Communicated by Martin Lindauer, University of Würzburg, Würzburg, Germany, September 2, 2005 (received for review April 5, 2005)
Glutamate antagonists limit the growth of human cancers in vitro. The mechanism of anticancer action of NMDA antagonists is not known, however. In this article, we report that the NMDA antagonist dizocilpine inhibits the extracellular signal-regulated kinase 1/2 pathway, an intracellular signaling cascade that is activated by growth factors and controls the proliferation of cancer cells. Dizocilpine reduces the phosphorylation of cAMP-responsive element binding protein, suppresses the expression of cyclin D1, up-regulates the cell cycle regulators and tumor suppressor proteins p21 and p53, and increases the number of lung adenocarcinoma cells in the G2 and S phases of the cell cycle. Silencing of the tumor suppressor protein p21 abolishes antiproliferative action of dizocilpine. Consistent with inhibition of the extracellular signal-regulated kinase 1/2-signaling cascade, dizocilpine reverses the stimulation of proliferation induced by epidermal, insulin, and basic fibroblast growth factors in lung adenocarcinoma cells. Furthermore, dizocilpine prolongs the survival of mice with metastatic lung adenocarcinoma and slows the growth of neuroblastoma and rhabdomyosarcoma in mice. These findings reveal the mechanism of antiproliferative action of dizocilpine and indicate that it may be useful in the therapy of human cancers.
One of every three people in the world contracts cancer; of those individuals, one in four die. Advances in cancer management with chemotherapy, bone marrow transplantation, radiotherapy, and surgery have had only a modest effect on mortality, and therefore, the search for chemotherapeutics for cancer continues (1). Anticancer drugs are cytotoxic and interfere with synthesis of DNA and cell division. Deregulated growth of cancers, however, may arise from mutations within the pathways that promote survival (2, 3).
Glutamate regulates the proliferation, migration, and survival of neuronal progenitors and immature neurons during development (4, 5). Glutamate activates ionotropic and metabotropic receptors. The ionotropic glutamate NMDA receptors assemble from subsets of two subunits, NR1 and NR2. The NR1 subunit imparts on heteromeric NMDA channels ion permeability, whereas the NR2 subunits A, B, C, and D determine electrophysiological properties of the channel (6–8). NMDA receptors are expressed both in neurons and in nonneuronal tissues (6). Activation of glutamate NMDA receptors in neurons is translated to the nucleus by the extracellular signal-regulated kinase (ERK1/2)-signaling cascade leading to the phosphorylation of the cAMP-responsive element binding protein (CREB) and activation of genes promoting survival (9–12).
Glutamate NMDA antagonists block the channel function in a competitive or noncompetitive manner. Dizocilpine blocks NMDA receptor-associated channels in a noncompetitive manner regardless of the subunit composition of the channel (8).
We determined that dizocilpine inhibits the ERK1/2 pathway in cancer cells and reduces the phosphorylation of CREB and expression of CREB-regulated genes. These actions translate into the slowing of cell cycle progression and proliferation of cancer cells in vitro and achieve an anticancer effect in mice in vivo.
Cell Culture. Human Caucasian lung carcinoma A549 cells (Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland) were incubated with (+)-dizocilpine (10–500 μM; Sigma). Cells were exposed for 3 h to 10 μM U0126 and 200 nM phorbol 12-myristate 13-acetate or for 6 h to 200 nM wortmannin, 50 μM KN62 (Calbiochem), insulin growth factor (IGF; 100 ng/ml; Sigma), epidermal growth factor (EGF; 10 ng/ml; Sigma), basic fibroblast growth factor (bFGF; 10 ng/ml; Sigma), or NMDA (10 μM; Tocris Cookson, Bristol, U.K.). Human neuroblastoma (SKNAS) and human rhabdomyosarcoma/medulloblastoma (TE671) cells were obtained from the European Collection of Cell Cultures (ECACC; Center for Applied Microbiology and Research, Salisbury, U.K.). SKNAS and TE671 cells were grown in a 1:1 mixture of DMEM (D 6421, Sigma; 1.05 mM Ca2+ concentration) and a nutrient mixture (Ham's F-12 medium, Sigma) supplemented with 10% FBS (GIBCO). For in vivo experiments, (+)-dizocilpine was chosen because of its high efficacy and selectivity as a noncompetitive NMDA antagonist, its safety, and its pharmacokinetic properties (bioavailability and half-life time) allowing for two daily systemic administrations in rodents (13). The plasma concentrations of (+)-dizocilpine in in vivo experiments are lower than those applied in in vitro settings; however, the exposure of the tumor cells to (+)-dizocilpine is longer in vivo.
