Cancer Letters 226 (2005) 37–47 www.elsevier.com/locate/canlet
Antitumor effect of gefitinib (‘Iressa’) on esophageal squamous cell carcinoma cell lines in vitro and in vivo Fumikata Hara*, Motoi Aoe, Hiroyoshi Doihara, Naruto Taira, Tadahiko Shien, Hirotoshi Takahashi, Seiji Yoshitomi, Kazunori Tsukuda, Shinichi Toyooka, Tetsuya Ohta, Nobuyoshi Shimizu Department of Cancer and Thoracic Surgery, Okayama University Graduate School Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan Received 19 August 2004; received in revised form 13 December 2004; accepted 15 December 2004
Abstract High expression of epidermal growth factor receptor (EGFR) is thought to be correlated with cell proliferation, invasion, metastasis, resistance to chemoradiotherapy, and poor prognosis in various kinds of human cancers. Blockade of EGFR signal transduction can be a promising strategy for cancer therapy. Approximately 40–70% of esophageal squamous cell carcinomas (ESCCs) show high expression of EGFR. In this study, we examined the antitumor effect of gefitinib, an EGFR tyrosine kinase inhibitor, against ESCC cells in vitro and in vivo. In three ESCC cell lines (TE8, T.T and T.Tn), cell proliferation had been inhibited in a dose-dependent manner and IC50 values (respectively, 8.49, 18.9 and 17.3 mM). Gefitinib inhibited EGF-induced autophosphorylation of EGFR and its downstream signaling pathways, Ras/Raf/MAPK and PI3K/Akt, and caused G1 arrest of cell cycle and apoptosis confirmed with flow cytometry. We examined the effect of gefitinib on nude mice bearing established TE8 and T.T xenografts. Gefitinib (100 or 200 mg/kg once-daily, p.o.) showed antitumor activity in a dose-dependent manner, resulting in a significantly improved survival of treated mice as compared with untreated mice. Immunohistochemical examination of the harvested tumor was performed to examine the status of phosphorylated EGFR, PCNA, Factor VIII and apoptosis. We found inhibition of EGFR phosphorylation, cell cycle arrest (by PCNA staining), decrease of microvessel density (Factor VIII) and induction of apoptosis by TUNEL staining. In conclusion, our findings demonstrate that gefitinib is effective
Abbreviations: ESCC, esophageal squamous cell cancer; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor; Erk, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3 0 -kinase; FACS, fluorescence-activated cellsorting; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotide transferase dUTP nick end labeling; VW, Von Willebrand; VEGF, vascular endothelial growth factor; MVC, microvessel count. * Corresponding author. Tel.: C81 86 235 7265; fax: C81 86 235 7269. E-mail address:
[email protected] (F. Hara). 0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.12.025
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for growth inhibition of ESCC cell lines in vitro and in vivo and suggest that gefitinib may be one of the new therapeutic options for ESSC. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Esophageal squamous cell carcinoma; EGFR; Gefitinib
1. Introduction Esophageal cancer is one of the most frequently occurring malignancies worldwide. Annually, approximately 400,000 patients were diagnosed and an estimated more than 300,000 died of this malignancy in 2000 [1]. Esophageal squamous cell carcinoma (ESCC) is a common problem in several Eastern and Asian countries. Approximately 95% of esophageal cancers are squamous cell carcinoma; adenocarcinoma is less common in Japan [2]. The poor prognosis reflects the fact that few esophageal cancers are diagnosed at an early stage and that, even in localized stages, early lymphatic metastasis occurs [3]. Though new cytotoxic agents and radiation have provided some improvement in the treatment of advanced esophageal cancer, response rate is low and patients’ survival remains poor [4]. Thus, novel approaches to esophageal cancer therapy are urgently needed. Much progress in understanding the molecular biology of carcinogenesis has been made over the past 20 years. EGFR was one of the receptor tyrosine kinases identified as being highly expressed in cancer cells. EGFR is a transmembrane protein consisting of an extracellular ligand binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain [5]. EGFR