CI-988 Inhibits Growth of Small Cell Lung Cancer Cells

  1. Terry W. Moody1 and
  2. Robert T. Jensen2
  1. 1Department of Cell and Cancer Biology, Medicine Branch, National Cancer Institute, Rockville, Maryland (T.W.M.); and 2Digestive Disease Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland (R.T.J.).
  1. Dr. Terry W. Moody, National Cancer Institute, Medicine Branch, Bldg. KWC, Rm. 300, 9610 Medical Center Dr., Rockville, MD 20850. E-mail:moodyt{at}bprb.nci.nih.gov
 
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Abstract

The effects of cholecystokinin (CCK) antagonists on small cell lung cancer (SCLC) cells were investigated. CI-988, L-365,260, and L-364,718 inhibited specific 125I-CCK-8 binding to NCI-H209 cells with IC50 values of 5, 2, and 200 nM. ([R-(R*,R*)]-4[[2-[[3-(1H-Indole-3-yl)-2-methyl-1-oxo-2-[[tricyclo[3.3.1.13,7]- dec-2-yloxy)carbonyl[amino]propyl]amino]-1-phenylethyl]amino]-4-oxobutanoic acid) (CI-988; 100 nM) inhibited the ability of 10 nM CCK-8 to elevate cytosolic Ca2+ in 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester-loaded NCI-H209 cells. By Western blot, CI-988 inhibited tyrosine phosphorylation of focal adhesion kinase and paxillin stimulated by CCK-8. Also, CI-988 inhibited tyrosine phosphorylation of mitogen-activated protein kinase stimulated by CCK-8. By Northern blot, CI-988 antagonized the ability of 10 nM CCK-8 to increase c-fos mRNA in NCI-H209 cells. Also, CI-988 inhibited the ability of CCK-8 to increase vascular endothelial cell growth factor mRNA. Using a [3-(4,5 dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide] and clonogenic assay, CI-988 inhibited the proliferation of NCI-H209 cells in vitro. Using nude mice, CI-988 inhibited the proliferation of NCI-H209 xenografts. These results suggest that CI-988 is a CCK2 receptor antagonist that inhibits the proliferation of SCLC cells.

Cholecystokinin (CCK) is biologically active in the central nervous system and periphery (Mutt and Jorpes, 1971). In the central nervous system, CCK alters neuronal activity, alters behavior, and causes satiety (Gibbs et al., 1973;Crawley 1988; Wang et al., 1988). In the periphery, CCK is localized to intrinsic neurons of the brain, myenteric plexus, and endocrine cells (Buchan et al., 1978; Beinfeld et al., 1989). When secreted, CCK has potent effects on the gallbladder, pancreas, and stomach (Jensen et al., 1980; Steigerwalt et al., 1984; Moran et al., 1985). CCK is derived from a 115 amino acid precursor protein to 58, 39, 33, and 8 amino acid forms, which have a sulfated tyrosine (Dockray, 1977;Takahashi et al., 1985; Chang and Lotti, 1986). CCK1 receptors, which are present in the pancreas, certain brain regions, and gallbladder, bind sulfated but not nonsulfated forms of CCK as well as the antagonist L-364,718 with high affinity (Makovec et al., 1985; Chang et al., 1989;Freidinger, 1989; Jensen et al., 1989). CCK2receptors, which are widely distributed in the central nervous system, bind gastrin, L-365,260, as well as sulfated and nonsulfated CCK with high affinity (Innes and Snyder, 1980; Saito et al., 1980; Lotti and Chang, 1984).