Immunofluorescence and Cell Staining. A549 cells were grown on coverslips, incubated with 250 μM dizocilpine for 3 h, fixed with 100% methanol (–20°C, 5 min), and probed with rabbit anti-phospho-ERK1/2 antibody (1:300 in 5% BSA/Tris-buffered saline containing Tween 20; Cell Signaling Technology, Beverly, MA) or with rabbit anti-NR1 antibody (2.0 μg/ml in 1% BSA/PBS; Chemicon International, Temecula, CA) and anti-NR2B antibody (1:250 in 1% BSA/PBS; Santa Cruz Biotechnology), followed by incubation with chicken anti-rabbit Alexa Fluor 594 secondary antibody (5 μg/ml in 1% BSA/TBS; Molecular Probes). Images were collected by using a two-photon confocal laser-scanning microscope (Leica, Vienna). Alexa Fluor 594 was excited by a mode-locked Ti/sapphire laser (Tsunami, Spectra-Physics) at wavelength 770 nm.
Flow Cytometry. A FACScan flow cytometer (Becton Dickinson) equipped with a 488-nm argon laser was used. Cells were stained with propidium iodide by using the CycleTEST PLUS DNA reagent kit (BD Pharmingen). Cell cycle analysis was performed by using noncommercial flow cytometry analyzing software (cylchred 1.0.2 for Windows; Department of Hematology, Cardiff University, Wales, U.K.) and winmdi 2.8 for Windows (Scripps Research Institute, La Jolla, CA). Cells were acquired and gated by using the dot plot fluorescence-2 width (x) vs. the fluorescence-2 area (y)-gate to exclude aggregates and analyzed in histograms displaying fluorescence-2 area (yellow–orange fluorescence, 585 nm).
Viability Assay. Cells were plated on 96-well microplates (Nunc) at a density of 1 × 104 A549 cells per ml and exposed to IGF (100 ng/ml; Sigma), EGF (10 ng/ml; Sigma), or bFGF (10 ng/ml; Sigma) alone or with 10 μM dizocilpine (Sigma). Cells were also exposed to 50 ng of p21 small interfering RNA (siRNA) per well, diluted in GeneSilencer reagent, or exposed to GeneSilencer reagent (Gene Therapy Systems, San Diego) in growing medium with 10% FCS, according to the manufacturer's protocol. Cell proliferation was assessed after 48–96 h by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method (14).
Proliferation Assay. Tumor cells were incubated with 10 mM BrdUrd (BrdUrd labeling and detection kit III, Roche Diagnostics) over 18 h, fixed with 0.5 M ethanol·HCl, and incubated with nucleases to digest DNA. Monoclonal anti-BrdUrd antibodies conjugated to peroxidase were added and detected by using a 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate. Quantitation was performed colorimetrically at 405–490 nm.
Cytotoxicity Assay. A cytotoxicity detection kit measuring the lactate dehydrogenase activity was used (Roche Diagnostics).
Western Blot Analysis. Cells were lysed in RIPA buffer (1% Nonidet P-40/0.5% sodium deoxycholate/0.1% SDS/1 mM EDTA/1 mM EGTA/1mMNa3VO4/20 mM NaF/0.5mMDTT/1 mM PMSF/protease inhibitor mixture in PBS, pH 7.4) and centrifuged at 3,000 × g for 10 min. Supernatants were solubilized in 3× Laemmli sample buffer (30% glycerol/3% SDS/0.19 M Tris·HCl, pH 6.8/0.015% bromophenol blue/3% 2-mercaptoethanol) and boiled for 5 min at 100°C. Equal amounts of cellular protein extract were electrophoresed on 10% SDS/PAGE under reducing conditions and transferred onto a nitrocellulose membrane (Schleicher and Schüll). After blocking for 1 h at room temperature with 5% nonfat dry milk/TBS/0.1% Tween 20, membranes were probed at 4°C with primary antibodies as follows: anti-phospho-ERK1/2, anti-phospho-mitogen-activated protein kinase (MEK), anti-phospho-p90RSK, anti-phospho-CREB (Ser-133), anti-ERK1/2, anti-phospho-Akt (Ser-473), anti-Akt (1:1,000 in 1% BSA/TBS/0.1% Tween 20; Cell Signaling Technology), or anti-phospho-calmodulin kinase (CaMK)II (Thr-286), and anti-CaMKII and anti-ERK1/2 (1:250 in 1% BSA/1% nonfat dry milk/TBS/0.1% Tween 20; Santa Cruz Biotechnology), incubated with the secondary antibody coupled to horseradish peroxidase (1:5,000 in 1% BSA/TBS/0.1% Tween 20; Amersham Pharmacia Biosciences) and visualized by using enhanced chemiluminescence (ECL, Amersham Pharmacia Biosciences). Serial exposures were made on autoradiographic film (Hyperfilm ECL, Amersham Pharmacia Biosciences). Densitometric analysis of the blots was performed with the image analysis program tina 2.09g (Raytest Isotopenmessgeräte, Straubenhardt, Germany). The sequence of oligonucleotide primers used in RT-PCR are shown in Table 1.
