Affiliations of authors: Department of Thoracic/Head and Neck Medical Oncology (HYL, YSC, KHC, KH, RL, WKH), Department of Thoracic Surgery (LJ), University of Texas M. D. Anderson Cancer Center, Houston, TX; Division of Biometry and Risk Assessment, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR (HM); Department of Hematology and Medical Oncology, Translational Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA (FRK)
Correspondence to: Ho-Young Lee, PhD, Unit 432, Department of Thoracic/Head and Neck Medical Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: hlee{at}mdanderson.org)
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ABSTRACT |
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INTRODUCTION |
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We previously found that expression of IGF binding protein-3 (IGFBP-3), a natural inhibitor of IGF-mediated lung cancer cell survival and growth (9), is frequently decreased in lung cancer and that this decrease is strongly associated with a poor prognosis in patients with stage I nonsmall-cell lung cancer (NSCLC) (10,11). An inverse relationship has been demonstrated between serum or plasma levels of IGFBP-3 and the risk of lung cancer (12,13). IGFBP-3 in the serum sequesters IGFs, reducing IGF availability to various cells and tissues. IGFBP-3 also has IGF-independent antiproliferative and proapoptotic effects. For example, IGFBP-3 overexpression inhibits the growth of IGF-1Rnull fibroblasts (14). The effects of IGFBP-3 are probably mediated by other cell surface receptors, such as the type V transforming growth factor (TGF-
) receptor (15). Recently, antitumor effects of recombinant human IGFBP-3 were observed in solid tumor models, including a lung carcinoma model (16). Thus, these findings provide a strong rationale for the use of IGFBP-3 as a therapeutic agent for the treatment of lung cancer.
One potential obstacle to using IGFBP-3 in the treatment of NSCLC, however, is the development of nonresponsiveness to IGFBP-3, which is believed to be mediated by oncogenic Ras (17). Ras-mediated signaling pathways can be activated by multiple events, including the overexpression of the epidermal growth factor receptor family and one of its ligands, TGF-; mutations in Ras; reduced expression of PTEN; and amplification of a region of chromosome 3q that includes the p110 catalytic subunit of phosphoinositide 3-kinase (PI3K), all of which have been found in 24%80% of lung tumor tissues from patients with NSCLC (1824). Because Ras-mediated signaling pathways are numerous and complex, blocking Ras before it localizes to the cell membrane may be an attractive and practical target. Indeed, inhibitors of farnesyltransferase, an enzyme required for posttranslational processing and membrane translocalization of Ras, have recently been introduced into clinical trials (25). Combinations of the farnesyltransferase inhibitor (FTI) SCH66336, one of the first such inhibitors to undergo clinical testing, with cyclophosphamide, 5-fluorouracil, vincristine, cisplatin, taxanes, or p53 gene therapy, have been reported to enhance antitumor activity in preclinical cancer models (2529). In this study, we hypothesized that Ras-mediated signaling is critical to blocking IGFBP-3 activity and that blocking Ras-mediated signaling would increase the antitumor activity of IGFBP-3 in NSCLC cells. We tested this hypothesis by targeting Ras activation with SCH66336 and evaluated the consequence of treating NSCLC cells with the combination of SCH66336 and IGFBP-3.
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METHODS |
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Tissue culture reagents and plasticware were from Nunc (Roskilde, Denmark). An adenoviral vector expressing IGFBP-3 (9), referred to here as Ad-IGFBP3; an adenoviral vector expressing constitutively active MEK1 (Ser217/221 to Glu) (kindly provided by Dr. J. D. Molkentin, Cincinnati, OH) (30), referred to here as Ad-MEK1; an adenoviral vector expressing constitutively active Akt (MyrAkt) (31), referred to here as Ad-MyrAkt; and an adenoviral vector expressing empty vector, referred to here as Ad-EV, were amplified as described previously (17). IGF and epidermal growth factor (EGF) were purchased from R & D Systems (Minneapolis, MN). SCH66336 ([+] 4-{2-[4-(8-chloro-3, 0-dibromo-6, 11-dihydro-5-benzocyclohepa {1,2-b} pyridin-11-yl)-1-piperidinyl]-2-oxoethyl}-1-piperidinecarboxamide) was obtained from Schering-Plough (Kenilworth, NJ). We confirmed the activity of SCH66336 in inhibiting farnesylation (32), measured as an enhancement in the level of unfarnesylated H-Ras in NSCLC cell lines (data not shown). LY294002, an inhibitor of PI3K, and PD98059, an inhibitor of mitogen-activated protein kinase MEK1, were purchased from Calbiochem-Novabiochem (Alexandria, New South Wales, Australia). Inhibitors were prepared as 20 mM stock solutions in dimethyl sulfoxide (DMSO) and stored at 20 °C.