is a part of the subfamily of four closely related receptors: EGFR, HER2, HER3 and HER4 [6]. After specific ligand binding, EGFR homodimerizes with itself or heterodimerizes with other HER receptors. Sequentially, specific tyrosine residues of the intracellular domain are autophosphorylated, which results in initiation of the intracellular signaling cascade, such as Ras/Raf/MAPK pathway and PI3K-Akt pathway. These signal transduction pathways lead to cell proliferation, cell differentiation, angiogenesis, metastasis and antiapoptosis [7,8]. High expression of EGFR has been reported in various kinds of malignancies such as head and neck
tumors, colorectal cancers, ovarian cancers, breast cancers, lung cancers and bladder cancers [9–12]. Approximately 40–70% of ESCC specimens expressed high levels of EGFR, assessed by EGF binding assay or immunohistochemistry [13,14]. These reports also demonstrated that there was a significant negative relationship between high expression of EGFR and survival [15–18]. In addition, high expression of EGFR was correlated with lymph node metastasis [19] and resistance to chemoradiotherapy [20]. Considering these facts, EGFR has become a promising target for novel anticancer therapies. Gefitinib is an orally active EGFR tyrosine kinase inhibitor, which has demonstrated antitumor activity in vitro or in vivo against various kinds of human cancer cell lines, including head/neck, ovary, breast, lung, bladder, malignant mesothelioma, and colon cancer [9–12]. Recently, the Food and Drug Administration approved gefitinib for previously treated, advanced non-small cell lung cancer and clinical trials in other tumors are ongoing [21]. Phase I and II studies indicate that gefitinib is generally well tolerated and has strong evidence of clinical antitumor activity [22,23]. However, the effect of gefitinib on esophageal cancers has not been reported. With the expectation of gefitinib as a new treatment for ESCC patients, we conducted a preclinical study to investigate the effect of gefitinib on the EGFR signaling pathway in ESCC cell lines and evaluated the antitumor effect of gefitinib on ESCC in vitro and in vivo.
2. Materials and methods 2.1. Drugs Gefitinib was kindly provided by AstraZeneca Pharmaceuticals (Macclesfield, United Kingdom) and was dissolved in dimethylsulfoxide (DMSO) at stock
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concentration 10 mM and stored at K20 8C for in vitro experiment. Control cells were treated with the medium containing an equal concentration of DMSO. For in vivo administration, gefitinib was dissolved in 1% Tween80. 2.2. Cell lines and culture conditions The human esophageal cancer cell line TE8 was obtained from the Cancer Cell Repository, Research Institute for Tuberculosis and Cancer, Tohoku University, Japan. T.T and T.Tn were obtained from Japanese Collection of Research Bioresources, Japan. TE8 cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 UI/ml) and streptomycin (100 mg/ml) (Sigma Chemical Co., St Louis) in a humidified atmosphere of 95% air and 5% CO2 at 37 8C. T.T and T.Tn were maintained in DMEM/F12 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 UI/ml) and streptomycin (100 mg/ml) in a humidified atmosphere of 95% air and 5% CO2 at 37 8C. 2.3. In vitro cytotoxicity assay The growth-inhibitory activity of gefitinib was determined using Cell Proliferation Reagent WST-1 (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, cancer cells were seeded on 96-well plates at a concentration of 5!103 cells/well with complete culture medium and allowed to adhere to the plate overnight. Then the cells were incubated in the presence of various concentrations of gefitinib (0.01–40 mM) for another 72 h at 37 8C in 5% CO2. After the treatment, tetrazolium salt WST-1 was added to each well and incubated another 4 h. The absorbance of the treated samples and the blank control was measured using an Immuno Mini NJ-2300 (Nalge Nunc International K.K., NY, USA). The wavelength for measuring absorbance of the formazan product was 440 nm and reference wavelength was 600 nm. Each condition was performed with 8 wells and each experiment was repeated three times. The antiproliferative activities of gefitinib are shown in terms of IC50s.
