-oncogene expression.

Growth hormone-releasing hormone (GHRH) is secreted by the hypothalamus and stimulates the release of growth hormone from the pituitary. Recent studies also indicate that in addition to its neuroendocrine function, GHRH may play a direct role in the proliferation of cancer cells, acting as growth factor for various human tumors. In the present study we investigated the effects of JI-38, an agonistic analog of GHRH, on the rate of proliferation of normal human diploid dermal fibroblasts (NHF) cultured in vitro. The effects of JI-38 on the levels of mRNA for c-myc proto-oncogene were also tested. Exposure to 10(-7) M JI-38 stimulated the rate of proliferation of early passage NHF by about 100%. Exposure of NHF cells to 10(-8)-5x10(-6) M JI-38 for 24 h resulted in about 0.5-3.5 fold increase in the levels of mRNA for c-myc proto-oncogene. The ability of JI-38 to stimulate the proliferation of NHF cells was abolished in cells cultured at late passage. Continuous exposure to 10(-7) M JI-38, over 6-7 passages (15-20 population doublings), progressively reduced the rate of proliferation of NHF compared with cells exposed to medium alone, indicating that GHRH agonist acted as a growth inhibitor. Our results suggest that at certain developmental stages, GHRH may act on various tissues, stimulating cell proliferation.

Effective treatment of metastatic MDA-MB-435 human estrogen-independent breast carcinomas with a targeted cytotoxic analogue of luteinizing hormone-releasing hormone AN-207.

A highly potent derivative of doxorubicin, 2-pyrrolinodoxorubicin (AN-201), was linked to [D-Lys6]luteinizing hormone-releasing hormone (LH-RH) to form a cytotoxic analogue, AN-207, that can be targeted to LH-RH receptors. The effects of AN-207 were investigated in MDA-MB-435 human estrogen-independent breast carcinomas, which express LH-RH receptors. In experiment 1, nude mice bearing orthotopically implanted tumors received a single i.v. injection of AN-207, AN-201, or the carrier at 250 nmol/kg. Five weeks after administration of AN-207, tumor volume was significantly decreased by 66% (P < 0.001) and tumor burden by 71% (P < 0.05) as compared with controls, but no significant effects occurred in other groups. Six of eight (75%) control animals and 37.5% of mice treated with AN-201 developed metastases in the lymph nodes, whereas no lymphatic spread was found in any of the mice that received injections of AN-207. The antitumor effect of AN-207 could be partially blocked by pretreatment of the tumor-bearing mice with high doses of agonist [D-Trp6]LH-RH, which suggests that AN-207 acts on LH-RH receptors on tumors. The mortality due to toxicity was 25% in the group receiving AN-201 and 12.5% in the AN-207-treated group. Radioligand binding assays revealed the presence of high-affinity binding sites for LH-RH on tumor membranes, and mRNA for LH-RH receptors was demonstrated by reverse transcription-PCR. In experiment 2, two i.v. injections of AN-207 or AN-201 at 150 nmol/kg were given on days 0 and 28 to mice bearing orthotopic xenografts of MDA-MB-435. The outcome of the treatment was similar to that observed in experiment 1, but without any toxicity-related deaths. Tumor growth inhibition and prevention of metastatic disease suggest that cytotoxic LH-RH analogue AN-207 could be considered for the treatment of human estrogen-independent breast cancers expressing receptors for LH-RH.

Inhibition of growth of OV-1063 human epithelial ovarian cancers and c- jun and c- fos oncogene expression by bombesin antagonists.

Receptors for bombesin are present on human ovarian cancers and bombesin-like peptides could function as growth factors in this carcinoma. Therefore, we investigated the effects of bombesin/gastrin-releasing peptide (GRP) antagonists RC-3940-II and RC-3095 on the growth of human ovarian carcinoma cell line OV-1063, xenografted into nude mice. Treatment with RC-3940-II at doses of 10 microg and 20 microg per day s.c. decreased tumour volume by 60.9% (P< 0.05) and 73.5% (P< 0.05) respectively, after 25 days, compared to controls. RC-3095 at a dose of 20 microg per day reduced the volume of OV-1063 tumours by 47.7% (P = 0.15). In comparison, luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix at a dose of 100 microg per day caused a 64.2% inhibition (P< 0.05). RT-PCR analysis showed that OV-1063 tumours expressed mRNA for bombesin receptor subtypes BRS-1, BRS-2, and BRS-3. In OV-1063 cells cultured in vitro, GRP(14-27) induced the expression of mRNA for c- jun and c- fos oncogenes in a time-dependent manner. Antagonist RC-3940-II inhibited the stimulatory effect of GRP(14-27) on c- jun and c- fos in vitro. In vivo, the levels of c- jun and c- fos mRNA in OV-1063 tumours were decreased by 43% (P< 0.05) and 45% (P = 0. 05) respectively, after treatment with RC-3940-II at 20 microg per day. Exposure of OV-1063, UCI-107 and ES-2 ovarian carcinoma cells to RC-3940-II at 1 microM concentration for 24 h in vitro, extended the latency period for the development of palpable tumours in nude mice. Our results indicate that antagonists of bombesin/GRP inhibit the growth of OV-1063 ovarian cancers by mechanisms that probably involve the downregulation of c- jun and c- fos proto-oncogenes.