Expression and Purification of TAT Fusion Protein
Proteins generated from the vector pTAT-HA have an N-terminal six-histidine leader sequence, which facilitates purification on nickel-based Sepharose columns; an 11amino-acid transduction domain of the human immunodeficiency virus type 1 TAT protein, which facilitates the intracellular delivery of mature proteins; and a nineamino-acid hemagglutinin tag (33). The full-length human IGFBP-3 coding region was inserted into the EcoRIXhoI restriction site of the pTAT-HA bacterial expression vector (kindly provided by Dr. S. F. Dowdy, St. Louis, MO) to produce in-frame TAT fusion proteins (TAT-BP3).
IGFBP-3 primers used in this study are listed as follows (restriction enzyme sites incorporated to facilitate cloning are underlined): pTAT-BP3 (sense) 5'-ACCGCTCGAGGGCGCGAGCTCGGGGGGC-3' (XhoI), (antisense) 5'-ACCGGAATTCCTACTTGCTCTGCATGCTGTAGC-3' (EcoRI). The presence of IGFBP-3 in pTAT-BP3 vector was confirmed by dideoxy-DNA sequencing. pTAT-BP3 vectors were expressed in Escherichia coli strain BL21 (DE3) pLysS (Novagen, Madison, WI), according to the manufacturers recommended protocol. To purify the fusion protein, we lysed the bacterial cells by sonication in a buffer consisting of 20 mM phosphate buffer (pH 7.8), 500 mM NaCl, 100 mg of lysozyme, and protease inhibitors (0.1M phenylmethylsulfonyl fluoride, 0.5 mg/mL leupeptin, 1 mg/mL pepstatin, and 1 mg/mL apropronin). E. coli lysates were denatured in 8 M urea, and IGFBP-3 proteins were purified by metal-affinity chromatography using Ni-NTA matrix (Qiagen, Chatsworth, CA) and gel filtration on a Sephadex G-25M (Amersham Pharmacia Biotech, Arlington Heights, IL) column, as previously described (33). Proteins were eluted from the Ni-NTA matrix by the addition of 500 mM imidazole and dialyzed against 1x phosphate-buffered saline (PBS) overnight at 4 °C.
The induction of IGFBP-3 expression in E. coli from pTAT-BP3 was examined by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis of the purified proteins through a 12% gel and Coomassie blue staining, and by western blot analysis for IGFBP-3. The binding of E. coliexpressed IGFBP-3 proteins to IGF-I was confirmed by western blot analysis (data not shown).
Cells and Cell Culture
Normal human bronchial epithelial (NHBE) cells were purchased from Clontech (Palo Alto, CA) and maintained in keratinocyte serum-free medium (Life Technologies, Gaithersburg, MD) containing EGF (2 µg/mL) and bovine pituitary extract (25 µg/mL) (31). Ten human NSCLC cell lines with high endogenous levels of IGFBP-3 (H596, A549, and H460), detectable levels of IGFBP-3 (H358 and H226B), and undetectable levels of IGFBP-3 (H1299, H441, H322, H226Br, and Calu-6) of IGFBP-3 (34) were used. Human NSCLC cell lines H1299, H226B, H596, A549, H460, and H358 were purchased from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (GIBCO-BRL, Gaithersburg, MD) in a humidified atmosphere with 5% CO2.
To determine the effects of IGFBP-3 or SCH66336 on NSCLC cell proliferation, the NSCLC cell lines were plated at concentrations of 1 x 103 to 2 x 103 cells per well in 96-well plates. The next day, cells were mock-infected or infected with various titers of Ad-IGFBP3 or Ad-EV for 2 hours and then incubated in complete medium for the assay period. To test antiproliferative effects of SCH66336, we treated cells with either 0.1% DMSO or various concentrations of SCH66336 (final DMSO concentration = 0.1%) in complete medium, which was changed daily during the assay period. At the end of the assay period, the proliferation of treated cells was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously described (31). Six replicate wells were used for each assay; data from replicate wells are presented as mean values with 95% confidence intervals (CIs). Three independent experiments were performed with similar results; representative results of one experiment are presented.
To test the effects of combined treatment with IGFBP-3 and SCH66336 or LY294002 on H460 cell proliferation, we infected cells with various titers of Ad-IGFBP3 or treated with TAT-BP3 protein (500 ng/mL) in the presence of 1 µM SCH66336 (the dose that blocks Ras farnesylation) or 10 µM or 25 µM LY294002 (doses that block Akt activation). After 3 days, proliferation was measured by using the MTT assay.
In experiments assessing the contribution of the MAPK and Akt signaling pathways, H460 cells, which have relatively high levels of endogenous MAPK and Akt, were infected first with 25 plaque-forming units (pfu) per cell with Ad-EV, Ad-MEK-1, or Ad-MyrAkt and then mock-infected or coinfected with Ad-EV (25 pfu/cell) or Ad-IGFBP3 (5 or 25 pfu/cell). After 2 hours of infection, cells were treated with 1 µM SCH66336 and then incubated for 3 days. All infections and treatments were carried out at these titers, times, and concentrations, unless otherwise stated. The viability of treated cells was measured by using the MTT assay. In experiments assessing the levels of phosphorylated IGF-1R and Akt, all NSCLC and NHBE cells were maintained in serum-free medium for 48 hours before lysis to remove exogenous activators of the IGF-1R pathway.
