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10.1245/s10434-006-9274-7
Annals of Surgical Oncology 14:1405-1415 (2007)
© 2007 Society of Surgical Oncology
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Original Article

STAT1 Activation-Induced Apoptosis of Esophageal Squamous Cell Carcinoma Cells In Vivo

Junichi Kaganoi, MD, PhD1,2, Go Watanabe, MD, PhD1, Michio Okabe, MD1, Shiro Nagatani, MD1, Atsushi Kawabe, MD, PhD1, Yutaka Shimada, MD, PhD1, Masayuki Imamura, MD, PhD3 and Yoshiharu Sakai, MD, PhD1

1 Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
2 Kitano Hospital, Tazuke Kofukai Medical Research Institute, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8480, Japan
3 Osaka Saiseikai Noe Hospital, Imafukuhigashi, Zyoto-ku, Osaka 536-0002, Japan

Correspondence: Address correspondence and reprint requests to: Go Watanabe, MD, PhD; E-mail: gowata{at}kuhp.kyoto-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The induction of apoptosis might be a promising treatment for cancers refractory to conventional therapies, such as esophageal cancer. In this study, we examined whether epidermal growth factor–induced growth inhibition results from apoptosis of esophageal squamous cell carcinoma (SCC) cells as a result of STAT1 activation and evaluated whether interferon gamma (IFN-{gamma}) can induce apoptosis of cancer cells in vivo.

Methods: To assess the function of STAT1, we established stable transfectants expressing dominant-negative STAT1. Apoptosis was assessed by several experimental techniques, including flow cytometry. Differentiation was evaluated by Western blot test with involucrin used as a marker. In vivo, cancer cells were injected into male BALB/c nu/nu mice. Two weeks later, the mice started to receive injections of IFN-{gamma} or saline into a tail vein four times per week. Concentrations of IFN-{gamma} in the tumors were analyzed by enzyme-linked immunosorbent assay. Apoptosis was evaluated by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining.

Results: Epidermal growth factor inhibited the growth of esophageal SCC cells by causing apoptosis through several pathways involving STAT1 activation. IFN-{gamma} induced the apoptosis of cancer cells, but it also promoted the differentiation (not apoptosis) of primary cultured cells derived from normal esophageal epithelium. IFN-{gamma} also inhibited the growth of xenograft tumors of esophageal SCC cells in vivo.

Conclusions: Our results suggest that IFN-{gamma} is one candidate for cytokine-based therapy of cancer. IFN-{gamma}–induced STAT1 activation might be involved in the apoptosis of esophageal SCC cells and in the terminal differentiation of normal squamous cells. Further studies of STAT1 signaling pathways may provide the basis for new targeted therapeutic strategies for esophageal SCC.

Key Words: Interferon • STAT1 • Epidermal growth factor • Esophageal cancer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidermal growth factor (EGF) is a cytokine first discovered in mouse submaxillary gland.1 It induces the growth and proliferation of many types of epithelial cells,2 and protects embryonic kidney epithelial cells (HEK293)3 and keratinocytes (HaCaT) from apoptosis.4,5 Recent studies have reported that EGF inhibits cell growth6 by activating STAT1 (signal transducer and activator of transcription 1).7 Unstimulated STAT1 proteins reside in the cytoplasm but dimerize on activation, translocate to nuclei, and transactivate specific genes.8–10 EGF has been shown to activate STAT1 in only a very few cultured cell lines (human epidermoid carcinoma cell line A43111,12 and human breast cancer cell line MDA-MB-468).13–15 We have previously reported that EGF strongly arrested the growth of 3 (KYSE-520, -590, and -1860) of 30 esophageal squamous cell carcinoma (SCC) cell lines (KYSE series) established in our laboratory from surgical specimens. Our studies also showed that the existence of the EGF-STAT1 pathway correlates well with a better clinical course in patients with esophageal SCC.16–18

