Suppression of growth of H-69 small cell lung carcinoma by antagonists of growth hormone releasing hormone and bombesin is associated with an inhibition of protein kinase C signaling

We investigated the effects of antagonists of growth hormone-releasing hormone (GHRH) alone and in combination with bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC-3940-II on the growth of H-69 human small cell lung carcinoma (SCLC) xenografted into nude mice. Since the activation of the signaling pathways involving protein kinase C (PKC) and the subsequent steps involving mitogen-activated protein kinase (MAPK) and c-fos and c-jun oncogenes are known to be important mechanisms implicated in cellular growth, we investigated how the blockade of tumoral GHRH receptor splice variants and BN/GRP receptors by these antagonists could interfere with these intracellular signaling pathways. Treatment with GHRH antagonists JV-1-65 or MZ-J-7-110 for 4 weeks significantly (p<0.05) decreased the tumor volume by 22.7+/-3.0% and 36.7 +/- 3.6%, respectively, as compared to controls. A larger decrease in tumor volume of 73.0 +/- 9.5% (p<0.01) was produced by BN/GRP antagonist RC-3940-II and its combination with JV-I-65 caused the greatest tumor reduction of 91.0 +/- 9.8% (p<0.01) vs. controls. H-69 SCLC tumors expressed alpha-, betaII-, delta- and eta-PKC isoforms. Antagonists of GHRH and BN/GRP decreased significantly (p<0.05) the expression of betaII- and delta-, but not of alpha- and eta-PKC isoforms. They also inhibited MAPK levels, the effects being significant (p<0.05) in the groups that received BN/GRP antagonist. In addition, expression of c-fos and c-jun mRNA was reduced after combined treatment with JV-1-65 and RC-3940-II. The proliferation of H-69 SCLC cells "in vitro" was also significantly inhibited after incubation of cells with GHRH antagonist, PKC inhibitors or MAPK inhibitor. These findings suggest that the anti-proliferative effects of antagonists of GHRH and BN/GRP on H69-SCLC involve an inhibition of the signaling pathways of specific PKC isoforms, MAPK and c-fos and c-jun oncogenes.     

Alterations of EGFR/HER, angiogenesis and apoptosis pathways after therapy with antagonists of growth hormone releasing hormone and bombesin in non-small cell lung cancer

New therapeutic strategies are necessary to improve the treatment of lung cancer. We investigated the effects of bombesin/gastrin-releasing peptide (GRP) antagonist, RC-3940-II, and growth hormone-releasing hormone (GHRH) antagonists, MZ-J-7-114 and MZ-J-7-118, on the expression of epidermal growth factor receptor (EGFR)/HER (-2, -3, and -4) family, angiogenic factors, VEGF-A and VEGF receptors (VEGF-R1 and VEGF-R2), and the apoptotic molecules Bax and Bcl-2, in H-460 and A-549 non-small cell lung carcinomas (NSCLC). Nude mice bearing xenografts of H-460 and A-549 NSCLC were treated daily with these peptide analogues for 4 weeks. The treatment resulted in growth inhibition of H-460 by 22-77% and A-549 NSCLCs by 64-84%. The inhibition of tumor growth was associated with a down-regulation of members of EGFR/HER family. A significant reduction of the levels of expression of EGFR/HER family on both tumors varied from 29-96%: the greatest inhibition being induced by RC-3940-II. Similarly, a significant decrease in the levels of VEGF-A in tumors by 19-60% and VEGF receptors (VEGF-R1, 24-74% and VEGF-R2, 25-50%) was detected after therapy. An up-regulation of Bax by 21-63% and a down-regulation of Bcl-2 by 23-39% was observed only for H-460 NSCLC. Our study demonstrates that human H-460 and A-549 NSCLC, express receptors for GHRH and bombesin/GRP, and respond to the respective antagonists. The antagonists of bombesin/GRP and GHRH could provide a new strategy for treatment of NSCLC through down-regulation of EGFR/HER family and an interference with the angiogenic and apoptotic pathways.     

Secretin/vasoactive intestinal peptide-stimulated secretion of bombesin/gastrin releasing peptide from human small cell carcinoma of the lung

Bombesin/gastrin releasing peptide-like immunoreactivity (BLI) is found in the majority of small cell carcinoma of the lung (SCCL) cell lines examined. Because BLI is present in high concentration in SCCL we studied the mechanism of BLI secretion from several SCCL cell lines and in patients with SCCL. In cell line NCI-H345 the structurally related polypeptide hormones secretin, vasoactive intestinal peptide, and peptide histidine isoleucine as well as theophylline, a phosphodiesterase inhibitor, N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate, a cyclic nucleotide analogue, increased BLI release by 16-120% and cyclic adenosine 3':5'-monophosphate by 36-350%. Similar results were obtained in SCCL cell line NCI-H209. i.v. injection of secretin (2 units/kg) significantly increased plasma BLI in 2 patients with extrapulmonary SCCL. These data suggest that SCCL cells possess receptors for secretin/vasoactive intestinal peptide and that receptor occupation stimulates in vitro and in vivo BLI secretion.     

