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ORIGINAL ARTICLE
Year : 2012  |  Volume : 8  |  Issue : 1  |  Page : 57-61

Overexpression of interferon regulatory factor 1 enhances chemosensitivity to 5-fluorouracil in gastric cancer cells


Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China

Date of Web Publication19-Apr-2012

Correspondence Address:
Jinbo Gao
Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan, Hubei Province 430022
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.95175

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 > Abstract 

Purpose: To investigate the effects of interferon regulatory factor 1 (IRF-1) gene overexpression on chemotherapeutic sensitivity of gastric cancer cells.
Materials and Methods: An AGS cell system with tetracycline-inducible IRF-1 expression (AGS/IRF-1) was established. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of the IRF-1 gene. Chemosensitivity to 5-fluorouracil (5-FU) was assessed by cell proliferation assay and cell apoptosis.
Results: IRF-1 mRNA and protein level were significantly increased in AGS/IRF-1 cells induced with tetracycline. Compared with control cells, the growth inhibition rate of cells with IRF-1 overexpression was significantly increased when treated with 5-FU (P<0.01). Treatment with 5 μmol/l 5-FU resulted in 12.6% apoptotic cells, whereas such treatment after overexpression of IRF-1 resulted in 39.4% apoptotic cells. Moreover, more poly(adenosine diphosphate-ribose) polymerase (PARP) cleavage was seen in cells with IRF-1 overexpression.
Conclusions: Overexpression of IRF-1 enhanced the chemosensitivity of gastric cancer cells to 5-FU through induction of apoptosis.

Keywords: Apoptosis, chemosensitivity, gastric cancer, interferon regulatory factor 1


How to cite this article:
Gao J, Tian Y, Zhang J. Overexpression of interferon regulatory factor 1 enhances chemosensitivity to 5-fluorouracil in gastric cancer cells. J Can Res Ther 2012;8:57-61

How to cite this URL:
Gao J, Tian Y, Zhang J. Overexpression of interferon regulatory factor 1 enhances chemosensitivity to 5-fluorouracil in gastric cancer cells. J Can Res Ther [serial online] 2012 [cited 2019 Dec 16];8:57-61. Available from: http://www.cancerjournal.net/text.asp?2012/8/1/57/95175


 > Introduction Top


Gastric cancer is the fourth most common cancer worldwide, with 934,000 cases per year. [1] It is a leading cause of death worldwide and is especially prevalent among Asian populations. Survival from gastric cancer is poor since patients are often diagnosed with advance disease. [1] To improve survival in patients with gastric cancer, the use of chemotherapy is increasing. Chemotherapy is used to relieve symptoms in advanced patients and to reduce the risk of recurrence in patients with localized disease after surgery.

Intrinsic and acquired chemotherapy resistances are the main causes for the limited efficacy of chemotherapy in gastric cancer patients. The resistance of the tumor cell may be inherent to the specific genetic background or result from mutations and epigenetic alterations after treatment. These include upregulation of the multidrug resistance gene product, mutation of the p53 tumor suppressor gene, and activation of nuclear factor-kappa B (NF-κB). The result of these genetic changes is augmented survival pathways and deregulated apoptosis signaling pathways. [2],[3]

Interferon regulatory factor 1 (IRF-1) is a transcriptional factor that binds to specific DNA sequences of genes and controls the transcription of genes involved in mediating antiviral, immunomodulatory, anti-proliferative effects and apoptosis. [4],[5] Emerging evidence suggests that IRF-1 plays an important role in apoptosis. IRF-1 not only induces apoptosis, but is also a necessary mediator of apoptosis induced by DNA damage or other stimuli in a number of cancer cell types. [6],[7],[8] Apoptosis is a major antitumor pathway for chemotherapy. The objective of this study has been to investigate whether IRF-1 is involved in cell sensitivity to chemotherapeutic agents in gastric cancer. To achieve this, we used the human gastric cancer cell line, AGS, and established stable clones in AGS cells with a tetracycline-inducible IRF-1 expression system.


