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Cancer Research 67, 1163, February 1, 2007. doi: 10.1158/0008-5472.CAN-06-2037
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

p53 Enhances Gefitinib-Induced Growth Inhibition and Apoptosis by Regulation of Fas in Non–Small Cell Lung Cancer

Jin Kyung Rho, Yun Jung Choi, Baek-Yeol Ryoo, Im II Na, Sung Hyun Yang, Cheol Hyeon Kim and Jae Cheol Lee

Department of Internal Medicine, Korea Institute of Radiological and Medical Science, Seoul, Korea

Requests for reprints: Jae Cheol Lee, Department of Internal Medicine, Korea Institute of Radiological and Medical Science, 215-4 Gongneung-dong, Nowon-gu, Seoul 139-706, Korea. Phone: 82-2-9701206; E-mail: jclee{at}kcch.re.kr.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment with gefitinib, a specific inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), has resulted in dramatic responses in some patients with non–small cell lung cancer (NSCLC). Most patients who respond to gefitinib have EGFR-TK mutations; however, >10% of patients with EGFR-TK mutations do not respond. Similarly, some patients without EGFR-TK mutations respond to this drug, suggesting that other factors determine sensitivity to gefitinib. Aberrations of the tumor suppressor gene p53 are frequently associated with drug resistance. In this study, we investigated the role of p53 in growth-inhibitory and apoptotic effects of gefitinib in the human NSCLC cell lines NCI-H1299 and A549, which have no EGFR-TK mutations. NCI-H1299 cells, which had a p53-null genotype, were more resistant to gefitinib compared with A549 cells, which were wild-type p53 (IC50, 40 µmol/L in NCI-H1299 and 5 µmol/L in A549). Treatment of A549 with gefitinib resulted in the translocation of p53 from cytosol to nucleus and the up-regulation of Fas, which was localized to the plasma membrane. In the stable H1299 cell line with tetracycline-inducible p53 expression, induced p53 enhanced growth inhibition and apoptosis by gefitinib through the up-regulation of Fas and restoration of caspase activation. A caspase inhibitor, Z-VAD-fmk, reduced these effects. Conversely, inhibition of p53 using antisense oligonucleotide in A549 caused a significant decrease in apoptosis by gefitinib and down-regulation of Fas under the same conditions. In conclusion, p53 may play a role in determining gefitinib sensitivity by regulating Fas expression in NSCLC. [Cancer Res 2007;67(3):1163–9]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is the leading cause of cancer deaths worldwide, representing more deaths than those from prostate, breast, and colorectal cancers combined (1). The overall 5-year survival rate of individuals with lung cancer remains at ~14% because only a minority of patients can be candidates for radical treatment with curative intent, and the effect of systemic therapies has been modest (2). Therefore, novel therapeutic strategies to improve the prognosis of lung cancer are urgently needed.

Recent advances in cancer biology have identified many therapeutic targets related to the growth and survival of cancer cells. One of these targets is the epidermal growth factor receptor (EGFR), which is highly expressed in numerous types of human cancer and acts as a strong prognostic indicator in head and neck, breast, ovarian, cervical, bladder, and esophageal cancers (3). Gefitinib (Iressa) is an orally active EGFR tyrosine kinase (EGFR-TK) inhibitor that shows good tolerability and antitumor activity in patients with non–small cell lung cancer (NSCLC) refractory to platinum-based chemotherapy (4, 5). Somatic mutations in the EGFR-TK domain are important predictors of an individual's response to gefitinib (6, 7). EGFR mutations are more prevalent in females, nonsmokers, adenocarcinoma, and Asian heritage (8, 9). Although most patients who respond to gefitinib have EGFR-TK mutations, >10% of patients with EGFR-TK mutations do not respond to gefitinib. Similarly, some patients in whom EGFR-TK mutations are absent respond to this drug. In addition, most patients achieving stable disease have wild-type EGFR, suggesting that other factors are involved in determining sensitivity to gefitinib (10).

p53 is a nuclear phosphoprotein that plays an important role in apoptosis, growth arrest, genomic stability, cell senescence, and differentiation. p53 mutations are the most common genetic changes found in human cancers, including lung cancer (~70% of small cell lung cancer and 50% of NSCLC; ref. 11). Mutations of this gene often result in loss of p53 function and p53 inactivation (12, 13), contributing to aggressive tumor behavior and therapeutic resistance (14). Several reports have shown that the expression of wild-type p53 is necessary for the cytotoxic response to chemotherapeutic agents (15–17), although there are some conflicting results (18, 19).