The CCK2 receptor is a 390 amino acid protein that is a member of the heptahelical superfamily (Wank, 1995). It interacts with G proteins such as Gq causing phosphatidylinositol turnover and G12,13 causing focal adhesion kinase tyrosine phosphorylation (Sethi and Rozengurt, 1991). Focal adhesion kinase interacts with adaptor proteins such as paxillin causing cytoskeletal reorganization, whereas phosphatidylinositol is metabolized to inositol-1,4,5-trisphosphate and diacylglycerol. The inositol-1,4,5-trisphosphate causes release of Ca2+ from intracellular organelles such as the endoplasmic reticulum, whereas the diacylglycerol activates protein kinase C. Protein kinase C phosphorylates protein substrates such as mitogen-activated protein kinase kinase, which phosphorylates mitogen-activated protein kinase (Whitmarsh and Davies, 1996). Phosphorylated mitogen-activated protein kinase enters the nucleus, leading to increased expression of the nuclear oncogene c-fos. After translation of the mRNA, c-fos protein forms heterodimers with c-jun binding to activating protein-1 (AP-1) sites in growth factor genes.

Previously, we found that CCK-8 bound with high affinity to SCLC cells and caused elevation of cytosolic Ca2+ (Yoder and Moody, 1987; Staley et al., 1990). CCK-8 or gastrin increased cytosolic Ca2+ and stimulated SCLC growth (Staley et al., 1990). The increase in cytosolic Ca2+ caused by CCK-8 or gastrin was blocked by L-365,260 (Staley et al., 1990). In the present study we report that CCK-8 increases mitogen-activated protein kinase and focal adhesion kinase tyrosine phosphorylation, and the effects of CCK-8 were antagonized by the CCK2receptor antagonist ([R-(R*,R*)]-4[[2-[[3-(1H-indole-3-yl)-2-methyl-1-oxo-2-[[tricyclo[3.3.1.13,7]dec-2-yloxy)carbonyl[amino]propyl]amino]-1-phenylethyl]amino]-4-oxobutanoic acid) (CI-988; PD134308) (Hughes et al., 1990). Also, CCK-8 increased c-fos and VEGF mRNA and the increase caused by CCK-8 was inhibited by CI-988. CCK-8 increased SCLC proliferation and the effects of CCK-8 were inhibited by CI-988. Also, CI-988 inhibited basal SCLC growth in vitro and in vivo. These results suggest that biologically active CCK2 receptors that are present in SCLC cells are coupled to multiple second messenger cascades whose activation results in proliferation.

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Materials and Methods

Cell Culture.

NCI-H82, H209, and H345 cells were cultured in RPMI-1640 containing 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) (Carney et al., 1985). The cells, which were nonadherent, were split weekly by 1:1 dilution. The cells were mycoplasma free and were used when they were in exponential growth phase after incubation at 37°C in 5% CO2, 95% air.

Receptor Binding.

The ability of a CCK1 receptor antagonist, L-364,718, and two CCK2 receptor antagonists, L-365,260 and CI-988, to inhibit specific 125I-CCK-8 binding to SCLC cells was investigated (Yoder and Moody, 1987). NCI-H209 or NCI-H345 cells (106) were incubated with125I-CCK-8 (2200 Ci/mmol; PerkinElmer Life Sciences, Boston, MA) in 100 μl of SIT medium (RPMI-1640 containing 3 × 10−8 M sodium selenite, 5 μg/ml bovine insulin, and 10 μg/ml transferrin) with 0.25% bovine serum albumin and 250 μg/ml bacitracin. After incubation at 25°C for 60 min, free 125I-CCK-8 was removed and the cells that contained bound 125I-CCK-8 were counted in an LKB gamma counter.

Cytosolic Ca2+.

Previously, we found that CCK caused elevation of cytosolic Ca2+ in NCI-H209 cells, which was antagonized by L-365,260 (Staley et al., 1990). NCI-H209 cells were harvested (2.5 × 106/ml) and incubated with 5 μM Fura-2 AM at 37°C for 30 min in SIT medium. The cells, which contained loaded Fura-2 (Calbiochem, La Jolla, CA), were centrifuged at 1500gfor 10 min and resuspended at the same concentration in new SIT medium containing 20 mM HEPES · NaOH, pH 7.0. The fluorescence intensity was continuously monitored using a PerkinElmer LS2 spectrofluorometer equipped with a magnetic stirring mechanism and temperature (37°C)-regulated cuvette holder before and after the addition of CCK-8 in the presence or absence of CI-988, and the Ca2+ concentrations were calculated as described (Staley et al., 1990).