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2.4. Western blot analysis Cells were rinsed twice with ice-cold PBS and then lysed on ice with lysis buffer (50 mM HEPESNaOH (pH 7.4), 250 mM NaCl, 1 mM EDTA, 1% Igepal, 1 mM DTT, 1 mM phenylmethylsulphonyl fluoride, 5 mg/ml leupeptin, 5 mg/ml aprotinin, 2 mM Na3VO4, 1 mM NaF, 10 mM b-GP). Protein concentrations were determined using the Bradford assay (Bio Rad, Hercules, CA). Cell lysates were denatured in SDS. Equal amounts of protein were separated on 8% SDS-polyacrylamide gel by electrophoresis and blotted onto polyvinylidene difluoride membrane. Membranes were blocked overnight at 4 8C in blocking buffer (0.1% TBST, 1% BSA, 0.05% NaN3) and then immunoblotted with primary antibodies overnight at 4 8C. The blots were then incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. Detection was performed using the enhanced chemiluminescence plus western blotting detection system (Amersham, IL), according to the manufacturer’s instructions. Primary antibodies were as follows: goat polyclonal anti-EGFR (1005), goat polyclonal anti-p-EGFR (Tyr1173), rabbit polyclonal anti-ERK1 (K-23), mouse monoclonal anti-p-ERK (E-4) which specifically reacts with Tyr-204 phosphorylated ERK1 and ERK2, rabbit polyclonal anti-Akt1/2 (H-136), rabbit polyclonal anti-neu (C-18), anti-p-neu (Tyr1248) and ErbB3 (C-17) (Santa Cruz Biotechnology, CA), and rabbit polyclonal anti-AKT/PKB [pS473] phosphospecific antibody. Secondary antibodies were peroxidase-labeled antibody against mouse, rabbit and goat (Arlington Heights, IL). 2.5. Flow cytometry Subconfluent cells were cultured in 60 mm dishes with 0.1 mM or 1 mM gefitinib for 24 h. The cells were trypsinized, fixed in 70% ice-cold ethanol, and stored at K20 8C for 48–72 h. After fixation, cells were suspended in 100 ml phosphate–citrate buffer (0.19 M Na2HPO4, 4 mM citric acid) and incubated for 30 min at room temperature and resuspended in 1 ml of PBS containing 10 mg/ml of propidium iodide and 10 mg/ml of RNase A. The propidium iodide-stained cell samples were analyzed using a FACSCalibur
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(Beckton Dickinson, San Jose, CA) and data analysis for the population of cells in sub-G1, G1, S, and G2-M phase of the cell cycle was performed with the CELLQuest software (Beckton Dickinson, San Jose, CA). Cells undergoing apoptosis were determined as a percentage of cells with sub-G1 population. 2.6. In vivo studies Female athymic BALB/c nu/nu mice (6 weeks of age) were purchased from Charles River Laboratories (Tokyo, Japan). The research protocol was approved and mice were maintained in accordance with the institutional guidelines of Okayama University Animal Care and Use Committee. Exponentially growing 1!106 TE8 and T.T cells suspended in 100 ml PBS were injected s.c. into the hind limb on day 0. When tumors were palpable, about 3–5 mm in diameter, the mice were randomized into the following treatment groups of 10–12 mice; Control: daily oral administration of vehicle solution for gefitinib (1% Tween80), Treatment: daily oral administration of gefitinib (100 or 200 mg/kg). Mice were monitored daily for signs of toxicity and were weighed regularly. Tumor size was measured with a caliper and tumor volume (TV) was calculated by the formula, TVZp/6!larger diameter!(smaller diameter)2 every 2 or 3 days. 2.7. Immunohistochemical analysis Two mice were sacrificed at the end of treatment to perform immunohistochemical analysis. Paraffin sections were placed on slides and were deparaffinized in xylene, rehydrated in graded ethanol, and endogenous peroxidase was blocked using 3% hydrogen peroxide in PBS. The slides were heated in a microwave for 10 min in a 10 mM citrate buffer solution at pH 6.0 and cooled to room temperature for 20 min. The tissues were incubated overnight at 4 8C with a 1:100 dilution of a polyclonal rabbit antiPCNA antibody (Biotechnology), 1:400 dilution of a polyclonal rabbit anti-Von Willebrand (VW) Factor antibody (Dako), or 1:50 dilution of a polyclonal rabbit anti-EGFR and phospho-EGFR antibody (SantaCruz). The samples were then incubated for 60 min at room temperature with the appropriate