Semiquantitative RT-PCR. RNA (500 ng) was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Promega) and oligo d(T)16 primer (Promega) in 25 μl of reaction mixture. The resulting cDNA (1–2 μl) was amplified by PCR for bcl-2, c-fos, c-jun, cyclin D1, p21, p53, NR1, NR2B, and β-actin. cDNA was amplified in 28–32 cycles, consisting of denaturing over 30 s at 94°C, annealing over 45 s at 52°C or 58°C, and primer extension over 45 s at 72°C. Amplified cDNA was subjected to polyacrylamide gel electrophoresis, silver staining, and densitometric analysis with the image analysis program biodoc analyze (Whatman Biometra, Göttingen, Germany).
siRNA Design and Transfection. p21 siRNA (5′-GACCAUGUGGACCUGUCAC-3′) was designed by using the Eurogentec siRNA design service (Brussels) and in vitro-synthesized with the Silencer siRNA construction kit (Ambion, Austin, TX). Cells were plated at a density of 60% in 24-well plates the day before transfection. Transfection was carried out by using GeneSilencer (Gene Therapy Systems).
p21 Real-Time PCR. Real-time PCR of p21 was carried out by using TaqMan universal master mix (Applied Biosystems), as described by Sifringer et al. (15). RNA samples were identical to those described above. The used p21 TaqMan probe (5′FAM-CTTCGGCCCAGTGGACAG-TAMRA3′) and primers (forward primer: 5′-ACCCATGCGGCAGCAA-3′; reverse primer: 5′-CGCCATTAGCGCATCACA-3′) were designed and synthesized by Applied Biosystems. Amplification of an endogenous reference (18S rRNA TaqMan ribosomal RNA control reagents, Applied Biosystems) was performed. Reactions were carried out in triplicate in a 20-μl volume and repeated twice by using the Applied Biosystems PRISM 7000 sequence detection system. The PCR amplification was performed in 96-well optical reaction plates for 40 cycles, each at 94°C for 20 s and 60°C for 1 min. Calibration curves for p21 and 18S rRNA were performed to determine the linear range of the assay. For relative comparison of the expression levels, the comparative CT method was used (Applied Biosystems, User Bulletin no. 2).
Tumor Growth in Rodents. In vivo studies in mice were approved by the Animal Use and Care Committee of Humboldt University. Animals were maintained under pathogen-free conditions. SCID/SCID mice (Charles River Breeding Laboratories, Sulzfeld, Germany), 4 weeks old, were inoculated with lung carcinoma (A549, 1 × 107 cells) into the peritoneal cavity and administered either dizocilpine (0.03 or 0.1 mg/kg, i.p.) or vehicle once a day. Nude mice (nu/nu; Charles River Breeding Laboratories), 5 weeks old, were inoculated into the right flank with human neuroblastoma (SKNAS; 1 × 107 cells per 0.1 ml of Hanks' balanced salt solution) or human rhabdomyosarcoma (TE671; 1 × 107 cells per 0.1 ml of Hank's balanced salt solution) and randomized. Dizocipline was dissolved in saline and administered i.p. at 0.3 mg/kg twice daily, beginning the day after tumor cell inoculation and continuing for 21 days. Tumor volume (V; cubed centimeters) was calculated by using the equation V = d2 × D/2, where d (squared centimeters) and D (centimeters) are the smallest and largest perpendicular diameters.
Statistics. One- and two-way ANOVA and Student's t test were used for comparisons. Survival data of tumor-bearing mice are presented as a Kaplan–Meier plot, and the log-rank test was applied for statistical analysis.