We examined whether sensitivity to gefitinib could be affected by the status of p53 expression in NSCLC lines without EGFR-TK mutations. In addition, the molecular mechanisms underlying the effect of p53 on gefitinib-induced cell growth inhibition and apoptosis were investigated.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents. The two human NSCLC cell lines used in this study, A549 and NCI-H1299, were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin (Invitrogen) at 37°C in an atmosphere of 5% CO2. Gefitinib was kindly provided by AstraZeneca Korea (Seoul, Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution and propidium iodide were purchased from Sigma Chemical Co. (St. Louis, MO). FITC-labeled goat/mouse anti-mouse IgG antibodies were purchased from Zymed (South San Francisco, CA). Z-VAD-fmk was purchased from Calbiochem (San Diego, CA).

EGFR DNA sequencing. Total genomic DNA was isolated from cell lines according to the protocol of the Puregene DNA purification kit (Gentra Systems, Plymouth, MN). DNA (100 ng) was amplified in a 20-µL reaction solution containing 2 µL 10x buffer (Roche, Mannheim, Germany), 1.7 to 2.5 mmol/L MgCl2, 250 µmol/L deoxynucleoside triphosphate, 2.5 units of DNA polymerase (Roche), and 0.3 µmol/L of each primer pair for EGFR. Amplification of EGFR was carried out using primers detailed in Table 1 . Fragments of EGFR were amplified with a 5-min initial denaturation at 94°C followed by 30 cycles of 1 min at 94°C, 1 min at 57°C, and 1 min at 72°C, with a final 10-min extension at 72°C. PCR of EGFR was targeted for exons 18, 19, 20, and 21 in the critical kinase domain. DNA sequencing was done as described previously (20). DNA purification and EGFR DNA sequencing were done in separate laboratories.


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  		Table 1. Primers used for amplification of genomic DNA sequences of EGFR

 
MTT assay. Briefly, cells in the logarithmic growth phase were harvested and seeded in 96-well plates overnight. Gefitinib was added and cells were incubated for 72 h. The viability of cells was determined using the MTT assay in accordance with the method described by Carmichael et al. (21).

Apoptosis assay. Apoptosis was quantified using the Annexin V-FITC apoptosis kit (BD Biosciences, San Diego, CA) in accordance with the manufacturer's instructions. Briefly, cells were trypsinized (Invitrogen), pelleted by centrifugation, and resuspended in Annexin V–binding buffer (150 mmol/L NaCl, 18 mmol/L CaCl2, 10 nmol/L HEPES, 5 mmol/L KCl, 1 mmol/L MgCl2). FITC-conjugated Annexin V (1 µg/mL) and propidium iodide (50 µg/mL) were added to cells and incubated for 30 min at room temperature in the dark. Analyses were done on a FACScan (Becton Dickinson, Mountain View, CA). The data were analyzed with CellQuest software (Becton Dickinson).

Western blot analysis. Cells were washed with PBS and solubilized in lysis buffer (25 mmol/L HEPES, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, 10 mmol/L EDTA, 10 mmol/L NaF, 125 mmol/L NaCl). The protein was then collected after centrifugation at 10,000 x g for 10 min at 4°C. The resulting supernatant (50 µg) was separated on 8% to 12% SDS-PAGE and transferred to nitrocellulose filters. The membranes were blocked with 5% skim milk-PBS-0.1% Tween 20 for 1 h at room temperature before being incubated overnight with primary antibodies specific for caspase-8 (4-1-20; 1:1,000; Pharmagene, San Diego, CA), caspase-9 (Poly1330; 1:1,000; Pharmagene), caspase-3 (E-8; 1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), ß-actin (AC-74; 1:4,000; Sigma Chemical), p53 (DO-7; 1:2,000; DakoCytomation, Carpinteria, CA), Ref-1 (N-16; 1:2,000; Santa Cruz Biotechnology), Fas (1:1,000; R&D Systems, Minneapolis, MN), Bax (1:2,000; DakoCytomation), or p21 (C-19; 1:2,000; Santa Cruz Biotechnology) diluted in 5% skim milk-PBS-0.1% Tween 20. The membranes were then washed thrice in 1x PBS-0.1% Tween 20 and incubated with horseradish peroxidase–conjugated secondary antibodies diluted to 1:2,000 in 5% skim milk-PBS-0.1% Tween 20 for 1 h. After successive washes, the membranes were developed using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ).