dilution of peroxidase-conjugated anti-rabbit IgG or anti-rat IgG. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine. A positive reaction was indicated by brown staining. For PCNA, densities of apoptotic cells were expressed as the average of the five highest areas of greatest intensity identified within a single !200 field. 2.8. Microvessel density count Each slide immunostained with VW factor antibody was firstly scanned at low power, and the area with the higher number of new vessels was identified (hot spot) and then scanned at high power. Stained vessels were counted in each of four different fields. For individual tumors, the microvessel count was scored by averaging the counts from five fields [24]. 2.9. TUNEL assay Cell death in tumors was determined by the TUNEL method. Paraffin-embedded tissue sections (5 mm thick) were deparaffinized and rehydrated as previously described. The slides were rinsed twice with PBS and treated with proteinase K (1:500, 10 mg/ml) for 15 min at 37 8C. Endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in PBS for 20 min. The tissue sections were then washed three times with PBS and incubated for 60 min at room temperature with TdT buffer (200 mM/l potassium cacodylate, 25 mM/l Tris–Cl (pH 6.5), 0.25 mg/ml BSA, 1 mM/l CoCl2, 0.01 mM/l Biotin-dUTP, 1120 U/ml TdT (Terminal Deoxynucleotidyl Transferase). The samples were rinsed four times with TB buffer (0.03 M sodium chloride, 0.03 M sodium citrate) and incubated for 30 min at 37 8C in a 1:400 dilution of peroxidase-conjugated streptavidin. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine. The sections were washed three times with PBS, counterstained with hematoxylin, and again washed three times with PBS. For TUNEL, densities of apoptotic cells were expressed as the average of the five highest areas of greatest intensity identified within a single ! 200 field.
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2.10. Statistical anaylsis In vivo experiments were analyzed by ANOVA. Survival was evaluated by Kaplan–Meier method. All statistical analyses were performed with StatView (Version 5.0; Abacus Concepts, Berkeley, CA). P! 0.05 was considered statistically significant.
3. Results 3.1. Gefitinib inhibited growth of esophageal squamous cell carcinoma cell lines We examined the growth inhibitory effect of gefitinib on ESCC cell lines TE8, T.T and T.Tn in vitro by WST assay. ESCC cell lines were treated with various concentrations of gefitinib (0.01–40 mM) (Fig. 1A). Gefitinib inhibited cell proliferation in a dose-dependent manner in all cell lines. IC50s of TE8, T.T and T.Tn were 8.49, 18.9 and 17.3 mM, respectively. The human squamous vulvar cancer cell line A431 is known to express high levels of EGFR and be highly sensitive to gefitinib. We also examined the IC50 value of A431 (IC50Z14.7 mM) to find there is no significant difference between A431 and ESCCs in IC50 value.
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3.3. Gefitinib inhibited phosphorylation of EGFR and downstream signal transduction pathway The addition of EGF into culture media at the concentration of 20 ng/ml caused autophosphorylation of EGFR and activation of downstream signals, including the PI3K/Akt and MEK/MAPK pathways, in a time-dependent manner. We examined whether the cellular inhibitory effect of gefitinib was due to the suppression of EGF-stimulated phosphorylation of EGFR in ESCC cell lines. EGFR was phosphorylated with 20 ng/ml EGF stimulation within 15 min in TE8 cells. The phosphorylation of EGFR induced with EGF treatment was inhibited by gefitinib at the concentration of 0.01 mM. EGFR expression level was not changed with gefitinib exposure. Phosphorylation of Erk and Akt was reduced by pretreatment
3.2. Correlation of gefitinib sensitivity with EGFR, HER2 and HER3 expression level We next investigated the relationship between gefitinib sensitivity and expression levels of the HER receptor family EGFR, HER2 and HER3. Expression levels of these receptors were analyzed by western blotting. TE8 expressed a high level of EGFR similar to that of A431, and also exhibited high HER2 expression. T.T showed the lowest expression of EGFR revealing resistance to gefitinib. An inverse relationship was found between the expression level of EGFR and IC50s (rZK0.962, PZ0.0492). In contrast, HER2 and HER3 expression levels did not relate to sensitivity to gefitinib (rZK0.921, PZ0.1107 and rZK0.559, PZ0.5279, respectively) (Fig. 1B).
