Characterization of VIP- and helodermin-preferring receptors on human small cell lung carcinoma cell lines

We investigated the molecular and pharmacologic characteristics of VIP receptors on two human SCLC cell lines: NCI-N592 and NCI-H345. With NCI-N592 cell, the order of potency of VIP-related peptides in inhibiting ¹²⁵I-VIP binding and in stimulating cAMP production was typical of the human VIP receptor. By covalent cross-linking, a polypeptide of Mr 62,300 was obtained. Conversely, the behavior of NCI-H345 cell line was totally different: helodermin was the most potent peptide, VIP and PHI were equipotent, while hGRF and secretin were totally ineffective. These results suggest that NCI-N592 cells possess a typical VIP receptor while NCI-H345 cells possess a helodermin-preferring receptor, and that the natural target of helodermin might not be the VIP receptor.     

Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.     

Modulation of bombesin-induced phosphatidylinositol hydrolysis in a small-cell lung-cancer cell line.

Bombesin is an amphibian tetradecapeptide whose mammalian homologue, gastrin-releasing peptide (GRP), is produced by many small-cell lung-cancer (SCLC) cells, and which can function in an autocrine growth-promoting manner in SCLC. Studies reported here show that [Tyr4]bombesin and its congeners increase inositol 1,4,5-trisphosphate within seconds in NCI-H345, a SCLC cell line that constitutively produces GRP. After 30 min in the presence of 0.01 M-Li+ and [Tyr4]bombesin, there is marked accumulation of inositol monophosphates and inositol tetrakisphosphate. Pretreatment with phorbol 12-myristate 13-acetate (PMA) for 20 min inhibited the ability of [Tyr4]bombesin to induce phosphatidylinositol (PtdIns) turnover and to increase intracellular free Ca2+ ([Ca2+]i). Pretreatment with PMA for 48 h attenuated the ability of subsequently added PMA to decrease the response to [Tyr4]bombesin. Pretreatment with pertussis toxin (PT; 1 microgram/ml for 18-24 h) decreased by less than 30% [Tyr4]bombesin-induced increases in [Ca2+]i and PtdIns metabolites. However, interpretation of this result is complicated by the inability of PT to ADP-ribosylate completely its substrates in intact NCI-H345 cells. In contrast, pretreatment with cholera toxin (1 microgram/ml for 18-24 h) lowered basal [Ca2+]i and basal inositol phosphate concentrations, attenuated the response of NCI-H345 to subsequently added [Tyr4]bombesin, and was not mimicked by treatments that increase cellular cyclic AMP. These data demonstrate the activation of phospholipase C in SCLC by bombesin congeners. In addition, the results suggest a regulatory role for protein kinase C, a cholera-toxin substrate, and perhaps a pertussis-toxin substrate in the response of SCLC to bombesin.     

High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer

The binding of a radiolabeled bombesin analogue to human small cell lung cancer (SCLC) cell lines was investigated. (125I-Tyr4)bombesin bound with high affinity (Kd = 0.5 nM) to a single class of sites (2,000/cell) using SCLC line NCI-H446. Binding was reversible, saturable and specific. The pharmacology of binding was investigated using NCI-H466 and SCLC line NCI-H345. Bombesin and structurally related peptides, such as gastrin releasing peptide (GRP), but not other peptides, such as substance P or vasopressin, inhibited high affinity (125I-Tyr4)BN binding activity. Finally, the putative receptor, a 78,000 dalton polypeptide, was identified by purifying radiolabeled cell lysates on bombesin or GRP affinity resins and then displaying the bound polypeptides on sodium dodecylsulfate polyacrylamide gels. Because SCLC both produces bombesin/GRP-like peptides and contains high affinity receptors for these peptides, they may function as important autocrine regulatory factors for human SCLC.     

Synthesis of peptide and pseudopeptide amides inhibiting the proliferation of small cell and epithelial types of lung carcinoma cells

