Lh-rh and its antagonist cetrorelix inhibit growth of jar human choriocarcinoma cells in-vitro

The effects of luteinizing hormone-releasing hormone (LH-RH), and LH-RH antagonist Cetrorelix, (SB-75, [Ac-D-Nal(2)(1),D-Phe(4-Cl)(2),D-Pal(3)(3),D-Cit(6),D-Ala(10)]LH-RH) on cell growth and the production of hCG and cAMP in JAR human choriocarcinoma cells were examined in vitro. Both LH-RH and its antagonist SE-75, at 1 mu g concentration, inhibited the growth of JAR cells in cultures. When SE-75 (1 mu M) was given in combination with different doses (0.1 nM to 1 mu M) of LH-RH, it was found that 0.1 nM LH-RH nullified the inhibitory effect of SE-75 on cell growth, however, the 100 nM and 1 mu M doses of LH-RH caused a greater inhibition of cell proliferation than SE-75 alone. Incubation with LH-RH slightly increased the hCG production and the cAMP release in the cultured tumor cells. SE-75 alone or in combination with LH-RH reduced the hCG as well as the cAMP release from JAR human choriocarcinoma cells; however, the magnitude of the decrease was smaller for hCG than for cAMP. The effect of different doses of LH-RH, administered simultaneously with 1 mu M SE-75, on the cAMP production, was similar to that on cell growth: 0.1 nM LH-RH in combination with 1 mu M SE-75 caused a smaller inhibition of cAMP than SE-75 alone. However, when LH-RH was given at concentrations from 1 nM to 1 mu M together with 1 mu M SE-75, we observed a greater inhibition of cAMP than after SE-75 alone. The presence of low affinity LH-RH receptors on JAR cells was also demonstrated and competitive binding studies showed that agonist D-Trp(6)-LH-RH and antagonist SE-75 could bind to these receptors. Our findings provide new information on the effect of LH-RH and antagonist SE-75 on the proliferation of JAR human choriocarcinoma cells and may offer a new insight on their mechanisms of action in the suppression of tumor cell growth and their influence on intracellular signal transduction pathways. Hormonal therapy based on Cetrorelix could be considered for the development of new approaches to treatment of patients with choriocarcinomas.     

Antagonists of bombesin/gastrin-releasing peptide inhibit growth of SW-1990 human pancreatic adenocarcinoma and production of cyclic AMP

We investigated the effects of bombesin/GRP antagonists RC-3095 and RC-3940-II on the growth of SW-1990 human pancreatic adenocarcinoma cells xenografted into nude mice or cultured in vitro. Nude mice implanted with SW-1990 tumors received s.c. injections of RC-3095 and RC-3940-II or the vehicle (control) for 28 days. Chronic administration of RC-3940-II inhibited the growth of SW-1990 tumors, as shown by a reduction in tumor volume during the treatment and a significant increase in tumor doubling time. RC-3940-II decreased final tumor volume by 57.7% and tumor growth rate by 65%. Final tumor weights in mice treated with RC-3940-II were 75% lower than in controls. Treatment with RC-3095 induced smaller, and not significant, decreases in tumor volume and weight. In cell cultures, both RC-3095 and RC-3940-II effectively inhibited the proliferation of SW-1990 cells, inducing a dose- and time-dependent decrease in the number of cells. RC-3940-II again suppressed in vitro growth of SW-1990 cells more effectively than RC-3095. After 72 hr of culture, RC-3940-II and RC-3095 at I μM concentrations decreased cell numbers by 45.7% and 27.7%, respectively. The estimated EC₅₀ value for RC-3940-II was I nM. When SW-1990 cells were cultured in the presence of I nM and 10 nM RC-3095 for 72 hr, cAMP levels in the incubation medium were decreased to 77.3% and 26.9% of the control value. Our results indicate that bombesin/GRP antagonist RC-3940-II can inhibit the proliferation of SW-1990 human pancreatic adenocarcinoma cells in vivo and in vitro. Our findings also suggest that this effect may involve the intracellular cAMP pathway.     

Stimulation by bombesin and inhibition by bombesin/gastrin-releasing peptide antagonist RC-3095 of growth of human breast cancer cell lines.