Fluorescent immunocytochemistry. The cells were cultured in 60-mm dishes in the presence or absence of gefitinib for 48 h. Collected cells were fixed with cold methanol for 10 min and washed thrice with PBS. Next, they were permeabilized with 0.2% Triton X-100 in PBS for 5 min followed by blocking with goat serum and 5% bovine serum albumin for 1 h. Cells were stained with monoclonal antibodies to p53 or Fas at 2 µg/mL for 6 h, washed twice with PBS, and incubated with FITC-conjugated goat anti-mouse IgG (1:20) or anti-rabbit IgG (1:50) for 1 h. The slides for translocation of p53 were counterstained with propidium iodide (0.5 µg/mL) to observe DNA and then washed and coverslipped. Slides were viewed under a confocal laser scanning microscope (TC SP2; Leica, Heidelberg, Germany) and green fluorescence, and the images were photographed.

Preparation of nuclear extracts. Nuclear extracts from cells were isolated using a nuclear extract buffer kit in accordance with the manufacturer's instructions (Pierce, Rockford, IL). Briefly, cells were treated with gefitinib for 48 h and then washed with cold PBS. The cellular membrane was then lysed by adding CER buffer and incubating on ice for 5 min. After centrifugation at 15,000 x g for 5 min, the pellets were resuspended in NER buffer and shaken for 2 h at 4°C to release the nuclear proteins. After centrifugation at 15,000 x g for 5 min, the supernatant was aliquoted and stored at –80°C until use.

Construction of stable cell lines. p53 cDNA was constructed by PCR using the pcDNA4/TO expression vector (Invitrogen). This placed the p53 open reading frames under the translational control of the phCMV/TetO2 promoter containing the tetracycline (Tet)-responsive element immediately upstream of the minimal cytomegalovirus (CMV) promoter. The pcDNA4/TO-p53 plasmid was cotransfected into NCI-H1299 cells with pcDNA6/TR, a plasmid that confers blasticidin resistance, to allow the selection of stable transfectants. Stable clones of NCI-H1299-pcDNA4/TO-p53-pcDNA6/TR (H1299/p53) constitutively express the Tet-controlled repressor TetR, which interacts with and inactivates the phCMV/TetO2 promoter in the absence of doxycycline in the culture medium. After transfection, H1299/p53 cells were exposed for 2 to 3 weeks in zeocin-containing (Invitrogen) and blasticidin-containing medium (800 µg/mL zeocin and 10 µg/mL blasticidin) to select cells that express resistance to this marker. Colonies were picked for their resistance to zeocin and blasticidin. The expression of several individual clones derived from each transfection was tested in the presence or absence of doxycycline.

Transfection of antisense oligodeoxynucleotides. Antisense oligodeoxynucleotides were synthesized by Bioneer Co. (Seoul, Korea) in the form of phosphorothioate oligodeoxynucleotides. The sequence of p53 antisense oligodeoxynucleotide was 5'-CCCTGCTCCCCCCTGGCTCC-3' and the control oligodeoxynucleotide was 5'-GGAGCCAGGGGGGAGCAGGG-3' (22). These oligodeoxynucleotides were used for transfection in cells. The cells (5 x 105 per p35 dish) were transfected with 100 nmol/L of control or antisense oligodeoxynucleotides by using LipofectAMINE 2000 (Invitrogen) in 1 mL of serum-free medium for 5 h at 37°C in a CO2 incubator in accordance with the manufacturer's recommendations. Then, 1 mL DMEM with 20% FBS was added without removing the transfection mixture, and incubation proceeded for an additional 24 h. After transfection, the cells were treated with gefitinib for 48 h and the apoptosis assay was conducted as described above.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A549 cells were more sensitive to gefitinib compared with NCI-H1299 cells. The effects of gefitinib on the viability of NCI-H1299 (p53-null genotype) and A549 (p53 wild-type genotype) cells were examined. Cells were treated with various concentrations of gefitinib for 72 h. As shown in Fig. 1A , gefitinib exhibited antiproliferative effects in a dose-dependent manner in A549 and NCI-H1299 cells; however, A549 cells were more sensitive to gefitinib than NCI-H1299 cells (IC50, 5 µmol/L in A549 cells and 40 µmol/L in NCI-H1299 cells). We further assessed apoptosis in cells that were exposed to 20 µmol/L gefitinib using flow cytometry after Annexin V-FITC and propidium iodide staining. Gefitinib induced more apoptosis in A549 cells than in NCI-H1299 cells in a time-dependent manner, with ~15% to 60% apoptosis in A549 cells and ~3% to 20% in NCI-H1299 (Fig. 1B). Western blot analysis showed that procaspases were markedly reduced in gefitinib-treated A549 cells; however, procaspase reduction did not occur in NCI-H1299 cells (Fig. 1C). In addition, the reduction of procaspases led to increase of cleaved forms (see Supplementary Fig. S1). A549 and NCI-H1299 cells do not have EGFR-TK mutations in the exon 18 to 21 sequence, showing that the difference in sensitivity to gefitinib between the two cell types was not caused by EGFR-TK mutations (Table 2 ).