Western Blot.

NCI-H209 cells were put in SIT containing 0.5% FBS overnight. Three hours before treatment cells were preincubated in ST media (RPM1-1640 containing 3 × 10−8 M Se2O3 and 10 ng/ml transferrin). For the focal adhesion kinase and paxillin assays, cells were treated with CCK-8 in the presence or absence of CI-988 for 1 min, washed with saline, lysed with 1 ml of buffer containing 50 mM Tris/HCl, pH 7.5; 150 mM NaCl; 1% (w/v) Triton X-100; 1% (w/v) deoxycholate; 1% (w/v) NaN3; 1 mM EGTA; 0.4 mM EDTA; 2.5 μg/ml aprotinin; 2.5 μg/ml leupeptin; 1 mM phenylmethylsulfonyl fluoride; and 0.2 mM Na3VO4 (Sigma Chemical, St. Louis, MO), sonicated for 5 s at 4°C, and centrifuged at 10,000g for 15 min. Protein concentration was measured by Bio-Rad protein assay reagent, and the volume was adjusted such that the cell lysates contained the same amount of protein (150 μg/ml). The lysates were incubated with 4 μg of anti-phosphotyrosine monoclonal antibody (PY20), 4 μg of goat anti-mouse IgG, and 30 μl of protein A-agarose overnight at 4°C.

Immunoprecipitates were fractionated by SDS-polyacrylamide gel electrophoresis by using 10% polyacrylamide gels (Novex, Carlsbad, CA), and proteins were transferred to nitrocellulose membranes. Membranes were blocked overnight at 4°C by using blotto [5% nonfat dried milk in 50 mM Tris/HCl, pH 8.0; 2 mM CaCl2; 80 mM NaCl; 0.05% (v/v) Tween 20; and 0.02% (w/v) NaN3] and incubated for 2 h at 25°C with 1 μg/ml focal adhesion kinase monoclonal antibody or 1 μg/ml paxillin monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). The membranes were washed twice for 10 min each with blotto and incubated for 40 min at 25°C with anti-mouse IgG-horseradish peroxidase conjugate. The membrane was washed for 10 min with blotto, followed by two 10-min treatments with washing solution [50 mM Tris/HCl, pH 8.0; 2 mM CaCl2; 80 mM NaCl; 0.05% (v/v) Tween 20; and 0.02% (w/v) NaN3], incubated with enhanced chemiluminescence detection reagent for 5 min, and exposed to Hyperfilm ECL (Amersham Pharmacia Biotech, Arlington Heights, IL). The density of bands on the film was measured using a scanning densitometer (Leyton et al., 2001).

The ability of CCK-8 to phosphorylate mitogen-activated protein kinase was investigated (Weber et al., 2001). NCI-H209 cells were cultured in 15-ml flasks. Cells were placed in SIT media containing 0.5% FBS overnight. Three hours before treatment cells were placed in fresh ST media. Cells were treated with CCK-8 for 2 min in the presence or absence of CI-988. Cells were lysed as described above and 150 μg/ml protein lysate incubated with 4 μg of mitogen-activated protein kinase antibody (Upstate Biotechnology), 4 μg of goat anti-rabbit IgG, and 30 μl of protein A-agarose (Sigma Chemical) overnight at 4°C. The immunoprecipitates were washed three times with phosphate-buffered saline and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Membranes were blocked overnight at 4°C by using blotto as described above and treated with anti-phospho mitogen-activated protein kinase antibody and incubated for 2 h at 25°C with anti-mouse IgG-horseradish peroxidase conjugate. The membrane was washed for 10 min with blotto and twice for 10 min with washing solution (50 mM Tris/HCl, pH 8.0; 2 mM CaCl2; 80 mM NaCl; 0.05% Tween 20; and 0.02% NaN3) (Sigma Chemical), and the blot was incubated with enhanced chemiluminescence detection reagent for 5 min and exposed to Hyperfilm ECL.

Northern Blot.