Recently, it was reported that bombesin/gastrin-releasing peptide (GRP) have mitogenic effects on some human breast cancer cell lines. In this study, we investigated the effects of bombesin/GRP and its receptor antagonist (RC-3095) on the proliferation of three breast cancer cell lines, MDA-MB-231, MCF-7 MIII, and MCF-7. Stimulation by bombesin and inhibition by RC-3095 of cell growth were found in MDA-MB-231 and MCF-7 MIII cells cultured in phenol red-free medium with 5% heat-inactivated and dextran-coated charcoal-treated fetal bovine serum (DCC-FBS). A stimulatory effect by bombesin was not observed in the presence of untreated FBS. [3H]Thymidine incorporation into DNA in these cells was suppressed by RC-3095. MCF-7 cells failed to respond to bombesin and RC-3095 in the presence of either FBS or DCC-FBS. GRP-like immunoreactivity was found in the cell extracts and FBS, but it was undetectable in DCC-FBS. It appears that the stimulatory effect of bombesin on cell proliferation of MCF-7 MIII and MDA-MB-231 cell lines could be obtained because of reduction in the levels of some serum factors in DCC-FBS. These results suggest that bombesin/GRP can act as growth factors through bombesin/GRP receptors in some human breast cancers.     

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.     

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.     

Systematic development of bombesin/gastrin-releasing peptide antagonists.

Several families of very potent bombesin (Bn) receptor antagonist analogues have recently been developed and their biological potencies evaluated in a number of in vitro systems including guinea pig and rat pancreatic acini and Swiss 3T3 cells. These studies showed that analogues can exhibit diverse properties ranging from full antagonists, partial agonists, or full agonists depending on the assay system and animal species employed. We have developed two classes of more potent, shorter chain antagonists based on [psi CH2NH(13-14)]Bn(6-14) and desMet14Bn(6-13)NH2 structures. [D-Phe6 psi Leu13-Leu14] Bn(6-14)NH2 was a potent antagonist (Ki 6nM) in Swiss 3T3 cells and guinea pig acini but exhibited 10% partial agonist activity and lower binding affinity (Ki 60 nM) in rat acini. The partial agonism could be eliminated by using p-Cl-Phe or D-Phe at the C-terminus and partially eliminated using D-4-Cl-Phe in position 6. With the antagonist [D-Phe6]Bn(6-13)NH2 (Ki 96 nM), alkyl substituents on the amide group increased affinity 25-fold with the propylamide being the most potent peptide (Ki 4 nM) in 3T3 cells or guinea pig acini. It did, however, have high 40% partial agonist activity in rat acini. Alkyl esters or hydrazide derivatives were, in contrast, pure antagonists in all systems tested with [D-Phe6]Bn(6-13)OMe having the highest affinity in all systems and also excellent in vivo properties. All of the potent antagonists examined had little affinity for neuromedin B--preferring bombesin receptors, which had entirely new ligand structure-activity relationships.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a bombesin/gastrin-releasing peptide receptor on a human gastric-cancer cell line

This study examined the expression of receptors of the bombesin (BBS) family in human gastric-cancer cell lines. Of 5 cell lines screened, only one, St42, demonstrated specific binding sites for ¹²⁵I-Tyr⁴-BBS, which have been further characterized. This binding was saturable, and temperature- and time-dependent. Scatchard analysis of displacement data performed at 37°C revealed 2 binding sites: a high-affinity, low-capacity site (K_D = 0.13 nM, B_max = 1500 sites/cell) and a lower-affinity, higher-capacity site (K_D = 11 nM, B_max = 35,000 sites/cell); the latter was lost when internalization of peptide was prevented, suggesting that it may be an artefact. Displacement assays with gastrin-releasing peptide (GRP) and neuromedin B (NMB) revealed that the receptor was of the GRP-preferring sub-type (GRP IC₅₀ = 0.35 nM; NMB IC₅₀ = 112 nM). Co-valent cross-linking of ¹²⁵I-Tyr⁴-BBS to the receptor demonstrated the presence of a single band corresponding to a molecular weight of 37 to 44 kDa on SDS-PAGE, similar to that of the cloned GRP receptor protein core. G-protein linkage of this receptor was demonstrated by selective inhibition of ¹²⁵I-Tyr⁴-BBS binding by guanosine nucleotides. The binding of BBS to the receptor resulted in a rise in intracellular calcium. Three of four structurally distinct BBS antagonists bound to the receptor with high affinity, but [DPhe¹², Leu¹⁴]-bombesin did not cause any displacement of ¹²⁵I-Tyr⁴-BBS even at 10 mM. The functional significance of GRP receptors on human gastric-cancer cells is as yet unknown, but further studies may determine whether such receptors have importance in the therapy of gastric cancer.     