Dizocilpine Inhibits Proliferation of Lung Carcinoma Cells in Vitro. (+)-Dizocilpine reduced viability of A549 cells in a concentration-dependent manner (Fig. 1a; exposure 96 h). (–)-Dizocilpine (Tocris Cookson), the less active stereoisomer of dizocilpine at NMDA receptors, was substantially less effective in preventing proliferation of tumor cells. Antiproliferative effect was elicited with memantine (Sigma), another NMDA receptor antagonist. Decreased BrdUrd incorporation in A549 cells and a mild increase in the lactate dehydrogenase activity in cultures exposed to (+)-dizocilpine were evident (Fig. 1b). Fluorescence confocal microscopy showed diffuse membrane and cytoplasmic staining for human NR1 and NR2B subunits (Fig. 1c), which conforms with RT-PCR analysis demonstrating the expression of NR1 and NR2B mRNA (Fig. 1c).
(+)-Dizocilpine suppresses the growth of lung adenocarcinoma cells (A549) and inhibits the ERK1/2 pathway. (a) A549 cells were treated with 10–500 μM (+)-dizocilpine, (–)-dizocilpine, the less potent blocker of NMDA receptor channels, or memantine, another noncompetitive NMDA receptor antagonist. Normalized cell viability, measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay at 96 h, is depicted as the mean ± SEM at each concentration. Lines represent the result of linear regression analysis. ANOVA revealed a significant effect of treatment with dizocilpine and memantine on the cell viability (P < 0.001) and clear concentration-dependency of anticancer action of both compounds (n = 6 at each concentration). (–)-Dizocilpine was significantly less active than (+)-dizocilpine in inhibiting tumor growth. (b) Decreased levels of incorporated BrdUrd and increased the lactate dehydrogenase activity in A549 cells after a 96-h treatment with 250 μM(+)-dizocilpine (normalized means ± SEM; n = 8 per column; ***, P < 0.001; Student's t test). (c) The expression of NR1 and NR2B subunits in A549 cells as revealed by immunocytochemistry (confocal images, 770-nm wavelength). RT-PCR analysis confirmed the expression of mRNA specific for NR1 and NR2B in A549 cells (Right). (Scale bar, 20 μm.) (d) A549 cells were treated with 10–500 μM(+)-dizocilpine for 15 min to 24 h. Where indicated, cells were treated with the specific inhibitor of MEK1/2 kinases U0126 (10 μM) or phorbol 12-myristate 13-acetate (PMA) (200 nM) for 3 h. Time-dependent inhibition of the phosphorylation of ERK1/2, MEK1/2, p90RSK, and CREB by (+)-dizocilpine was detected. Equal loading was confirmed by antibody against total ERK1/2 and total CREB. Experiments were performed in quintuplicate with identical results. (e) Concentration-dependent inhibition of ERK1/2by(+)-dizocilpine. Equal loading was confirmed by using antibody against total ERK1/2. Experiments were performed in quintuplicate. (f) Confocal imaging of immunofluorescence staining of A549 cells treated with control (VEH) and (+)-dizocilpine (DIZ) (250 μM) by using antibody against phosphorylated ERK1/2. Note the more intense staining of control cells, as well as the inhibited translocation of phospho-ERK1/2 kinases into the nucleus in (+)-dizocilpine-treated cells. The lower photographs were taken at higher magnifications. (Scale bar, 20 μm.)
Dizocilpine Inhibits the ERK1/2 Pathway. In A549 cells treated with 250 μM (+)-dizocilpine for 15 min to 24 h, decreased levels of phosphorylated ERK1/2, MEK1/2, and p90RSK became evident after 30 min and remained low for up to 24 h (Fig. 1d). The level of total ERK1/2 proteins was unaltered (Fig. 1d). Confocal imaging confirmed a decrease of ERK1/2 activity after dizocilpine treatment in A549 cells, resulting in more intense staining of unexposed cells compared with (+)-dizocilpine-exposed cells (Fig. 1f). Quantification of the relative intensity of staining revealed that relative intensity decreased from 148.1 ± 29.7 pixels per cell (n = 18) in control cultures to 80.3 ± 20.6 pixels per cell (n = 15) in cultures exposed to (+)-dizocilpine (P < 0.001; Student's t test). Translocation of phosphorylated ERK1/2 into the nucleus was abolished in cells incubated with 250 μM (+)-dizocilpine over 3 h (Fig. 1f).