The ability of CCK to stimulate nuclear oncogene expression was investigated (Weber et al., 2001). For the c-fos experiments, NCI-H209 cells were cultured with SIT medium containing 0.5% fetal bovine serum. After 1 h, the cells were treated with 10 nM CCK-8 in the presence or absence of CI-988 for 60 min. Total RNA was isolated using guanidinium isothiocyanate (Fluka, Buchs, Switzerland). Ten micrograms of denatured RNA was separated in a 0.66 M formaldehyde 1% agarose gel. The gel was treated with ethidium bromide to assess RNA integrity. The RNA was blotted onto a nytran membrane overnight and the membrane hybridized with DNA probes labeled with [32P]dCTP by using an Invitrogen random priming kit. The membrane was exposed to Kodak XAR-2 film at −80°C for 1 day and the autoradiogram developed. The autoradiograms were analyzed using a Molecular Dynamics (Sunnyvale, CA) densitometer. For the VEGF experiments, the reaction was stopped 8 h after addition of CCK-8 (Casibang et al., 2001).

Proliferation Assays.

The ability of CI-988 to alter the growth of SCLC cells was investigated in vitro and in vivo by using (4,5 dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide] (MTT) colorimetric assays. SCLC cells (104/well) were placed in SIT medium and varying concentrations of CI-988 added. After 4 days, MTT (Sigma Chemical) was added. After 4 h, 150 μl of dimethyl sulfoxide was added. After 16 h, the optical density at 540 nm was determined using an enzyme-linked immunosorbent assay reader. Also, the effects of CI-988 were investigated using a clonogenic assay (Mahmoud et al., 1991). The base layer consisted of 3 ml of 0.5% agarose (FMC Bioproducts, Rockland, ME) in SIT medium containing 5% fetal bovine serum in six-well plates. The top layer consisted of 3 ml of SIT medium in 0.3% agarose, CI-988, and 5 × 104 lung cancer cells. Triplicate wells were plated and after 2 weeks, 1 ml of 0.1%p-iodonitrotetrazolium violet was added and after 16 h at 37°C, the plates were screened for colony formation. The number of colonies larger than 50 μm in diameter were counted using an Omnicon (Fairfax, VA) image analysis system.

The ability of the CI-988 to inhibit SCLC growth was also investigated in vivo. Female athymic Balb/c nude mice, 4 to 5 weeks old, were housed in a pathogen-free, temperature-controlled isolation room and the diet consisted of autoclaved rodent chow and autoclaved water given ad libitum. NCI-H209 cells (1 × 107) were injected into the right flank of each mouse by subcutaneous injection. Palpable tumors were observed in approximately 90% of the mice after 1 week. CI-988, 10 μg/0.1 ml in polyethylene glycol-400 (Aldrich Chemical, Milwaukee, WI), was administered by gavage during weeks 2 through 5. The tumor volume (height × width × depth) was determined weekly by calipers and recorded. When the tumor became necrotic (>2000 mm3 in volume), the growth studies were terminated.

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Results

CI-988 Binds with High Affinity to SCLC Cells.

The binding of CCK receptor antagonists was investigated. Figure1 shows that 0.1 nM CI-988 had little effect on 125I-CCK-8 binding to NCI-H209 cells, whereas 100 nM CI-988 inhibited almost all specific125I-CCK-8 binding. The IC50 was 5 nM for CI-988 and 2 nM for CCK-8. Table 1 shows that L-365,260, CI-988, and L-364,718 inhibited specific 125I-CCK-8 binding to NCI-H209 cells with IC50 values of 2, 5, and 200 nM. Similar data were obtained using NCI-H345 cells. These results indicate that CCK-8, L-365,260, and CI-988 bind with high affinity to SCLC cells.

Figure 1
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Figure 1

CCK-8 binding. The ability of CI-998 (○) and CCK-8 (●) to inhibit specific binding of 125I-CCK-8 to NCI-H209 cells was determined. The mean value ± S.D. of three determinations each repeated in quadruplicate is indicated.

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Table 1

CCK antagonist binding

CI-988 Is an SCLC CCK2 Receptor Antagonist.