Figure 1
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  		Figure 1. A549 cells were more sensitive to gefitinib compared with NCI-H1299 cells. A, A549 and NCI-H1299 cells were treated with the indicated doses of gefitinib for 72 h. The viability of cells was determined using the MTT assay. B, cells were treated with or without 20 µmol/L gefitinib for the indicated times. Cells were harvested, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry. The percentage of apoptotic cells was expressed as the sum of the bottom right (early state of apoptosis) and top right quadrants (late stage of apoptosis). Columns, mean of three experiments; bars, SE. C, under identical conditions of apoptotic analysis, proform levels of caspase-3, caspase-8, and caspase-9 were detected by Western blot analysis.

 

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  		Table 2. Cell line characteristics and sensitivity to gefitinib

 
p53 was translocated to the nucleus and increased Fas expression in gefitinib-induced apoptosis. Wild-type p53 is a critical transcription factor that responds to signals from a wide range of cellular stresses. To investigate the possible roles of p53 in the gefitinib-mediated cytotoxicity of NSCLC, A549 cells were treated with gefitinib for 48 h, and then total protein and nuclear extracts were separated on a SDS-PAGE gel and Western blot analysis was done. Furthermore, duplicated samples were measured to localize p53 in the cell. Although the amount of p53 did not change in gefitinib-treated A549 cells, p53 accumulated in the nucleus (Fig. 2A and B ). Unequal protein loading was not the cause of this apparent difference in p53 distribution between gefitinib-treated and nontreated A549 cells because no difference in ß-actin, or the nuclear protein marker Ref-1, was observed. The low amount of ß-actin observed in nuclear extracts indicated that very little cytoplasmic protein contamination of the nuclear extracts had occurred. Nuclear translocation of p53 can lead to up-regulation of the Fas gene as well as several other genes, including Bax and p21. Therefore, the expression of these genes was quantified using immunostaining and Western blotting. There was no difference in p21 and Bax expression in A549 cells treated with gefitinib for 48 h (Fig. 3A ). However, the level of Fas increased on gefitinib treatment and Fas was localized to the plasma membrane (Fig. 3A and B). These data show that the translocation of p53 to the nucleus and the up-regulation of Fas are involved in gefitinib-induced apoptosis.


Figure 2
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  		Figure 2. Gefitinib-induced apoptosis was accompanied by accumulation of p53 in the nucleus. A549 cells were treated with the indicated doses of gefitinib for 48 h. A, total protein lysates or nuclear extracts of A549 cells were treated as indicated and Western blot analyses for p53, Ref-1, and ß-actin were done. B, the treated A549 cells were fixed with 95% methanol, immunostained with anti-p53 (green fluorescence) and propidium iodide (red fluorescence), and analyzed by confocal microscopy to determine the intracellular localization of p53. Each photograph is representative of three independent experiments.

 

Figure 3
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  		Figure 3. The expression level of Fas was enhanced in gefitinib-induced apoptosis. A549 cells were treated with gefitinib for 48 h. A, total protein was extracted and the levels of p21, Bax, and Fas were analyzed by Western blotting. B, treated cells were stained with anti-Fas for determination of Fas expression and localization.