To investigate whether the observed ERK1/2 inhibition was concentration-dependent, A549 cells were incubated with different concentrations of (+)-dizocilpine (10–500 μM) for 3 h. A concentration-dependent decrease of ERK1/2 phosphorylation was observed between 100 and 500 μM (Fig. 1e).
Decreased CREB Phosphorylation After Treatment with Dizocilpine. CREB controls transcription of immediate early genes (9, 16) and plays a role in tumor progression (17, 18). Phosphorylation on Ser-133 by p90RSK kinase is critical for positive regulation of the transcriptional activity of CREB. Decrease of the p90RSK activity was followed by the hypophosphorylation of CREB in A549 cells (Fig. 1d). To evaluate the possible involvement of CaM kinases in CREB hypophosphorylation (19), CaMKII phosphorylation was analyzed by immunoblotting. No significant changes in the activities of CaMKII were detected when A549 cells were incubated with 250 μM(+)-dizocilpine for 15 min every 24 h (Fig. 2a). KN62 (50 μM), which directly binds to the calmodulin binding site of the enzyme, inhibited the activity of CaMKII (Fig. 2a). Inhibition of CaMKII and CaMKIV with KN62 had no impact on CREB phosphorylation (Fig. 2a). No changes in the phosphorylation of Akt kinase, which has also been shown to phosphorylate CREB (20), were detected. Akt phosphorylation was inhibited when cells were incubated with 200 nM wortmannin for 6 h (Fig. 2a). Wortmannin did not inhibit CREB phosphorylation, whereas (+)-dizocilpine inhibited CREB phosphorylation markedly (Fig. 2a). These results indicate that inhibition of ERK1/2 kinases by (+)-dizocilpine results in inhibition of CREB phosphorylation at Ser-133.
(+)-Dizocilpine inhibits the expression of CREB-regulated genes and affects the cell cycle. (a) (+)-Dizocilpine does not affect the phosphorylation of CaMKII and Akt kinases. In the blots shown in a, A549 cells were exposed to (+)-dizocilpine (250 μM) for 15 min to 24 h. No change of phosphorylated CaMKII (pCaMKII) is evident in Western blot after exposure to (+)-dizocilpine. KN62 reduced the phosphorylation of the kinase. No change of phosphorylated Akt (pAkt) is induced by (+)-dizocilpine (DIZ). The Akt inhibitor wortmannin (W; 6-h exposure) reduced Akt phosphorylation. The CaMKII/CaMKIV inhibitor KN62 (6-h exposure) and the Akt inhibitor wortmannin did not influence levels of phosphorylated CREB (pCREB), whereas (+)-dizocilpine (250 μM; 3-h exposure) markedly reduced CREB phosphorylation. Equal loading was confirmed by antibodies against total CaMKII, Akt, and CREB (Lower). Experiments were performed in quintuplicate. (b–d)(+)-Dizocilpine induces changes in the expression of genes that regulate the cell cycle. A549 cells were incubated with 250 μM(+)-dizocilpine for 24 h. (c) Representative bands for bcl-2, c-fos, c-jun, cyclin D1, p53, and p21 detected by RT-PCR in control A549 cells (C) and (+)-dizocilpine-exposed A549 cells. The housekeeping gene β-actin was coamplified as an internal control (Lower). Quantification of the expression of bcl-2, c-fos, and c-jun (b) or cyclin D1, p21, and p53 (d) detected by semiquantitative RT-PCR in control and dizocilpine-exposed A549 cells. Density ratios were calculated in relation to the internal standard β-actin and are expressed as means ± SEM (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student's t test). (e)(+)-Dizocilpine reduces numbers of A549 cells entering the cell cycle. Flow cytometry analysis of propidium iodide-stained cells revealed reduced percentages of cells in the G2 and S phase of the cell cycle after exposure to dizocilpine (500 μM; filled columns) compared with respective controls (open columns).