Effects of bombesin on growth of human small cell lung carcinoma in vivo

Bombesin-like peptides are found in many different human tumors and are thought to function as an autocrine growth factor for small cell lung cancer in humans. In this study, a human small cell lung carcinoma (NCI-H69) was s.c. implanted bilaterally into the flanks of 12 nude mice. The mice were randomized and divided into two groups and given either bombesin (20 micrograms/kg) or saline i.p. 3 times a day. Tumor areas were measured twice weekly for 6 wk. At sacrifice, the tumors and normal pancreas were excised, weighed, and assayed for DNA, RNA, and protein content. Significant stimulation of tumor growth was observed at weeks 4, 5, and 6. Tumor weight at sacrifice was significantly elevated (77%) above the control, as was DNA content (78%). Bombesin significantly increased the weight (42%), DNA (48%), and protein (61%) contents of the normal mouse pancreas. We conclude that bombesin may act as an autocrine growth factor, or indirectly through the release of other growth factors, on human small cell lung carcinoma.     

Psi 13,14] bombesin analogues inhibit growth of small cell lung cancerin vitro and in vivo.

Bombesin/gastrin releasing peptide (BN/GRP) functions as an autocrine growth factor in small cell lung cancer (SCLC). Previously, this autocrine growth cycle was disrupted by a monoclonal antibody which binds to the carboxyl terminal of BN and neutralizes the peptide so that it is unable to interact with the BN/GRP receptor. Here a series of BN analogues were synthesized which have a reduced peptide bond near the carboxyl terminal. The analogues inhibited specific binding of 125I-GRP to SCLC cell line NCI-H345 in a dose-dependent manner and the analogue [D-Nal6, Psi13,14, Phe14] BN6-14 was approximately 6-fold more potent than was (Psi13,14, Leu14)BN with a 50% inhibition concentration value of 5 nM. [DNal6, Psi13,14, Phe14]BN6-14 and [Psi13,14, Leu14]BN had no effect on the cytosolic Ca2+ levels but antagonized the increase in cytosolic Ca2+ caused by 10 nM BN. [Psi13,14, Leu14]BN (1 microM) inhibited the growth of SCLC in vitro using a clonogenic assay by approximately 70% Also, injection of [Psi13,14, Leu14]BN (10 micrograms, s.c.) inhibited the growth of SCLC xenografts in nude mice in vivo by approximately 50%. These data suggest that the autocrine growth cycle of BN/GRP in SCLC may also be disrupted by peptide antagonists which bind to the BN receptor.     

Amurubicinol-induced eotaxin-3 expression in human NCI-H69 small cell lung carcinoma cells

We previously demonstrated the doxorubicin-induced expression of urokinase-type plasminogen activator (uPA), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha in human RC-K8 lymphoma cells and NCI-H69 small cell lung carcinoma cells in which reactive oxygen species might be involved. Amurubicin hydrochloride (AMR), a novel derivative drug of doxorubicin, was recently introduced to clinical practice for treatment of lung cancer in Japan. Therefore, we investigated the effects of AMR on the expression of uPA and chemokines in NCI-H69 cells. AMR and its active form, amurubicinol hydrochloride (AMROH), both induced the expression of uPA, IL-8 and MCP-1 in H69 cells in a dose-dependent manner. When the cultured supernatant obtained from AMR-treated H69 cells was subcutaneously injected into rabbits, migration of a significant number of eosinophils was observed around the injected site. Antigen levels of eotaxin-3, a major migration-factor of eosinophils, were increased in AMROH-treated cells in parallel with the mRNA levels. The induction was observed below the clinically achievable concentration of AMR or AMROH. Thus, the simultaneous induction of uPA, IL-8, MCP-1 and eotaxin-3 may play a role in the pharmacological action of AMR through induction of the interaction between proinflammatory cells and lung carcinoma cells.     

Antitumoral activity of bombesin analogues on small cell lung cancer xenografts: relationship with bombesin receptor expression.

