Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14373–14378, November 1998 Medical Sciences

Vasoactive intestinal inhibits human small-cell lung cancer proliferation in vitro and in vivo (neuropeptides͞cAMP͞cell culture͞athymic 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 ␮g͞ml , 10 neoplasm. We report that vasoactive intestinal peptide inhib- ␮g͞ml transferrin, 10 nM 17␤-estradiol, 30 nM sodium selen- its the proliferation of SCLC cells in culture and dramatically ite, 100 units͞ml penicillin, and 100 ␮g͞ml 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 ϫ 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 ␮M. 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 Ϫ80°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 ␮M), isobutyl methylxanthine (IBMX; 1.0 ␮Mto1.0 The neuroendocrine nature of SCLC is well known; the mM), or either of these agents together with 1.0 ␮M 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 such as - to 1.0 ␮M) 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 ␮M). 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 (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 ϫ 104 in 180 ␮l 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 ␮l 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 ␮M. Forskolin or IBMX was added in 25 ␮l of HITES its mediation by cAMP and cAMP-dependent protein kinase medium in four concentrations of 100 nM to 100 ␮Mor1.0␮M (protein kinase A), and report that VIP also suppresses the to 1.0 mM, respectively, with or without 1.0 ␮M VIP. After a growth of SCLC tumor implants in athymic nude mice in vivo. 20-h incubation at 37°C, 25 ␮l of [methyl-3H]thymidine solu- tion [DuPont͞NEN; 5.0 Ci͞mmol, originally in ethanol͞water MATERIALS AND METHODS (1:1, vol͞vol), 0.1 ␮Ci 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 0027-8424͞98͞9514373-6$2.00͞0 ‡To whom reprint requests should be addressed. e-mail: ssaid@mail. PNAS is available online at www.pnas.org. som.sunysb.edu.

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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 ␮M (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 ϫ 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 (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 ϫ 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 ␮M), the washings were added to the supernatant of each sample. with forskolin (100 nM to 100 ␮M), with IBMX (1.0 ␮Mto1.0 The combined solution was evaporated to dryness, and the ␮ 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 DuPont͞NEN. vated intracellular cAMP levels by 103% at 1.0 nM, 149% at In Vivo Experiments. Female athymic BALB͞c nude mice, 10 nM, 188% at 100 nM, and 200% at 1.0 ␮M. 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 ϫ 107) were injected subcutaneously lar cAMP levels by up to 290% (Fig. 2A); combined with VIP, into the right flank of each mouse. There were four experi- it stimulated cAMP production by up to 2120% (Fig. 2A). mental groups, of four mice each, three of which received VIP Forskolin dose-dependently reduced the increase in NCI- (1.0, 5.0, or 10 ␮g͞day) in PBS; as a control, the fourth received H345 cell counts by up to 90% (Fig. 2B); combined with 1.0 only PBS. All solutions were infused for 8 weeks, beginning 1 ␮M VIP, it reduced the cell counts by up to 97% (Fig. 2B). week after injection of the cells, and delivered by Alzet osmotic Forskolin dose-dependently reduced [3H]thymidine incorpo- pumps (model 2002; Palo Alto, CA) placed aseptically under ration into NCI-H345 cells by up to 36% (Fig. 3); combined