The ability of CI-988 to antagonize the actions of CCK was investigated. In a cytosolic Ca2+ assay, 10 nM CCK-8 increased the cytosolic Ca2+ in Fura-2 AM-loaded NCI-H209 cells from 150 to 180 nM (Fig. 2) within 15 s. The response to CCK-8 slowly declined and returned to baseline after 2 min. CI-988 (10 nM) and 100 nM CI-988 weakly and strongly antagonized the increase in cytosolic Ca2+ caused by CCK-8. These results indicate that CI-988 antagonizes the actions of CCK-8 in a concentration-dependent manner. Similar results were obtained using NCI-H345 cells (data not shown).

Figure 2
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Figure 2

Cytosolic Ca2+. The ability of 10 nM CCK-8 to cause increased cytosolic Ca2+ was determined in the absence or presence of 10 or 100 nM CI-988 by using Fura-2 AM-loaded NCI-H209 cells. This experiment is representative of three others.

CI-988 antagonized the tyrosine phosphorylation of mitogen-activated protein kinase caused by CCK-8. Figure3 shows that 2 min after the addition of 10 nM CCK-8 to NCI-H209 cells, phosphorylation of the 42- and 44-kDa forms of mitogen-activated protein kinase increased 3-fold. The response to CCK-8 slowly declined returning to baseline after 10 min (data not shown). The transient increase in mitogen-activated protein kinase tyrosine phosphorylation caused by CCK-8 was inhibited if 100 nM but not 10 nM CI-988 was present.

Figure 3
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Figure 3

Mitogen-activated protein kinase and CI-988. The ability of 10 nM CCK-8 to cause tyrosine phosphorylation of mitogen-activated protein kinase was determined after 2 min in the absence or presence of 10 or 100 nM CI-988. This experiment is representative of four others.

CCK-8 increased focal adhesion kinase tyrosine phosphorylation 1 min after addition to NCI-H209 cells (Fig.4). CCK-8 (100 nM) increased focal adhesion kinase tyrosine phosphorylation 2-fold and the response returned to baseline after 20 min (data not shown). CI-988 (1000 nM) had little effect of basal focal adhesion kinase tyrosine phosphorylation but antagonized the increase in focal adhesion kinase tyrosine phosphorylation caused by CCK-8. Similarly, CCK-8 caused increased tyrosine phosphorylation of paxillin and the effects of CCK-8 were antagonized by CI-988.

Figure 4
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Figure 4

Focal adhesion kinase (FAK) and CI-988. The ability of 100 nM CCK-8 to cause tyrosine phosphorylation of FAK and paxillin was determined after 1 min in the absence or presence of 1000 nM CI-988. This experiment is representative of four others.

CI-988 inhibited gene expression induced by CCK-8. Figure5, top, shows that 10 nM CCK-8 increased c-fos mRNA 2-fold 1 h after addition to NCI-H209 cells. One hundred nanomolar but not 10 nM CI-988 inhibited the increase in c-fos mRNA caused by 10 nM CCK-8. Equal amounts of RNA were loaded onto the gel based on the Northern blot analysis of the housekeeping gene actin (Fig. 5, bottom). Also, 10 nM CCK-8 increased VEGF mRNA 2-fold after 8 h. Figure 6, top, shows that 100 nM CI-988 inhibited the increase in VEGF mRNA caused by CCK-8. Equal amounts of RNA were loaded onto the gel based on ethidium bromide staining of the 18S and 28S rRNA bands (Fig. 6, bottom).

Figure 5
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Figure 5

C-Fos mRNA and CI-988. Top, ability of 10 nM CCK-8 to cause elevated c-fos mRNA was determined after 60 min in the absence or presence of 100 nM CI-988. Bottom, equal amounts of actin mRNA were loaded onto the gel based on Northern analysis. This experiment is representative of three others.

Figure 6
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Figure 6

VEGF mRNA. Top, ability of 10 nM CCK-8 to cause elevated VEGF mRNA was determined after 8 h in the absence or presence of 100 nM CI-988. Bottom, equal amounts of mRNA were loaded onto the gel based on ethidium bromide staining of the 28S and 18S rRNA bands. This experiment is representative of three others.