Esophageal cancer, the seventh most frequent cancer worldwide,19 carries a very poor prognosis. Most patients respond poorly to surgery or chemo-radiation. Effective new therapies are therefore needed. In general, chemotherapy or radiotherapy induces apoptosis of cancer cells; however, normal cells are also damaged to some extent. Selective induction of apoptosis of cancer cells without injuring normal cells would potentially be an ideal cancer therapy. Recent studies that used cDNA microarray analysis have shown that the STAT1 gene mediates radioresistance and that the response to 5-fluoro-uracil correlates with interferon-induced gene expression, suggesting that the STAT1 signaling pathway has an important role in signal transduction related to the clinical response to treatment for SCC.20–22 These findings encouraged us to reexamine whether STAT1 activation can induce apoptosis of esophageal carcinoma cells, possibly leading to clinical applications such as cytokine therapy.

This study showed that EGF induced apoptosis by activating STAT1 in esophageal SCC cells and activated various factors, such as caspase-3, Bax, and Bcl-2. Interferon gamma (IFN-{gamma}) also induced apoptosis of SCC cells in vitro, but not of primary cultured cells derived from normal esophageal epithelium. Systemic intravenous administration of IFN-{gamma} strongly induced STAT1-activation-dependent inhibition of cell growth in a xenograft tumor model in vivo. Our findings suggest that further investigations of STAT1 signaling pathways may provide the basis for new therapeutic strategies for esophageal SCC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
Cancer cells were cultured in Ham F12 (Nissui, Tokyo, Japan)/RPMI 1640 (Gibco, Grand Island, NY) medium supplemented with 2% fetal calf serum (FCS), penicillin (100 U/mL), and gentamicin (2.5 µg/mL) at 37°C in a 5% CO2 atmosphere.18 Primary normal human esophageal keratinocytes (KNEC-2) were prepared as previously reported.23 Cell count was determined by the trypan blue dye exclusion method.

Transfection and Stable Cell Lines
The DN-Stat1 vector was the gift of Dr. K. Nakajima and Dr. T. Hirano (Department of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, Osaka, Japan),24 and the pCAG vector was the gift of Dr. J. Miyazaki (Department of Nutrition and Physiological Chemistry, Osaka University Medical School).25 Stable transfectants were selected by adding 800 µg/mL of geneticin (G418) to the medium (Nacalai Tesque, Kyoto, Japan).

Morphologic Examination
After 1 x 107 KYSE-590 cells were cultured in medium without FCS for 24 hours, they were incubated for another 24 hours in the presence of different concentrations (0, 1, 10, and 100 ng/mL) of human recombinant EGF (Higeta Shoyu, Tokyo, Japan). Unstained cells were photographed with a phase-contrast microscope (Carl Zeiss, Jena, Germany).

Immunocytochemistry
A total of 1 x 105 KYSE-70 and -590 cells were cultured on coverslips in medium without FCS for 24 hours and were then incubated with or without 100 ng/mL of EGF for 2 hours. The cells were fixed in 99.5% cold methanol for 5 minutes and cold acetone for 2 minutes and were then permeabilized in .2% Triton X-100. After blocking nonspecific reactions with 10% FCS in phosphate-buffered saline (PBS), the cells were incubated with anti-STAT1{alpha} p91 antibody (c-24; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour, stained with fluorescein anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA), and photographed with a fluorescent microscope (Carl Zeiss).

Hoechst Staining
A total of 1 x 106 KYSE-70 and -590 cells were cultured in medium without FCS for 24 hours in two culture dishes. A total of 10 ng/mL of EGF was added to one dish, and the cells were incubated for 48 hours; the other dish served as a control. The cells were collected by trypsinization, centrifuged at 1500 rpm for 5 minutes, washed once with PBS, resuspended in 100 µL of 1% glutaraldehyde in PBS, incubated for 30 minutes at room temperature, washed twice in PBS, and stained with 24 µL of 16.7 µM bis-benzamide (Hoechst 33258; Sigma Chemical, St. Louis, MO). After incubation for 30 minutes at room temperature to allow DNA binding, 20 µL of the cell suspension was placed on a slide glass and examined for apoptotic chromatin changes with a fluorescence microscope (Carl Zeiss).