the skin of the back of the mice. The pump released its contents ␮ 3 ␮ ͞ with 1.0 M VIP, the inhibition of [ H]thymidine incorpora- at a rate of 0.5 l h for a duration of 2 weeks. The spent pumps tion reached 58% (Fig. 3). IBMX alone increased cAMP levels were removed every 2 weeks, and new pumps, containing fresh by 75%. In combination with VIP, the effects closely paralleled solutions, were implanted (11); this procedure was repeated those of VIP and forskolin on cAMP, cell counts, and [3H]thy- three times. The tumors were measured with calipers, and the mice were weighed weekly for 8 weeks. Tumor volume was midine incorporation into NCI-H345 cells. The inhibition of ϫ cell counts and [3H]thymidine incorporation correlated highly calculated for an ellipsoid as maximal length maximal ϭ height ϫ maximal width ϫ ␲͞6. On the last day of the with the increase in intracellular cAMP levels (r 0.97 and 3 experiment, blood was sampled from the retroorbital plexus 0.98 for cell counts and [ H]thymidine incorporation with ϭ into chilled heparin-containing tubes rinsed with 0.05% forskolin and VIP, respectively, and r 0.995 and 0.96 with NaEDTA and containing three protease inhibitors, 10 ␮g͞ml IBMX and VIP, respectively). soybean trypsin inhibitor, 100 TIU͞ml aprotinin, and 10 ␮g͞ml phosphamidon; all from Sigma), as well as 0.1 mM IBMX for measurement of plasma VIP and cAMP levels. The mice were then euthanized. The tumors were excised, weighed, and frozen in liquid nitrogen for subsequent extraction (in meth- anol) and for measurement of protein content (12); a portion of the tumor was fixed in 10% neutral buffered formalin for morphologic examination. In another experiment, six groups of mice received, via two pumps in each mouse, 123 ␮g͞day of forskolin in dimethyl sulfoxide (DMSO) and PBS (n ϭ 6), 0.5 ␮g͞day of VIP in PBS and DMSO (n ϭ 5), 1.0 ␮g͞day of VIP in PBS and DMSO (n ϭ 6), 0.5 ␮g͞day of VIP in PBS and forskolin in DMSO (n ϭ 5), 1.0 ␮g͞day of VIP in PBS and forskolin in DMSO (n ϭ 7), or only PBS and DMSO daily (n ϭ 6) as a control group. The pumps were replaced every 2 weeks, as described above. The tumors were measured with calipers, and the mice were weighed weekly for 8 weeks. On the last day of the experiment, blood was sampled, and the tumors were weighed and then frozen in liquid nitrogen. Statistical Analysis. Data are expressed as mean Ϯ SD. Statistical differences were examined by ANOVA and Tukey’s protected t test. In vivo data were transferred to the logarithmic scale before statistical analysis.

FIG. 1. VIP dose-dependently inhibits NCI-H345 and NCI-H69 RESULTS cell proliferation in culture, whereas glucagon does not. The cells (2.0 ϫ 104 per ml) were cultured for 4 days in the presence of between In Vitro Experiments. Selective, dose-dependent inhibition of 1.0 ϫ 10Ϫ9 and 1.0 ϫ 10Ϫ6 M VIP (NCI-H345, closed circles, and cell proliferation by VIP. In concentration-response experi- NCI-H69, open circles) or glucagon (NCI-H345, closed triangles, and .P Ͻ 0.01 vs. untreated cells ,ءء .(ments limited to 4 days, VIP dose-dependently inhibited the NCI-H69, open triangles Downloaded by guest on September 29, 2021 Medical Sciences: Maruno et al. Proc. Natl. Acad. Sci. USA 95 (1998) 14375