 
Reconstitution of p53 in NCI-H1299 cells enhanced sensitivity to gefitinib. To determine whether the restoration of p53 influences gefitinib-induced apoptosis, we used the lung cancer cell line NCI-H1299, which lacks p53 expression because of homozygous deletion of the gene (23). For the expression of p53, we introduced full-length p53 into the pcDNA4/TO expression vector. In this vector, p53 was placed downstream of the Tet-responsive element, which can turn the CMV promoter on and off. The pcDNA4/TO-p53 plasmid was cotransfected into NCI-H1299 cells with pcDNA6/TR, which encodes a Tet repressor, and stable cell lines were obtained following antibiotic selection. After treatment with the Tet analogue doxycycline, H1299/p53 cells exhibited induction of p53 in a dose-dependent manner at 24 h (Fig. 4A ). In particular, the level of p53 expression was saturated by doxycycline (1 µg/mL). A similar increase was observed in the expression of the classic p53 target genes p21WAF-1/CIP-1. The observed changes in cell growth and cell responses to gefitinib after restoration of p53 are displayed in Table 2. No significant difference, in accordance with the reconstitution of p53, was observed in in vitro cell growth (doubling time of 15.8 h in NCI-H1299 cells and 16.2 h in H1299/p53 cells; P < 0.02) and microscopic morphology. However, restoration of p53 enhanced the sensitivity to gefitinib (IC50, 40 µmol/L in NCI-H1299 cells and 12 µmol/L in H1299/p53 cells). The incubation of cells for the indicated times with 20 µmol/L gefitinib resulted in a marked increase in time-dependent cell death in H1299/p53 (+Dox) cells (from ~10% to 52%; Fig. 4B), which was much less obvious in H1299/p53 (–Dox) cells (from ~6% to 17%; Fig. 4B). Under the same conditions, an increase in Fas expression was found only in p53-induced cells in a time-dependent manner (Fig. 4C). Furthermore, the activation of caspases, which can be regulated by the Fas/FasL system, was restored, like the apoptosis in A549 cells, by gefitinib (Fig. 4C). To assess whether the activated caspases are necessary for apoptosis induced by gefitinib, cell-permeable Z-VAD-fmk was used to inhibit the activation of caspases (24). As shown in Fig. 4D, Z-VAD-fmk significantly inhibited gefitinib-mediated apoptosis in H1299/p53 (–Dox) and H1299/p53 (+Dox) cells (from ~17% to 10% and 30% to 5%, respectively). Together, these findings indicate that the restored p53 enhanced gefitinib-induced apoptosis via the activation of caspases by the Fas/FasL signaling pathway.


Figure 4
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  		Figure 4. Reconstitution of p53 in NCI-H1299 cells enhanced sensitivity to gefitinib via activation of caspases. A, H1299/p53 cells were treated with indicated doses of doxycycline (Dox) for 24 h. Lysates were separated by SDS-PAGE and immunoblotted using anti-p53, anti-p21, and anti-ß-actin antibodies. B, cells were treated with 20 µmol/L gefitinib in the presence or absence of doxycycline (1 µg/mL) for the indicated times. Apoptotic cells were determined by flow cytometry. C, under identical conditions of apoptotic analysis, proforms of caspase-3, caspase-8, caspase-9, and Fas were analyzed by Western blotting. D, after treatment with doxycycline (1 µg/mL), cells were incubated with 20 µmol/L gefitinib for 48 h with or without preincubation in 20 µmol/L Z-VAD-fmk. Columns, mean of three experiments; bars, SE. P values were calculated to assess the effect of Z-VAD-fmk. *, P < 0.04; **, P < 0.02.

 
Silencing of p53 reduced gefitinib-induced apoptosis via down-regulation of Fas. We examined whether the increase in Fas depends on p53 in gefitinib-induced apoptosis and whether inhibition of p53 influences sensitivity to gefitinib. Here, a p53 antisense oligodeoxynucleotide was used to block the effect of p53 in gefitinib-induced apoptosis. The level of p53 and p21 expression decreased when A549 cells were treated with 10 nmol/L p53 antisense oligodeoxynucleotide for 24 h (Fig. 5A ). After transfection of cells with the p53 antisense oligodeoxynucleotide, gefitinib-induced apoptosis decreased from 53% to 15% (Fig. 5B and C). Moreover, this decrease was accompanied by a decrease in Fas expression (Fig. 5C). Taken together, these results indicate that the regulation of Fas by p53 affects the cellular response to gefitinib.