In Vitro Drug Interaction Studies
In this study, Dm is the drug dose required to produce the median effect, which is analogous to the concentration required to inhibit growth by 50% (i.e., IC50). Dm was determined by calibration after fitting data via a nonparametric regression (35). A subset of NSCLC cells (H1299, H596, A549, H460, H322, H226B, and H358 cells) were infected with Ad-IGFBP3 or Ad-EV and treated with SCH66336 simultaneously at fixed ratios of dosages corresponding to one-half, five-eighths, three-fourths, seven-eighths, one, and fivefold the IC50. The fractional survival rate (f) was calculated by dividing absorbance values from the MTT assay for the drug-treated wells by those from the control wells. The data were analyzed by the method of Chou and Talalay (36). In brief, log [(1 f)/f] was plotted against log [D/Dm]. From the resulting median effect curves, the x-intercept (log) and the slope, , were calculated for each drug individually and for the combination. These parameters were then used to calculate doses of the individual drugs and of the combination required to produce various levels of cytotoxicity (f = 0.95, 0.90, 0.85,..., 0.05), according to the equation D = Dm[(1f)/f]1/
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The dose of the combination required to produce fractional survival was divided into the component doses (D)1 and (D)2 of drugs 1 and 2, respectively. For each level of cytotoxicity, the combination index (Cx) was calculated according to the following equation:
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where (Dalone)1 and (Dalone)2 were doses of the individual drugs 1 and 2, respectively, required for a given effect (f); (Dcomb)1 and (Dcomb)2 were doses of the combination required to produce survival f; and was equal to 1 or 0, depending on whether the drugs were assumed to be mutually nonexclusive or mutually exclusive, respectively. For two drugs that have the same or similar modes of action, the effects of two drugs are mutually exclusive; the plots for parent drugs and their mixtures of median-effect plot of log[(fa)1 1]1 versus log drug concentration are parallel. For two drugs that have different modes of action or act differently, the effects of two drugs are mutually nonexclusive. Cx is the parameter that indicates whether the doses of the two agents required to produce a given degree of cytotoxicity were greater than (Cx>1), equal to (Cx = 1), or less than (Cx<1) the doses that would be required if the effects of the two drugs were strictly additive (36). In this method, therefore, synergy was indicated if Cx were less than 1, and less than synergy if Cx were 1 or greater.
Western Blot Analysis
Whole-cell, nuclear, and cytosolic extracts from 106 cells were prepared in lysis buffer as described previously (31,34). Equivalent amounts of protein were resolved by SDSpolyacrylamide gels (7.5%12%) and transferred to nitrocellulose membranes. After the membranes were blocked in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) and 5% (wt/vol) nonfat powdered milk, the membranes were incubated with primary antibodies diluted in TBS5% nonfat milk at 4 °C for 16 hours. Membranes were then washed multiple times with TBST and incubated with the appropriate horseradish peroxidaseconjugated secondary antibody for 1 hour at room temperature. The proteinantibody complexes were detected by using the enhanced chemiluminescence (ECL) kit (Amersham, Arlington Heights, IL), according to the manufacturers recommended protocol. Representative results from two separate experiments are shown.
The following antibodies and working dilutions, suggested by the manufacturers, were used for the western blots: rabbit polyclonal anticaspase-3 (1:2000) and rabbit polyclonal antiBax antibodies (1:2000) (Pharmingen, San Diego, CA); rabbit polyclonal antiBcl-2 (1:1000), rabbit polyclonal antiBcl-xL, and rabbit polyclonal antipoly(ADP-ribose) polymerase (PARP) antibody (1:1000) (VIC 5; Roche Molecular Biochemicals, Indianapolis, IN); mouse monoclonal antiphosphorylated MAPK (ppP44/42 MAPK) (Thr202/Tyr204) (1:500), rabbit polyclonal antibodies against phosphorylated JNK (pJNK) (1:1000), phosphorylated p38 MAPK (pp38 MAPK) (1:1000), p38 MAPK (1:1000), phosphorylated Akt (pAkt) (Ser473) (1:1000), phosphorylated glycogen synthase kinase 3/
(pGSK-3
/
) (Ser21/9) (1:1000), phosphorylated phospho-IGF-1R (Tyr131) (1:1000), or IGF-1R
(1:1000) (Cell Signaling Technology, Beverly, MA); rabbit polyclonal antiGSK-3
antibody (1:1000) (BD Transduction Laboratories, Lexington, KY); goat polyclonal antibodies against extracellular elated kinase 1 (p44 MAPK) (1:1000) or JNK (1:1000), rabbit polyclonal antiAkt1 and anti
-actin (1:4000) antibodies (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit antimouse immunoglobulin G (IgG)horseradish peroxidase conjugate (1:2000) (DAKO, Carpinteria, CA); and donkey antirabbit IgGhorseradish peroxidase conjugate (1:2000) and rabbit antigoat IgGhorseradish peroxidase conjugate (1:2000) (Amersham Pharmacia Biotech).