Small cell lung cancer (SCLC) cell lines produce and secrete various peptide hormones, e.g. bombesin (BN)/gastrin releasing peptide (GRP) like peptides that are proposed to function as their autocrine growth factors. To inhibit the proliferative effect of these hormones we have synthesized short chain BN[7-14]-analogues replacing the C-terminal peptide bond by a methylene-amino (-CH₂NH-) unit and introducing d -Phe or d -Ser into position 12. As several substance P (SP) analogues were found to inhibit the growth of SCLC cells, some short chain SP-analogues have been synthesized. (Pseudo)octapeptides were synthesized in solution, by fragment condensation using the DCC/HOPfp method. Fragments and SP-analogues were synthesized stepwise using pentafluorophenyl esters. The resistance to hydrolysis of the reduced peptide bond made permitted exact quantification of the Leuψ(CH₂NH)Leu pseudopeptide in hydrolysates. The binding ability of both types of peptides to BN-receptors on Swiss 3T3 mouse fibroblast cells and their antiproliferative effect on NCI-H69 human SCLC cell line have been tested and compared with a short chain SP-antagonist pHOPA-d -Trp-Phe-d -Trp-Leu-Leu-NH₂ ( R ) previously described as a potent inhibitor of SCLC proliferation. While BN-analogues showed weak activity in inhibition of proliferation of SCLC cells, SP-analogues 6 : d -MePhe-d-T rp-Phe-d -Trp-Leuψ(CH₂NH)-Leu-NH₂ and 7 : d -MePhe-d -Trp-Phe-d -Trp-Leu-MPA, in spite of greatly diminished affinity towards the BN-receptor, inhibited SCLC proliferation more effectively than R ( 6 : IC₅₀=2 μm , 7 : IC₅₀=5 μm and R : IC₅₀=10 μm ). Moreover, 6 inhibited the respiratory activity of SK-MES 1 epithelial type of lung carcinoma cells in proliferating but not in the quiescent state, suggesting that the antiproliferative effect of these compounds is not due to simple cytotoxicity. These short chain analogues of SP might be promising candidates as therapeutic agents in the treatment of SCLC. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

SR48692 is a neurotensin receptor antagonist which inhibits the growth of small cell lung cancer cells

Neurotensin (NT) is an autocrine growth factor for some small cell lung cancer (SCLC) cells. In this communication, the effects of a non-peptide NT receptor antagonist, SR48692, were investigated using SCLC cells. (3)H-SR48692 bound with high affinity (IC(50) = 20 nM) to NCI-H209 cells. Also, NT and SR48692 inhibited specific (125)I-NT binding with high affinity (IC(50) values of 2 and 200 nM). In contrast, the NT(2) receptor agonist, levocabastine, had little effect on specific (125)I-NT binding, second messenger production and proliferation using NCI-H209 cells. SR48692 (5 microM) antagonized the ability of NT (10 nM) to cause elevated cytosolic Ca2+ in Fura-2 AM loaded NCI-H209 cells. SR48692 antagonized the ability of NT to cause elevation of c-fos mRNA in these cells. Using a MTT proliferation assay, SR48692 inhibited NCI-H209 and H345 proliferation in a concentration-dependent manner. Using a clonogenic assay, 1 microM SR48692, reduced NCI-H209 colony number. Also, SR48692 (0.4 mg/kg per day) inhibited NCI-H209 xenograft proliferation in nude mice. These results suggest that SR48692 is a NT(1) receptor antagonist which inhibits SCLC growth.     

Metabolic stability and tumor inhibition of bombesin/GRP receptor antagonists.

Small cell lung cancers (SCLC) synthesize and secrete bombesin/gastrin releasing peptide (BN/GRP). The autocrine growth cycle of BN/GRP in SCLC can be disrupted by BN/GRP receptor antagonists such as [Psi13,14]BN. Here several BN analogues were solid-phase synthesized and incubated with intact SCLC cells at 37 degrees C in RPMI medium in a time-course fashion (0-1080 minutes) to determine enzymatic stability. The proteolytic stability of the compounds was determined by subsequent HPLC analysis. The metabolic half-life ranged from 154 minutes to 1388 minutes for the six analogues studied. [Psi13,14]BN was found to be very stable to metabolic enzymes (T1/2 = 646 mm) and also inhibited SCLC xenograft formation in vivo in a dose-dependent manner. When [Psi13,14]BN was incubated with NCI-H345 cells, it inhibited 125I-GRP binding with an IC50 value of 30 nM. These data suggest that BN/GRP receptor antagonists such as [Psi13,14]BN may be useful for the treatment of SCLC.     