Fig. 1. (A) Effect of gefitinib on cell proliferation in ESCC cell lines. Cells were incubated with various concentrations of gefitinib for 72 h, and cell proliferation was determined by WST assay. Data represent cell number during treatment relative to control untreated cells (100%). The averages: bars, SD. The antiproliferative activities of gefitinib are shown in terms of IC50s. (B) Expression level of HER family in ESCC cell lines and A431 SCC cell line was analyzed by using Western blot. Cells were incubated in CM with 10% FBS. b-Actin was used as internal control.
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with 0.1 mM gefitinib without affecting the total amount of expression of these proteins. Interestingly, phosphorylation of HER2 was induced by EGF stimulation and inhibited by gefitinib at the dose of 0.01 mM, suggesting the strong effect of gefitinib to inhibit HER2 phosphorylation compared with that for EGFR (Fig. 2). 3.4. Flow cytometric analysis of cell cycle distribution and of induction of apoptosis We assessed the effect of gefitinib on cell cycle progression. TE8 cells were harvested after 24 h exposure to different dose of gefitinib and stained with PI for flow cytometry analysis. The percentage of G1 phase cells increased from 45.10 to 72.03 or 71.70% after treatment with 0.1 or 1 mM gefitinib, respectively (Fig. 3). The sub-G1 apoptotic population (7.83%) after 1 mM gefitinib treatment increased approximately 3-fold compared with control (2.73%) (Fig. 3). Fig. 2. Effects of gefitinib on EGFR and HER2 phosphorylation and activation of PI3K/Akt and Ras/Mek/MAPK pathways in TE8 cells. Western blots of lysates from TE8 cells after serum starvation and stimulation with EGF were probed with antibodies to EGFR, phospho-EGFR, HER2, phospho-HER2, MAPK, phospho-MAPK Akt, and phospho-Akt. Equal amounts of protein were loaded in each lane.
3.5. Antitumor effect of gefitinib on ESCC in vivo We evaluated the antitumor effect of gefitinib in vivo using the model of nude mice bearing TE8 and T.T xenografts. When the size of established tumors reached w60 mm3, groups of 10–12 mice were
Fig. 3. Cell cycle analysis of ESCC cells treated with gefitinib. TE8 cells were stained with PI after 24 h exposure to gefitinib (0.1 or 1 mM) or control and analyzed by flow cytometry. DNA histograms of untreated cells (control), or gefitinib (0.1 or 1 mM) treated cells are shown. Percentages of the cell population in the different phase of cell cycle were analyzed by cell quest. Percentages of apoptosis were measured as sub-G1 content of the histogram. Similar results were obtained in three independent experiments.
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treated orally with once daily 100 or 200 mg/kg gefitinib or vehicle solution for gefitinib (1% Tween 80) as control for 11–14 days. In T.T tumor xenografts, administration of gefitinib significantly inhibited tumor growth compared with controls in a dose-dependent manner (P!0.0001 for control versus 100 and 200 mg/kg gefitinib, PZ0.0247 for 100 mg/kg gefitinib versus 200 mg/kg gefitinib; Fig. 4A). In TE8 tumor xenografts, gefitinib also significantly shrank tumors (P!0.0001 for control versus 100 and 200 mg/kg gefitinib; Fig. 4A). Shortly after the end of treatment, tumors resumed growth. However, regrowth of tumors in the treatment groups was substantially delayed compared with control groups. The delayed TE8 tumor growth in the gefitinib treatment group was accompanied by a prolonged life span of mice that was significantly different from that of untreated controls. Gefitinib significantly prolonged the survival of TE8 tumorbearing mice (P!0.05, Mantel-Cox log-rank test) (Fig. 5). In addition, we investigated whether gefitinib reduced the size of well-established tumor xenografts (allowed to reach a volume of 400–500 mm3 before treatment). In large size tumors, gefitinib also significantly inhibited tumor growth compared with control as shown Fig. 4B (P!0.0001). 3.6. Immunohistochemistry in vivo Two mice were sacrificed at the end of treatment to perform immunohistochemical analysis. Immunohistochemistry using antibody against specific antiEGFR and phosphorylated EGFR demonstrated that tumors from all groups expressed similar levels of EGFR. In contrast, only tumors from the control group stained positive for phosphorylated EGFR (Fig. 6A–F). We next evaluated cell proliferation and apoptosis using anti-PCNA antibody and TUNEL assay, respectively. The mean proportion of PCNA positive tumor cells in control tumor was 81.6G3.7%. 3
Fig. 4. (A) Antitumor effect of gefitinib in ESCC xenografts. We examined antitumor effect of gefitinib (100, 200 mg/kg/day) or vehicle (1% tween80) on the growth of tumor xenografts. ESCC cells (TE8 or T.T) were injected s.c. into the flank of nude mice. Each group consisted of 10–12 mice. Data represent the averages:
bars, SD. Gefitinib significantly inhibited tumor growth compared with control (P!0.0001 in TE8 and T.T xenografts) in a dosedependent manner (100 versus 200 mg/kg PZ0.0247 in T.T xenografts). (B) Effect of gefitinib on well-established TE8 tumor xenografts. Gefitinib reduced well-established tumor xenografts allowed to reach a volume of 400–500 mm3 before treatment. In large size tumors, gefitinib also significantly inhibited tumor growth compared with control as shown Fig. 4B (P!0.0001).
