High levels of intracellular bombesin characterize human small-cell lung carcinoma.

"Small cells" or "oat cells" characterize a virulent form of lung cancer and share many biochemical properties with peptide-secreting neurones. The neuropeptide bombesin is present in all small-cell lines examined, but not in other lung cancer cell lines, suggesting that bombesinergic precursor cells in lung may give rise to this disease.

Modulation of angiogenesis by dithiolethione-modified NSAIDs and valproic acid.

BACKGROUND AND PURPOSE: Angiogenesis involves multiple signaling pathways that must be considered when developing agents to modulate pathological angiogenesis. Because both cyclooxygenase inhibitors and dithioles have demonstrated anti-angiogenic properties, we investigated the activities of a new class of anti-inflammatory drugs containing dithiolethione moieties (S-NSAIDs) and S-valproate. EXPERIMENTAL APPROACH: Anti-angiogenic activities of S-NSAIDS, S-valproate, and the respective parent compounds were assessed using umbilical vein endothelial cells, muscle and tumor tissue explant angiogenesis assays, and developmental angiogenesis in Fli:EGFP transgenic zebrafish embryos. KEY RESULTS: Dithiolethione derivatives of diclofenac, valproate, and sulindac inhibited endothelial cell proliferation and induced Ser(78) phosphorylation of hsp27, a known molecular target of anti-angiogenic signaling. The parent drugs lacked this activity, but dithiolethiones were active at comparable concentrations. Although dithiolethiones can potentially release hydrogen sulphide, NaSH did not reproduce some activities of the S-NSAIDs, indicating that the dithioles regulate angiogenesis through mechanisms other than release of H(2)S. In contrast to the parent drugs, S-NSAIDs, S-valproate, NaSH, and dithiolethiones were potent inhibitors of angiogenic responses in muscle and HT29 tumor explants assessed by 3-dimensional collagen matrix assays. Dithiolethiones and valproic acid were also potent inhibitors of developmental angiogenesis in zebrafish embryos, but the S-NSAIDs, remarkably, lacked this activity. CONCLUSIONS AND IMPLICATION: S-NSAIDs and S-valproate have potent anti-angiogenic activities mediated by their dithiole moieties. The novel properties of S-NSAIDs and S-valproate to inhibit pathological versus developmental angiogenesis suggest that these agents may have a role in cancer treatment.

Neuropeptides: brain messengers of many faces.

Neuropeptides 2001, 2nd Joint Meeting of the European Neuropeptide Club and the American Summer Neuropeptide Conference (11th Annual Meeting). 6-11 May 2001 with Satellite Symposium, Israeli-French Symposium, Israel Ministry of Science, Culture and Sport, 6 May 2001, held at Maale Hachmicha and Tel Aviv University, Israel.

Peptide hormones and lung cancer.

Several peptide hormones have been identified which alter the proliferation of lung cancer. Small cell lung cancer (SCLC), which is a neuroendocrine cancer, produces and secretes gastrin releasing peptide (GRP), neurotensin (NT) and adrenomedullin (AM) as autocrine growth factors. GRP, NT and AM bind to G-protein coupled receptors causing phosphatidylinositol turnover or elevated cAMP in SCLC cells. Addition of GRP, NT or AM to SCLC cells causes altered expression of nuclear oncogenes, such as c-fos, and stimulation of growth. Antagonists have been developed for GRP, NT and AM receptors which function as cytostatic agents and inhibit SCLC growth. Growth factor antagonists, such as the NT1 receptor antagonist SR48692, facilitate the ability of chemotherapeutic drugs to kill lung cancer cells. It remains to be determined if GRP, NT and AM receptors will served as molecular targets, for development of new therapies for the treatment of SCLC patients. Non-small cell lung cancer (NSCLC) cells also have a high density of GRP, NT, AM and epidermal growth factor (EGF) receptors. Several NSCLC patients with EGF receptor mutations respond to gefitinib, a tyrosine kinase inhibitor. Gefitinib relieves NSCLC symptoms, maintaining stable disease in patients who are not eligible for systemic chemotherapy. It is important to develop new therapeutic approaches using translational research techniques for the treatment of lung cancer patients.

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.

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.

Thymosin alpha 1 down-regulates the growth of human non-small cell lung cancer cells in vitro and in vivo.