Gastrin releasing peptide (GRP), the human homologue of bombesin (BN), is an autocrine growth factor for small cell lung cancer (SCLC) cells. The synthetic octapeptides [D-cpa1-beta-Leu8-des-Met9]litorin (BIM 26182) and [D-Phe6-Leu13-CH2NH-Cpa14]bombesin(6-14)NH2 (BIM 26189) are potent GRP/BN antagonists of the proliferation of 3T3 and rat pancreas cells. The effect of these analogues on the proliferation of four SCLC cell lines (SCLC 6, SCLC 41, SCLC 75, SCLC 74R) was tested in vitro and in vivo. Two of these SCLC lines (SCLC 41M and SCLC 75) had receptors for BN/GRP and expressed the prepro-GRP mRNA. BIM 26182 and BIM 26189 inhibited [3H]thymidine incorporation into the DNA of SCLC 41 cells, stimulated [3H]thymidine incorporation in SCLC 6, and had no effect on the two other cell lines. The SCLC implanted s.c. in nude mice were treated with either BIM 26182 or BIM 26189. BIM 26182 and BIM 26189 injected at the doses of 50 micrograms twice a day (s.c.) around the tumor for 10 to 21 days delayed the growth of SCLC 41 and of SCLC 75. The maximal effect was observed during the treatment period, after which the tumors regrew, suggesting a cytostatic effect of these peptides. No inhibitory effect of the peptides on SCLC 74R or SCLC 6 growth was observed. These data suggest that GRP antagonists are able to inhibit the in vitro and in vivo growth of BN/GRP receptors-positive SCLC.     

Molecular evidence for the lack of epidermal growth factor receptor gene expression in small cell lung carcinoma cells

It has been shown that none of the small cell lung carcinoma (SCLC) cell lines possess epidermal growth factor (EGF) binding activity on their surface. We have examined several SCLC cell lines for the possibility that they may have EGF receptors but that the receptors are masked by an EGF-like protein factor(s), which may be produced by an autocrine mechanism. No evidence, however, was found for the production of such factors. We then used an EGF receptor complementary DNA to determine the state of the EGF receptor gene by Southern blot analysis. The receptor gene appears to be present in these cells in an intact, unrearranged form. These cells, however, were found to lack detectable levels of EGF receptor mRNA, suggesting a possible reason for the absence of EGF receptors on the cell surface. Furthermore, karyotype analysis revealed that SCLC cell lines Lu134 and H69 contained a morphologically normal chromosome 7, which carries the EGF receptor gene. Also, these SCLC cells contained the apparently normal chromosome 3 and exhibited the presence of c-raf-1 gene in an unrearranged form. Thus, the previously noted partial deletion of chromosome 3 is not necessarily common to the SCLC cells. Instead, the lack of EGF receptor is frequently found in SCLC cell lines and is distinct from the other types of lung cancer. We postulate that SCLC cells have some active regulatory mechanism which prevents the expression of EGF receptor gene.     

Inverse relationship of epidermal growth factor receptor and HER2/neu gene expression in human renal cell carcinoma

Expression of the oncogenes, epidermal growth factor (EGF) receptor, HER2/neu, c-myc, and c-fos, in renal cell carcinoma and corresponding nonneoplastic kidney tissue of 30 patients has been analyzed by Northern blot analysis. In renal cell carcinoma an inverse relationship of EGF receptor and HER2/neu gene expression was detected, with high expression of the EGF receptor gene in 22 of 30 (73%) cases and low expression of the HER2/neu gene in 28 of 30 (93%) cases. Furthermore, altered expression of the oncogenes c-myc and c-fos was detected in renal cell carcinoma, which appears to be related to the tumor grade of malignancy. Additional Southern blot analysis of six renal cell carcinomas gave no indication of chromosomal rearrangement events or gene amplification.     

Antagonists of bombesin/gastrin-releasing Peptide inhibit growth of jar human choriocarcinoma cells and production of cyclic-amp in-vitro

The effects of bombesin/GRP antagonists RC-3095 and RC-3940-II on the in vitro proliferation of JAR human choriocarcinoma cells were evaluated. Antagonists RC-3095 and RC-3940-II effectively inhibited growth of cultured JAR cells, inducing a dose- and time-dependent decrease in the number of treated cells. RC-3940-II was more potent than RC-3095 in inhibiting the growth of JAR cells. Addition of RC-3940-II to JAR cell cultures significantly inhibited the cell proliferation at concentrations as low as 1 nM, while 10 nM RC-3095 was required for a similar effect. Receptor binding studies demonstrated the presence of a single class of binding sites for bombesin on JAR cells. RC-3940-II displaced [I-125]Tyr(4)-bombesin bound to the receptors. When JAR cells were cultured in the presence of 10 nM RC-3095 or RC-3940-II for 72 h, cAMP levels in the incubation medium were decreased by 70-80%, compared to the controls. These results suggest that bombesin/GRP antagonists RC-3095 and RC-3940-II inhibit the proliferation of JAR human chorionic adenocarcinoma cells in vitro and that these effects may involve intracellular cAMP pathway.     