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Fig. 5. Effect of gefitinib treatment on the survival of TE8 tumor bearing mice. Five mice/group were monitored for survival. Differences in animal survival among groups were evaluated using the Mantel-Cox log-rank test. The survival of mice was significantly different between gefitinib 100 mg/kg-treatment group and control group (P!0.0001); gefitinib 200 mg/kg-treatment group and control group (P!0.0001); gefitinib 100 mg/kg-treatment group and gefitinib 200 mg/kg-treatment group (P!0.03).
After therapy with gefitinib at 100 or 200 mg/kg, it was 39.5G2.8 or 26.5G8.3%, respectively (control versus 100 mg/kg gefitinib, P!0.001; control versus 200 mg/kg gefitinib, P!0.001, Fig. 6G–I). The mean number of TUNEL-positive cells was 3.0G0.7 in control tumors, whereas, it was 14.8G5.8 or 19.4G 6.9 in gefitinib at 100 or 200 mg/kg treated tumors, respectively (control versus 100 mg/kg gefitinib, P!0.05; control versus 200 mg/kg gefitinib, P! 0.001; Fig. 6J–L and Table 1). To evaluate whether gefitinib caused inhibition of angiogenesis, we quantified the tumor-induced vascularization as microvessel count in the most intense areas of neovascularization using anti-factor VIII related antigen Mab. We found a significant reduction in tumor microvessel density per field after treatment with 100 mg/kg gefitinib (4.8G0.8) or treatment with 200 mg/kg gefitinib (3.4G1.8) as compared with control tumors (8.4G1.5) (control versus 100 mg/kg gefitinib, P!0.05; control versus 200 mg/kg gefitinib, P!0.001; Fig. 6M–O and Table 1).
4. Discussion We have shown that gefitinib prevented EGFmediated activation of its receptor and MAPK and Akt downstream signaling pathways, in TE8 ESCC cell
line in a dose-dependent manner. These mechanisms of antiproliferative effects of gefitinib in ESCC are consistent with those in other kinds of human cancers which were reported recently [25–30]. The inhibition of MAPK pathway reduces cyclin-CDK2 kinase activity resulting in reduction of Rb phosphorylation and G1 arrest [26,27]. On the other hand, inhibition of the PI3K/Akt pathway that mediates the anti-apoptosis signal induces apoptosis [28–30]. In our in vivo study, immunohistochemical analyses showed that treatment with gefitinib at the dose of 100 mg/kg completely inhibited phosphorylation of EGFR and induced cell cycle arrest and apoptosis in TE8 tumor xenograft, confirming the effect of orally administrated gefitinib on an animal model with the same mechanisms as in vitro. Furthermore, inhibition of neovascularization was also shown to be another antitumor mechanism of gefitinib. Hirata et al. has shown that gefitinib activity might inhibit tumor angiogenesis through direct effects on microvascular endothelial cells that express EGFR and through inhibition of proangiogenic factor production by tumor cells [32]. Recent results have suggested that in vivo treatment with gefitinib causes a decrease in expression of the proangiogenic molecule VEGF and induction of apoptosis in endothelial cells [31]. Taken together, these facts suggest that antiangiogenic activity of gefitinib might contribute to its antitumor activity. Many studies have reported that expression of EGFR is increased in ESCC as well as in various human malignancies and elevated EGFR level is a significant poor prognostic indicator for ESCC [13–18]. It is assumed that there is a relationship between high expression of EGFR and sensitivity to gefitinib. All ESCC cell lines in this study expressed intermediate to high levels of EGFR and expression of EGFR correlated inversely with sensitivity to gefitinib. However, there is room for argument on this point, because sample number in this study is too small. Further investigations will be needed to evaluate more clearly the relationship between expression of EGFR and sensitivity to gefitinib in ESCC. Magne et al. reported significant inverse correlation between sensitivity to gefitinib and EGFR content in colon cancer cell lines [33]. However, correlation between EGFR expression and effect of gefitinib is still controversial [34,35].
