Tumor Biol. DOI 10.1007/s13277-014-1697-3

RESEARCH ARTICLE

Increased expression of IL17A in human gastric cancer and its potential roles in gastric carcinogenesis Xiaoqin Wu & Zhirong Zeng & Lixia Xu & Jun Yu & Qinghua Cao & Minhu Chen & Joseph J. Y. Sung & Pinjin Hu

Received: 16 October 2013 / Accepted: 26 January 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Inflammatory cytokines modulate immune responses in the tumor microenvironment during progression. The role of interleukin (IL) 17A in cancer is currently under debate. We aim to investigate the expression of IL17A in situ tumors as well as in nontumor gastric mucosa tissues and further explore the functional significance of IL17A on gastric cancer cells in vitro. We found that compared with nontumor regions, the expression of IL17A were increased significantly in tumors of gastric cancer patients (P=0.007). The immunoreactivity for IL17A was found only in cytoplasm of inflammatory cells as well as vascular endothelial cells but not in tumor cells. Consistently, IL17A transcription was silenced in a variety of gastric cancer cell lines. In vitro, recombinant human IL17A protein promotes cell proliferation and monolayer wound healing of both AGS and SGC7901cells, in a dose-dependent manner. Besides, IL17A inhibits H2O2-induced cell apoptosis. Expression of IL6 and MMP13 mRNA was increased significantly after IL17A stimulation. These

data suggest that accumulation of intratumoral IL17Aproducing cells may promote gastric cancer progression directly or by inducing key signal transduction pathways implicated in gastric carcinogenesis. Keywords IL17A . Gastric cancer . Intratumoral expression . Cell proliferation . Migration . Apoptosis Abbreviations IL Interleukin MMP Matrix metalloproteinase Th17 T-helper cells type 17 TNF Tumor necrosis factor ERK Extracellular signal-regulated protein kinase MAPK Mitogen-activated protein kinase

Introduction Electronic supplementary material The online version of this article (doi:10.1007/s13277-014-1697-3) contains supplementary material, which is available to authorized users. X. Wu Department of Gastroenterology, Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, China X. Wu : Z. Zeng (*) : M. Chen : P. Hu Department of Gastroenterology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China e-mail: [email protected] L. Xu : J. Yu : J. J. Y. Sung Institute of Digestive Disease and Department of Medicine and Therapeutics, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China Q. Cao Department of Pathology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

Gastric cancer is one of the most common types of cancer globally, which remains an important public health burden in Asian countries such as Japan and China [1]. To date, the exact mechanisms of gastric cancer development remains unclear although multiple factors have been proposed to play a role in gastric carcinogenesis, including Helicobacter pylori infection and host genetic susceptibility as well as host immune status [2]. More than 100 years have past since Virchow hypothesized that the origin of cancer was at sites of chronic inflammation [3]. Today, the causal relationship between inflammation and cancer is more widely accepted [4, 5]. Persistent inflammation secondary to H. pylori infection is associated with increased risk of gastric cancer [6]. The inflammatory cells, and the cytokines and chemokines they produced, influence the migration and differentiation of all cell types in the tumor

Tumor Biol.

microenvironment [4]. Cytokines in tumor microenvironment may induce malignant transformation, tumor growth, invasion, and metastasis. For example, an important role for interleukin (IL)1β and TNFα has been established in the pathogenesis of inflammation-associated cancer [7, 8]. IL17A, a proinflammatory cytokine produced by a new lineage of CD4+ T-helper cells, named Th17 cells, is proved to have a key role in many inflammatory and autoimmune diseases and may be involved in inflammation-related tumor formation [9–11]. Several studies have found excess expressing of IL17A in various tumor tissues, including prostate cancer, ovarian cancer, colorectal cancer, breast cancer, and gastric cancer [12–16]. Moreover, increasing evidences suggest the role of IL17A in H. pylori-related gastric diseases [17]. However, results of IL17A in cancer are more heterogeneous. IL17A may promote tumor growth by stimulating angiogenesis and invasive capacity of tumor cells, as well as inhibit apoptosis [18]. In contrast, other studies suggest that IL17A have a protective effect against tumor development by enhancing the acquired immune response [19, 20]. We have confirmed from clinical epidemiology that IL17A 197 AG genotype increased risks of certain subtypes of gastric cancer [21]. In this study, we examined the expression of IL17A in gastric cancer patients and further explored the impact of recombinant human IL17A on the biological behavior of gastric cancer cells in vitro.