Homologous and heterologous regulation of the helodermin/vasoactive-intestinal-peptide response in the murine radiation leukemia-virus-induced lymphoma cell line BL/VL3

1. Functional vasoactive intestinal peptide (VIP)/helodermin receptors and beta 2-adrenoceptors coexist in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus (see preceding paper in this journal). 2. Short-term (5-30 min) exposures of BL/VL3 cells to VIP or isoproterenol induced both homologous and heterologous desensitization. The potency of VIP and isoproterenol to desensitize was similar to their potency to occupy receptors and activate adenylate cyclase. 3. Long-term (16-h) exposure of BL/VL3 cells to VIP induced homologous down regulation only, whereas isoproterenol induced both homologous and heterologous down regulation. The potency of VIP, peptide histidine isoleucinamide, helodermin, helospectin, and [D-Phe2]VIP on the one hand, and of isoproterenol on the other hand, to decrease homologous responses was comparable to their potency for receptor occupancy and adenylate cyclase activation.     

Octapeptide analogues of somatostatin inhibit the clonal growth and vasoactive intestinal peptide-stimulated cyclic AMP formation in human small cell lung cancer cells.

Two endocrinologically active octapeptide analogues (BIM-23014 C and BIM-23034) of somatostatin (SRIF) containing either an N- or C-terminal 3-(2-naphthyl)-D-Ala residue were examined for their ability to inhibit the in vitro receptor binding, clonal growth, and vasoactive intestinal peptide (VIP)-stimulated cyclic AMP formation in human small cell lung cancer cell (SCLC) line NCI-H345. Both SRIF peptides inhibited [125I]SRIF(Tyr11)-14 binding with IC50 values in the low nM range. Colony formation in the in vitro SCLC growth assay was also inhibited in the same concentration range, as was VIP-stimulated cyclic AMP formation. Therefore, octapeptide analogues of SRIF function as SCLC SRIF receptor agonists.     

Specific labelling by [125I]helodermin of high-affinity VIP receptors in rat liver membranes

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.     

Is helodermin produced by medullary thyroid carcinoma cells and normal C-cells? Immunocytochemical evidence

Helodermin is a VIP/secretin-like 35-amino acid peptide originally isolated from the venom of the lizard Gila monster. Recently, helodermin-immunoreactive material was demonstrated in mammalian salivary glands, brain and gut. In the present study 8 human medullary thyroid carcinomas as well as 4 normal thyroid glands were examined immunocytochemically for the presence of helodermin using an antiserum raised against helodermin-(5-35) that does not cross-react with VIP or secretin. Cells displaying helodermin-like immunoreactivity were found in all tumours examined except one. On the whole the helodermin-immunoreactive cells had the same distribution as those storing calcitonin, suggesting coexistence of the two peptides in most of the tumour cells. Also normal human C-cells displayed helodermin immunoreactivity. The results suggest that a peptide chemically related to helodermin is a constituent of human medullary thyroid carcinoma cells as well as of normal C-cells.     

Activation of muscarinic acetylcholine receptors inhibits cell cycle progression of small cell lung carcinoma.

We previously reported that activation of muscarinic acetylcholine receptors (mAChR) of M3 subtype causes hydrolysis of phosphoinositides and inhibits voltage-gated Ca2+ channel activity in small cell lung carcinoma (SCLC) cells. We now report that mAChR activation causes exponentially growing SCLC cells to arrest in S and G2/M phases of the cell cycle, concomitant with a decrease in DNA synthesis. Cell cycle progression and DNA synthesis resume when mAChR are down-regulated. In serum-starved SCLC cells, mAChR activation inhibits DNA synthesis induced by serum, bombesin, insulin, or insulin-like growth factor-I. The finding that DNA synthesis is inhibited even when mAChR are activated after exposure of cells to growth factors indicates that decreased signal transduction by growth factor receptors is not the mechanism of mAChR-mediated growth inhibition. Our data suggest that mAChR activation disrupts a common event that is induced by different growth factors and is fundamental for cell cycle progression.     