Dizocilpine Decreases the Expression of CREB-Regulated Genes. CREB has been implicated in the transcriptional control of genes involved in cell proliferation, such as c-fos and cyclin D1 (21). Activation of NMDA receptors in neuronal cells results in CREB-dependent expression of the prosurvival gene bcl-2 (11). We studied the expression of bcl-2, c-fos, and c-jun mRNA by using RT-PCR analysis in A549 cells exposed to (+)-dizocilpine. After a 24-h exposure to 250 μM (+)-dizocilpine, all three gene products decreased in A549 cells (c-fos reduced to 50% and c-jun and bcl-2 to 40% of basal levels; Fig. 2 b and c). Members of Fos and Jun families form the AP1 transcription factor, which regulates cell proliferation and cell cycle progression through induction of cyclin D1 transcription and repression of p53 gene expression followed by repression of p21Cip1 transcription (22, 23). Therefore, it is possible that (+)-dizocilpine, by down-regulating c-fos/c-jun (AP1) expression, also affects the expression of AP1-regulated genes. Changes in the expression of cyclin D1, p53, and p21 after 24 h of exposure to (+)-dizocilpine were found (Fig. 2 c and d). Cyclin D1 expression was reduced to 60% of the basal level, and p53 and p21 gene transcription was increased 2- and 3-fold, respectively. Consequently, (+)-dizocilpine affected the cell cycle (Fig. 2e). The percentage of cells in the G2 and S phase decreased significantly from 53.2 ± 0.3% to 44.9 ± 0.8% or 39.0 ± 1.5% after 250 μMor 500 μM (+)-dizocilpine, respectively (P < 0.001; Student's t test). Concomitantly, the percentage of resting cells (G1) increased from 46.8 ± 0.3% to 55.1 ± 0.8% (250 μM dizocilpine) or 61.0 ± 1.5% (500 μM dizocilpine) (P < 0.001; Student's t test).
Silencing of p21 Ameliorates Antiproliferative Action of Dizocilpine. To confirm the functional significance of p21 up-regulation, we investigated whether p21-silencing would reverse the antiproliferative action of (+)-dizocilpine. siRNA oligos were designed complementary to human p21 mRNA. At 48 h after transfection of A549 cells, reduction of the p21 protein was confirmed by Western blot analysis (Fig. 3a). Real-time PCR analysis revealed decreased levels of p21 mRNA (Fig. 3a). A significant increase (P < 0.001; Student's t test) in the cell viability after silencing of p21 was detected compared with nontransfected A549 cells exposed to transfection reagent only (gene-silencing reagent) (data not shown). Exposure to 250 μM (+)-dizocilpine over 48 h resulted in an additional significant reduction of the cell viability. (+)-Dizocilpine slightly reduced the cell viability of p21 siRNA-transfected A549 cells (P < 0.05), but the level of inhibition was lower compared with nontransfected cells (Fig. 3b).
RNA interference reduces p21 expression and reverses the antiproliferative effect of (+)-dizocilpine (250 μM; 48 h) in A549 cells. (a) Western blot for p21 in control A549 cells (C) and cells transfected with p21 siRNA for 48 h demonstrating reduction of p21 protein. p21 mRNA expression determined by real-time PCR is shown. Each bar represents the relative gene expression (2–ΔΔCT) of p21 gene related to the endogenous reference 18S rRNA (means ± SEM; n = 4) (***, P < 0.001; Student's t test). (b) The percent inhibition of proliferation by 250 μM(+)-dizocilpine after p21-silencing (p21+DIZ) or exposure to dizocilpine (DIZ) is shown. After p21-silencing, the growth inhibition by dizocilpine after a 48-h exposure is reduced from 14% to 6% (***, P < 0.001; Student's t test). (c and d) (+)-Dizocilpine reverses the stimulation of proliferation and ERK-phosphorylation by the growth factors in vitro.(c) A549 cells were exposed for 96 h to EGF (10 ng/ml), IGF (100 ng/ml), bFGF (10 ng/ml), or (+)-dizocilpine (10 μM) alone, or in combination with the growth factors. Growth factors stimulated the proliferation of lung adenocarcinoma cells. This effect was reversed by (+)-dizocilpine. Columns represent mean normalized cell viability ± SEM of six measurements (**, P < 0.01; ***, P < 0.001; Student's t test). (d) A549 cells were exposed for 6 h to EGF (10 ng/ml), IGF (100 ng/ml), or bFGF (10 ng/ml) alone or in combination with (+)-dizocilpine (10–100 μM). The growth factors stimulated the phosphorylation of ERK1/2 and CREB. (+)-Dizocilpine at concentrations that reversed the proliferation-promoting effect of the growth factors reversed the stimulation of ERK1/2 and CREB phosphorylation in a concentration-dependent manner. NMDA (10 μM) stimulated ERK1/2 phosphorylation. This effect was reversed by (+)-dizocilpine. Experiments were performed in quintuplicate. (e and f) (+)-Dizocilpine suppresses cancer growth in vivo. (e) Kaplan–Meier survival curves in SCID/SCID mice with metastatic lung adenocarcinoma. Lung carcinoma cells (A549; 1 × 107) were injected into the peritoneal cavity of mice. (+)-Dizocilpine (0.03 mg/kg) significantly prolonged the survival in mice carrying A549 tumors (P < 0.05; log-rank test) compared with vehicle-treated controls (C). (+)-Dizocilpine (0.1 mg/kg) had more pronounced effects on survival (P < 0.001; log-rank test). (f) Nude mice (nu/nu) were inoculated into the right flank with neuroblastoma (SKNAS; 1 × 107 cells) or rhabdomyosarcoma (TE671; 1 × 107 cells) cells, and tumor growth was monitored over 21 days. Shown are means ± SEM of tumor volumes in vehicle-treated (n = 8) or drug-treated mice over time. In mice treated twice daily with (+)-dizocilpine (0.3 mg/kg) (n = 8), tumor growth was suppressed [F(1, 176) = 13.01, P < 0.0001 for SKNAS; F(2, 467) = 2.11, P < 0.0001 for TE671; ANOVA]. The time effect was significant, suggesting that tumor growth depended on the duration of treatment with (+)-dizocilpine [F(10, 176) = 31.67, P < 0.0001 for SKNAS; F(10, 467) = 54.52, P < 0.0001 for TE671].