The effect of thymosin alpha 1 (THN alpha 1) and its NH2-terminal fragment (THN1-14) and COOH-terminal fragment (THN15-28) on non-small cell lung cancer (NSCLC) growth was evaluated. Using an anti-THN alpha 1 antibody, receptors were identified on NSCLC cells that were pretreated with 10(-6) M THN alpha 1. [3H]Arachidonic acid was readily taken up by NSCLC cells and THN alpha 1 significantly increased the rate of arachidonic acid release. THN1-15 slightly stimulated but THN15-28 and THN beta 4 did not alter arachidonic acid release from NCI-H1299 cells. In clonogenic growth assays in vitro, THN alpha 1 (10(-6) M) significantly decreased NSCLC colony number whereas THN1-14, THN15-28, and THN beta 4 were less potent. Using growth assays in vivo, THN alpha 1 (10 micrograms s.c./day) but not THN1-14, THN15-28, or THN beta 4 inhibited significantly NSCLC xenograft formation in nude mice. These data suggest that biologically active THN alpha 1 receptors are present on NSCLC cells and that native THN alpha 1 inhibits the growth of human NSCLC.

Neuromedin B is present in lung cancer cell lines.

Previously, high levels of gastrin-releasing peptide and its mRNA were detected in classic small cell lung cancer cell lines. Here the ability of lung cancer cell lines to synthesize neuromedin B (NMB), a structurally similar mammalian bombesin-like peptide, was investigated. By radioimmunoassay, NMB (0.1-0.7 pmol/mg of protein) was detected in 23 of 33 lung cancer cell lines. In contrast, gastrin-releasing peptide (0.1-12.9 pmol/mg of protein) was detected in 16 of 32 cell lines. Using gel filtration and high pressure liquid chromatography techniques, the main peak of immunoreactive NMB coeluted with synthetic NMB. By Northern analysis, a 0.8-kilobase mRNA species was present, using poly(A) mRNA derived from two of three lung cancer cell lines. Using a more sensitive S1 nuclease protection assay, NMB mRNA was present in most of the 15 lung cancer cell lines examined. These data suggest that NMB may be a regulatory peptide in lung cancer.

[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.

Synthesis and pharmacological characterization of 4-[125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide: a high affinity sigma receptor ligand for potential imaging of breast cancer.

The synthesis of [125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide (4-[125I]BP), a novel radiopharmaceutical that possesses high affinity for both sigma-1 and sigma-2 receptor subtypes, and its binding characteristics to MCF-7 breast cancer cells are described. To obtain high yields (with high specific activity) of radioiodinated ligand, (N-benzylpiperidin-4-yl)-4-tri-butylstannyl benzamide was synthesized. Radiolabeled 4-[125I]BP was prepared from tri-butylstannyl precursor with the use of chloramine-T or hydrogen peroxide as an oxidizing agent in high yields (71-86%). The competition binding studies of 4-[125I]BP in MCF-7 breast tumor cells with haloperidol and DTG (known sigma ligands) showed a dose-dependent displacement and high affinity binding (Ki = 4.6 and 56 nM, respectively), demonstrating that sigma receptors are expressed in MCF-7 breast tumor cells. Scatchard analysis of 4-[125I]BP binding in MCF-7 cells revealed saturable binding, with a Kd = 26 nM and a Bmax = 4000 fmol/mg protein. Furthermore, the Scatchard analysis of [3H]DTG binding in MCF-7 cells gave a Kd of 24.5 nM and a Bmax of 2071 fmol/mg of protein. The biodistribution and clearance of 4-[125I]BP was studied in rats. The radiopharmaceutical cleared quickly from the blood pool but rather slowly from the hepatobiliary system. The in vivo specificity was demonstrated by blocking the receptor binding in the presence of haloperidol. A decrease of 55, 63, 43, and 68% was found at 1 h postinjection in brain, kidney, heart, and lung, respectively. These results demonstrate that a high density of sigma receptors are expressed in MCF-7 cells and that radioiodinated 4-IBP may be useful for imaging breast cancer by targeting sigma sites.

Pituitary adenylate cyclase activating peptide receptors regulate the growth of non-small cell lung cancer cells.