Antagonists of growth hormone releasing hormone and bombesin inhibit the expression of EGF/HER receptor family in H-69 small cell lung carcinoma

Effects of in vivo treatment with antagonists of growth hormone-releasing hormone (GHRH), JV-1-65 and MZ-J-7-110, and bombesin/gastrin-releasing peptide antagonist RC-3940-II, on the EGF receptor (EGFR) family, were investigated in H-69 SCLC. Tumors were analyzed by RT-PCR, immunoblotting and binding assays. Treatment with these analogs reduced the binding capacity of EGFR by 18-64%, and inhibited the mRNA expression for EGFR, HER-2 and -3 by 27-75.4, 17-26.3, and 13.8-46.6%, respectively. The antagonists also decreased the protein levels for EGFR by 21-34%, HER-2 by 36-68% and HER-3 by 43-49%. This is the first demonstration that antiproliferative effects of GHRH antagonists are associated with a downregulation of EGF/HER receptors.     

Selective stimulation of small cell lung cancer clonal growth by bombesin and gastrin-releasing peptide

Human small cell lung cancers (SCLC) produce and secrete the regulatory peptide bombesin (BN) or its mammalian counterpart gastrin-releasing peptide (GRP). In addition, several SCLC tumor lines have been shown to express high affinity receptors for BN/GRP. On the basis of these findings, we investigated the effect of exogenously added BN and GRP on the soft agarose colony growth of a panel of human cell lines. In serum-free defined medium, colony formation of 9 of 10 SCLC cell lines was stimulated up to 150-fold by BN or GRP, with peak colony stimulation observed at 50 nM BN. In contrast, no stimulatory effect of BN was observed on nine non-SCLC cell lines. Although no stimulation of colony growth by BN was seen in serum-supplemented medium, addition of BN to the serum-free medium increased cloning efficiency to that achieved by serum in most of the SCLC cell lines. GRP 1-27, the active mammalian analogue of Bn, stimulated colony growth of SCLC cells similar to the manner of BN, while the physiologically inactive BN analogue, des-Leu (13)-Met (14)-BN, had no effect on colony growth. No correlation was observed in SCLC cell lines between the response of these cells to exogenous BN and the amount of cellular BN/GRP produced or the presence of BN receptors. These data suggest that BN/GRP may in some instances function as an autocrine growth factor for SCLC and indicate new ways for modulating SCLC growth in patients with this tumor.     

New antagonists of bombesin gastrin-releasing Peptide with C-terminal leu-psi-(ch2n)tac-nh2

Several new pseudononapeptide bombesin/GRP analogs containing C-terminal Leu Psi(CH2N)Tac-NH2 with variations at the N-terminus, corresponding to position 6 of bombesin, have been synthesized in order to develop more potent Bn antagonists for the hormonal therapy of cancers. The biological activities of the new compounds were evaluated in vitro by investigating their ability to inhibit the binding of [I-125-Tyr(4)]Bn and to suppress the GRP(14-27)-stimulated DNA synthesis in quiescent Swiss 3T3 cells. All compounds investigated inhibited the binding of [I-125-Tyr(4)]Bn, suppressed the GRP(14-27)-induced proliferation of Swiss 3T3 cells in a dose-dependent manner and proved to act as Bn antagonists without agonistic activity. Two of the newly synthesized pseudononapeptides [Hca(6), Leu(13)Psi(CH2N)-Tac(14)]Bn(6-14) (RC-3940-II) and [D-Nal(6), Leu(13)Psi (CH2N)Tac(14)]Bn(6-14) (RC-3965-II) exhibited higher binding affinities to Swiss 3T3 cells than the Bn/GRP antagonist RC-3095 and the recently developed compound [D-Phe(6), Leu(13)Psi(CH2N) Tac(14)]Bn(6-14) (RC-3950-II). RC-3940-II caused 50% inhibition of the specific binding of [I-125-Tyr(4)]Bn to Swiss 3T3 cells at concentrations less than 1 pM and suppressed by 50% the GRP(14-27)-induced proliferation of Swiss 3T3 cells at doses one order of magnitude lower than RC-3095. This study demonstrates the importance of the nature of the N-terminus in addition to the C-terminal Leu Psi(CH2N)Tac-NH. The elimination of the free amino group in the aromatic residue in position 6 appears to increase the antagonistic activity. These findings suggest the merit of further investigations of this class of Bn/GRP antagonists for their antitumor activities in various cancers.     