Apoptosis Analysis
H460 cells were plated at a concentration of 2 x 105 cells/well in six-well plates. The next day, cells were mock-infected or infected with Ad-EV or Ad-IGFBP3 for 2 hours and then treated with SCH66336 (final DMSO concentration = 0.1%) or DMSO (0.1%) for 3 days. Apoptosis was assessed using a flow cytometrybased modified terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) assay. For TUNEL analysis, both adherent and nonadherent cells were harvested and pooled, fixed with 1% paraformaldehyde and 70% ethanol, and processed using the APO-BrdU staining kit (Phoenix Flow Systems, San Diego, CA), as described previously (31). Cells treated with DMSO were used to gate the control nonapoptotic populations, and cells treated with deguelin (31) were used as a positive control. An internal control (HL-60 cells treated with camptothecin to induce apoptosis) provided in the apoptosis detection kit was also used to ensure that the TUNEL reaction occurred during the staining procedure. Two independent experiments were performed; representative results of one experiment are presented.
Metabolic Labeling and Immunoprecipitation
Metabolic labeling was performed with H460 cells (2 x 106 cells in 100-mm3 plates) that were infected first with Ad-EV (25 pfu/cell) or Ad-IGFBP3 (25 pfu/cell) for 2 hours and then treated with 0.1% DMSO or SCH66336 (1 µM) for 3 days. Cells were washed in PBS and incubated in RPMI medium without methionine and cysteine (Sigma, St. Louis, MO) for 1 hour. Trans-35S label (0.5 mCi; ICN) was added to the culture medium, and the cells were incubated for 1 hour. After the pulse, the medium containing the label was removed and replaced with fresh medium containing methionine and cysteine added to final concentrations of 150 mg/L. After 3 hours, cells were washed in ice-cold PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer. Lysates containing equal amounts of protein (30 µg) were used in immunoprecipitation analysis using 1 µg of antibody detecting Akt (Santa Cruz Biotechnology) and 40 µL of a 50% slurry of Sepharose protein A beads (Pharmacia-LKB Biotechnology, Piscataway, NJ). The immunoprecipitates were washed five times with lysis buffer, separated by SDSpolyacrylamide gel electrophoresis, and analyzed by autofluorography, as described previously (37). A preimmune serum was used as a negative control for immunoprecipitation. Cell extracts were also subjected to Western blot analysis for -actin to ensure that equal amounts of protein were used. Two independent experiments were performed with similar results; representative results of one experiment are presented.
In Vivo Tumor Model
The effect of the combination of IGFBP-3 and SCH66336 on established subcutaneous tumor nodules was studied in athymic nude mice (Harlan-Sprague-Dawley, Indianapolis, IN) in a defined pathogen-free environment. Briefly, 4-weekold female nude mice were irradiated with 350 rad from a cesium-137 source and then were injected subcutaneously with 107 H1299 cells in 100 µL of growth medium at a single dorsal site. The mice were randomly assigned to one of six treatment groups, with each group containing five mice. Group 1 received PBS and hydroxyl-propyl-betacyclodexatrin (HPCD), a vehicle for SCH66336; group 2 received PBS and SCH66336; group 3 received Ad-EV and HPCD; group 4 received Ad-EV and SCH66336; group 5 received Ad-IGFBP3 and HPCD; and group 6 received Ad-IGFBP3 and SCH66336. After the tumor volume reached approximately 75 mm3 (day 0), mice received a single intratumoral injection of 2 x 109 particles of Ad-IGFBP3 or Ad-EV in 100 µL of PBS or 100 µL of PBS (as a control). On days 2 through 10, mice received SCH66336 (40 mg/kg of body weight) or 20% HPCD (100 µL) delivered orally twice a day. Tumor size was measured every day for 16 days, when the mice in groups 14 were killed because of excessive tumor burden. Tumor growth was quantified by measuring the tumors in three dimensions with calipers. Mice with necrotic tumors or tumors 1.5 cm or greater in diameter were immediately killed. The results were expressed as the mean tumor volume (n = 5) with 95% confidence intervals. The statistical significance of differences in tumor growth between the combination treatment group and the single-agent treatment groups was analyzed using two-way analysis of variance.