Flow Cytometric Analysis
A total of 1 x 106 cells were cultured in medium without FCS for 24 hours and incubated under various conditions. A total of 20 µM of Z-VAD-fmk was added to the inhibitor group before EGF stimulation, and the cells were incubated with Z-VAD-fmk for 24 hours. Under various conditions, the cells were treated with .02% ethylenediaminetetraacetic acid and 200 U/mL of trypsin (Mochida, Tokyo, Japan) in PBS. After centrifugation at 1500 rpm for 5 minutes, the cell pellet was fixed in 70% ethanol for 4 hours on ice, washed in PBS, centrifuged at 2500 rpm for 5 minutes, and resuspended in 60 µL of phosphate-citrate buffer (200 mM of sodium phosphate and 4 mM of citrate) by pipetting. The cells were incubated at room temperature for 30 minutes, centrifuged at 2500 rpm for 5 minutes, and resuspended in 10 µg/mL of RNase A in PBS at 37°C for 30 minutes. Finally, the cells were stained with 50 µg/mL of propidium iodide19 for 30 minutes on ice, with protection from light. Stained cells were filtered through a nylon mesh and immediately subjected to flow cytometry (Becton Dickinson, Mountain View, CA).26,27

Immunoprecipitation
A total of 1 x 106 KYSE-70 and -590 cells and transfectants (KYSE-590/pCAG and KYSE-590/Stat1F-1, -2, -3) were cultured in medium without FCS for 24 hours and then in the presence of 100 ng/mL of EGF for 15 minutes. The cells were washed twice in cold PBS and lysed in lysis buffer (20 mM Tris-HCl [pH 7.5] containing 1% Triton X-100, 1 of mM ethylenediaminetetraacetic acid, 1 mM of sodium ortho vanadate [Na3VO4], 1 mM of phenyl-methylsulfonyl fluoride, 10 µg/mL of leupeptin, 10 µg/mL of aprotinin, 50 mM of NaF, and .1% deoxycholate sodium) on ice. The extracts were cleared by centrifugation (10,000 rpm) at 4°C for 20 minutes and incubation with protein G-Sepharose beads for 1 hour at 4°C. The cleared supernatants were incubated with the indicated antibodies for 12 hours at 4°C. Finally, immune complexes were collected by incubation with protein G-Sepharose beads and eluted by boiling in sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris-HCl [pH 6.8], 4% SDS, 10% sucrose, 10% ß-mercaptoethanol, .004% brom-ophenol blue). Immunoprecipitates were subjected to 8% SDS–polyacrylamide gel electrophoresis and transferred to an Immobilon polyvinylidene diflouride microporous membrane (Millipore, Bedford, MA). Washing, blocking, and detection were performed as described for Western blot analysis.

Western Blot Analysis
Protein under various conditions was subjected to electrophoresis on 8% SDS-polyacrylamide gels. The gels were electrotransferred to polyvinylidene diflouride membranes. The membranes were incubated overnight at 4°C with 5% skim milk (Difco Laboratories, Detroit, MI) or 10% FCS in .1% Tween-20/Tris-buffered saline to block any nonspecific reaction, followed by incubation with the indicated primary antibodies for 60 minutes at room temperature. The membranes were washed three times briefly and twice for 10 minutes with fresh .1% Tween-20/Tris-buffered saline washing buffer at room temperature. The membranes were then incubated for 60 minutes with peroxidase-conjugated secondary antibodies (Zymed Laboratories, San Francisco, CA). After washing the membranes, they were incubated in mixed enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL) for 1 minute at room temperature, and signals were detected on exposed X-ray films (Fuji, Tokyo).