We have identified pituitary adenylate cyclase activating peptide (PACAP) receptors on small cell lung cancer cell line NCI-N417 in a previous study. In this study, the role of PACAP in the growth and signal transduction of non-small cell lung cancer cells was investigated. Northern blot analysis with a full-length human PACAP receptor cDNA probe revealed a major 7.5-kb hybridizing transcript when total RNA extracted from NCI-H838 cells was used. PACAP bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 14,000/cell) when NCI-H838 cells were used. Specific 125I-labeled PACAP binding was inhibited with high affinity by PACAP-27 and PACAP-38, with moderate affinity by PACAP(6-38), and with low affinity by vasoactive intestinal polypeptide, PACAP(28-38), and PACAP(16-38). PACAP-27 elevated cAMP in a dose-dependent manner, and the increase in cAMP caused by PACAP was reversed by PACAP(6-38). PACAP-27, but not vasoactive intestinal polypeptide, elevated cytosolic Ca2+ in individual NCI-H838 cells. PACAP-27 stimulated arachidonic acid release, and the increase caused by PACAP was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in NCI-H838 cells, whereas the PACAP antagonist PACAP(6-38) reduced colony formation in the absence or presence of exogenous PACAP-27. In nude mice bearing NCI-H838 xenografts, PACAP(6-38) slowed tumor growth significantly. These data suggest that biologically active type 1 PACAP receptors are present on human non-small cell lung cancer cells, which exhibit dual signal transduction pathways and regulate cell proliferation.

Breast cancer growth is inhibited by vasoactive intestinal peptide (VIP) hybrid, a synthetic VIP receptor antagonist.

Breast cancer vasoactive intestinal peptide (VIP) receptors were characterized. Using in vitro autoradiographic techniques, 125I-labeled VIP bound with high affinity to breast biopsy sections. 125I-labeled VIP bound specifically to give breast cancer cell lines examined using receptor-binding techniques. Specific 125I-labeled VIP binding to MDA-MB-231 cells was inhibited with high affinity by VIP and pituitary adenylate cyclase-activating polypeptide (IC50, = 2 nM) and with moderate affinity by the VIP hybrid (IC50 = 0.5 microM). VIP elevated the cAMP in a dose-dependent manner, and VIP hybrid (10 microM) inhibited the increase in cAMP caused by VIP. Using Northern blot analysis, VIP (10 nM) stimulated c-fos and c-myc mRNA, and the increase caused by VIP was reversed by the VIP hybrid. The VIP hybrid inhibited breast cancer growth in vitro and in vivo using nude mice bearing breast cancer xenografts. These data suggest that the VIP hybrid is a breast cancer VIP receptor antagonist.

Establishment and identification of small cell lung cancer cell lines having classic and variant features.

Using a chemically defined medium containing hydrocortisone, insulin, transferrin, 17 beta-estradiol and selenium, with or without serum supplementation (2.5% v/v), continuous cell lines can be established from 72% of all fresh biopsy specimens of small cell lung cancer (SCLC) containing tumor cells. No differences were observed in the rate of establishing cell lines from newly diagnosed untreated patients, or from patients who have relapsed from prior therapy, or from a variety of different organ sites. Biochemical characterization of 50 SCLC cell lines for the expression of L-dopa decarboxylase; bombesin-like immunoreactivity; neuron-specific enolase, and the brain isozyme of creatine kinase, revealed that SCLC cell lines can be subdivided into two distinct classes: classic SCLC cell lines (35 lines), which express elevated levels of all four biomarkers; and variant SCLC cell lines (15 lines) which have undetectable levels of L-dopa-decarboxylase and bombesin-like immunoreactivity, but continue to express neuron-specific enolase and the brain isozyme of creatine kinase. The presence of the latter two markers distinguishes variant lines fron non-SCLC cell lines. In addition, four distinct classes were identified morphologically. The biomedical differences among established SCLC cell lines may account for the differences in response rates to cytotoxic therapy observed in newly diagnosed SCLC patients. A prospective study of biomarker characterization of SCLC tumors will determine if clinical differences exist between classic and variant SCLC tumors.

The gastrin-releasing peptide receptor is differentially coupled to adenylate cyclase and phospholipase C in different tissues.