CI-988 Inhibits Proliferation of SCLC.

Figure7 shows that 3000 nM but not 100 or 300 nM CI-988 significantly inhibited the proliferation of NCI-H82, H209, or H345 cells in vitro using the MTT assay. Also, 1000 nM CI-988 significantly inhibited the proliferation of NCI-H82 cells. Table2 shows that 1000 nM CI-988 or L-365,260 but not L-364,718 significantly inhibited NCI-H209 proliferation in the clonogenic assay. In contrast, 10 nM CCK-8 increased colony number 2-fold. CI-988 (1000 nM) reversed the increase in colony number caused by CCK-8.

Figure 7
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Figure 7

MTT assay. Ability of CI-988 as a function of concentration to inhibit NCI-H82 (○), H345 (●), and H209 (▴) proliferation was determined after 3 days. The mean value ± S.D. of eight determinations is indicated; p < 0.05 using Student's t test. ∗, experiment is representative of two others.

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Table 2

Clonogenic assay

CI-988 inhibited SCLC proliferation in vivo. Figure8 shows that after 2 weeks, NCI-H209 xenografts formed in nude mice. The tumors grew exponentially in the absence of additions achieving a mean volume of 1713 mm3 after 5 weeks. If the animals were treated with CI-988 (10 μg/day), however, xenograft proliferation was dramatically slowed and the tumor volume was only 49 mm3 after 5 weeks.

Figure 8
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Figure 8

Xenograft proliferation. The ability of CI-988 to inhibit NCI-H209 xenograft proliferation in nude mice was determined in the absence (●) or presence (○) of CI-988 (10 μg by gavage daily in polyethylene glycol-400). The mean value ± S.D. of four determinations is indicated; p < 0.01 using Student's t test. ∗∗, experiment is representative of two others.

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Discussion

SCLC cells have CCK2 receptors. CCK-8 and gastrin inhibit 125I-CCK-8 binding to NCI-H209 cells with high affinity (IC50 = 2 and 6 nM, respectively; Yoder and Moody, 1987). CCK-8 elevates cytosolic Ca2+ and the increase caused by CCK-8 was inhibited by L-365,260 but not L-364,718 (Staley et al., 1990). Here the effects of the potent CCK2 receptor antagonist CI-988 were investigated on SCLC cells.

CI-988 inhibited specific 125I-CCK-8 binding to NCI-H209 cells with high affinity (IC50 = 5 nM). Similarly, L-365,260 (IC50 = 2 nM) but not L-364,718 (IC50 = 200 nM) inhibited125I-CCK-8 binding to NCI-H209 cells with high affinity. Using Chinese hamster ovary cells transfected with CCK2 receptors, L-365,260 and CI-988 had similar potency to inhibit the ability of CCK to elevate cytosolic Ca2+ (Kopin et al., 2000). These results suggest that CCK2 receptors are present on NCI-H209 cells. Surprisingly, L-365,260 binds with 20-fold lower affinity to canine CCK2 than human CCK2receptors. Subsequent studies showed that if canine Leu355 was changed to Val, L-365,260 bound with high affinity to the CCK2 receptors (Dunlop et al., 1996). These results indicate that species polymorphisms can occur in CCK2 receptors, which affect receptor antagonist binding. In contrast, agonists such as gastrin and CCK-8 bind with high affinity to both human and canine CCK2 receptors. These results suggest that CCK agonists and antagonists bind to different sites on CCK2 receptors.