nude mice was enhanced by forskolin; see Fig. 7. In PBS and DMSO-treated mice, the tumor volume increased exponen- tially during weeks 1–9, from 11 mm3 to 840 mm3. In mice treated with 123 ␮g͞day of forskolin, the rate of tumor growth was slowed by up to 70% at week 6 and 62% at week 9 (P Ͻ 0.01). In mice treated with either 0.5 or 1.0 ␮g͞day of VIP, the rate of tumor growth was dose-dependently slowed by up to 81% at week 6 in the 0.5 ␮g͞day group and by up to 91% at week 9 in the 1.0 ␮g͞day group (P Ͻ 0.01). Maximal tumor- suppression rate at week 9 was 91% at the 1.0 ␮g͞day dose level. In mice treated with the combination of VIP (0.5 or 1.0 ␮g͞day) and 123 ␮g͞day of forskolin, the rate of tumor growth was attenuated by up to 96% at week 5 and 97% at week 9. Tumor volume was significantly smaller in all VIP- or forsko- lin-treated mice than in PBS and DMSO-treated mice after week 4 (P Ͻ 0.01); after week 4 (except for week 6) tumor volume in mice treated with 1.0 ␮g͞day of VIP was signifi- cantly smaller than in mice treated with 0.5 ␮g͞day of VIP (P Ͻ 0.05). After week 5, tumor volume was significantly smaller in mice treated with VIP (0.5 or 1.0 ␮g͞day) and forskolin than in mice treated with VIP alone (P Ͻ 0.01); after week 7, tumor volume was smaller in mice treated with 1.0 ␮g͞day of VIP and forskolin than in those treated with 0.5 ␮g͞day of VIP and forskolin (P Ͻ 0.05). Treatment with 1.0, 5.0, and 10 ␮g͞day of VIP, dose- dependently decreased tumor weights from 617 Ϯ 233 g (buffer only), respectively to 229 Ϯ 91.8, 155 Ϯ 20.1, and 91.5 Ϯ 15.7 g (P Ͻ 0.05 or Ͻ 0.01 among groups). At the same time, FIG.2.(A) Dose-related stimulation of cAMP production in ␮ tumor protein content decreased dose-dependently from NCI-H345 cells by forskolin alone (0.1–100 M) or combined with 1.0 Ϯ Ϯ Ϯ ␮M VIP. The cells (1.0 ϫ 106 per ml) were incubated for 10 min in the 49.3 15.6 mg (buffer only), respectively to 24.0 8.16, 16.0 .(P Ͻ 1.16, and 9.53 Ϯ 1.77 mg (P Ͻ 0.05 or Ͻ 0.01 among groups ,ء .(presence of forskolin alone (open bars) or with VIP (solid bars P Ͻ 0.01 vs. values in absence of forskolin. (B) Dose- In another group of experiments, tumor weights decreased ,ءء and 0.05 related inhibition of NCI-H345 cell proliferation by forskolin alone from 484 Ϯ 227 (buffer only) to 231 Ϯ 52.0 g with 123 ␮g͞day (0.1–100 ␮M), or combined with 1.0 ␮M VIP. The cells (2.0 ϫ 104 per of forskolin, 157 Ϯ 67.6 g with 0.5 ␮g͞day of VIP, 58.5 Ϯ 16.2 g ml) were cultured for 4 days in the presence of forskolin alone (open ␮ ͞ Ϯ ␮ ͞ Ͻ with 1.0 g day of VIP, 64.8 38.6 g with 0.5 g day of VIP ءء Ͻ ء bars) or with forskolin and VIP (closed bars). , P 0.05 and , P and forskolin, and 19.6 Ϯ 12.8 g with 1.0 ␮g͞day of VIP and 0.01 vs. values without forskolin. forskolin (P Ͻ 0.05 or Ͻ0.01 among groups). In these exper- Role of protein kinase A. To examine the role of protein iments too, tumor protein content decreased in parallel with kinase A in inhibiting the proliferation of SCLC cells, NCI- tumor weight. H345 cells were incubated for 4 days with KT5720, a selective Plasma VIP levels. In the first group of in vivo experiments, inhibitor of this enzyme, with or without VIP. Combined with plasma VIP levels, determined in blood collected 14 days after the last pump replacement (n ϭ 4), were 23.9 Ϯ 7.73 pM in 1.0 ␮M VIP, KT5720 (1.0 nM to 1.0 ␮M) dose-dependently mice receiving buffer, and 29.9 Ϯ 3.37 pM, 58.0 Ϯ 4.24 pM reversed the inhibition of NCI-H345 cell counts by VIP, (P Ͻ 0.01), and 63.6 Ϯ 21.0 pM (P Ͻ 0.01) in those treated, abolishing it at a concentration of 1.0 ␮M (Fig. 4). The same respectively, with 1.0, 5.0, and 10 ␮g͞day of VIP. In the second concentrations of KT5720 alone did not alter the cell counts. group of experiments, conducted in the same way, plasma VIP In Vivo Experiments. Growth of tumor-cell implants and levels (n ϭ 5–7) were 27.1 Ϯ 7.61 pM in mice treated with suppression by VIP. The injected