Animals and Tumor Formation In Vivo
Male BALB/c nu/nu mice were obtained from Clea Japan (Tokyo, Japan). The mice were kept in sterilized cages equipped with an air filter and sterile bedding materials. Sterilized water and food were provided throughout the study. When the mice were 4 weeks old, 5 x 106 of either KYSE-510/pCAG or KYSE-510/DN6J cells in 40 µL of PBS was injected subcutaneously in the back of the neck to induce tumors.

In Vivo Treatment with IFN-{gamma}
Two weeks later after injection of cancer cells, tumors reached 5 mm in diameter, and IFN-{gamma} treatment was started. A total of 100 IU of IFN-{gamma} or saline was injected into a tail vein four times per week. Control mice received saline alone in the same volume as the IFN-{gamma} treatment group (KYSE-510/pCAG [IFN-{gamma}]: n = 7, KYSE-510/pCAG [saline]: n = 7, KYSE-510/DN6J [IFN-{gamma}]: n = 7, KYSE-510/DN6J [saline]: n = 7). Tumor growth was monitored daily by measuring two perpendicular tumor diameters with calipers, and tumor volume was calculated according to the formula V = 52 x a x b x (a + b)/2, where a is the smallest superficial diameter and b is the largest superficial diameter. Concentrations of IFN-{gamma} 24 hours after injection were measured by enzyme-linked immunosorbent assay of three tumors.

TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling) Assay
Tumor blocks were frozen with cold isopentane. Frozen sections were protease digested and incubated with terminal transferase mixture with an in situ apoptosis detection kit (Takara Bio, Shiga, Japan) in accordance with the instructions of the manufacturer. Each tissue section was examined by light microscopy, and apoptosis was analyzed in at least 10 areas by two masked investigators.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
EGF-Induced Growth Inhibition Associated with Stat1 Activation in Esophageal SCC Cell Lines
We previously reported that EGF inhibited the growth of 3 (KYSE-520, -590, and -1860) of 30 KYSE series SCC cell lines studied. The 50% inhibitory concentration of EGF against KYSE-590 cells was 6.62 ng/mL (data not shown). To assess the mechanism of EGF-induced growth arrest in esophageal SCC cells, we first stained the cells with Hoechst stain. Nuclear morphological changes, such as chromatin condensation, nuclear fragmentation, and nuclear shrinkage, were observed in KYSE-590 cells in association with translocation and phosphorylation of STAT1, indicating STAT1 activation (Fig. 1C, D, G, H, IGo). Similar findings were observed in KYSE-520 and -1860 cells (data not shown). In contrast, no nuclear morphological changes, translocation of Stat1, tyrosine phosphorylation, or growth inhibition was observed in KYSE-70 cells (Fig. 1A, B, E, F, IGo).16 We confirmed that EGF receptors were similarly activated in both KYSE-70 cells and KYSE-590 cells in response to EGF stimulation (Fig. 1IGo), whereas there was no difference between these cell lines in mitogen-activated protein kinase activation induced by EGF stimulation.16 These findings suggested that EGF-induced growth arrest was caused by apoptosis in association with STAT1 activation.


Figure 1
Figure 1
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  		FIG. 1. Correlation of epidermal growth factor (EGF)-induced growth inhibition with Stat1 activation in esophageal squamous cell carcinoma (SCC) cells. (A–D) Nuclear structural changes on staining with fluorochrome bis-benzamide (Hoechst 33258) stain. Cells were stimulated or not stimulated with EGF at a concentration of 100 ng/mL for 24 hours. Chromatin condensation, nuclear shrinkage, and nuclear fragmentation were identified (white arrows). (E–H) Translocation of STAT1 to the nucleus in response to EGF stimulation. Cells were treated with or without 100 ng/mL EGF for 2 hours before fixation. Immunocytochemical analysis was performed with the use of anti-STAT1{alpha} p91 antibody. (I) Tyrosine phosphorylation of Stat1 and EGF receptor in KYSE-70 and -590. After EGF stimulation, cells were collected with the use of lysis buffer. Immunoprecipitation was performed with anti-STAT1{alpha} p91 antibody or anti-EGFR antibody. Blotting with anti-phosphotyrosine antibody (PY20) and reblotting with anti-STAT1{alpha} p91 antibody or anti-EGFR antibody were performed using the same membrane. (J–O) Flow cytometric analysis of apoptosis. Dot plots depict DNA content quantified by propidium iodide19 staining of ethanol-permeabilized KYSE-590 cells. The cells were fixed after incubation in the presence of EGF at various concentrations for 24 hours, with or without preincubation with Z-VAD-fmk for 24 hours. Figures are the histograms of the cell cycle measured on the basis of DNA volume; the number of cells is indicated on the vertical axis, and the DNA volume is indicated on the horizontal axis. M1 represents the apoptotic range of cells in the pre-G0/G1 (sub-G1) phase ([M1/(M1 + M2) x 100] [%]; mean ± SEM), and M2 represents the nonapoptotic range of cells in G0/G1 and G2/M phase ([M2/(M1 + M2) x 100] [%]; mean ± SEM). Averages were calculated from the results of three independent experiments (n = 4).