After addition of CCK to NCI-H209 cells Gq may be activated, resulting in phosphatidylinositol turnover (Sethi and Rozengurt, 1991). The resulting inositol 1,4,5-trisphosphate causes release of Ca2+ from intracellular organelles such as the endoplasmic reticulum. Previously, we found that nanomolar concentrations of CCK-8 half maximally increased cytosolic Ca2+ in Fura-2 AM-loaded NCI-H209 cells within 15 s. Because the Kd for CCK-8 binding to NCI-H209 cells is 2 nM, occupation of approximately 50% of the CCK2 receptors causes a strong Ca2+ response, whereas no response was observed using 0.1 nM CCK-8. Here 10 nM CCK-8 caused a strong Ca2+ response. Because an order of magnitude more CCK-8 was used relative to the Kd, 10 nM CI-988 had little effect, whereas 100 nM CI-988 blocked the increase in cytosolic Ca2+ caused by CCK-8. Thus, it is necessary to use approximately 1 order of magnitude more CI-988 than CCK-8 to block SCLC CCK2 receptors.

When phosphatidylinositol is metabolized, diacylglycerol is released, which activates protein kinase C. Protein kinase C may phosphorylate protein substrates such as mitogen-activated protein kinase kinase, which in turn phosphorylates mitogen-activated protein kinase. CI-988 (100 nM) had little effect on basal mitogen-activated protein kinase phosphorylation but antagonized the increase in tyrosine phosphorylation of the 42- and 44-kDa bands caused by 10 nM CCK-8 after 2 min. Phosphorylated mitogen-activated protein kinase may enter the nucleus of SCLC cells and alter gene expression.

Ten nanomolar CCK-8 increased the c-fos mRNA 2-fold in NCI-H209 cells after 1 h. CI-988 (100 nM) had little effect on basal c-fos mRNA but antagonized the increase in c-fos mRNA caused by CCK-8. Preliminary data (T. Moody, unpublished data) indicate that CCK-8 increased c-jun mRNA. The c-fos and c-jun proteins may form heterodimers and activate AP-1 sites in growth factor genes. Previously, we showed that agents that cause elevated cAMP, such as prostaglandin E2 and vasoactive intestinal peptide, increased VEGF mRNA in lung cancer cells. Also, prostaglandin E2 and vasoactive intestinal peptide increase secretion of VEGF from lung cancer cells (Casibang et al., 2001). The VEGF may diffuse and activate receptors present on endothelial cells, facilitating angiogenesis of lung cancer tumors. The increase in VEGF mRNA after 8 h caused by 10 nM CCK-8 was antagonized by 100 nM CI-988. Preliminary data (T. Moody, unpublished data) indicate that 10 nM CCK-4 had little effect on VEGF mRNA; CCK-4 is a weak agonist for CCK2 receptors, which binds with approximately 2 orders of magnitude lower affinity than does CCK-8. It remains to be determined whether CI-988 inhibits angiogenesis of lung cancer tumors.

CCK-8 (100 nM) caused increased tyrosine phosphorylation of focal adhesion kinase and paxillin 1 min after addition to NCI-H209 cells. The samples were immunoprecipated using an anti-phosphotyrosine antibody and blotted using anti-focal adhesion kinase and anti-paxillin antibodies, which recognized both phosphorylated and unphosphorylated forms of the protein. Preliminary data (T. Moody, unpublished data) indicate that the increases in p125FAK and paxillin tyrosine phosphorylation caused by CCK-8 were accompanied by reorganization of the actin cytoskeleton and by the assembly of SCLC focal adhesion plaques where both p125FAK and paxillin are localized.

CI-988 inhibited the proliferation of SCLC cells. In the MTT assay, CI-988 at 3000 nM significantly inhibited the proliferation of NCI-H82, H209, and H345 cells. In the clonogenic assay 10 nM CCK-8 stimulated the proliferation of NCI-H209 cells, which was antagonized by 1000 nM CI-988. The clonogenic assay may be more sensitive to CI-988 than the MTT assay due to the fact that the NCI-H209 cells are exposed to the CI-988 for 2 weeks in the clonogenic assay as apposed to 4 days in the MTT assay. These results indicate that CI-988 is a CCK2 receptor antagonist whose action can be reversed by addition of exogenous CCK-8. Also, pancreatic cancer has CCK2 receptors that are antagonized by CI-988; however, CI-988 caused gastric gland degeneration and mucosal atrophy consistent with its role of stimulating stomach mucosal proliferation (Smith and Watson, 2000). Also, CI-988 and L-365,260 but not L-364,718 inhibited the clonal growth of NCI-H209 cells. These results suggest that SCLC cells may synthesize and secrete endogenous CCK-8-like peptides that function as autocrine growth factors. Previously, we did not detect CCK-8-like immunoreactivity in extracts derived from SCLC cells (Yoder and Moody, 1987). Gastrin-like peptides have been detected in colon carcinoma cells (Dockray, 1977), however, and it remains to be determined whether SCLC cells make gastrin.