SCLC cells grew into palpable buffer and DMSO, and 27.2 Ϯ 3.07 pM and 58.7 Ϯ 29.8 pM tumors in the nude mice within 1 week; see Figs. 5 and 6. In (P Ͻ 0.01) in those treated with 0.5 and 1.0 ␮g͞day of VIP, mice treated with buffer only, the tumors grew exponentially, ␮ ͞ 3 respectively. In the two groups of mice treated with 1.0 g day increasing in volume during weeks 1 to 9 from 1.5 mm to 1100 of VIP, tumor growth at week 9 was suppressed by 71% and 3 ␮ ͞ mm . In mice treated with VIP (1.0, 5.0, or 10 g day), the rate 91%. The greater inhibition of tumor growth correlated with of tumor growth was dose-dependently slowed by up to 80% a higher mean plasma VIP level: 58.7 Ϯ 29.8 pM vs. 29.9 Ϯ 3.37 at week 5 in the 1.0 ␮g͞day group, by up to 84% at week 6 in Ͻ ␮ ͞ pM (P 0.05). Plasma cAMP levels did not increase in mice the 5.0 g day group, and by up to 95% at week 6 in the 10 treated with VIP or with VIP and forskolin. ␮ ͞ g day group. The maximal tumor suppression rate at week 9 No change in body weight. Total body weight was the same ␮ ͞ was 88%, at 10 g day. Tumor volume was significantly in solvent-treated mice as in mice treated with VIP or VIP and smaller in all VIP-treated mice than in PBS-treated mice after forskolin, suggesting that no systemic toxicity was associated week 4. After week 6, tumor volume was significantly smaller with even the highest dose regimen used. in mice treated with 10 ␮g͞day of VIP than in those treated with 1.0 ␮gor5.0␮g͞day of VIP but was not different between mice treated with 1.0 ␮g͞day of VIP and those treated with 5.0 DISCUSSION ␮g͞day of VIP. Dramatic, dose-dependent suppression of Our results show that VIP inhibits the growth and multiplica- SCLC tumor growth was observed after treatment with any of tion of human SCLC cells NCI-H345 and NCI-H69 in culture, the doses of VIP, whereas PBS-treated mice experienced rapid and markedly suppresses the growth of SCLC tumor-cell tumor growth. Histological examination of the tumors showed implants in vivo. The specificity of this effect is supported by no increased necrosis with VIP treatment, suggesting that VIP the lack of any inhibition by glucagon, a peptide that is related slowed cell proliferation, rather than causing cell death. structurally to VIP. In an earlier study, we found that glucagon Potentiation of VIP effect by forskolin. As in the cultured cells failed to compete with VIP binding to SCLC cells (4). How- in vitro, the inhibitory effect of VIP on the growth of SCLC in ever, helodermin, another VIP-like peptide, which binds to Downloaded by guest on September 29, 2021 14376 Medical Sciences: Maruno et al. Proc. Natl. Acad. Sci. USA 95 (1998)

FIG. 3. Dose-related inhibition of [3H]thymidine incorporation into NCI-H345 cells by forskolin alone (0.1–100 ␮M) or combined with 1.0 ␮M VIP. The cells (5.0 ϫ 104 per well) were incubated for 24 h in the presence of forskolin alone (open bars) or of forskolin and VIP (solid bars). .P Ͻ 0.01 vs. values without forskolin ,ءء P Ͻ 0.05 and ,ء

helodermin-preferring receptors on NCI-H345 cells (4), in- acteristic SCLC morphology as well as amine-precursor- hibited their proliferation(5). By contrast, the proliferation of uptake and decarboxylation (APUD) cell characteristics (7, 8). NCI-H128 cells, which lack VIP receptors (10), was not Apparently, the inhibition of SCLC cell proliferation by VIP inhibited by VIP (data not shown). and other agents was mediated by increased intracellular The proliferation of NCI-H69 cells was inhibited more than cAMP levels. (i) Forskolin and IBMX, which elevated intra- that of