 > Materials and Methods Top


The human gastric cancer AGS cell lines were obtained from the American Type Culture Collection, Manassas, USA. A T-REx TM system, which includes the regulatory vector pcDNA6/TR and the expression vector pcDNA4/TO/myc-His, was from Invitrogen, Carlsbad, USA. RPMI 1640 and fetal bovine serum were purchased from GIBCO, Carlsbad, USA. SuperFect® transfection reagent was the product of QIAGEN,Valencia, USA. 5-fluorouracil (5-FU) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were procured from Sigma, St. Louis, USA. An FITC Annexin V Apoptosis Detection Kit was purchased from BD Pharmingen, San Jose, USA, and iScript One-Step RT-PCR Kit with SYBR Green was from Bio-Rad,Hercules,USA. Rabbit polyclonal antibody against IRF-1 and mouse polyclonal antibody against β-actin were from Santa Cruz, Santa Cruze, USA. Rabbit polyclonal antibody against poly(adenosine diphosphate-ribose) polymerase (PARP) was from Cell Signaling Technology, Danvers, USA. PCR primers were synthesized by Shanghai Shenggong Co. Ltd, Shanghai, China.

Cells were propagated in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cell cultures were maintained in 5% CO 2 at 37°C in a humidified environment. To establish an inducible IRF-1 expression system, we used the T-REx TM system, which includes the regulatory vector pcDNA6/TR and the expression vector pcDNA4/TO/myc-His. The full-length human IRF-1 gene was cloned into the expression vector pcDNA4/TO/myc-His. AGS cells were plated onto 60 mm plates at a density of about 5×10 5 cells per plate and incubated overnight. Cells were co-transfected with pcDNA6/TR and pcDNA4-IRF-1 using SuperFect transfection reagent according to the manufacturer's instructions. For stable clone selection, the transfected cells were cultured in the presence of 10 μg/ml blasticidin and 100 μg/ml zeocin. Stably transfected clones were evaluated by detecting the IRF-1 mRNA with RT-PCR and immunoblotting for IRF-1 protein expression. The stable transfected cells were named AGS/IRF-1.

Total cellular RNA was extracted with Trizol reagent. The quantitative reverse transcription polymerase chain reactions (qRT-PCRs) were carried out using an iScript One-Step RT-PCR Kit with SYBR Green. The following primers were used for IRF-1 (forward 5'-CCTGGCTAGAGATGCAGATT-3' and reverse 5'-TTCCTCTTGGCCTTGCTCTT-3') and beta-actin (forward 5'-ACAGAGCCTCGCCTTTGCCG-3' and reverse 5'-TGGGCCTCGTCGCCCACATA-3'). The reactions contained 1× SYBR green QPCR reaction mix, each primer at 300 nM, 100 ng RNA, and 1 μl iScript reverse transcriptase mixture in a 25 μl reaction. The reaction mixture was first incubated at 50°C for 10 min to allow for reverse transcription. PCR was initiated with one cycle of 95°C for 5 min to activate the Taq polymerase followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. The average threshold cycle for each sample was determined from triplicate reactions. To normalize the amount of input RNA, PCR reactions were done with probe and primers for β-actin.

To determine cell growth, 10,000 cells were seeded in triplicate on 96-well plates and incubated overnight. 5-FU was then added so that the final concentrations were 1, 5 and 10 μmol/l, with dimethyl sulfoxide (DMSO) as a control. Cells were incubated in a humidified incubator in 5% CO 2 at 37°C for 24 h. After the exposure period, the medium was removed and the cells were incubated for 1 h at 37°C with fresh growth medium containing 0.5 mg/ml MTT. The supernatant was then carefully removed and 100 μl of DMSO was added to dissolve the formazan crystals. The absorption (A) was measured at 570 nm in a microplate reader. Growth inhibition rate (%) = (1- A of experimental group/A of control group)×100%.

The untreated AGS/IRF-1 cells were treated with 5 μmol/l 5-FU or treated with 5 μmol/l 5-FU and 1 μg/ml tetracycline for 48 h, after which apoptotic cells were detected using an FITC Annexin V Apoptosis Detection kit according to the manufacturer's instructions. Briefly, after treatment, cells were washed twice with cold phosphate buffered saline (PBS) and then resuspended in 1×binding buffer at a concentration of 1×10 6 cells/ml. Then, 100 μl of the solution (1×10 5 cells) was transferred to a 5 ml culture tube, and 5 μl of FITC Annexin V and 5 μl propidium iodide(PI) were added to the solution. After incubation for 15 min at room temperature in the dark, 400 μl of 1×binding buffer was added to each tube. The stained cells were then analyzed using flow cytometry.