In Vivo Apoptosis and Immunohistochemistry
To determine whether the combination of Ad-IGFBP3 and SCH66336 induced apoptosis in tumor nodules in vivo, we collected tumor tissues from each treatment group, fixed them with 10% formaldehyde, embedded them in paraffin, and sectioned them. For some tumors, sections (5-µm thick) were subjected to the TUNEL assay to detect apoptosis-induced DNA fragmentation, as previously described (38). For other tumors, sections were processed for immunohistochemistry and deparaffinized through a series of xylene baths and rehydrated through a series of graded ethanol baths. The sections were immersed in methanol containing 0.3% hydrogen peroxidase for 20 minutes to block endogenous peroxidase activity and then incubated in 2.5% blocking serum for 1 hour to reduce nonspecific antibody binding. Sections were incubated overnight at 4 °C with primary antibody against Akt (diluted 1:100 in 2.5% blocking serum) or Bcl-xL (diluted 1:100). The sections were then processed using standard avidinbiotin immunohistochemical techniques according to the manufacturers recommendations (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as a chromogen, and commercial hematoxylin was used as a counterstain. H1299 cells were stained at the same time to serve as a positive control. Akt and Bcl-xL labeling indices were defined as the percentage of tumor cells displaying membranous or cytoplasmic immunoreactivity, respectively, and were calculated by counting the number of stained tumor cells among more than 1000 tumor cells from multiple representative areas of each tissue section. In this study, we used a 5% labeling index as a cutoff point. We evaluated the slides based on five score levels. We chose a 5% labeling index as a cutoff point for statistically significant results (P<.05). On the basis of the immunohistochemical staining results, we considered tumors in which less than 5% of the cells stained positive to have decreased Akt and Bcl-xL expression. All slides were evaluated and scored independently by two pathologists who were blinded to the clinical information of the subjects.
Statistical Analysis
Cell survival comparisons among groups were analyzed by the two-way analysis of variance for 2 x 2 factorial design. All means from triplicate samples and 95% confidence intervals were calculated using SAS software (Release 8.02; SAS Institute, Cary, NC). In all statistical analyses, two-sided P values less than .05 were considered statistically significant.
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RESULTS |
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To investigate the role of the IGF pathway in NSCLC cells, we first examined the level of expression and the phosphorylation status of IGF-1R and its signaling component Akt in 10 NSCLC cell lines and in NHBE cells by western blot analysis (Fig. 1, A). The level of IGF-1R was associated with its level of phosphorylation; i.e., cell lines that expressed detectable levels of IGF-1R also expressed phosphorylated IGF-1R. Seven of 10 NSCLC cell lines expressed higher levels of IGF-1R and phosphorylated IGF-1R than NHBE cells, indicating that the IGF-1R pathway was activated in most of the NSCLC cell lines tested. Phosphorylated Akt levels were also higher in nine of 10 NSCLC cell lines than in NHBE cells, although Akt levels were similar among all cell lines. Only five of 10 NSCLC cell lines (H1299, H596, H460, H226B, and Calu6) expressed both phosphorylated Akt and phosphorylated IGF-1R.
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Response of NSCLC Cells to IGFBP-3 and SCH66336
The level of IGF-1R is associated with the mitogenic response to IGF-I (39). We found that inhibiting Akt and MAPK activation was associated with a decrease in IGF-1induced IGF-1R phosphorylation in the NSCLC cells infected with Ad-IGFBP3 [Lee et al. (9) and data not shown]. Because activated Rasmediated signaling could limit the therapeutic efficacy of IGFBP-3, we investigated the effects of single-agent Ad-IGFBP3 or cotreatment with SCH66336 on the proliferation of the NSCLC cell lines H1299, H596, A549, H460, H358, H226B, and H322. The IC50s, which are analogous to Dm, for Ad-IGFBP3 and SCH66336 in these cell lines, ranged from 50 to 100 pfu/cell and 2 to 6 µM, respectively (data not shown). Growth was reduced by no more than 50% in cells infected with Ad-IGFBP3 or in cells treated with SCH66336 for 72 hours (Fig. 2, A). However, growth of cells infected with Ad-IGFBP3 and treated with SCH66336 was reduced by at least 75%. A combination effect in cells infected with Ad-IGFBP3 and treated with SCH66336 was statistically significant at a 5% level in H1299 (P<.001), H596 (P = .02), A549 (P = .006), H460 (P<.001), and H358 (P<.001) cells, suggesting that attenuation of Ras-mediated signaling augmented the antiproliferative effects of IGFBP-3 for these five NSCLC cell lines.
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TUNEL staining and flow cytometry analysis were used to determine whether H460 cells infected with Ad-IGFBP3 and treated with SCH66336 underwent apoptosis. Approximately 1% of control H460 cells, 23% of H460 cells infected with Ad-IGFBP3, and 19% of H460 cells treated with SCH66336 underwent apoptosis (Fig. 2, C). By contrast, 83% of H460 cells infected with Ad-IGFBP3 and treated with SCH66336 underwent apoptosis.