端粒酶RNA反义寡核苷酸对人绒毛膜癌裸鼠移植瘤的生长抑制作用

目的探讨端粒酶RNA反义寡聚核苷酸(anti-hTR)对绒癌的治疗作用。方法采用绒癌JAR细胞移植裸鼠成瘤,行anti-hTR的低浓度和高浓度治疗,并设立生理盐水组、随机序列组及药物放线菌素D(Act-D)对照组,动态观察并测定肿瘤的生长。采用TRAP-ELISA方法测定端粒酶活性。采用Western blot法测定hTERT蛋白的表达。结果anti-hTR低浓度组、高浓度组和药物Act-D组的抑瘤率分别为76．6％、93．8％和85．4％,三组对肿瘤生长的抑制,与随机序列组和生理盐水组相比,差异均有统计学意义,而三组之间差异无统计学意义。三组的端粒酶活性及hTERT蛋白表达下降,较随机序列组和生理盐水组相比,差异均有统计学意义,而三组之间差异无统计学意义。结论anti-hTR能够抑制绒癌移植瘤的生长,可能成为肿瘤治疗的新方法。     

Expression of matrix metalloproteinase-9 and bombesin/gastrin-releasing peptide in human prostate cancers and their lymph node metastases

Neuroendocrine differentiation and subsequent excretion of neuropeptides have been demonstrated to be associated with progression of human prostate cancer. Among neuropeptides found to exist in the prostate, bombesin/gastrin-releasing peptide has been shown to upregulate matrix metalloproteinase-9 (MMP-9) in human prostate cancer cell lines. Expression levels of bombesin, MMP-9, and neuron-specific enolase were examined by immunohistochemistry in 41 cases of clinically organ-confined prostate cancers including 9 with microscopic lymph node metastases. Twenty-seven (64%) of the 41 radical prostatectomy specimens were positive for both MMP-9 and bombesin. Expression of these molecules was observed in almost the same population of the cancer cells. The remaining 14 cases were negative for both MMP-9 and bombesin. High-grade tumors (Gleason sum ≥7) were more likely to express MMP-9 and bombesin (21/24 : 88%) than low-grade tumors (Gleason sum ≥6) (7/17 : 41%). In eight of the nine cases with pathological lymph node metastases, expression of MMP-9 and bombesin was also noted in metastatic sites. Neuron-specific enolase was positive in 16 cases (39%) and not always associated with the expression of bombesin. Expression of bombesin and expression of MMP-9 are common in human prostate cancers and may be related to an aggressive phenotype.     

Antagonists of growth hormone-releasing hormone inhibit the growth of U-87MG human glioblastoma in nude mice