Muscarinic signaling in carcinoma cells

We previously reported that activation of M(3) muscarinic acetylcholine receptors (mAChR) generates anti-proliferative signals and stimulates cadherin-mediated adhesion in the SCC-9 small cell lung carcinoma (SCLC) cell line. The current study was undertaken to determine the frequency of functional mAChR expression among different SCLC cell lines, and to test the ability of mAChR to generate anti-proliferative signals in different SCLC cell lines. The potential role of Rac1 in SCLC cell-cell adhesion was also investigated. Exposure to the mAChR agonist carbachol induces robust Ca(2+) mobilization (indicated by intracellular fluorescence of the Ca(2+)-binding dye Indo-1) in three SCLC cell lines (SCC-9, SCC-15, and NCI-H146), modest Ca(2+) mobilization in one SCLC cell line (NCI-H209), and no detectable Ca(2+) mobilization in two SCLC cell lines (SCC-18 and NCI-H82). The M(3) mAChR-selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibits Ca(2+) mobilization in all SCLC cell lines responding to carbachol. Incubation with carbachol for four hours significantly inhibits [3H]thymidine uptake in three of the four SCLC cell lines expressing functional mAChR (SCC-9, SCC-15, and NCI-H146 cells), but does not significantly alter [3H]thymidine uptake in the other SCLC cell lines examined. These results indicate that SCLC cell lines often express functional mAChR which elicit anti-proliferative signals when activated. To investigate the role of Rac1 in SCLC adhesion, SCC-9 cells were transiently transfected with cDNA constructs coding for Rac1, constitutively active Rac1(Val-12), or dominant negative Rac1(Asn-17) tagged to green fluorescent protein (GFP). SCC-9 cells expressing GFP-tagged constitutively active Rac1(Val-12) exhibit increased cell-cell adhesion in comparison to cells expressing GFP-Rac1 or GFP-Rac1(Asn-17). Constitutively active GFP-Rac1(Val-12), but not GFP-Rac1 or GFP-Rac1(Asn-17), accumulates at cell-cell junctions in SCC-9 cells. These results indicate that activated Rac1 increases SCLC cell-cell adhesion, consistent with the possibility that Rac1 activation contributes to increased SCLC cell-cell adhesion induced by mAChR stimulation. These findings indicate that activation of mAChR may play a significant role in regulating the proliferation and adhesion of SCLC cells. The demonstration by other investigators that acetylcholine is expressed by a variety of cells in the airways supports the possibility that acetylcholine may activate mAChR expressed by SCLC cells in primary tumors.     

BW1023U90: A new GRP receptor antagonist for small-cell lung cancer cells

Gastrin-releasing peptide (GRP) receptor antagonists were synthesized and their ability to interact with small-cell lung cancer (SCLC) cells determined. [¹²⁵I]BW1023U90, bound with high affinity (K_d = 2 nM) to a single class of sites (Bmax = 55 fmol/mg protein) using SCLC cell line NCI-H345. [¹²⁵I]BW1023U90 binding was time dependent and reversible even at 37°C as the ligand was minimally internalized. Specific [¹²⁵I]BW1023U90 binding was inhibited with high affinity by GRP as well as bombesin (BB) but not neuromedin B (NMB). BW1023U90 inhibited the ability of BB to elevate cytosolic Ca²⁺ and increase the growth of SCLC cells. A BW1023U90 analogue, BW2258U89 (10 μg/day, SC) slowed SCLC xenograft formation in nude mice and [¹²⁵I]BW1023U90 localized to SCLC tumors 1 h after injection into nude mice. BW2258U89 (4% by weight) was placed in microspheres and slowly released over a 3-week period in nude mice bearing SCLC xenografts. The microspheres containing BW2258U89 strongly inhibited SCLC growth in vivo. A radioimmunoassay was developed for the GRP receptor antagonists and the rabbit antiserum cross-reacted totally with BW2258U89 or BW1023U90. BW2258U89 immunoreactivity (5 nM) was detected in the plasma of nude mice containing the microspheres after 1 week. These data suggest that GRP receptor antagonists bind to receptors on SCLC tumors.     

Small cell lung cancer bombesin receptors are antagonized by reduced peptide bond analogues

The potency of 3 reduced peptide bond analogues of bombesin (BN) was investigated using small cell lung cancer (SCLC) cell lines. (Psi13,14, Leu14)BN, (Psi9,10, Leu14)BN and (Psi12,13, Leu14)BN inhibited specific binding of 125I-GRP with IC50 values of 15, 90, and 600 nM. (Psi13,14, Leu14)BN and (Psi9,10, Leu14)BN did not elevate cytosolic Ca2+ levels but antagonized the increase in cytosolic Ca2+ caused by BN. (Psi13,14, Leu14)BN antagonized the clonal growth of SCLC cells caused by BN. These data indicate that reduced peptide bond analogues may disrupt the autocrine growth cycle of SCLC cells by functioning as BN receptor antagonists.     