Protein expressions were analyzed by Western blotting. Cells were lysed and equal amounts of protein lysates (40 μg) were electrophoresed on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in tris-buffered saline Tween-20 (TBST) for 1 h at room temperature, and incubated with the primary antibody diluted at 1:1000 in TBST at 4°C overnight. Membranes were washed three times with TBST, and then incubated with the secondary antibody at a dilution of 1:5000 for 1 h at room temperature. Signal detection was performed using an enhanced chemiluminescence reaction. To ensure that equal amounts of protein were loaded, the membranes were stripped and probed with β-actin antibodies.

All data are presented as the mean ± standard deviation (SD). Student's t-test was performed for intergroup comparison. A probability of P<0.05 was considered to be statistically significant.


 > Results Top


[Figure 1] shows clear induction of IRF-1 mRNA expression after stimulation with tetracycline in AGS/IRF-1 cells. When stimulated with 1 μg/ml tetracycline, the IRF-1 mRNA level significantly increased by about 13-fold (P<0.001). The expression of IRF-1 in AGS/IRF-1 cells was confirmed by Western blotting. As shown in [Figure 2], the level of IRF-1 protein was elevated markedly when stimulated with tetracycline for the indicated times. This suggests that tetracycline could effectively induce IRF-1 gene expression in AGS/IRF-1 cells.
Figure 1: IRF-1 mRNA expression levels determined by qRT-PCR in AGS/IRF-1 cells and AGS/IRF-1 cells induced by 1 μg/ml tetracycline (TET) (*P<0.001)

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Figure 2: IRF-1 protein expression levels detected by Western blotting. Column 1: AGS/IRF-1 cells; Column 2: AGS/IRF-1 induced by 1 μg/ml tetracycline for 24 h; Column 3: AGS/IRF-1 cells induced by 1 μg/ml tetracycline for 48 h

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To examine whether overexpression of IRF-1 enhances 5-FU-mediated growth inhibition, cell proliferation assays were performed. The growth of AGS/IRF-1 cells was inhibited in a dose-dependent manner when treated with 1, 5 and 10 μmol/l 5-FU for 24 h. The inhibition rates were 8.4, 19.1 and 31.5%, respectively [Figure 3]. When AGS/IRF-1 cells expressed IRF-1 by induction with tetracycline, the growth inhibition rate was increased to 18.7, 35.2 and 60.3%, respectively, after treatment with 5-FU for 24 h (P<0.01; [Figure 3]).
Figure 3: Cell growth inhibition rates of AGS/IRF-1 in the presence (TET on) or absence (TET off) of 1 μg/ml tetracycline 24h after treatment with various concentrations of 5-FU (*P<0.01)

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Apoptosis is a major antitumor pathway for chemotherapy. To investigate whether overexpression of IRF-1 increases apoptosis induced by 5-FU, we measured the apoptotic cell death in AGS/IRF-1 cells following IRF-1 overexpression and treatment with 5-FU or treatment with 5-FU alone. As shown in [Figure 4], the apoptotic cell fraction was increased following IRF-1 overexpression and treatment with 5 μmol/l 5-FU, as compared with 5-FU treatment only. Treatment with 5 μmol/l 5-FU resulted in 12.6% total apoptotic cells. Overexpression of IRF-1 by induction with tetracycline, followed by 5 μmol/l 5-FU resulted in 39.4% total apoptotic cells [Figure 5].
Figure 4: Apoptosis detected by fl ow cytometry. AGS/IRF-1 cells were untreated, treated with 5 μmol/l 5-FU or treated with 5 μmol/l 5-FU and 1 μg/ml tetracycline (TET) for 48h, then apoptotic cells were detected by fl ow cytometry

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Figure 5: AGS/IRF-1 cells treated with nothing (control), 5 μmol/l 5-FU only or 1 μg/ml tetracycline (TET) and 5 μmol/l 5-FU. Percentages of apoptotic cells are shown as the mean ± SD (error bar) of three experiments. *P< 0.01