Recent studies suggest that in some tissues GRP receptor activation can both stimulate phospholipase C and the adenylate cyclase pathway and that activation of the latter pathway may be important in mediating some of its well-described growth effects. However, other studies suggest GRP-R may not be coupled to adenylate cyclase. To investigate this possibility, in the present study we determined the coupling of the GRP receptors to each pathway in mouse, rat, and guinea pig pancreatic acini and compared it to that in mouse Swiss 3T3 cells and human SCLC cells, all of which possess well-characterized GRP receptors. Moreover, we tested the effect of PKC activation on the ability of GRP-related peptides to increase cAMP accumulation in these tissues. Changes in cAMP levels were determined with or without IBMX present, with or without forskolin, or both to amplify small increases in cAMP. In mouse, rat and guinea pig pancreatic acini, murine Swiss 3T3 cells and human SCLC cells, GRP-related peptides caused a 600%, 500%, 250%, 300% and 60% increase, respectively, in [3H]IP with 1-3 nM causing a half-maximal effect. In murine Swiss 3T3 cells, IBMX, forskolin, and IBMX plus forskolin caused a 300%, 3500% and 10500% increase in cAMP, respectively. GRP-related peptides and VIP caused an additional 70% increase in cAMP with GRP causing a half-maximal (EC50) increase in cAMP at 2.1 +/- 0.5 nM, which was not significantly different from the EC50 of 3.1 +/- 0.9 nM for increasing [3H]IP in these cells. GRP-related peptides did not stimulate increases in cAMP in mouse, rat or guinea pig pancreatic acini or in SCLC cells either alone, with IBMX or forskolin or both. However, in pancreatic acini IBMX, forskolin or both increased cAMP 3 to 8-, 10 to 500-, and 100 to 1000-fold increase and the addition of VIP caused an additional 20-, 2-, and 3-fold increase in cAMP in the different species. In mouse pancreatic acini with TPA alone or IBMX plus TPA, neither bombesin nor GRP increased cAMP. Furthermore, in mouse pancreatic acini, neither TPA nor TPA plus IBMX altered basal or VIP-stimulated increases in cAMP. In mouse Swiss 3T3 cells TPA significantly increased cAMP stimulated by Bn, GRP or VIP. These results demonstrated that GRP receptor activation in normal tissues from three different species and a human tumoral cell line do not result in adenylate cyclase activation, whereas in Swiss 3T3 cells it causes such activation. The results suggest that the difference in coupling to adenylate cyclase is likely at least partially due to a difference in coupling to an adenylate cyclase subtype whose activation is regulated by PKC. Therefore, the possible growth effects mediated by this receptor in different embryonic or tumoral cells through activation of adenylate cyclase are not likely to be an important intracellular pathway for these effects in normal tissues.

Regulation of insulin secretion and blood glucose metabolism by adrenomedullin.

Adrenomedullin (AM), a recently discovered hypotensive peptide, is expressed in the endocrine pancreas of different species, as demonstrated by immunocytochemistry. Electron microscopic studies with double immunogold showed colocalization of AM and pancreatic polypeptide. A homogeneous expression of AM receptor was found throughout the islet using in situ hybridization. Six different insulin- producing cell lines have been analyzed by reverse transcription-PCR and showed expression of both AM and its receptor. Two experimental models have been used to study the effects of AM in pancreatic physiology. 1) Analysis of isolated rat islets shows that AM inhibits insulin secretion in a dose-dependent manner. The monoclonal antibody MoAb-G6, which neutralizes AM bioactivity, was able to increase insulin release 5-fold; this effect was reversed by the addition of synthetic AM. 2) Oral glucose tolerance tests showed that iv injection of AM reduces the levels of insulin in the bloodstream with a concomitant increase in circulating glucose. These studies implicate AM as a newly defined factor of the insulin regulatory system that could be involved in disorders such as diabetes and obesity.

Expression of adrenomedullin and its receptor in normal and malignant human skin: a potential pluripotent role in the integument.

Adrenomedullin (AM) is a multifunctional peptide involved in a variety of physiological functions, including growth regulation and antimicrobial activity. We have determined by immunohistochemistry and in situ hybridization that AM and its receptor are present in all the epithelial cells of the normal skin, including keratinocytes of the epidermis and hair follicles, as well as cells of the glands and secretory ducts. We also have detected AM in the sweat, by RIA. In addition, AM and its receptor were found in skin tumors of different histologies. The presence of AM and its receptor in normal and neoplastic skin was confirmed by RT-PCR and Western blot analysis performed on cell extracts from human skin cell lines. Radiolabeled AM bound to specific sites in cultured cells with a Kd of 9 nM. This binding was blocked by the addition of cold AM but not by related peptides such as AM 22-52, pro-AM 20 N-terminal peptide, calcitonin gene-related peptide, calcitonin gene-related peptide 8-37, or amylin. Finally, exposure to synthetic AM resulted in an increase of thymidine intake by skin cells. These results implicate AM as a potential player in skin defense against infectious microorganisms and as a possible autocrine growth factor in normal skin physiology and tumor development.

Epidermal growth factor receptor monoclonal antibodies inhibit the growth of lung cancer cell lines.