Antagonists of growth hormone-releasing hormone(GH-RH)inhibit the growth of various cancers by mechanisms that involve the suppression of the insulin-like growth factor (IGF)-I and/or IGF-II. In view of the importance of the IGF system in glioma tumorigenesis, the effects of GH-RH antagonists MZ-5-156 and JV-1-36 were investigated in nude mice bearing subcutaneous and orthotopic xenografts of U-87MG human glioblastomas. After 4 weeks of therapy with MZ-5-156 or JV-1 -36 at the dose of 20 microg/day per animal, the final volume of subcutaneous U-87MG tumors was significantly (P < .01) decreased by 84% and 76%, respectively, as compared with controls. Treatment with GH-RH antagonists also reduced tumor weight and the levels of mRNA for IGF receptor type I (IGFR-I). A reduction in the mRNA levels for IGF-II was found in tumors of mice treated with MZ-5-156. Treatment with MZ-5-156 or JV-1 -36 also extended the survival of nude mice implanted orthotopically with U-87MG glioblastomas by 81% (P < .005) and 18%, respectively, as compared with the controls. Exposure in vitro to GH-RH antagonists MZ-5-156 or JV-1 -36 at 1 microM concentration for 24 hours decreased the tumorigenicity of U-87MG cells in nude mice by 10% to 30% and extended the latency period for the development of subcutaneous palpable tumors by 31% to 56%, as compared with the controls. Exposure of U-87MG cells to GH-RH antagonists in vitro also resulted in a time-dependent increase in the mRNA levels of IGFR-II or a decrease in the mRNA levels of IGFR-I. mRNA for GH-RH was detected in U-87MG cells and xenografts implying that GH-RH may play a role in the pathogenesis of this tumor. Our results suggest that GH-RH antagonists MZ-5-156 and JV-1-36 inhibit the growth of U-87MG human glioblastoma by mechanisms that involve the suppression of IGF system. Antagonistic analogs of GH-RH merit further development for the treatment of malignant glioblastoma.     

Effects of somatostatin analogue RC-160 and bombesin/gastrin-releasing peptide antagonists on the growth of human small-cell and non-small-cell lung carcinomas in nude mice.

We investigated the effects of our synthetic bombesin/gastrin-releasing peptide (GRP) antagonists and somatostatin analogue RC-160 on the growth of human small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (non-SCLC) lines in nude mice. Athymic nude mice bearing xenografts of the SCLC NCl-H69 line or non-SCLC NCl-H157 line were treated for 5 and 4 weeks, respectively, with somatostatin analogue RC-160 or various bombesin/GRP antagonists. RC-160, administered s.c. peritumorally at a dose of 100 micrograms per animal per day, inhibited the growth of H69 SCLC xenografts as shown by more than 70% reduction in tumour volumes and weights, as compared with the control group. Bombesin/GRP antagonists, RC-3440, RC-3095 and RC-3950-II, given s.c. peritumorally at a dose of 20 micrograms per animal per day, also inhibited the growth of H69 SCLC tumours. RC-3950-II had the greatest inhibitory effect and decreased tumour volume and weights by more than 80%. The growth of H-157 non-SCLC xenografts was significantly reduced by treatment with RC-160, but not with bombesin/GRP antagonist RC-3095. In mice bearing either tumour model, administration of RC-160 significantly decreased serum growth hormone and gastrin levels. Specific high-affinity receptors for bombesin and somatostatin were found on membranes of SCLC H69 tumours, but not on non-SCLC H157 tumours. Receptor analyses demonstrated high-affinity binding sites for epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the membranes of H69 and H157 tumours. EGF receptors were down-regulated on H69 tumours after treatment with RC-160 and bombesin/GRP antagonists. The concentration of binding sites for EGF and IGF-I on the H157 tumours was decreased after treatment with RC-160, but bombesin/GRP antagonist RC-3095 had no effect. These results demonstrate that bombesin/GRP antagonists inhibit the growth of H-69 SCLC, but not of H-157 non-SCLC xenografts in nude mice, whereas somatostatin analogue RC-160 is effective in both tumour models. This raises the possibility that these peptide analogues could be used selectively in the treatment of various subclasses of lung cancer.     