Dizocilpine reverses the stimulatory effect of the growth factors. The ERK1/2 pathway mediates the cell proliferation and survival in response to the growth factors. We investigated how A549 cells respond to the growth factors and whether (+)-dizocilpine influences their effect. IGF (100 ng/ml), EGF (10 ng/ml), or bFGF (10 ng/ml) stimulated tumor cell proliferation, which is revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (96-h exposure). In the presence of (+)-dizocilpine (10 μM), no such growth stimulatory effect was seen (Fig. 3c). Of note is that these concentrations of the NMDA antagonist did not influence proliferation or levels of phosphorylated ERK and CREB when given in the absence of the growth factors (Fig. 3c). Increased phosphorylation of ERK1/2 and CREB was found in A549 cells stimulated with the growth factors (Fig. 3d). Similar to the antagonistic effect of dizocilpine on growth stimulation induced by EGF, FGF, or IGF, reduction of p-ERK1/2 and p-CREB was detected in cultures coexposed to the growth factors and low concentrations (10–100 μM) of (+)-dizocilpine (Fig. 3d). NMDA (10 μM) stimulated ERK1/2 phosphorylation. This effect was reversed by (+)-dizocilpine at concentrations between 10 and 50 μM (Fig. 3d).
Dizocilpine Suppresses Tumor Growth in Vivo. To assess whether dizocilpine has anticancer activity in vivo, SCID/SCID mice (Charles River Breeding Laboratories), 4 weeks old, were subjected to inoculation of lung carcinoma cells (A549; 1 × 107 cells) into the peritoneal cavity and once daily administration of either dizocilpine (0.03 or 0.1 mg/kg, i.p.) or vehicle. (+)-Dizocilpine prolonged the survival of SCID/SCID mice with metastatic disease (Fig. 3e). (+)-Dizocilpine (0.03 mg/kg) prolonged the median survival time to 41 days as opposed to 32.5 days in vehicle-treated animals (P < 0.05; log-rank test). (+)-Dizocilpine (0.1 mg/kg) prolonged the median survival time of SCID/SCID mice to 59 days (P < 0.001; log-rank test). (+)-Dizocilpine (0.03–0.1 mg/kg) did not induce neurological side effects, caused no mortality, and did not unfavorably affect feeding and weight gain in comparison with vehicle-treated SCID/SCID mice. To determine whether dizocilpine may influence proliferation of other tumor types in vivo, we implanted neuroblastoma (SKNAS; 1 × 107 cells) or rhabdomyosarcoma (TE671; 1 × 107 cells) cells into the right flank of nude mice and monitored tumor growth over 3 weeks. Both tumor cell lines display impaired growth in vitro in the presence of NMDA antagonists (14). Treatment with (+)-dizocilpine (0.3 mg/kg) twice daily significantly impaired tumor growth (Fig. 3f). Treatment with (+)-dizocilpine induced mild increases in the locomotor activity, oral stereotypes, and sniffing lasting for up to 1 h after administration. No mortality was observed in mice subjected to treatment with (+)-dizocilpine.