Antagonists of growth hormone-releasing hormone and vasoactive intestinal peptide inhibit tumor proliferation by different mechanisms: evidence from in vitro studies on human prostatic and pancreatic cancers.

Antagonists of GH-releasing hormone (GHRH) and vasoactive intestinal peptide (VIP) inhibit the proliferation of various tumors in vitro and in vivo, but a comparison of their antitumor effects and mechanisms of action has not been reported to date. We recently synthesized and characterized a series of analogs, some of which are primarily GHRH antagonists (JV-1-36, JV-1-38, and JV-1-42), whereas others are more selective for VIP receptors (VPAC-R; JV-1-50, JV-1-51, JV-1-52, and JV-1-53). LNCaP human prostatic cancer cells express VPAC-R, with predominant subtype 1 determined by RT-PCR. Our studies show that GHRH antagonists significantly inhibit the proliferation of both VPAC-R positive LNCaP cells (P < 0.001) and VPAC-R negative MiaPaCa-2 human pancreatic cancer cells cultured in vitro (P < 0.05 to P < 0.001). Growth inhibition of LNCaP cells is accompanied by a proportional reduction in prostate-specific antigen (PSA) secretion (P < 0.001). In a superfusion system, the inhibitory activities of the analogs on the rate of VIP and GHRH-induced PSA secretion correlate well with their VPAC-R binding affinities to LNCaP cell membranes. Antagonists more selective for VPAC-R display a stronger inhibition of inducible PSA release than GHRH antagonists, but have smaller effects or no effects on proliferation and PSA secretion in culture. Collectively, our findings demonstrate that the antiproliferative activity of the analogs on cancer cells is not correlated to their VPAC-R antagonistic potencies. Because GHRH antagonists inhibit the proliferation of LNCaP cells more powerfully than VPAC-R antagonists and also suppress the growth ofVPAC-R-negative MiaPaCa-2 cells, it can be concluded that their antiproliferative effect is exerted through a mechanism independent of VPAC-R.

Antagonists of growth hormone-releasing hormone arrest the growth of MDA-MB-468 estrogen-independent human breast cancers in nude mice.

Since antagonists of growth hormone-releasing hormone (GH-RH) inhibit proliferation of various tumors, in this study we investigated the effects of GH-RH antagonists MZ-5-156 or JV-1-36 on growth of estrogen-independent MDA-MB-468 human breast cancers xenografted into nude mice. Both GH-RH antagonists administered at a dose of 20 microg/day induced regression of some and growth-arrest of other tumors, while control tumors continued to grow. After 5 weeks of therapy with MZ-5-156 or JV-1-36, final volume and weight of MDA-MB-468 tumors were significantly decreased (all p values < 0.001) and serum IGF-I levels as well as tumor IGF-I mRNA expression were reduced as compared with controls. High affinity binding sites for IGF-I were detected by the ligand binding method. Gene expression of human IGF-I receptors, as measured by the RT-PCR, was not significantly different in control and treated MDA-MB-468 tumors. In cell culture, IGF-I did not stimulate, GH-RH slightly stimulated, while MZ-5-156 and JV-1-36 inhibited proliferation of MDA-MB-468 cells known to possess defective insulin and IGF-I receptor signaling. The expression of mRNA for human GH-RH was found in five of 8 tumors treated with GH-RH antagonists, and in one of the five control tumors. These results suggest that GH-RH antagonists inhibit MDA-MB-468 breast cancers possibly through mechanisms involving interference with locally produced GH-RH.

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 microg intraperitoneally or 5 microg 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 microg 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.

Inhibition of growth of human malignant glioblastoma in nude mice by antagonists of bombesin/gastrin-releasing peptide.