The ability of monoclonal antibody (MAb 108), an immunoglobulin G (IgG)2a against the epidermal growth factor receptor (EGF-R), to interact with lung cancer cell lines was investigated. 125I-EGF bound with high affinity to non-small-cell lung cancer (NSCLC) cells, and MAb 108 inhibited specific binding of nine NSCLC cell lines in a dose-dependent manner (IC50 = 0.3-3 micrograms per ml). 125I-MAb 108 bound with high affinity (kd = 2 nM) to a single class of sites (Bmax = 70,000 per cell) using NSCLC neuroendocrine cell line NCI-H460. Specific 125I-MAb 108 binding was inhibited with high affinity by MAb 108 but not by a control antibody IgG using large-cell carcinoma cell line NCI-H1299. 125I-MAb 108 binding was not internalized at 37 degrees C using NSCLC neuroendocrine cell line NCI-H460 and adenocarcinoma cell line NCI-H23. Also, 1 microgram per ml of MAb 108 but not of a control IgG inhibited the clonal growth of NCI-H23 and squamous cell carcinoma cell line NCI-H157 in vitro. Also, MAb 108 inhibited xenograft formation of cell lines NCI-H460, NCI-H157, and NCI-H727 in nude mice in vivo. After a palpable tumor had formed using NCI-H460 cells, injection of 100 micrograms of MAb 108 (intraperitoneally three times weekly) inhibited xenograft volume in nude mice by approximately 50%. These data suggest that MAb 108 may interact with EGF receptors on lung cancer cell lines and inhibit NSCLC proliferation.

Binary drugs: conjugates of purines and a peptide that bind to both adenosine and substance P receptors.

A "functionalized congener" approach to adenosine receptor antagonists has provided a means to synthesize highly potent peptide conjugates of 1,3-dialkylxanthines. The antagonist XAC, such a functionalized xanthine amine congener, has been attached to a segment derived from the neurotransmitter peptide substance P (SP) to form a binary drug that binds to both receptors with Ki values of 35 nM (central A1-adenosine) and 300 nM (striatal SP). Coupling of the functionalized adenosine agonist N6-[p-(carboxymethyl)phenyl]adenosine to an SP C-terminal peptide also resulted in a binary drug that binds to both receptors. The demonstration that the biochemical properties of two unrelated drugs, both of which act through binding at extracellular receptors, may be combined in the same molecule suggests a novel strategy for drug design. In principle, a combined effect of the two different substances that produce the same final effect (e.g., hypotension by adenosine agonists and by SP analogues) might occur in vivo. Adenosine analogues have analgesic properties, and the binary drug derived from substance P and adenosine agonists or antagonists might provide useful tools for probing interrelationships of SP pathways and sites for the antinociceptive action of adenosine.

Thymosinalpha1 is chemopreventive for lung adenoma formation in A/J mice.

The effects of thymosin (THN) alpha1 were investigated using the urethane injection carcinogenesis A/J mouse model. Lung adenomas were observed 2.5, 3, and 4 months after urethane injection (400 mg/kg i.p.) into female A/J mice. Daily administration of THNalpha1 (0.4 mg/kg, s.c.) reduced lung adenoma multiplicity significantly, by approximately 45, 40, and 17%, respectively, 2.5, 3, and 4 months after urethane injection. Animals treated with THNalpha1 had a significantly greater white cell density than control A/J mice. Endogenous THNalpha1-like peptides were detected in the mouse lung. By radioimmunoassay and by Western blot, prothymosin alpha was detected in the mouse lung. By immunocytochemistry, THNalpha1-like peptides were detected in all lung compartments including the bronchus, adenoma, bronchioles, and alveoli. These results indicate that exogenous THNalpha1 prevents lung carcinogenesis in A/J mice.

PACAP(6-38) inhibits the growth of prostate cancer cells.

The effects of pituitary adenylyl cyclase activating polypeptide (PACAP) analogs on prostate cancer cell lines was investigated. 125I-PACAP-27 bound with high affinity to PC-3 cells (Kd = 10 nM) to a single class of sites (Bmax = 30000/cell). By RT-PCR, a major 305 bp band was observed using cDNA derived from PC-3, LNCaP or DU-145 cells. Specific 125I-PACAP binding was inhibited with high affinity by PACAP-27, PACAP-38 and PACAP(6-38) (IC50 values of 15, 10 and 300 nM, respectively) but not by PACAP(28-38). PACAP elevated cAMP and the increase caused by PACAP-27 was reversed by PACAP(6-38). PACAP transiently increased c-fos gene expression and the increase in c-fos mRNA was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in PC-3 cells, whereas PACAP(6-38) reduced colony number and size. In nude mice bearing PC-3 xenografts, PACAP(6-38) significantly slowed tumor growth. These data suggest that biologically active type 1 PACAP receptors are present on human prostate cancer cells and that prostate cancer cell growth is inhibited by PACAP(6-38).