In peripheral cancers, gastrin expression is an early event in the adenoma-carcinoma sequence. Normal colonic mucosa does not express gastrin or CCK2 receptors, whereas 78 and 81% of polyps expressed gastrin and CCK2-R immunoreactivity. In particular, 91% of the human specimens had progastrin immunoreactivity, 80% had glycine extended gastrin, 47% had amidated gastrin (Hellmich et al., 2000), and 96% of the polyps had CCK2 receptors. Also, colonic cancer has a splice variant of the CCK2 receptor that is constitutively active (Goetze et al., 2000). In pancreatic cancer, amidated gastrin was detected in 75% of the specimens, whereas CCK2 receptors were detected in 100% of the specimens (Caplin et al., 2000). Normal pancreas had little detectable gastrin or CCK2 receptors (Dethloff et al., 1999). CCK-8 and amidated gastrin stimulated but L-365–260 inhibited the growth of many pancreatic cancer cell lines (Ohlsson et al., 1999). These results indicate that CCK2 receptors may regulate the growth of colon and pancreatic cancer cells.

CI-988 inhibited the growth of NCI-H209 tumors in nude mice. Preliminary data (T. Moody, unpublished data) indicate the 10 μg/day but not 0.1 μg/day CI-988 in polyethylene glycol-400 by gavage inhibited NCI-H209 xenograft proliferation. Although CI-988 slowed xenograft proliferation by over 95%, tumor regression was not observed. The effects of CI-988 were reversible in that if CI-988 administration was discontinued the xenografts rapidly grew. These results suggest that CI-988 can be administered orally and absorbed from the gastrointestinal tract into the blood. Because CI-988 is an organic chemical, it is not readily degraded by stomach enzymes. Previously, it was found that i.p. administration of 1 mg/kg CI-988 prevented morphine tolerance in rats, enhanced the locomotor response to phencyclidine and dizocilpine maleate, and enhanced the reflex-depressive effect of morphine in axotomized rats (Xu et al., 1992, 1994). It remains to be determined whether CI-988 crosses the blood-brain barrier. Because the body weight of animals treated with CI-988 was similar to that of the control, whereas tumor weight was significantly reduced, there was little evidence of CI-988 toxicity in the nude mice bearing NCI-H209 xenografts (T. Moody, unpublished data).

In summary, CI-988 is a CCK2 receptor antagonist for SCLC cells. It remains to be determined whether CI-988 will be a therapeutic agent for lung cancer.

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Acknowledgments

We thank A. Guzzone and M. Casibang for technical assistance and the Parke-Davis Neuroscience Research Center (Cambridge, UK) for the CI-988.

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Footnotes

  • Abbreviations:
    CCK
    cholecystokinin
    SCLC
    small cell lung cancer
    CI-988
    ([R-(R*,R*)]-4[[2-[[3-(1H-indole-3-yl)-2-methyl-1-oxo-2-[[tricyclo[3.3.1.13,7]dec-2-yloxy)carbonyl[amino]propyl]amino]-1-phenylethyl]amino]-4-oxobutanoic acid)
    VEGF
    vascular endothelial cell growth factor
    FBS
    fetal bovine serum
    Fura-2 AM
    1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester
    MTT
    [3-(4,5 dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide]
    L-364,718
    3S(−)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl)-1H-indole-2 carboxamide
    L-365,260
    3R-(+)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N′-(3-methylphenyl) urea
    • Received June 19, 2001.
    • Accepted September 5, 2001.
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References

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