 
EGF-Induced Growth Inhibition of KYSE-590 Cells Resulted From Apoptosis
To confirm that EGF-induced growth inhibition caused apoptosis, we analyzed DNA fragmentation with the use of genomic DNA extracted from KYSE-590 cells stimulated by EGF. We observed DNA ladder formation in KYSE-590 cells in response to EGF stimulation (data not shown). We also analyzed the apoptotic ratio by flow cytometry. The cell number in the M1 fraction (sub-G1 ratio) increased in an EGF-dose-dependent manner (Fig. 1J–MGo). To investigate the caspase cascade, we performed cas-pase-3 colorimetric protease assay. Caspase-3 activity was found to increase in response to EGF stimulation in a dose-dependent manner (data not shown). Activation was then blocked with the pan-caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) in the caspase-3 colorimetric protease assay (data not shown). The results were consistent with those of flow cytometry (Fig. 1N, OGo), showing almost complete inhibition, as well as those of proliferation assay (data not shown). Furthermore, we found that cleavage of BAX was caused by EGF stimulation and showed that the Bax/Bcl-2 ratio, which represents the apoptotic tendency, increased by 24 hours after treatment with EGF, suggesting that the BAX/Bcl-2 pathway was also activated (data not shown). As previously reported, the cell cycle inhibitor p21WAF1/CIP1 was also induced by EGF stimulation (data not shown). These results suggested that EGF-induced growth inhibition of KYSE-590 cells was caused by apoptosis.

Activation of Stat1 was Essential for EGF-Induced Apoptosis of Esophageal SCC Cells
To investigate whether STAT1 activation was essential for EGF-induced apoptosis of KYSE-590 cells, we established dominant-negative Stat1 (DN-Stat1, pCAGGS-Neo-HA-Stat1F),24 three transfectants with different expression levels of DN-Stat1 proteins, and vector control transfectants (pCAGGS-Neo). We found that the EGF-induced cell death percentage was markedly suppressed depending on the expression level of DN-Stat1 (Fig. 2A, BGo), and flow cytometric analysis showed that the increase in the M1 phase was inhibited in KYSE-590/Stat1F-2 cells (Fig. 2CGo). To address whether DN-STAT1 can suppress endogenous STAT1, we analyzed the phosphorylation level of STAT1 in KYSE-590/Stat1F-2 cells. Tyrosine phosphorylation in response to EGF stimulation was almost completely suppressed in KYSE-590/Stat1F-2 cells (Fig. 2DGo). Activation of caspase-3 and induction of p21WAF1 in response to EGF stimulation were blocked in KYSE-590/Stat1F-2 cells (data not shown). These findings suggested that STAT1-activation was essential for EGF-induced apoptosis of esophageal SCC cells.