Figure 5
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  		Figure 5. Blocking of p53 expression suppressed gefitinib-induced apoptosis in A549 cells. A, p53 antisense oligodeoxynucleotide was transfected into A549 cells. After 24 h, lysates were separated by SDS-PAGE and immunoblotted to detect p53 expression. B, cells were incubated with 20 µmol/L gefitinib for 48 h after transfection of control or p53 antisense oligodeoxynucleotide. The percentage of apoptotic cells was determined using the method described in Fig. 1. Diagrams of FITC-Annexin V/propidium iodide flow cytometry in a representative experiment are presented above the graphs. C, under identical conditions of apoptotic analysis, the level of Fas was detected by Western blot analysis. Columns, mean of three experiments; bars, SE. P value was calculated to assess the difference between control and p53 antisense oligodeoxynucleotide–transfected cells after gefitinib treatment. *, P < 0.001.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many previous studies have reported that viral or inducible p53 gene delivery could lead to a significant level of cell death in many cancer cell lines (25, 26). We conducted reconstitution of p53 through Tet-inducible transfection in NCI-H1299 cells, which do not express wild-type p53. However, cell death was not detected by p53 expression itself within the experimental time of our study (data not shown). This may be due to the different cell types and the amount of p53 expression. It was interesting that sensitivity to gefitinib was enhanced by reintroduction of p53 in NCI-H1299 cells and the inhibition of p53 in A549 cells by antisense oligonucleotide reduced gefitinib-induced apoptosis. Moreover, the reintroduction of p53 enhanced Fas expression and led to activation of caspases (caspase-9, caspase-8, and caspase-3), which can induce effective apoptosis. These findings imply that p53 is required for the enhancement of gefitinib-induced apoptosis.

An intact apoptotic pathway is necessary for chemotherapy-induced cell death, and conversely, abnormality in the ability of a cell to undergo apoptosis may lead to chemotherapy resistance (27). Several previous studies have shown that activation of caspase system is involved in gefitinib-induced apoptosis, although the kind of caspase activation could be different according to the investigated cell type (28–30). We found that 20 µmol/L gefitinib did not activate caspases in NCI-H1299 cells, a relatively resistant cell line proven by MTT assay, which contrasts with our results for A549 cells. Thus, the restoration of caspase activation might increase sensitivity to gefitinib in NCI-H1299 cells. In the present study, reintroduction of p53 enhanced apoptosis and activated caspase cascade in NCI-H1299 cells. Moreover, enhanced apoptosis and caspase activation after gefitinib treatment were suppressed by pretreatment with pan-caspase inhibitor (Z-VAD-fmk). Therefore, it is likely that activation of caspases via reintroduction of p53 results in enhanced apoptosis.

Our study showed that gefitinib treatment resulted in nuclear translocation of p53 and enhancement of apoptosis through reintroduction of p53. These results suggest that p53 acts as a necessary factor for apoptosis induced by gefitinib treatment. Although the exact mechanism by which p53 promotes apoptosis is not fully understood, the p53-dependent expression of proapoptotic genes, such as Bax and Fas, has been suggested to be a possible mechanism (31, 32). In fact, it was reported that gefitinib induced apoptosis through activation of Bax in human gallbladder adenocarcinoma cells (33). In addition, they showed that activation of Bax was independent on p53 as significant change of p53 was not detected. However, our study showed that Bax was not involved in p53-dependent apoptosis induced by gefitinib in lung cancer cells. Although the difference of investigated cell type might contribute to these results, other determining factors to activate the pathway leading to cell death should be clarified.

Several studies have shown that p53 (reintroduction of p53 by viral vector or activation of p53 by stress) is associated with up-regulation of Fas (34, 35), and the Fas promoter contains a p53-responseive element (36). Thus, the loss of wild-type p53 protein expression has been widely believed to also result in the loss of Fas-induced apoptosis (37). A recent study showed that gefitinib treatment up-regulated Fas in lung cancer cells (38). However, the mechanism of gefitinib-induced Fas expression was not elucidated. Our data showed that wild-type p53 increased the expression of Fas in endogenous or exogenous p53 expression model. In addition, enhanced Fas expression was localized to plasma membrane. Interestingly, this phenomenon was caused by treatment of gefitinib in the presence of p53, not by induced p53 expression alone. Conversely, the inhibition of p53 in A549 cells by antisense oligonucleotide reduced Fas expression and apoptosis, suggesting that Fas is an important transcriptional target of p53 mediating gefitinib-induced apoptosis. Fas-mediated apoptosis may explain the short response time of gefitinib in patients with NSCLC compared with other chemotherapeutic agents, although the major antitumor effect of gefitinib is cytostatic (39).