Western blot analysis of apoptosis-related enzymes (caspase-3 and PARP) and proteins (Bcl-2, Bax, and Bcl-xL) showed that, after 72 hours, H460 cells infected with Ad-IGFBP3 and treated with SCH66336 had decreased levels of the inactive form of procaspase-3 (relative molecular mass = 32) and concomitant increased cleavage of the 113-kd poly ADP-ribose polymerase (PARP) to the 89-kd fragment (Fig. 2, D). Compared with levels in control cells, levels of Bcl-2 and Bcl-xL were lower in H460 cells infected with Ad-IGFBP3 and even lower or undetectable in H460 cells infected with Ad-IGFBP3 and treated with SCH66336. By contrast, levels of Bax were unchanged by either agent or the combination. These results suggest that the ratio of antiapoptotic proteins (i.e., Bcl-2, Bcl-xL) to proapoptotic proteins (i.e., Bax) is altered by IGFBP-3 and SCH66336 in NSCLC cells.
Effect of Ad-IGFBP3 and SCH66336 on MAPK and Akt Pathways in NSCLC Cells
We next investigated the mechanism by which Ad-IGFBP3 and SCH66336 increased apoptosis in H460 cells. FTIs inactivate Ras and can inhibit the in vitro growth of transformed cells with wild-type ras gene. Because the response of NSCLC cell lines to Ad-IGFBP3 and SCH66336 was not associated with mutations in the ras gene (Table 1), we determined the effects of Ad-IGFBP3 and SCH66336, both alone and in combination, on the downstream mediators of Ras signaling pathways, including P44/42 MAPK, stress-induced MAPK (JNK), P38 MAPK, and Akt.
Compared with levels of phosphorylated p44/42 MAPK, Akt, and GSK-3/
(a downstream mediator of Akt) in vehicle-treated control cells, levels of these proteins were lower in H460 cells infected with Ad-IGFBP3 or treated with SCH66336, and even lower in H460 cells infected with Ad-IGFBP3 and treated with SCH66336 (Fig. 3, A). Although levels of phosphorylated JNK or p38 MAPK were slightly lower in cells infected with Ad-IGFBP3 than in control cells, the addition of SCH66336 to the infected cells had no consistent effect on the levels of these phosphorylated proteins. Each agent alone and their combination showed no effect on the basal levels of p44/42 MAPK, JNK, p38MAPK, or GSK-3
. However, levels of Akt were decreased in H460 cells infected with Ad-IGFBP3 and treated with SCH66336.
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Effect of p44/42 MAPK and Akt on Apoptosis Induced by Ad-IGFBP3 and SCH66336
Because the combination of Ad-IGFBP3 and SCH66336 inhibited the activation of the p44/42 MAPK and PI3K/Akt pathways, we first tested whether the inhibition of these pathways with the MEK inhibitor PD98059 and the PI3K inhibitor LY294002, respectively, would increase the antiproliferative effects of Ad-IGFBP3 and SCH66336 on H460 cells. We used inhibitor doses that were sufficient to reduce the IGF- or EGF-stimulated phosphorylation of p44/42 MAPK or Akt (data not shown). The mean difference in proliferation of cells infected with Ad-IGFBP3 and treated with LY294002 (66.19%, 95% CI = 53.19% to 79.19%) was statistically significantly greater than the sum of the mean difference of cells infected with Ad-IGFBP3 alone (26.79%, 95% CI = 13.78% to 39.79%) and that of cells treated with LY294002 alone (16.42%, 95% CI = 3.41% to 29.42%) (Fig. 4, A) (P = .005). The mean difference in proliferation of cells treated with Ad-IGFBP3 and PD98059 was not statistically significantly greater than the sum of the mean difference of cells treated with each agent alone.
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We tested whether overexpression of constitutively active Akt would rescue H460 cells from apoptosis induced by the expression of Ad-IGFBP3 and treatment with SCH66336. Infection with Ad-IGFBP3 and treatment with SCH66336 induced apoptosis in approximately 80% of uninfected H460 cells or cells infected with Ad-EV but in less than 30% of cells infected with Ad-MyrAkt (Fig. 4, C). In addition, loss of pAkt and of Akt by single-agent treatment or the combination of Ad-IGFBP3 and SCH66336 in H460 cells was completely blocked by infection with Ad-MyrAkt. Because Akt can induce expression of the antiapoptotic protein Bcl-xL (43), we assessed the effect of constitutive Akt activation on Bcl-xL expression. The level of Bcl-xL expression decreased in H460 cells infected with Ad-IGFBP3 and treated with SCH66336 but was similar among cells infected with Ad-MyrAkt, regardless of coinfection with Ad-IGFBP3 and treatment with SCH66336 (Fig. 4, D). Thus, increased apoptosis by infection with Ad-IGFBP3 and treatment with SCH66336 in H460 cells results, in part, from an inhibition of levels and activities of prosurvival Akt and antiapoptotic Bcl-xL proteins.