Figure 2
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  		FIG. 2. Dominant negative STAT1 inhibited epidermal growth factor (EGF)-induced apoptosis. (A) Establishment of stable transfectants of DN-Stat1 in KYSE-590 cells. Several stable transfectants of the vectors, pCAG (control) and Stat1Fs (DN-Stat1), were isolated. HA-DN-STAT1 expression was checked by Western blot analysis with anti-HA antibody followed by immunoprecipitation with anti-STAT1{alpha} p91 antibody. (B) Correlation between DN-Stat1 expression and inhibition of EGF-induced apoptosis. Cells were counted by trypan blue exclusion assay after EGF stimulation of the indicated stable transfectants for 24 hours. Cell death percentage was calculated as the total number of alive cells relative to the control value (100%) for each cell line. Four independent experiments were performed (mean ± SEM) (n = 3). (C) Apoptosis was inhibited by DN-Stat1 on flow cytometric analysis. KYSE-590/pCAG cells and KYSE-590/Stat1F-2 cells were stimulated with 10 ng/mL EGF for 24 hours before fixation for flow cytometry. Average values were obtained from two independent experiments (mean ± SEM) (n = 4). (D) Tyrosine phosphorylation of STAT1 with EGF-stimulation. Phosphorylation of STAT1 was detected by Western blot analysis using anti-phosphotyrosine antibody (PY-20), followed by immunoprecipitation with anti-STAT1{alpha} p91 antibody. Cells were incubated with EGF for 15 minutes before lysis.

 
IFN-{gamma}–Induced Apoptosis in Esophageal SCC Cells, Not in Normal Esophageal Epithelial Cells
EGF is not suitable for clinical application because EGF is a strong growth factor for various types of epithelial cells, including cancer cells, and only 3 (10%) of 30 esophageal cancer cell lines were sensitive to EGF. In contrast, IFN-{gamma}, one of the strongest activators of STAT1, inhibited the growth of 10 esophageal cancer cell lines (32%) by at least 50% and did not simulate marked cell growth in any cell line (Fig. 3AGo). To assess the toxicity of normal esophageal epithelial cells to IFN-{gamma}, we stained the cells with Hoechst stain and performed flow cytometric analysis. Chromatin condensation, nuclear shrinkage, and nuclear fragmentation were observed only in the KYSE-510 cancer cells, and the M1 fraction did not increase in response to IFN-{gamma} stimulation in normal epithelial cells derived from resected esophagus (KNEC-2)23; however, STAT1 activation was detected in the KNEC-2 cells in response to IFN-{gamma} stimulation (Fig. 3B–DGo). These findings suggested that IFN-{gamma} induced different phenotypes in KNEC-2 cells and cancer cells. Previous studies showed that IFN-{gamma} induced the expression of involucrin, a differentiation marker of squamous cells. We therefore sought to analyze the phenotype with respect to differentiation.28 Interestingly, we found that IFN-{gamma} stimulation induced the expression of involucrin in KNEC-2 cells, but not in cancer cells (Fig. 3EGo). This difference in response between cancer cells and normal cells suggested that IFN-{gamma} therapy might selectively and automatically provoke cytotoxic effects depending on cell type, provided that the STAT1 signaling pathway is intact.


Figure 3
Figure 3
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  		FIG. 3. Interferon gamma (IFN-{gamma})-induced apoptosis in squamous cell carcinoma (SCC) cancer cells, not in normal cells. (A) IFN-{gamma} sensitivity of esophageal SCC cells. Growth inhibition rate was calculated after stimulation with IFN-{gamma} at a concentration of 100 U/mL in 31 esophageal SCC cell lines. Cells were counted by trypan blue exclusion assay after IFN-{gamma} stimulation for 24 hours. The control for each cell line was the number of cells without stimulation. Four independent experiments were performed (mean ± SEM) (n = 3). (B) Translocation of STAT1 to the nucleus in response to IFN-{gamma} stimulation. Cells were treated with or without 10 ng/mL IFN-{gamma} for 2 hours before fixation. Immunocytochemical analysis was performed with the use of anti-STAT1{alpha} p91 antibody. (C) Nuclear structural changes on staining with fluorochrome bis-benzamide (Hoechst 33258) stain. Cells were stimulated or not stimulated with IFN-{gamma} at a concentration of 10 ng/mL for 24 hours. Chromatin condensation, nuclear shrinkage, and nuclear fragmentation were identified (white arrows). (D) IFN-{gamma}–induced apoptosis was inhibited in KNEC-2 cells on flow cytometric analysis. KYSE-510 cells and KNEC-2 cells were stimulated with 10 ng/mL IFN-{gamma} for 24 hours before fixation for flow cytometry. Average values were obtained from two independent experiments (mean ± SEM) (n = 4). (E) IFN-{gamma} induced involucrin expression in KNEC-2 cells, not in KYSE-510 cancer cells. After IFN-{gamma} stimulation for 72 hours, indicated cells were collected with the use of lysis buffer. Western blot assay was performed with anti-involucrin antibody, and the same membrane was reblotted with anti–ß-actin antibody.