NCI-H345 cells by VIP alone (Fig. 1). Because the cellular cAMP content in tumor cells, inhibited SCLC growth inhibition of proliferation of NCI-H345 cells by VIP was and potentiated the inhibitory effect of VIP in vitro and in vivo. comparable to that by helodermin (5), we focused on these (ii) In all instances, the inhibition of SCLC cell counts and of 3 cells in our studies of inhibition in vitro, including cell counting, [ H]thymidine incorporation into SCLC cells was highly cor- [3H]thymidine incorporation, and the stimulation of cAMP related with augmentation of cAMP levels. (iii) The VIP- production. Consistent with an earlier report (13), we were induced inhibition of SCLC cell counts was reversed totally by unable to implant NCI-H345 cells in nude mice; therefore, our KT5720, a selective inhibitor of protein kinase A. The results further suggest that the activation of protein kinase A is in vivo experiments were limited to NCI-H69 cells. Both cell lines belong to the classic subclass of SCLC and retain char-

FIG. 5. SCLC tumor growth in nude mice and its dose-dependent suppression by VIP. A week after injection of NCI-H69 cells into mice, FIG. 4. Dose-related attenuation by KT5720 (0.001–1.0 ␮M) of the four groups of four mice each were treated with PBS (closed circles), inhibition of NCI-H345 cell proliferation by 1.0 ␮M VIP. The cells 1.0 ␮g͞day of VIP (open circles), 5.0 ␮g͞day of VIP (closed triangles), (2.0 ϫ 104 per ml) were cultured for 4 days in the presence of KT5720 or 10 ␮g͞day of VIP (open triangles), via Alzet osmotic pumps. Tumor P Ͻ 0.01 vs. volume was measured at weekly intervals, as described in Materials and ,ءء .(alone (open bars) or of KT5720 and VIP (solid bars values without KT5720. Methods. For analysis of results, please see text. Downloaded by guest on September 29, 2021 Medical Sciences: Maruno et al. Proc. Natl. Acad. Sci. USA 95 (1998) 14377

FIG. 6. Representative nude mice 9 weeks after implantation of NCI-H69 cells and treatment for 8 weeks with PBS (mouse A), 1.0 ␮g͞day of VIP (mouse B), 5.0 ␮g͞day of VIP (mouse C), and 10 ␮g͞day of VIP (mouse D). The largest tumor is seen in mouse A, with progressively smaller tumor masses in the mice treated with increasingly larger doses of VIP (mice B–D). The cylindrical object under the skin of the back is the infusion pump (Alzet model 2002). (Bar ϭ 10 mm.)

required and may completely account for the antimitogenic concentrations of the peptide to the SCLC cells. (ii) The effect of VIP on SCLC cell proliferation. concentration of VIP within a solid tumor may be variable and The in vivo inhibition by VIP was evident at picomolar impossible to simulate in vitro.(iii) The environment of the concentrations of plasma VIP, whereas in vitro inhibition cells in vitro may lower their sensitivity to VIP, relative to in required at least 100-fold higher concentrations. Several fac- vivo conditions. (iv) VIP, given in vivo, may release other, more tors may account for this large difference in effective concen- potent peptides with similar activity, such as the recently trations. (i) A continuous supply of VIP, as provided in the in proposed activity-dependent neurotrophic peptide, found to vivo experiments, may be essential for delivering inhibitory possess highly potent neuroprotective properties (14). For systemic delivery of peptides in vivo, constant infusion by osmotic pumps increases efficacy and reduces side effects relative to bolus injection. Daily intraperitoneal injections over 3 weeks of the D-Phe5-substituted analogue of the neuropep- tide had no effect on the