Apoptotic Effects of Recombinant IGFBP-3 Protein and SCH66336
To confirm that the effects we observed with Ad-IGFBP3 were not related to the adenoviral vector itself, we generated a recombinant IGFBP-3 protein fused to a Tat protein, which permits heterologous proteins to enter cells (44). We monitored localization of TAT-BP3 protein into H1299 cells, which have no detectable endogenous IGFBP-3. TAT-BP3 translocation into H1299 cells was evident within 10 minutes, reached a maximum between 10 and 30 minutes, and then rapidly decreased by 3 hours (Fig. 5, A). Nuclear translocation of TAT-BP3 was observed at 10 minutes and reached a maximum at 1 hour. The levels of -actin and PARP, which were used as cytosolic and nuclear marker proteins, respectively, showed marginal or no detectable change.
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Effects of SCH66336 on IGFBP-3Induced Antitumor Activities in NSCLC
To determine whether Ad-IGFBP3 and SCH66336 had antitumor activities in vivo, we tested the effect of Ad-IGFBP3 and SCH66336 on NSCLC growth using H1299 xenograft tumors established in athymic nude mice. Mice received a single intratumoral injection of Ad-EV or Ad-IGFBP3, which had no detectable effect on H1299 tumor growth (data not shown). The mice were then treated with SCH66336 or HPCD (a vehicle) for 9 days (Fig. 6, A). Tumor size was measured every day for 16 days. Tumor growth in mice that received Ad-IGFBP3 or SCH66336 alone was not statistically significantly different from tumor growth in the appropriate control groups. By contrast, mice that received the combination of Ad-IGFBP3 and SCH66336 had statistically significantly smaller tumors than mice in the other treatment groups (Fig. 6, A). At day 16, the mean tumor volume in mice that received Ad-IGFBP3 and SCH66336 was approximately 45.2% (95% CI = 36.6% to 53.8%) of that in the mice that received PBS and HPCD (Fig. 6, B). The mean difference in tumor volume for mice who received the combination of Ad-IGFBP3 and SCH66336 (54.8%, 95% CI = 46.2% to 63.4%) was statistically significantly greater than the sum of mean differences in tumor volume for mice who received Ad-IGFBP3 and mice who received SCH66336 (14.3%, 95% CI = 0% to 15.9%) (P<.001), indicating that, in H1299 cells, the combination of Ad-IGFBP3 and SCH66336 had increased antitumor activity in vivo.
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DISCUSSION |
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Several studies (24) have suggested that interventions leading to the disruption of the IGF pathway should induce apoptosis, reverse the transformed phenotype, and have antitumor effects. Among the inhibitors of the IGF pathway, IGFBP-3 might be an effective therapeutic agent for lung cancer because it regulates cell growth through dual mechanisms: IGF-dependent extracellular regulation of antiproliferation and survival pathways and IGF-independent intracellular effects on proliferation and apoptosis (45). We demonstrated that overexpression of IGFBP-3 inhibits NSCLC cell proliferation in vitro and in vivo (9). Recently, recombinant IGFBP-3 protein had single-agent and combinatorial antitumor activity (additive or synergistic) with chemotherapeutic agents (16), indicating therapeutic efficacy.
Despite the potential of IGFBP-3 to be used as a therapeutic agent for lung cancer, several NSCLC cell lines showed mild or no sensitivity to IGFBP-3. In our effort to determine the mechanism underlying nonresponsiveness of these NSCLC cell lines to IGFBP-3, we studied the involvement of Ras-mediated signaling pathway because a recent study (17) implicated a Ras-mediated signaling mechanism in the development of resistance to IGFBP-3. We therefore investigated whether inhibition of the Ras-mediated pathway could increase the antiproliferative effects of IGFBP-3. Indeed, we observed a synergistic antiproliferative effect between Ad-IGFBP3 and SCH66336 in several NSCLC cell lines.
Our in vitro results indicate that the combination of Ad-IGFBP3 and SCH66336 increases apoptosis. The combination of these two agents suppressed expression of antiapoptotic proteins Bcl-2 and Bcl-xL without substantially changing the expression of the proapoptotic protein Bax. The balance between antiapoptotic and proapoptotic proteins has been shown to determine whether a cell survives or undergoes apoptosis (46), suggesting that the increased antiapoptotic activities of Ad-IGFBP3 and SCH66336 resulted, in part, from changes in the regulation of the antiapoptotic-to-proapoptotic protein ratio. Because Bcl-xL also functions independently in regulating cell death (47), regulation of the Bcl-xL protein expression could, in part, account for the increased apoptosis in NSCLC cells treated with Ad-IGFBP3 and SCH66336.