 
Systemic Administration of IFN-{gamma} Inhibited Growth of Esophageal SCC Cells
Finally, IFN-{gamma} was used instead of EGF as an STAT1 activator for preclinical experiments with esophageal SCC cells. We injected 100 IU of human IFN-{gamma} into a tail vein four times per week, after allowing the xenograft tumors to grow for 2 weeks. The growth of KYSE-510 cells harboring control vector pCAG was markedly inhibited by treatment with human IFN-{gamma}, and TUNEL staining was positive in most areas of the tumors. In contrast, little effect of IFN-{gamma} treatment was observed in KYSE-510 cells harboring the dominant-negative STAT1 gene (Fig. 4A, BGo). After the completion of IFN-{gamma} treatment, no regrowth was evident for 3 months (data not shown); however, regrowth was observed when IFN-{gamma} was given by continuous injection therapy for 14 days. These results demonstrated that IFN-{gamma} effectively induced cell death in vivo and showed that this effect depended on STAT1 activation. Further-more, the concentrations of human IFN-{gamma} in both types of tumors were maintained at similar levels, indicating that the effect of treatment did not depend on secondary conditions such as angiogenesis around the xenograft, which could affect the concentration of IFN-{gamma} in cancer cells (Fig. 4CGo).


Figure 4
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  		FIG. 4. Interferon gamma (IFN-{gamma})-inhibited growth of squamous cell carcinoma (SCC) cells in vivo. (A) IFN-{gamma} inhibited growth of SCC cells in vivo. A total of 100 IU of IFN-{gamma} or saline was injected into a tail vein 4 times per week. Control mice received saline alone in the same volume as the IFN-{gamma} treatment group. Volume ratio was calculated by the formula V/V0, where V is volume at each point and V0 is the volume on day 0. Arrows indicate the injection of IFN-{gamma}. (B) Apoptosis was analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay with 3,3'-diaminobenzidine staining. (C) Concentration of human IFN-{gamma} in the tumors. There were no statistically significant differences between the groups. Error bars indicate standard deviations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
EGF exists in human saliva and milk in high concentrations.29 It is thought to play physiological roles in the upper digestive tract. Our study confirmed that EGF-induced growth inhibition is caused by apoptosis in association with activation of various apoptotic pathways of esophageal SCC cells. Our results also showed that STAT1 activation was necessary for such apoptosis. However, EGF cannot be used clinically because this factor induces cell growth arrest with STAT1 activation in very few cultured cell lines, such as the human epidermoid carcinoma cell line A431,11,12 the human breast cancer cell line MDA-MB-468,14,15 and three esophageal SCC cell lines used in this study. Another drawback to clinical application is that EGF functions as a strong growth factor for many cell types, potentially promoting the development and progression of cancer. In contrast to EGF, IFN-{gamma}, another activator of STAT1, basically functions as an inducer of cell growth arrest and apoptosis by activating STAT1 in many cell lines.11,30 Indeed, we found that IFN-{gamma} induced apoptosis in 10 (32%) of 31 esophageal SCC cell lines, whereas EGF induced apoptosis in only 3 cell lines (10%). These findings suggest that IFN-{gamma} would be a more effective STAT1 activator than EGF in the clinical setting.