The NMDA antagonist (+)-dizocilpine influences pathways that control proliferation of cancer cells and suppresses tumor growth. These actions of (+)-dizocilpine arise from inhibition of the ERK1/2-signaling cascade. Exposure of lung carcinoma cells to (+)-dizocilpine results in decreased concentrations of phosphorylated CREB without involvement of CaMKII, CaMKIV, or Akt kinases. In addition, dizocilpine induces down-regulation of c-fos, c-jun, bcl-2, and cyclin D1, as well as up-regulation of p21 and p53. These changes in gene expression explain the antiproliferative effect of (+)-dizocilpine in human lung carcinoma cells and are consistent with reduction in CREB phosphorylation because CREB has been shown to alter the expression of cyclin D1, bcl-2, and c-fos (11, 17, 19, 21, 24, 25).
Studies applying inhibition of c-fos and c-jun expression demonstrated that these proteins are required for cell proliferation and cell cycle progression (22). Amelioration of the antiproliferative effect of (+)-dizocilpine by p21-silencing indicates that the observed changes in gene expression are of functional importance in mediating the anticancer effect of dizocilpine.
Down-regulation of c-fos/c-jun (AP1) expression inhibits the growth of breast cancer cells both in vitro and in vivo without inducing apoptosis (26). Inhibition of AP1 target genes is a strategy for cancer chemoprevention (23) and reveals NMDA antagonists as potential chemotherapeutic/chemopreventive agents in cancer therapy. Cyclin D1 has been shown as a chemopreventive target in lung cancer (26). Cyclin D1 is frequently overexpressed in lung cancers and serves as a negative biomarker (26). Depression of cyclin D1 and increased expression of p21 and p53 genes after (+)-dizocilpine treatment provides a link to the cell cycle machinery. Cyclin D1 is necessary for the G1 phase of the cell cycle, and p21 is a cyclin-dependent kinase inhibitor that is responsible for the antiproliferative activity of the tumor suppressor protein p53 (17). (+)-Dizocilpine exerts an inhibitory effect on cell cycle progression.
Growth factors stimulate the ERK1/2 pathway, resulting in CREB phosphorylation at Ser-133 and transcription of CREB-dependent genes (16, 21, 22). Lung adenocarcinoma cells responded to EGF, IGF, and FGF with increased growth. Inhibition of ERK1/2-signaling by (+)-dizocilpine reversed the stimulation of proliferation of cancer cells by these growth factors. (+)-Dizocilpine may inhibit the ERK1/2-signaling cascade in A549 cells by blockade of channels bearing NR2B subunits in their assembly. The NR2B subunit, however, is not expressed in all cancer cell lines that respond to (+)-dizocilpine with decreased proliferation. This phenomenon implies that mechanisms independent of NR2B subunits may be engaged. A549 cells express α7-nicotinic cholinergic receptors, which also have been linked to activation of the ERK1/2-signaling cascade (27, 28), and (+)-dizocilpine interacts with α7-nicotinic cholinergic receptor-dependent channels (29). Yet, (+)-dizocilpine inhibits ERK1/2 activation induced by NMDA, indicating that the NMDA receptor channel-dependent component predominates its inhibitory effects on proliferation.
In vivo,(+)-dizocilpine prolonged the survival of mice bearing metastatic lung adenocarcinoma at doses that were well tolerated. A daily dose of dizocilpine (0.1 mg/kg) doubled the survival of mice. Furthermore, an in vivo antiproliferative effect was observed with (+)-dizocilpine in two additional tumor types, neuroblastoma and rhabdomyosarcoma, indicating that the anticancer effect is not limited to a single cell line and that different tumors are susceptible to treatment with (+)-dizocilpine.
Our findings reveal prospects for (+)-dizocilpine in cancer therapy. If the anticancer effects of (+)-dizocilpine and other NMDA antagonists can be confirmed in cancer patients, then these drugs could expand armamentarium for cancer therapy in humans.
↵§§ To whom correspondence should be addressed. E-mail: hrissanthi.ikonomidou{at}uniklinikum-dresden.de.
Author contributions: A.S., C.B., M.S.-Z., L.T., and C.I. designed research; A.S., M.S., W.R., S.E., A.G., E.E.P., P.B., U.F.-M., A.M.K., H.H.H., L.T., and C.I. performed research; A.S., M.S., C.B., and C.I. analyzed data; and A.S., L.T., and C.I. wrote the paper.
Abbreviations: ERK, extracellular signal-regulated kinase; CREB, cAMP-responsive element binding protein; IGF, insulin growth factor; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; MEK, mitogen-activated protein kinase; CaMK, calmodulin kinase; siRNA, small interfering RNA.