肺癌细胞和蛙皮素抑制树突状细胞的产生和功能

目的：了解肺癌细胞株及蛙皮素对树突状细胞（DC）产生及功能的影响。方法：树突状细胞由健康人外周血单个核细胞CD14^ ，在完全细胞培养液中加入1000U／ml GM-CSF和1000U／ml IL-4,培养7天获得，肺癌细胞株CRL－5815，CRL－5826，Bombesin和Bombesin受体拮抗剂加入培养液中，Annxin V检测DC凋亡；流式细胞仪检测CD40，CD86，CD83，CD80，HLA－DR阳性表达。结果：培养7－10天后的DC前体细胞表达高水平的HLA－DR CD80 CD86 CD83 CD40。肺癌及蛙皮素可导致DC前体细胞凋亡，而蛙皮素受体拮抗剂可部分保护蛙皮素致DC凋亡的作用。加入肺癌细胞株或蛙皮素与DC共培养时明显抑制上述细胞表型的表达和DC刺激同种异体T细胞的增殖能力，当加入蛙皮素受体拮抗剂时，DC细胞表达HLA－DR CD80 CD86 CD83 CD40明显增加，接近DC正常对照组。结论：肺癌细胞株及蛙皮素可导致DC的凋亡，抑制树突状细胞的产生和功能。     

Effect of bombesin,gastrin-releasing peptide (GRP)(14–27) and bombesin/GRP receptor antagonist RC-3095 on growth of nitrosamine-induced pancreatic cancers in hamsters

Female Syrian golden hamsters with N-nitroso-bis (2-oxopropyl) amine (BOP)-induced pancreatic cancers were treated for 2 months with bombesin/gastrin-releasing peptide (GRP) antagonist D-Tpi⁶, Leu¹³Ψ(CH₂NH)Leu¹⁴ bombesin(6-14) (RC-3095). Bombesin and GRP(14–27) were also administered alone and in combination with the antagonist RC-3095. RC-3095 exerted a dose-dependent inhibitory effect on growth of pancreatic cancers. The number of animals with pancreatic cancers was significantly lower in the group treated with 60 μg/day of RC-3095 and the weight of tumorous pancreata was reduced. Administration of bombesin or GRP alone did not stimulate the growth of pancreatic tumors and, in fact, had a slightly suppressive effect on cancers which was significant only in Experiment I. Bombesin and GRP (14–27) given together with RC-3095 did not nullify the inhibitory effect of the antagonist on pancreatic cancer growth. Actually, a greater inhibition of pancreatic tumors was observed after administration of RC-3095 together with bombesin or GRP, than with RC-3095 alone. The mechanism of action of bombesin, GRP, and bombesin antagonists on pancreatic cancers appears to be complex. The inhibitory effect of bombesin antagonists on pancreatic cancer growth was accompanied by a decrease in the binding capacity of EGF receptors in tumor membranes. Administration of bombesin also caused a down-regulation of EGF receptors and the greatest decrease in binding capacity of EGF receptors was observed after treatment with RC-3095 in combination with GRP. Inhibition of pancreatic cancer can thus be tentatively explained by some common pathways in the action of bombesin, GRP and their antagonists, that could be mediated by interference with EGF-receptor mechanisms.     

Verotoxins inhibit the growth of and induce apoptosis in human astrocytoma cells

Verotoxin 1 (VT1) is an E. coli toxin comprising an A subunit with N-glycanase activity, and five smaller B subunits capable of binding to the functional receptor globotriaosylceramide (Gal1-4-Gal1-4-Glcceramide-Gb3). VT is implicated in hemorrhagic colitis and the more serious hemolytic uremic syndrome. VT1 is active against various tumor cell lines in vitro and in vivo. To extend the anti-cancer spectrum of activity of VT to human brain tumors, in the present analysis we studied the effects of VT on the growth of 6 permanent human astrocytoma cell lines. All astrocytoma cell lines analyzed express Gb3 and were sensitive to VT-1 at a dose of 50 ng/ml, but sensitivity was not proportional to the relative Gb3 concentration. VT induced apoptosis in these cells was shown by electron microscopy. Morphological evidence (nuclear shrinkage and chromatin condensation) of apoptosis could be clearly distinguished 1.5 hrs after toxin addition. Ultrastructural preservation of organelles was observed in conjunction with blebbing of the plasma membrane, condensation of chromatin within the nucleus and nuclear shrinkage. Apoptosis was also induced by the recombinant toxin B subunit alone, suggesting that the ligation of Gb3 is the primary induction mechanism. These studies indicate that verotoxin/Gb3 targetting may provide a novel basis for the inhibition of astrocytoma tumour cell growth.