Recent clinicopathological studies have shown that overexpression of EGF receptors does not correlate with the outcomes of patients with esophageal SCC.31 We previously reported that the existence of the EGF-STAT1 pathway in esophageal SCC cell lines correlates well with a better clinical course and found no major difference between KYSE-70 and -590 cells in the phosphorylation of EGF receptors or the expression level of STAT1. However, the expression level of EGF receptors in KYSE-70 cells is slightly less than that in KYSE-590. The expression level of EGF receptors might thus be involved in STAT1 activation in esophageal SCC cells. Differences in EGF receptor expression might account for the difference between cultured cells and tissues in vivo. EGF phosphorylates EGF receptors but cannot activate STAT1 in most esophageal SCC cell lines. However, EGF can induce STAT1 activation in primary cultures of normal esophageal epithelial cells.16 On the other hand, among the 31 KYSE-series cell lines tested, 10 were sensitive to IFN-{gamma} (more than 50% inhibition of growth as compared with control). The fact that IFN-{gamma} can activate STAT1 in many esophageal cancer cell lines (data not shown) suggested that the STAT1-activating mechanism and STAT1 itself are intact. Studies of ligand-dependent activation and inactivation of STAT1 are few. Such studies may provide important clues to understanding the pathogenesis of esophageal cancer.

Interestingly, the induced phenotype distinctly differed between these two cell types—that is, apoptosis occurred in cancer cells but not in normal cells, consistent with the results of flow cytometric analysis. Furthermore, we found that KNEC-2 cells spread in response to IFN-{gamma} stimulation (data not shown), and the promoter region of the involucrin gene has been reported to contain interferon-gamma-activated sequence binding sites.28 As expected, IFN-{gamma} induced involucrin expression in KNEC-2 cells, but not in cancer cells. Esophageal SCC cells retained involucrin genomic DNA (data not shown). Downregulation of involucrin expression may potentially result from epigenetic alterations of the involucrin gene, such as methylation of the promoter region.

In our in vivo model, tumor growth of xenografts in the KYSE-510/pCAG control group was markedly inhibited by IFN-{gamma} therapy, and apoptosis was histologically confirmed in suppressed tumors. Dominant-negative STAT1 stable transfectants were used to inactivate STAT1 in the cells. IFN-{gamma}–induced apoptosis was markedly inhibited depending on the expression level of the DN-STAT1 gene (data not shown). These findings suggested that STAT1 activation was also essential for IFN-{gamma}–induced apoptosis in esophageal SCC cells.

In conclusion, our results suggest that IFN-{gamma} is one candidate for cytokine-based therapy of cancer. IFN-{gamma}–induced STAT1 activation might be involved in the apoptosis of esophageal SCC cells and in the terminal differentiation of normal squamous cells. On the basis of the mechanism of STAT1 activation, IFN-{gamma} therapy may be an ideal cancer therapy. Further studies of the IFN-{gamma}–STAT1 pathway may provide the basis for new targeted therapeutic strategies for esophageal SCC.


   ACKNOWLEDGMENTS
 
Supported by grants-in-aid from the Japanese Ministry of Education, Science and Culture (10470241, 11877197, G.W.) and by the Public Trust Haraguchi Memorial Cancer Research Fund and the Uehara Memorial Life Science Fund. All animal experiments were performed in accordance with Kyoto University guidelines. We thank Dr. K. Nakajima and Dr. T. Hirano (Department of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, Osaka, Japan) for providing DN-Stat1F vector; Dr. J. Miyazaki (Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Osaka) for providing pCAG vector; T. Welte, A. Kano, and J. Moth for reading the article in manuscript and for their interesting discussion; S. Shimada for culturing cells; and Y. Nishimura and Y. Moriwaki for assistance.

Received for publication July 31, 2006. Accepted for publication October 18, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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