In the era of targeted and tailored therapy, it is very important to identify the predictive factors for response to several agents directing molecular pathways involved in cancer development and progress. Although EGFR-TK mutations mainly contribute to the response to gefitinib, we believe that p53 also may be a useful variable determining clinical response, especially in NSCLC without EGFR-TK mutations.

In summary, we have shown that p53 enhances gefitinib-induced growth inhibition and apoptosis by modulating Fas and the caspase system in NSCLC cells without EGFR-TK mutations. However, our data do not show the mechanism by which gefitinib inhibits EGFR signaling, which leads to the activation of p53. Further detailed studies are required to clarify this mechanism and the clinical usefulness of p53 as a marker for gefitinib sensitivity in NSCLC.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 6/ 2/06. Revised 9/22/06. Accepted 12/ 5/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2006. CA Cancer J Clin 2006;56:106–30.[Abstract/Free Full Text]
  2. Spiro SG, Silvestri GA. One hundred years of lung cancer. Am J Respir Crit Care Med 2005;172:523–9.[Abstract/Free Full Text]
  3. Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001;37 Suppl 4:S9–15.
  4. Kris MG, Natale RB, Herbst RS, et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small-cell lung cancer: a randomized trial. JAMA 2003;290:2149–58.[Abstract/Free Full Text]
  5. Fukuoka M, Yano S, Giaccone G, et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (the IDEAL 1 trial). J Clin Oncol 2003;21:2237–46.[Abstract/Free Full Text]
  6. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39.[Abstract/Free Full Text]
  7. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–500.[Abstract/Free Full Text]
  8. Janne PA, Engelman JA, Johnson BE. Epidermal growth factor receptor mutations in non-small-cell lung cancer: implications for treatment and tumor biology. J Clin Oncol 2005;23:3227–34.[Abstract/Free Full Text]
  9. Pao W, Miller VA. Epidermal growth factor receptor mutations, small-molecule kinase inhibitors, and non-small-cell lung cancer: current knowledge and future directions. J Clin Oncol 2005;23:2556–68.[Abstract/Free Full Text]
  10. Chan SK, Gullick WJ, Hill ME. Mutations of the epidermal growth factor receptor in non-small cell lung cancer—search and destroy. Eur J Cancer 2006;42:17–23.[CrossRef][Medline]
  11. Viktorsson K, De Petris L, Lewensohn R. The role of p53 in treatment responses of lung cancer. Biochem Biophys Res Commun 2005;331:868–80.[CrossRef][Medline]
  12. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49–53.[Abstract/Free Full Text]
  13. Braithwaite AW, Royds JA, Jackson P. The p53 story: layers of complexity. Carcinogenesis 2005;26:1161–9.[Abstract/Free Full Text]
  14. El-Deiry WS. The role of p53 in chemosensitivity and radiosensitivity. Oncogene 2003;22:7486–95.[CrossRef][Medline]
  15. Perdomo JA, Naomoto Y, Haisa M, et al. In vivo influence of p53 status on proliferation and chemoradiosensitivity in non-small-cell lung cancer. J Cancer Res Clin Oncol 1998;124:10–8.[CrossRef][Medline]
  16. Lai SL, Perng RP, Hwang J. p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells. J Biomed Sci 2000;7:64–70.[CrossRef][Medline]
  17. Vogt U, Zaczek A, Klinke F, et al. p53 gene status in relation to ex vivo chemosensitivity of non-small cell lung cancer. J Cancer Res Clin Oncol 2002;128:141–7.[CrossRef][Medline]
  18. Perez-Soler R, Kemp B, Wu QP, et al. Response and determinants of sensitivity to paclitaxel in human non-small cell lung cancer tumors heterotransplanted in nude mice. Clin Cancer Res 2000;6:4932–8.[Abstract/Free Full Text]