M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion

The importance of acetylcholine as a neurotransmitter in the nervous system is well established, but little is yet known about its recently described role as an autocrine and paracrine hormone in a wide variety of nonneuronal cells. Consistent with the expression of acetylcholine in normal lung, small cell lung carcinoma (SCLC) synthesize and secrete acetylcholine, which acts as an autocrine growth factor through both nicotinic and muscarinic cholinergic mechanisms. The purpose of this study was to determine if interruption of autocrine muscarinic cholinergic signaling has potential to inhibit SCLC growth. Muscarinic receptor (mAChR) agonists caused concentration-dependent increases in intracellular calcium and mitogen-activated protein kinase (MAPK) and Akt phosphorylation in SCLC cell lines. The inhibitory potency of mAChR subtype-selective antagonists and small interfering RNAs (siRNAs) on acetylcholine-increased intracellular calcium and MAPK and Akt phosphorylation was consistent with mediation by M3 mAChR (M3R). Consistent with autocrine acetylcholine secretion stimulating MAPK and Akt phosphorylation, M3R antagonists and M3R siRNAs alone also caused a decrease in basal levels of MAPK and Akt phosphorylation in SCLC cell lines. Treatment of SCLC cells with M3R antagonists inhibited cell growth both in vitro and in vivo and also decreased MAPK phosphorylation in tumors in nude mice in vivo. Immunohistochemical staining of SCLC and additional cancer types showed frequent coexpression of acetylcholine and M3R. These findings suggest that M3R antagonists may be useful adjuvants for treatment of SCLC and, potentially, other cancers.     

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.     

Effects of bombesin antagonists on the growth of small cell lung cancer cells in vitro

Small cell lung cancer (SCLC) produces several neuroendocrine peptides, including gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin. There is some evidence to support the suggestion that GRP is an autocrine regulator of SCLC growth. Therefore, we have tested the effect of bombesin and two antagonists of bombesin on SCLC cell growth in a serum-free liquid tissue culture system. The antagonists used were analogues of substance P: spantide and (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P. The cell lines used in this study all produced GRP-related peptides and one line had demonstrable GRP receptors. Exogenous bombesin did not cause any stimulation of growth in the liquid culture assay. The bombesin antagonists inhibited SCLC cell growth, but apparently not via the bombesin receptor. The bombesin used was biologically active because it stimulated the proliferation of Swiss 3T3 fibroblasts. The antagonists caused inhibition of this bombesin-induced proliferation, which was reversed by addition of excess bombesin. In addition, the antagonists and substance P alone stimulated proliferation of 3T3 cells, indicating that they may interact with another growth factor receptor on 3T3 cells. We conclude that growth of SCLC cells is not dependent on bombesin under all in vitro culture conditions because bombesin failed to stimulate growth in liquid cultures and the growth inhibition caused by bombesin antagonists was probably not mediated by the bombesin receptor.     

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.     

HER family receptor and ligand status in thymic carcinoma

Overexpression and gene amplification of the HER family of receptors and their ligands are important prognostic factors in many solid tumors and treatment targeting these molecules has recently become available. The role of this group of receptors has only rarely been described in thymic epithelial neoplasms and never before in a series of cases consisting exclusively of thymic carcinoma. Twenty-four primary squamous cell carcinomas of the thymus were examined for immunohistochemical expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR (pEGFR), HER2, phosphorylated HER2 (pHER2), HER3, phosphorylated HER3 (pHER3) and their ligands epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin and epiregulin. Fluorescence in situ hybridization (FISH) analysis for amplification of the EGFR and HER2 genes was performed including assessment of the copy numbers of EGFR and HER2 gene per cell and the ratio of EGFR and HER2 to centromere 7 and 17, respectively. Significant immunohistochemical expression was observed for EGFR (33.3%), pEGFR (33.3%), HER2 (58.3%), HER3 (45.8%), TGF-α (54.1%), amphiregulin (25.0%) and epiregulin (91.7%). A single case showed HER2 gene amplification by FISH. Increased EGFR and HER2 gene copy numbers were observed in 2 (8.4%) and 18 cases (75%), respectively. Eight cases (33.3%) showed an increased HER2:CEP17 ratio. The results of this study indicate that EGFR and HER2 amplification is a rare event in thymic carcinoma, however, protein expression for HER receptors as well as their ligands is a common finding indicating that targeted therapy directed against these molecules may be considered in the treatment of these tumors.