Properties of vasoactive-intestinal-peptide receptors and beta-adrenoceptors in the murine radiation leukemia-virus-induced lymphoma cell line BL/VL3

1. Based on radioligand binding and adenylate cyclase activation, functional receptors to vasoactive intestinal peptide(VIP)/helodermin, were shown to coexist with beta 2-adrenoceptors and prostaglandin receptors in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus. 2. The relative potency of VIP-related peptides to stimulate adenylate cyclase activity was: helodermin greater than VIP greater than peptide histidine isoleucinamide. Five VIP analogs inhibited 125I-iodo-VIP binding and stimulated adenylate cyclase activity, their decreasing order of potency being: VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-Ala4]VIP = [D-His1]VIP = [D-Phe2]VIP. [D-Phe2]VIP acted as a partial agonist (with an intrinsic activity of 0.1 as compared to that of VIP = 1.0) and competitively inhibited helodermin- and VIP-stimulated adenylate cyclase activity with a similar Ki (0.07-0.10 microM). These data suggest the existence, in this murine T-cell lymphoma, of VIP receptors of the 'helodermin-preferring' subtype that are coupled to adenylate cyclase.     

Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-2J

We characterized highly selective receptors for PACAP, the pituitary adenylate cyclase activating peptide, in the tumoral acinar cell line AR 4-2J derived from the rat pancreas. PACAP, a novel hypothalamic peptide related to vasoactive intestinal peptide (VIP), was tested as the full natural 38-residue peptide (PACAP-38) and as an N-terminal amidated 27-residue derivative (PACAP-27). The binding sites showed considerable affinity for [125I]PACAP-27 (Kd = 0.4 nM) and PACAP-38, while their affinity for VIP and the parent peptide helodermin was 1000-fold lower. These receptors were coupled to adenylate cyclase, the potency of PACAP-38 and PACAP-27 (Kact = 0.2 nM) being much higher than that of VIP (Kact = 100 nM) and helodermin (Kact = 30 nM). Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed a specifically cross-linked peptide with an Mr of 68,000 (including 3000 for one PACAP-27 molecule).     

Sensitization of pulmonary chemosensitive neurons by bombesin-like peptides in rats

Small cell lung cancer (SCLC) patients suffer from pulmonary stresses such as dyspnea and chest pain, and the pathogenic mechanisms are not known. SCLC cells secrete a variety of bioactive neuropeptides, including bombesin-like peptides. We hypothesize that these peptides may enhance the sensitivity of the pulmonary chemosensitive nerve endings, contributing to the development of these pulmonary stresses in SCLC patients. This study was therefore carried out to determine the effects of bombesin and gastrin-releasing peptide (GRP), a major bombesin-like peptide, on the sensitivities of pulmonary chemoreflex and isolated pulmonary vagal chemosensitive neurons. In anesthetized, spontaneously breathing rats, intravenous infusion of bombesin or GRP significantly amplified the pulmonary chemoreflex responses to chemical stimulants such as capsaicin and ATP. The enhanced responses were completely abolished by perineural capsaicin treatment of both cervical vagi, suggesting the involvement of pulmonary C-fiber afferents. In isolated pulmonary vagal chemosensitive neurons, pretreatment with bombesin or GRP potentiated the capsaicin-induced Ca(2+) transient. This sensitizing effect was further demonstrated in patch-clamp recording studies; the sensitivities of these neurons to both chemical (capsaicin and ATP) and electrical stimuli were significantly enhanced by the presence of either bombesin or GRP. In summary, our results have demonstrated that bombesin and GRP upregulate the pulmonary chemoreflex sensitivity in vivo and the excitability of isolated pulmonary chemosensitive neurons in vitro.     

Secretory actions of vasoactive intestinal polypeptide, peptide histidine isoleucine and helodermin in rat small intestine: the effects of putative VIP antagonists upon VIP-induced ion secretion

Vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), and helodermin stimulate electrogenic anion secretion in preparations of rat jejunum stripped of muscularis propria. Concentration-response curves to exogenously applied peptides yielded EC50 values of 12 nM, 12 nM and 100 nM for VIP, PHI and helodermin respectively. These secretory responses were most probably mediated via the same receptor population given that cross-desensitisation was observed between all 3 analogues. Four putative VIP antagonists, namely, two growth hormone releasing factors (GRF); [AcTyr1, D-Phe2]GRF-(1-29)-NH2 and [AcTyr1]hGRF-(1-40)-OH as well as [4Cl-D-Phe6,Leu17]VIP and VIP-(10-28) were tested for their ability to inhibit VIP induced electrogenic ion secretion. None of the above exhibited any intrinsic agonist activity nor were they competitive antagonists, although some inhibition was observed with [AcTyr1]hGRF-(1-40)-OH and VIP-(10-28). Their use as selective VIP antagonists is therefore limited in rat jejunal mucosa.