The effects of antagonists of bombesin/gastrin-releasing peptide (GRP) on the growth of human malignant glioblastoma cell line U-87MG xenografted into nude mice were evaluated. Nude mice bearing s.c. implanted U-87MG tumors were treated with bombesin/GRP antagonists RC-3095 and RC-3940-II. RC-3095 and RC-3940-II administered s.c. at a dose of 20 micrograms/day for 4 weeks decreased the volume of U-87MG xenografts by 60 and 74%, respectively, compared with controls. RT-PCR analysis showed that U-87MG xenografts expressed mRNA for bombesin receptor subtype (BRS)-1 (GRP receptor) and BRS-2 (neuromedin-B receptor), but the mRNA for GRP ligand was not detected in U-87MG cells suggesting that GRP may stimulate the growth of U-87MG glioblastomas by a paracrine mechanism. The levels of mRNA for c-fos oncogene were decreased by 30-40% in U-87MG tumors treated with RC-3095 or RC-3940-II. In U-373MG glioblastoma cells, which also express BRS-1, and U-87MG cells, cultured in vitro, GRP(14-27) induced the expression of c-fos mRNA, and some c-jun mRNA, in a time-dependent manner with the maximal effect occurring 2 h after the stimulation and a return to basal levels after 8 h. Antagonist RC-3940-II inhibited the stimulation of c-fos by GRP(14-27). Our results indicate that antagonists of bombesin/GRP inhibit the growth of U-87MG glioblastomas by a mechanism that may involve the downregulation of c-fos oncogene.

Growth hormone-releasing hormone: an autocrine growth factor for small cell lung carcinoma.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers in vivo. This effect is thought to be exerted through suppression of the pituitary growth hormone-hepatic insulin-like growth factor I (IGF-I) axis and direct inhibition of autocrine/paracrine production of IGF-I and -II in tumors. However, other evidence points to a direct effect of GHRH antagonists on tumor growth that may not implicate IGFs, although an involvement of GHRH in the proliferation of cancer cells has not yet been established. In the present study we investigated whether GHRH can function as an autocrine/paracrine growth factor in small cell lung carcinoma (SCLC). H-69 and H-510A SCLC lines cultured in vitro express mRNA for GHRH, which apparently is translated into peptide GHRH and then secreted by the cells, as shown by the detection of GHRH-like immunoreactivity in conditioned media from the cells cultured in vitro. In addition, the levels of GHRH-like immunoreactivity in serum from nude mice bearing H-69 xenografts were higher than in tumor-free mice. GHRH(1-29)NH(2) stimulated the proliferation of H-69 and H-510A SCLCs in vitro, and GHRH antagonist JV-1-36 inhibited it. JV-1-36 administered s.c. into nude mice bearing xenografts of H-69 SCLC reduced significantly (P < 0.05) tumor volume and weight, after 31 days of therapy, as compared with controls. Collectively, our results suggest that GHRH can function as an autocrine growth factor in SCLCs. Treatment with antagonistic analogs of GHRH may offer a new approach to the treatment of SCLC and other cancers.

Targeted cytotoxic analogue of bombesin/gastrin-releasing peptide inhibits the growth of H-69 human small-cell lung carcinoma in nude mice.

Recently, we developed a powerful cytotoxic analogue of bombesin AN-215, in which the bombesin-like carrier peptide Gln-Trp-Ala-Val-Gly-His-Leu-psi(CH2-NH)-Leu-NH2 (RC-3094) is conjugated to a potent derivative of doxorubicin, 2-pyrrolinodoxorubicin (AN-201). Small-cell lung carcinomas (SCLCs) are known to express high levels of bombesin receptors. We evaluated whether these receptors could be used for targeting cytotoxic bombesin analogue to H-69 SCLC cells. H-69 cells were xenografted into male nude mice, which then received an intravenous injection of AN-215, cytotoxic radical AN-201, the carrier peptide RC-3094 alone or unconjugated mixture of RC-3094 and AN-201. The levels of mRNA for bombesin receptor subtypes were evaluated by reverse transcription-polymerase chain reaction. In vitro, both the analogue AN-215 and the radical AN-201 showed strong antiproliferative effects on H-69 cells, AN-215 requiring more time to exert its action at 10(-8) M concentration than AN-201. In vivo, the growth of H-69 SCLC tumours was significantly inhibited by the treatment with 200 nmol kg(-1) of AN-215, while equimolar doses of the cytotoxic radical AN-201 or the mixture of AN-201 and the carrier peptide were toxic and produced only a minor tumour inhibition as compared with control groups. mRNA for bombesin receptor subtypes 2 (BRS-2) and 3 (BRS-3) was detected in H-69 tumours. The mRNA levels for BRS-3, but not for BRS-2, were lower in the AN-215-treated tumours as compared with controls. Our results demonstrate that the cytotoxic bombesin analogue AN-215 could be considered for targeted therapy of tumours, such as SCLC, that express bombesin receptors.

Targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 inhibits growth of OV-1063 human epithelial ovarian cancers in nude mice.

OBJECTIVE: The aim of the study was to investigate the effects of the cytotoxic analog of luteinizing hormone-releasing hormone AN-207 on the growth of the OV-1063 human epithelial ovarian cancers, which express luteinizing hormone-releasing hormone receptor. AN-207 consists of doxorubicin derivative 2-pyrrolinodoxorubicin (AN-201) linked with the carrier [D-lysine6 ]luteinizing hormone-releasing hormone. STUDY DESIGN: Female nude mice bearing xenografts of OV-1063 ovarian cancers were treated with analog AN-207, cytotoxic radical AN-201, or agonist [D-lysine6 ]luteinizing hormone-releasing hormone. The levels and expression of messenger ribonucleic acid of receptors for luteinizing hormone-releasing hormone and epidermal growth factor were evaluated. RESULTS: The growth of OV-1063 tumor was significantly inhibited by 3 to 5 nmol AN- 207 but not by [D-lysine6 ]luteinizing hormone-releasing hormone. Cytotoxic radical AN-201 was toxic at these doses. After treatment with AN-207 receptors for luteinizing hormone-releasing hormone were not detectable, epidermal growth factor receptor levels declined, and expressions of their respective messenger ribonucleic acids were decreased. CONCLUSIONS: Targeted cytotoxic luteinizing hormone-releasing hormone analog AN-207 is less toxic than equimolar doses of its radical 2-pyrrolinodoxorubicin and effectively inhibits ovarian tumor growth. Targeted chemotherapy may improve management of ovarian cancer.

Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro.

Antagonistic analogs of growth hormone-releasing hormone (GHRH) suppress growth of various tumors in vivo. This effect is exerted in part through inhibition of the GHRH-GH-insulin-like growth factor (IGF)-I axis. Nevertheless, because autocrine/paracrine control of proliferation by IGF-II also is a major factor in many tumors, the interference with this growth-stimulating pathway would offer another approach to tumor control. We thus investigated whether GHRH antagonists MZ-4-71 and MZ-5-156 also act on the tumor cells directly by blocking the production of IGF-II. An increase in the IGF-II concentration in the media during culture was found in 13 of 26 human cancer cell lines tested. Reverse transcription-PCR studies on 8 of these cell lines showed that they also expressed IGF-II mRNA. Antagonists of GHRH significantly inhibited the rate of proliferation of mammary (MDA-MB-468 and ZR-75-1), prostatic (PC-3 and DU-145), and pancreatic (MiaPaCa-2, SW-1990, and Capan-2) cancer cell lines as shown by colorimetric and [3H]thymidine incorporation tests and reduced the expression of IGF-II mRNA in the cells and the concentration of IGF-II secreted into the culture medium. Growth and IGF-II production of lung (H-23 and H-69) and ovarian (OV-1063) cancer cells that express mRNA for IGF-II and excrete large quantities of IGF-II also was marginally suppressed by the antagonists. These findings suggest that antagonistic analogs of GHRH can inhibit growth of certain tumors not only by inhibiting the GHRH-GH-IGF-I axis, but also by reducing the IGF-II production and by interfering with the autocrine regulatory pathway.

Expression of mRNA for luteinizing hormone-releasing hormone receptors and epidermal growth factor receptors in human cancer cell lines.

Conventional chemotherapy produces varying degrees of response in patients with many advanced cancers and has significant side effects. Receptors for luteinizing hormone-releasing hormone (LH-RH) are present in a high percentage of human ovarian, prostatic, breast and endometrial tumors and targeted chemotherapy based on cytotoxic analogs of LH-RH might yield better results. The present study was undertaken to determine whether human cancer cell lines express mRNA for LH-RH receptors and epidermal growth factor (EGF) receptors. Using radioligand binding studies, we showed the presence of high-affinity binding sites for LH-RH and EGF in the membranes of human ovarian, prostatic, breast and endometrial cancer cell lines as well as in the JAR choriocarcinoma cell line. The expression of the mRNA for LH-RH receptors and EGF receptors in these cell lines was demonstrated by RT-PCR using specific primers and by subsequent Southern blot analysis. The PCR products obtained were of the expected size, 319 bp for LH-RH receptors and 400 bp for EGF receptors. These findings support the view that cytotoxic analogs of LH-RH could be used for targeted chemotherapy of these cancers. Moreover, the results suggest that these human cancer cell lines might have local regulatory systems for their proliferation based on LH-RH and EGF. Further investigations are required to elucidate the signal transduction pathways involved in the effects of cytotoxic LH-RH analogs on human tumors.