growth of human SCLC tumor in nude mice (15). Administered by constant infusion via osmotic pump, however, the analogue inhibited tumor growth (15). Similarly, continuous infusions of several antag- onists of substance P were tolerated better in 5-fold higher doses by tumor-bearing nude mice than were bolus injections (16). Further, the constant, low plasma levels obtained with continuous infusion may not be cytotoxic to normal cells (17). The greater therapeutic effect of continuously infused VIP may be caused by a sustained plasma level that suppresses tumor-cell growth but spares normal cells. The absence of any weight loss by the animals in these experiments supports a relatively low systemic toxicity of VIP as administered. Our results seem to be at variance with recent reports that VIP stimulated colony formation by NCI-H345 SCLC cells (18) and that a VIP antagonist͞VIP hybrid inhibited colony formation by NCI-H209 and NCI-H345 SCLC cells (19) and suppressed non-SCLC tumor growth in athymic nude mice (20). In clonogenic assays, however, only a small number of the cells originally seeded into the layer of agarose form colonies in the assay. Plating efficiencies are low in clonogenic assays FIG. 7. SCLC tumor growth and its potentiated suppression by (21, 22), and proliferation is evaluated from colony-forming combined treatment with VIP and forskolin. The experiment was cells only, rather than the entire cell population (23). Clono- designed as described for Fig. 5, except that six treatment groups were genic assays may also be affected by a clumping artifact and cell examined: buffer only (closed circles), forskolin (123 ␮g͞day, open circles), VIP (0.5 ␮g͞day, closed squares), VIP 1.0 ␮g͞day, open migration (21, 23, 24). Further, no one has described an squares), VIP (0.5 ␮g͞day) and forskolin (closed triangles), and VIP inhibitory effect of these analogues on the stimulatory action (1.0 ␮g͞day) and forskolin (open triangles). For analysis of results, of VIP on SCLC colony formation or a direct stimulation of please see text. SCLC xenograft growth by VIP in vivo. Downloaded by guest on September 29, 2021 14378 Medical Sciences: Maruno et al. Proc. Natl. Acad. Sci. USA 95 (1998)

There is a great need for developing new and more success- 11. Szende, B., Redding, T. W. & Schally, A. V. (1990) Proc. Natl. ful strategies for the treatment of SCLC. Although it is Acad. Sci. USA 87, 901–903. premature to predict whether VIP will pass the test of clinical 12. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. trials, our findings suggest that VIP, or a related analogue or (1951) J. Biol. Chem. 192, 265–275. peptide mimetic, holds promise as an effective and relatively 13. Mahmoud, S., Staley, J., Teylor, J., Bogenden, A., Moreau, J. P., nontoxic anti-SCLC agent. The SCLC-inhibitory effect of VIP Coy, D., Avis, I., Cuttitta, F., Mulshine, J. L. & Moody, T. W. (1991) Cancer Res. 51, 1798–1802. reported here may be similar to that recently reported for 14. Brenneman, D. E. & Gozes, I. (1996) J. Clin. Invest. 97, 2299– cytotoxic analogs of (25). Our data have addi- 2307. tional implications in light of recent observations on the 15. Everard, M. J., Macaulay, V. M., Millar, J. L. & Smith, I. E. (1993) expression and localization of the human VIP type I receptor Eur. J. Cancer. 