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PARP is a key substrate associated with the activation of the biochemical pathway of apoptosis and is a hallmark of apoptosis. To further confirm that IRF-1 overexpression increases apoptosis induced by 5-FU, PARP cleavage was detected. As seen in [Figure 6], more PARP cleavage was seen in cells treated with 5-FU and tetracycline than in those treated with 5-FU only.
Figure 6: PARP cleavage was detected by Western blotting. Column 1: AGS/IRF-1 cells; Column 2: AGS/IRF-1 treated with 5 μmol/l 5-FU for 48h; Column 3: AGS/IRF-1 cells treated with 5 μmol/l 5-FU and 1
μg/ml tetracycline (TET) for 48h


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 > Discussion Top


Chemotherapy, together with surgery, is the main option for the treatment of gastric cancer. The clinical utility of anticancer drugs is limited by their toxicity and the resistance of cancer cells. Growing evidence suggests that chemotherapy induces apoptosis for its therapeutic effect and that defective apoptosis regulation is a major contributor to the development of acquired resistance to anticancer therapies. Restoring apoptosis in cancer cells could therefore reverse chemotherapy resistance and improve the therapeutic response to chemotherapy. [9],[10]

IRF-1, the founding member of the IRF family, regulates a variety of cell biological processes, including growth regulation, immune activation and inflammation. Accumulating evidence suggests that IRF-1 plays an important role in apoptosis. Tanaka et al. reported that wild-type, but not IRF-1-/-, mouse embryonic fibroblasts (MEFs) with expression of c-Ha-Ras gene undergo apoptosis when treated with anticancer drugs and ionizing radiation. [11] DNA damage-induced apoptosis in mitogenically activated mature T lymphocytes is also dependent on IRF-1. [12] Moreover, IRF-1 is also a mediator of apoptosis induced by other stimuli, such as interferons (IFNs), retinoids and tumor necrosis factor alpha (TNF-α) in different cell types. [13],[14],[15]

In human tumors, IRF-1 inactivation prevents apoptosis and promotes tumor development. [4] Frequent loss of heterozygosity at the IRF-1 locus and a point mutation in the IRF-1 gene has been found in gastric cancer patients. [16],[17] In addition to the genetic alterations of the IRF-1 gene, several mechanisms lead to the loss of function of IRF-1. High levels of aberrant spliced IRF-1 mRNAs with a subsequent reduction in the levels of full-length IRF-1 mRNA have been observed in chronic myeloid leukemia (CML) patients. [18] Another study has demonstrated that the level of SUMOylated IRF-1 is elevated in tumor cell lines and tumor tissues. [19] The SUMOylated protein represses IRF-1-mediated transcriptional activation and apoptosis.

In our present study, we investigated whether overexpression of IRF-1 could enhance the chemotherapeutic sensitivity of gastric cancer cells. 5-FU is the most frequently used chemotherapy drug for gastric cancer. Inhibition of cell proliferation has been attributed to the antitumor effects of chemotherapy. Our study showed that 5-FU could inhibit AGS cell growth in a dose-dependent manner, and that the rate of growth inhibition was markedly increased in cells with IRF-1 overexpression after treatment with 5-FU, compared with control cells. Our results suggest that overexpression of IRF-1 enhances the inhibitory effect of 5-FU on AGS cells.

Control of cancer progression by chemotherapy relies on the induction of cellular apoptosis. In cells with IRF-1 overexpression, we observed dramatically increased apoptosis in response to 5-FU. PARP is known to be a key substrate associated with apoptosis at the level of the nucleus and is a more terminal hallmark of apoptosis in this context. [20] More PARP cleavage was seen in cells treated with 5-FU and tetracycline than in those treated with 5-FU only. Our current data show that IRF-1 overexpression enhances 5-FU-induced apoptosis in AGS cells. This result is in agreement with a previous study which showed that IFN-α sensitized melanoma cells to chemotherapy agent by IRF-1-mediated cell apoptosis. [21] Similarly, it has also been reported that IRF-1 plays a pivotal role in the IFN-γ-mediated chemosensitivity to etoposide in human choriocarcinoma cells. [22] These findings along with ours suggest that IRF-1 is critical for sensitizing cancer cells to chemotherapy-induced apoptosis. It would be interesting to further investigate whether IRF-1 is an effective chemosensitizer in other cancer types. IRF-1 has been shown to regulate the transcription of many genes involving in apoptosis, such as caspase-1, [23] caspase-8 [24] and Fas ligand. [25] Further investigation is required to study the mechanisms by which IRF-1 enhances the sensitivity to anticancer drugs in gastric cancer.