  19. Oizumi S, Isobe H, Ogura S, et al. Topoisomerase inhibitor-induced apoptosis accompanied by down-regulation of bcl-2 in human lung cancer cells. Anticancer Res 2002;22:4029–37.[Medline]
  20. Han SW, Kim TY, Hwang PG, et al. Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib. J Clin Oncol 2005;23:2493–501.[Abstract/Free Full Text]
  21. Carmichael J, DeGraff WG, Gazdar AF, et al. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 1987;47:936–42.[Abstract/Free Full Text]
  22. Kuo PC, Liu HF, Chao JI. Survivin and p53 modulate quercetin-induced cell growth inhibition and apoptosis in human lung carcinoma cells. J Biol Chem 2004;279:55875–85.[Abstract/Free Full Text]
  23. Chen JY, Funk WD, Wright WE, et al. Heterogeneity of transcriptional activity of mutant p53 proteins and p53 DNA target sequences. Oncogene 1993;8:2159–66.[Medline]
  24. Zhu H, Fearnhead HO, Cohen GM. An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells. FEBS Lett 1995;374:303–8.[CrossRef][Medline]
  25. Yonish-Rouach E, Grunwald D, Wilder S, et al. p53-mediated cell death: relationship to cell cycle control. Mol Cell Biol 1993;13:1415–23.[Abstract/Free Full Text]
  26. Prives C, Hall PA. The p53 pathway. J Pathol 1999;187:112–26.[CrossRef][Medline]
  27. Strobel T, Swanson L, Korsmeyer S, Cannistra SA. BAX enhances paclitaxel-induced apoptosis through a p53-independent pathway. Proc Natl Acad Sci U S A 1996;93:14094–9.[Abstract/Free Full Text]
  28. Hopfner M, Sutter AP, Huether A, et al. Targeting the epidermal growth factor receptor by gefitinib for treatment of hepatocellular carcinoma. J Hepatol 2004;41:1008–16.[CrossRef][Medline]
  29. Teraishi F, Kagawa S, Watanabe T, et al. ZD1839 (gefitinib, ‘Iressa’), an epidermal growth factor receptor tyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma. FEBS Lett 2005;579:4069–75.[CrossRef][Medline]
  30. Piechocki MP, Yoo GH, Dibbley SK, et al. Iressa induces cytostasis and augments Fas-mediated apoptosis in acinic cell adenocarcinoma overexpressing HER2/neu. Int J Cancer 2006;119:441–54.[CrossRef][Medline]
  31. Miyahsita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995;80:293–9.[CrossRef][Medline]
  32. Friesen C, Herr I, Krammer PH, Debatin KM. Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells. Nat Med 1996;2:574–7.[CrossRef][Medline]
  33. Ariyama H, Qin B, Baba E, et al. Gefitinib, a selective EGFR tyrosine kinase inhibitor, induces apoptosis through activation of Bax in human gallbladder adenocarcinoma cells. J Cell Biochem 2006;97:724–34.[CrossRef][Medline]
  34. Shinoura N, Yoshida Y, Asai A, et al. Adenovirus-mediated transfer of p53 and Fas ligand drastically enhances apoptosis in gliomas. Cancer Gene Ther 2000;7:732–8.[CrossRef][Medline]
  35. Martin LJ, Chen K, Liu Z. Adult motor neuron apoptosis is mediated by nitric oxide and Fas death receptor linked by DNA damage and p53 activation. J Neurosci 2005;25:6449–59.[Abstract/Free Full Text]
  36. Munsch D, Watanabe-Fukunaga R, Bourdon JC, et al. Human and mouse Fas (APO-1/CD95) death receptor genes each contain a p53-responsive element that is activated by p53 mutants unable to induce apoptosis. J Biol Chem 2000;275:3867–72.[Abstract/Free Full Text]
  37. Egle A, Villunger A, Marschitz I, et al. Expression of Apo-1/Fas (CD95), Bcl-2, Bax, and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status. Br J Haematol 1997;97:418–28.[CrossRef][Medline]
  38. Chang GC, Hsu SL, Tsai JR, et al. Molecular mechanisms of SD1839-induced G1-cell cycle arrest and apoptosis in human lung adenocarcinoma A549 cells. Biochem Pharmacol 2004;68:1453–64.[CrossRef][Medline]
  39. Giocanti N, Hennequin C, Rouillard D, et al. Additive interaction of gefitinib (‘Iressa’, ZD1839) and ionizing radiation in human tumour cells in vitro. Br J Cancer 2004;91:2026–33.[CrossRef][Medline]



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