29A, 1450–1453. (26). This receptor, expressed with highest prevalence in the 16. Langdon, S., Sethi, T., Ritchie, A., Muir, M., Smyth, J. & lung (26, 27), has been localized to the short arm of chromo- Rozengurt, E. (1992) Cancer Res. 52, 4554–4557. some 3 (3p22) where allele loss has been linked to SCLC and 17. Siegall, C. B., Kreitman, R. J., FitzGerald, D. J. & Pastan, I. other cancers (28–30). The marked inhibition of SCLC by VIP (1991) Cancer Res. 51, 2831–2836. is consistent with the view that this chromosomal region 18. Moody, T. W., Zia, F. & Makheia, A. (1993) Peptides (Tarrytown, contains a functional tumor-suppressor gene and suggests that NY) 14, 241–246. VIP and its receptor may have a role in regulating the genesis 19. Moody, T. W., Zia, F., Goldstein, A. L., Naylor, P. H., Sarin, E., of SCLC malignancy (29, 30). Brenneman, D., Koros, A. M. C., Reubi, J. C., Korman, L. Y., Fridkin, M., et al. (1992) Biomed. Res. 13, Suppl. 2, 131–135. 20. Moody, T. W., Zia, F., Draoui, M., Brenneman, D. E., Fridkin, We thank Rosalind Antoniazzi for preparation of the manuscript. M., Davidson, A. & Gozes, I. (1993) Proc. Natl. Acad. Sci. USA This work was supported by National Institutes of Health Grant 90, 4345–4349. HL-30450 and by research funds from the Department of Veterans 21. Grenman, R., Burk, D., Virolainen, E., Buick, R. N., Church, J., Affairs, for which S.I.S. was a Medical Investigator. Schwartz, D. R. & Carey, T. E. (1989) Int. J. Cancer 44, 131–136. 22. Tanigawa, N., Kern, D. H., Hikasa, Y. & Morton, D. L. (1982) 1. Chia, M. M., Gazdar, A. F., Carbone, D. P. & Minna, J. P. (1994) Cancer Res. 42, 2159–2164. in Textbook of Respiratory Medicine, eds. Murray, J. & Nadel, J. 23. Selby, P., Buick, R. N. & Tannock, I. (1983) N. Engl. J. Med. 308, (Saunders, Philadelphia), 2nd Ed., pp. 1485–1503. 129–134. 2. Cuttitta, F., Carney, D. N., Mulshine, J., Moody, T. W., Fedorko, 24. Sondak, V. K., Berlelsen, C. A., Tanigawa, N., Hildebrande- J., Tischler, A. & Minna, J. D. (1985) Nature (London) 316, 823–826. Zanki, S. U., Morton, D. L., Korn, E. L. & Kern, D. H. (1984) 3. Said, S. I. & Mutt, V. (1970) Science 169, 1217–1218. Cancer Res. 44, 1725–1728. 4. Luis, J. & Said, S. I. (1990) Peptides (Tarrytown, NY) 11, 25. Nagy, A., Schally, A. V., Halmos, G., Armatis, P., Cai, R.-Z., 1239–1244. Csernus, V., Kova´cs,M., Koppa´n,M., Szepesha´zi,K. & Kaha´n, 5. Maruno, K. & Said, S. I. (1993) Life Sci. 52, PL267–PL271. Z. (1998) Proc. Natl. Acad. Sci. USA 95, 1794–1799. 6. Korman, L. Y, Carney, D. N., Citron, M. L. & Moody, T. W. 26. Sreedharan, S. P., Huang, J.-X., Cheung, M.-C. & Goetzl, E. J. (1986) Cancer Res. 46, 1214–1218. (1995) Proc. Natl. Acad. Sci. USA 92, 2939–2943. 7. Gazdar, A. F., Carney, D. N., Russell, E. K., Sims, H. L., Baylin, 27. Virgolini, I., Raderer, M., Kurtaran, A., Angelberger, P., Banyai, S. B., Bunn, P. A., Jr., Guccion, J. G. & Minna, J. D. (1980) S., Yang, Q., Li, S., Banyai, M., Pidlich, J., Niederle, B., et al. Cancer Res. 40, 3502–3507. (1994) N. Engl. J. Med. 331, 1116–1121. 8. Carney, D. N., Gazdar, A. F., Bepler, G., Guccion, J. G., 28. Brauch, H., Johnson, B., Hovis, J., Yano, T., Gazdar, A., Pet- Marangos, P. J., Moody, T. W., Zweig, M. H. & Minna, J. D. tengill, O. S., Graziano, S., Sorenson, G. D., Poiesz, B. J., Minna, (1985) Cancer Res. 45, 2913–2923. J., et al. (1987) N. Engl. J. Med. 317, 1109–1113. 9. Simms, E., Gazdar, A. F., Abrams, P. G. & Minna, J. D. (1980) 29. Naylor, S. L., Johnson, B. E., Minna, J. D. & Sakaguchi, A. Y. Cancer Res. 40, 4356–4363. (1987) Nature (London) 329, 451–456. 10. Shaffer, M. M., Carney, D. N., Korman, L. Y., Lebovic, G. S. & 30. Killary, A. M., Wolf, M. E., Giambernardi, T. A. & Naylor, S. L. Moody, T. W. (1987) Peptides (Tarrytown, NY) 8, 1101–1106. (1992) Proc. Natl. Acad. Sci. USA 89, 10877–10881. Downloaded by guest on September 29, 2021