 > Conclusion Top


This study showed that IRF-1 overexpression could increase the sensitivity of AGS cells to 5-FU by induction of apoptosis. These results will further our understanding of the role of IRF-1 in apoptosis and provide new strategies for improving chemosensitivity in human gastric cancer cells.

 
 > References Top

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11.Tanaka N, Ishihara M, Kitagawa M, Harada H, Kimura T, Matsuyama T, et al. Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1. Cell 1994;77:829-39.  Back to cited text no. 11
    
12.Tamura T, Ishihara M, Lamphier MS, Tanaka N, Oishi I, Aizawa S, et al. An IRF-1 dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes. Nature 1995;376:596-9.  Back to cited text no. 12
    
13.Kim EJ, Lee JM, Namkoong SE, Um SJ, Park JS. Interferon regulatory factor-1 mediates interferon-gamma-induced apotosis in ovarian carcinoma cells. J Cell Biochem 2002;85:369-80.  Back to cited text no. 13
    
14.Clarke N, Jimenez-Lara AM, Voltz E, Gronemeyer H. Tumor suppressor IRF-1 mediates retinoid and interferon anticancer signaling to death ligand TRAIL. EMBO J 2004;23:3051-60.  Back to cited text no. 14
    
15.Suk K, Chang I, Kim YH, Kim S, Kim JY, Kim H, et al. Interferon gamma (IFNgamma) and tumor necrosis factor alpha synergism in ME-180 cervical cancer cell apoptosis and necrosis. IFNgamma inhibits cytoprotective NF-kappa B through STAT1/IRF-1 pathways. J Biol Chem 2001,276:13153-9.  Back to cited text no. 15
    
16.Tamura G, Ogasawara S, Nishizuka S, Sakata K, Maesawa C, Suzuki Y, et al. Two distinct regions of deletion on the long arm of chromosome 5 in differentiated adenocarcinomas of the stomach. Cancer Res 1996;56:612-5.  Back to cited text no. 16
    
17.Nozawa H, Oda E, Ueda S, Tamura G, Maesawa C, Muto T, et al. Functionally inactivating point mutation in the tumor-suppressor IRF-1 gene identified in human gastric cancer. Int J Cancer 1998;77:522-7.  Back to cited text no. 17
    
18.Tzoanopoulos D, Speletas M, Arvanitidis K, Veiopoulou C, Kyriaki S, Thyphronitis G, et al. Low expression of interferon regulatory factor-1 and identification of novel exons skipping in patients with chronic myeloid leukaemia. Br J Haematol 2002;119:46-53.  Back to cited text no. 18
    
19.Park J, Kim K, Lee EJ, Seo T, Jang IS, Choi JS, et al. Elevated level of SUMOylated IRF-1 in tumor cells interferes with IRF-1-mediated apoptosis. Proc Natl Acad Sci U S A 2007;104:17028-33.  Back to cited text no. 19
    
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21.Upreti M, Koonce NA, Hennings L, Chambers TC, Griffin RJ. Pegylated IFN-á sensitizes melanoma cells to chemotherapy and causes premature senescence in endothelial cells by IRF-1 mediated signaling. Cell Death Dis 2010;1:e67.  Back to cited text no. 21
    
22.Sun QH, Peng JP, Xia HF. IFNgamma pretreatment sensitizes human choriocarcinoma cells to etoposide-induced apoptosis. Mol Hum Reprod 2006;12:99-105.  Back to cited text no. 22
    
23.Bowie ML, Dietze EC, Delrow J, Bean GR, Troch MM, Marjoram RJ, et al. Interferon-regulatory factor-1 is critical for tamoxifen-mediated apoptosis in human mammary epithelial cells. Oncogene 2004;23:8743-55.  Back to cited text no. 23
    
24.Ruiz-Ruiz C, Ruiz de Almodovar C, Rodríguez A, Ortiz-Ferrón G, Redondo JM, López-Rivas A. The up-regulation of human caspase-8 by interferon-gamma in breast tumor cells requires the induction and action of the transcription factor interferon regulatory factor-1. J Biol Chem 2004;279:19712-20.  Back to cited text no. 24
    
25.Chow WA, Fang JJ, Yee JK. The IFN regulatory factor family participates in regulation of Fas ligand gene expression in T cells. J Immunol 2000;164:3512-8.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


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