Vasoactive Intestinal Peptide Inhibits Human Small-Cell Lung Cancer Proliferation in Vitro and in Vivo (Neuropeptides͞camp͞cell Culture͞athymic Nude Mice)
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14373–14378, November 1998 Medical Sciences Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo (neuropeptidesycAMPycell cultureyathymic nude mice) KANAME MARUNO*, AFAF ABSOOD†, AND SAMI I. SAID‡ Department of Medicine, Northport Veterans Affairs Medical Center Stony Brook, NY 11768-2290, and State University of New York, Health Sciences Center 17-040, Stony Brook, NY 11794-8172 Communicated by Susan E. Leeman, Boston University School of Medicine, Boston, MA, September 23, 1998 (received for review March 15, 1998) ABSTRACT Small-cell lung carcinoma (SCLC) is an ag- cell lines (7, 8). The cells were maintained in RPMI medium gressive, rapidly growing and metastasizing, and highly fatal 1640 containing 10 nM hydrocortisone, 5.0 mgyml insulin, 10 neoplasm. We report that vasoactive intestinal peptide inhib- mgyml transferrin, 10 nM 17b-estradiol, 30 nM sodium selen- its the proliferation of SCLC cells in culture and dramatically ite, 100 unitsyml penicillin, and 100 mgyml streptomycin suppresses the growth of SCLC tumor-cell implants in athy- (HITES medium; ref. 9). As a control, the SCLC cell line mic nude mice. In both cases, the inhibition was mediated NCI-H128 (American Type Culture Collection), which lacks apparently by a cAMP-dependent mechanism, because the VIP receptors (10), was also tested. The cells were incubated inhibition was enhanced by the adenylate cyclase activator in tissue-culture flasks in a humidified atmosphere of 5% CO2 forskolin and the phosphodiesterase inhibitor 3-isobutyl-1- in air at 37°C and grown as floating cell aggregates. All methylxanthine in proportion to increases in intracellular chemicals needed for cell culture were purchased from Sigma. cAMP levels, and the inhibition was abolished by selective Cell Counts. Cells (2.0 3 104 per ml) in the logarithmic inhibition of cAMP-dependent protein kinase. If confirmed in growth phase were harvested and plated into 24-well cluster clinical trials, this antiproliferative action of vasoactive in- plates in 2.0 ml of HITES medium. VIP was added to four final testinal peptide may offer a new and promising means of concentrations from 1.0 nM to 1.0 mM. In other experiments, suppressing SCLC in human subjects, without the toxic side the structurally related peptide glucagon was added to the effects of chemotherapeutic agents. same final concentrations, and, in a third group of experi- ments, only diluent was added. Both VIP and glucagon were Small-cell lung carcinoma (SCLC) constitutes 20–25% of cases stored in 0.01 N acetic acid at 280°C before use to minimize of lung cancer, currently the leading cause of death from proteolytic degradation. Different concentrations of the pep- malignant disease among men and women in the U.S. (1). tides were prepared in the culture medium and then added to SCLC is also a highly fatal cancer; it metastasizes early and the plates. rapidly, and it is rarely curable (1). Therefore, means of To assess the role of cAMP in inhibiting the proliferation of controlling the growth and multiplication of SCLC are needed SCLC cells, NCI-H345 cells were incubated with forskolin (100 urgently. nM to 100 mM), isobutyl methylxanthine (IBMX; 1.0 mMto1.0 The neuroendocrine nature of SCLC is well known; the mM), or either of these agents together with 1.0 mM VIP. To tumor cells produce a variety of hormones and neurotrans- examine the role of protein kinase A in mediating the action mitters and are in turn influenced by their secretory products, of cAMP, we tested the effect of four concentrations (1.0 nM some of which (e.g., bombesin-like peptides such as gastrin- to 1.0 mM) of KT5720, a selective inhibitor of this enzyme, on releasing peptide) act as autocrine growth factors (2). We the inhibition of cell proliferation by VIP (1.0 mM). The recently reported that vasoactive intestinal peptide (VIP), a culture medium was partially (1.0 ml) replaced on the second naturally occurring 28-aa residue neuropeptide (3), binds to day with fresh medium containing freshly prepared peptides, specific adenylate cyclase-linked receptors on SCLC cell lines, forskolin, IBMX, or KT5720. The viable cells (those not NCI-H345 and NCI-H69 (as designated by the National Can- stained by trypan blue) were counted in triplicate on the fourth cer Institute; ref. 4), and inhibits the growth and multiplication day with a hemocytometer. of these cell lines in vitro (5), especially if the growth- [3H]Thymidine Incorporation. Cultured NCI-H345 cells promoting action of endogenously produced gastrin-releasing (5.0 3 104 in 180 ml of HITES medium) were seeded into a peptide (6) is blocked with an anti-bombesin monoclonal 96-well plate. VIP was added to the wells in 25 ml of HITES antibody (5). In this paper we further document the antipro- medium to achieve final concentrations in the wells of 1.0 nM liferative activity of VIP against SCLC cells in vitro, establish to 1.0 mM. Forskolin or IBMX was added in 25 ml of HITES its mediation by cAMP and cAMP-dependent protein kinase medium in four concentrations of 100 nM to 100 mMor1.0mM (protein kinase A), and report that VIP also suppresses the to 1.0 mM, respectively, with or without 1.0 mM VIP. After a growth of SCLC tumor implants in athymic nude mice in vivo. 20-h incubation at 37°C, 25 ml of [methyl-3H]thymidine solu- tion [DuPontyNEN; 5.0 Ciymmol, originally in ethanolywater MATERIALS AND METHODS (1:1, volyvol), 0.1 mCi per well] was added to each well. The Cell Culture. SCLC cell lines NCI-H345 and NCI-H69, belonging to the classic subclass of SCLC, were obtained from Abbreviations: protein kinase A, cAMP-dependent protein kinase; DMSO, dimethyl sulfoxide; IBMX, 3-isobutyl-1-methylxanthine; NCI, Adi F. Gazdar and colleagues (National Cancer Institute, designator of cell lines developed by the National Cancer Institute; Bethesda), who originally established these and other SCLC SCLC, small-cell lung cancer; VIP, vasoactive intestinal peptide. *Present address: Department of Surgery, Teikyo University Hospital, The publication costs of this article were defrayed in part by page charge 3-8-3 Mizonokuchi, Takatsu-ku, Kawasaki, Kanagawa 213-8507, Japan. payment. This article must therefore be hereby marked ‘‘advertisement’’ in †Present address: Department of Surgery, University of Michigan, accordance with 18 U.S.C. §1734 solely to indicate this fact. Ann Arbor, MI 48109-0329. © 1998 by The National Academy of Sciences 0027-8424y98y9514373-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: ssaid@mail. PNAS is available online at www.pnas.org. som.sunysb.edu. 14373 Downloaded by guest on September 29, 2021 14374 Medical Sciences: Maruno et al. Proc. Natl. Acad. Sci. USA 95 (1998) cells were harvested 4 h later on a filter paper by exhaustive basal increase of NCI-H69 and NCI-H345 cell counts, respec- elution with PBS and exposed to ice-cold 10% trichloracetic tively, by up to 84% and 75%, over a concentration range of acid in a semiautomatic cell harvester. The dried filter was from 1.0 nM to 1.0 mM (Fig. 1). Equimolar concentrations of counted in triplicate in a liquid scintillation counter. glucagon did not affect the cell count of either cell line. In 3 6 Intracellular cAMP Levels. NCI-H345 cells (1.0 10 per addition to inhibiting cell counts, VIP also dose-dependently ml) were incubated in 1.0 ml of DMEM, pH 7.3, containing 2% reduced [3H]thymidine incorporation into NCI-H345 cells by BSA and four concentrations of forskolin or IBMX in the same 23% at 1.0 nM, 31% at 10 nM, 44% at 100 nM, and 45% at 1.0 concentration range as described above, with or without 1.0 m m M (data not shown in figure). M VIP (4). After incubation for 10 min at 25°C, 1.0 ml of Enhancement of inhibition by other cAMP-promoting agents. methanol was added to stop the reaction and extract cAMP. In experiments designed to clarify the relationship between the Cell samples were then sonicated and centrifuged for 5 min at inhibition of cell proliferation and intracellular cAMP levels, 3,000 3 g. The pellet was washed with 1.0 ml of methanol, and NCI-H345 cells were incubated with VIP (1.0 nM to 1.0 mM), the washings were added to the supernatant of each sample. with forskolin (100 nM to 100 mM), with IBMX (1.0 mMto1.0 The combined solution was evaporated to dryness, and the m residue was dissolved in 0.5 ml of cAMP assay buffer. After mM), or with forskolin or IBMX together with 1.0 M VIP. In centrifugation, supernatants were assayed for cAMP in trip- these experiments, we measured cAMP content, cell counts, 3 licate by radioimmunoassay, with the use of a RIANEN kit and [ H]thymidine incorporation. VIP dose-dependently ele- from DuPontyNEN. vated intracellular cAMP levels by 103% at 1.0 nM, 149% at In Vivo Experiments. Female athymic BALByc nude mice, 10 nM, 188% at 100 nM, and 200% at 1.0 mM. The cAMP 4–5 weeks old, were housed in filter-top cages in a pathogen- elevation caused by VIP alone was small and transient, prob- free, temperature-controlled, laminar-flow, filtered-air, iso- ably because cAMP is metabolized quickly by phosphodies- lated room and were exposed to light from 7:00 a.m. to 7:00 terase. Alone, forskolin dose-dependently elevated intracellu- p.m. NCI-H69 cells (1.0 3 107) were injected subcutaneously lar cAMP levels by up to 290% (Fig.