The level of IGFBP-3 expression (34) did not affect the responsiveness of NSCLC cells to SCH66336 (Fig. 2). IGFBP-3 activity is altered by posttranslational modifications, including proteolysis; proteases such as kallikrein-like serine proteases, cathepsins, and matrix metalloproteinases (MMPs) cleave IGFBP-3 into small fragments that have reduced affinity for IGFs (4850). Increased MMP activity has been consistently detected in malignant tissues, including lung tumors (51), suggesting that activated MMPs may cleave and inactivate IGFBP-3 in lung cancer. IGFBP-3 has also been detected as a phosphoprotein that influences ligand binding (52,53). IGFBP-3 has potential phosphorylation sites for casein kinase II, cyclic adenosine monophosphatedependent protein kinase, calcium/phospholipiddependent protein kinase, and MAPK, all of which can be activated by the Ras-mediated signaling pathway (52). Therefore, it is reasonable to speculate that, in some NSCLC cell lines, endogenously expressed IGFBP-3 could be inactivated by these modifications and thus SCH66336 would have shown no stimulating effects on IGFBP-3-mediated apoptosis. Although overexpressed IGFBP-3 induced by the adenoviral vector would presumably be modified by the same proteases and kinases, it is possible that IGFBP-3 levels in such cells reached cytotoxic levels and induced apoptosis.
We investigated the mechanism underlying the increased apoptosis mediated by Ad-IGFBP3 and SCH66336. Although SCH66336 targets the Ras signaling pathway and most FTIs block the in vitro growth of transformed cells with wild-type Ras (26), the response of NSCLC cell lines to Ad-IGFBP3 and SCH66336 was not associated with Ras gene mutations (Table 1). Thus, inhibition of Ras activation by SCH66336 may not adequately account for the increased apoptotic effects of Ad-IGFBP3 and SCH66336 in NSCLC cell lines. In support of this notion, recent studies (26,27) have demonstrated that some FTIs have antitumor activities that are independent of Ras. Jiang et al. (54) demonstrated that FTIs block the PI3K/Akt-mediated growth factorand the adhesion-dependent survival pathway and induce apoptosis in human cancer cells that overexpress Akt. In that study, overexpression of Akt2 but not of oncogenic H-ras sensitized NIH/3T3 cells to the FTI. Therefore, we studied the involvement of downstream mediators of Ras-mediated signaling pathways to understand the mechanism of action of SCH66336. We found that, in H460 cells infected with Ad-IGFBP3 and treated with SCH66336, Akt expression was inhibited as a result of decreased protein stability and that inhibition of Akt expression increased apoptosis. This conclusion is supported by several lines of evidence. First, overexpression of constitutively active Akt but not constitutively active MEK1 blocked H460 cells from undergoing apoptosis induced by Ad-IGFBP3 and SCH66336. Second, the combination of Ad-IGFBP3 and SCH66336 blocked expression of the antiapoptotic protein Bcl-xL, which is regulated by Akt (43), except in cells that overexpressed constitutively active Akt. Third, the inhibitory effects on NSCLC cell growth, increased apoptosis, and decreased expression of Akt and Bcl-xL by Ad-IGFBP3 and SCH66336 were also observed in vivo in established NSCLC xenograft tumors established, suggesting the therapeutic efficacy of this combination.
In conclusion, our results demonstrate that the apoptotic activities of IGFBP-3 are increased by SCH66336 in NSCLC cells both in vitro and in vivo. Although inhibition of Ras activation by SCH66336 and blockade of IGF signaling by IGFBP-3 could contribute to cell death, decreased Akt stability seems to be one mechanism underlying the increased apoptotic activities of the IGFBP-3 and SCH66336 combination. LY294002, a PI3K/Akt inhibitor that blocks ATP binding to the p110 PI3K catalytic domain (55), was also effective in increasing the antiproliferative activities of Ad-IGFBP3 and of TAT-BP3. These findings suggest that the PI3K/Akt pathway might be involved in regulating antiproliferative activities of intracellular IGFBP-3, and they implicate IGFBP-3 in a mechanism regulating Akt stability. Akt is constitutively active in most NSCLC cells (9,56), probably by multiple mechanisms (1824), and could interfere with the functions of intracellular IGFBP-3. Therefore, suppression of PI3K/Akt activity might be required in designing IGFBP-3based therapeutic strategies for NSCLC. In support of this idea, recombinant IGFBP-3 protein (TAT-BP3) had enhanced antiproliferative effects on NSCLC cells, when combined with SCH66336 or LY294002.
The role of the IGFBP-3 and SCH66336 combination as an inhibitor of Akt expression has clinical implications, especially for NSCLC, in which constitutive Akt activation occurs frequently (56). Ongoing work is directed toward delineating the optimal clinical protocol in which to translate the activity of IGFBP-3 observed in vitro and in vivo. In addition, because tumor growth was not completely suppressed by simultaneous treatment of Ad-IGFBP3 and SCH66336, additional studies on optimal dosing schedule of the combination treatment and the sequence of administration of two agents, which affect the degree of synergism and thus the therapeutic efficacy (36), are warranted.
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Manuscript received January 20, 2004; revised July 26, 2004; accepted August 20, 2004.
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