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Fig. 6. Immunohistochemical analyses. TE8 tumor xenograft tissue sections harvested from mice treated with vehicle or gefitinib (100 or 200 mg/kg). (A–F) Tissue sections were stained for expression of EGFR and phospho-EGFR. Tumors from all treatment groups stained positive for EGFR, whereas only tumors from control mice stained positive for phospho-EGFR (!400). (G–O) The sections were immunostained for expression of PCNA (to show cell proliferation, bars; 40 mm), TUNEL (to show cell death, bars; 40 mm), and Factor VIII (to show angiogenesis, bars; 80 mm). Tumors from mice treated with gefitinib had a decrease in PCNA positive cells and increase in TUNEL positive cells. Tumors from mice treated with gefitinib had a decrease in microvessel density.
In several Phase II clinical trials of EGFR-TKIs, degree of EGFR expression as measured by immunohistochemistry was not found to be a determinant of antitumor activity [36]. Thus, the prospect of gefitinib-sensitivity based on the expression of EGFR disregarding heterodimerization with other HER family receptors seems to be oversimplistic. Indeed, earlier studies have shown that breast cancer cells that express high levels of HER2, even in
the presence of a low number of EGFR, are exquisitely sensitive to gefitinib [37]. Though in this study, we failed to show the relationship between sensitivity to gefitinib and expression of HER2 or HER3 in ESCCs, gefitinib 0.1 mM inhibited EGFinduced HER2 phosphorylation. It is assumed that by inhibiting EGFR kinase activity, gefitinib also blocks signaling from EGFR/HER2 heterodimers [38]. Recent data indicate that HER2 overexpression was
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Table 1 Immunohistochemical evaluation of ESCC xenografts Gefitinib
Proliferation (PCNA%)
Apoptosis (TUNEL)
Angiogenesis (Factor VIII)
Control 100 mg/kg 200 mg/kg
81.6G3.7 39.5G2.8* 26.5G8.3*
3.0G0.7 14.8G5.8# 19.4G6.9#
8.4G1.5 4.8G0.8# 3.4G1.8#
The percent (GSD) of PCNA positive cells was measured and scored by averaging four field counts for each group as described in Section 2. The number of apoptotic cells and microvessels (GSD) were measured and scored by averaging four field counts for each group as described in Section 2 (*P!0.001, #P!0.05 as compared with control).
related to resistance to chemoradiotherapy and unfavorable prognosis of ESCC [39,40]. Our data may suggest that inhibition of HER2 plays a potential role in antiproliferation of tumor as well as that of EGFR and gefitinib could be useful in EGFR and HER2 overexpressing ESCC. In summary, we demonstrate that the EGFR tyrosine kinase inhibitor gefitinib (‘Iressa’) has strong antitumor activity against ESCC in vitro and in vivo. Our results indicate the possibility of gefitinib as a promising therapeutic intervention for the treatment of ESCC.
Acknowledgements We thank AstraZeneca Pharmaceuticals for the supply of gefitinib (‘Iressa’). ‘Iressa’ is a trademark of the AstraZeneca group of companies. We also thank Youichi Ishibe for helpful discussions and Mayumi Okada for expert experimental assistance.
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