Material and methods Tissue specimens Tissues of primary gastric tumors and their corresponding adjacent nontumor tissues were collected from 54 gastric cancer patients who underwent surgery during 2004–2005 in the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. All diagnoses were pathologically confirmed. None of these subjects had a history of autoimmune or inflammatory diseases, such as systemic lupus erythematosus, diabetes mellitus, rheumatoid arthritis, or inflammatory bowel disease. None of them had received preoperative chemotherapy or radiotherapy. Tissue samples were fixed in 10 % neutralized formalin and embedded in paraffin for histological processing. The research protocol was approved by the Clinical Research Ethics Committee of the Sun Yat-sen University of Medical Sciences. Immunohistochemistry Paraffin-embedded samples were cut into 5-μm sections and processed for immunohistochemistry. Each tissue section was dewaxed in xylene and rehydrated in graded alcohols. For antigen retrieval, the slides were boiled in citrate buffer

(0.01 mol/L, pH 6.0) for 20 min in a microwave oven (750 W). Endogenous peroxidase activity was blocked with a 0.3 % hydrogen peroxide solution for 10 min at room temperature, and nonspecific staining was reduced using a blocking serum for 20 min. The sections were then incubated with goat antihuman IL17A polyclonal antibody (R&D systems; dilution 1/400) for 2 h at 37 °C. After three washes in PBS, sections were incubated with biotinylated antigoat or secondary antibody (Zhongshan Golden Bridge Biotech., Beijing, China) for 30 min at room temperature. Immunostaining was performed using the EnVision System with DAB (DakoCytomation, Glostrup, Denmark). Hematoxylin was used as a counterstain. Negative controls were carried out by not adding the first antibodies. Analysis was performed by two experienced histopathologists who were blinded to the clinical data. Tissue sections were screened at low power field (100×), and five most representative fields were selected at high power field (400×). Density was quantified according to the mean number of IL17A-producing cells in five hot spots. Cell culture Six gastric cancer cell lines—AGS, SGC7901, MKN45, MKN87, SUN1, and SUN16—were kindly provided by Professor Jun Yu (The Chinese University of Hong Kong, Hong Kong, China) or obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). All the cell lines were maintained in RPMI-1640 medium (Invitrogen, USA) supplemented with 10 % fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 u/ml penicillin at 37 °C in a humidified incubator in an atmosphere of 5 % CO2. Reverse transcription-PCR Cells were harvested from the exponential growth phase for examining IL17A mRNA expression. For testing downstream factors, IL6, COX2, VEGF, MMP9, MMP13 secretion, SGC7901 cells were serum starved in RPMI-1640 for 24 h, then treated with IL17A in a series of concentration (0, 1, 10, 50, 100, and 200 ng/ml) for 6 h. Total RNA in cells was extracted using the Qiagen RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Complementary DNA was synthesized with 1 μg total RNA using reverse transcriptase, ReverTra AceTM (Toyobo Co., Osaka, Japan) under the following conditions: 30 °C for 10 min, 42 °C for 20 min, 99 °C for 5 min, and 4 °C for 5 min. PCR of cDNA was carried out in a reaction mixture (25 μl) containing 2 μl of template cDNA, 12.5 μl of PCR Mix (Takara, Japan), 1 μl primer F and R, and 8.5 μl of ddH2O. Amplification was performed using the following conditions: 95 °C for 5 min, followed by 35 cycles (denaturation for 30 s at 95 °C, annealing for 30 s, and extension for 30 s at 72 °C), and

Tumor Biol.

then 72 °C for 5 min. Details of primers, annealing temperature, amplification cycles, and PCR product size for each gene are listed in Table 1. The PCR products were electrophoresed on 15 g/L agarose gel, stained with ethidium bromide, and visualized with an UV transilluminator.

and 100 ng/ml), and cell images are taken at regular intervals over the course of 24–48 h of both areas. Images are analyzed by digitally drawing lines (using Adobe Photoshop) averaging the position of the migrating cells at the wound edges. The migration of the cells is determined by measuring the width of the wound divided, described previously [23].

Real-time TaqMan PCR assay Invasion assay SGC7901 and AGS cells were serum-starved in RPMI-1640 for 24 h, then treated with IL17A in a series of concentration (0, 10, and 100 ng/ml) for different time intervals (3, 6, and 12 h). Total RNA was extracted from these cells with TRIzol reagent (Invitrogen, USA), and the cDNA was generated with an oligo (dT) primer followed by TaqMan PCR assays using ABI 7500 automated fluorescent quantitative PCR system, which has been described previously [22]. The PCR amplification was performed in a total volume of 50 μl mixture containing 5 μl cDNA, 10 pmol of each primer, 5 pmol of probe, 1 μl of dNTPs, 3 U Taq DNA polymerase, and 10 μl 5×PCR buffer using the ABI 9700 PCR system. The primer sequences and probes were listed in Table 2. Following the guidelines of the manufacturer, PCR was performed under the following conditions: 93 °C for 3 min, 40 cycles for 30 s at 93 °C, and 45 s at 55 °C.

Cell invasive ability was examined using a 24-well transwell plate with 8-μm pore polycarbonate membrane inserts according to the manufacturer’s protocol (Corning, NY, USA). The matrigel (14.8 μg/ml) employed for the invasion assays was applied to the upper surface of the membranes. Twelve hours after treatment with IL17A, 5×104 cells per well were seeded into the top chamber in serum-free media. Cells that invaded through the surface of the membrane were fixed with methanol and stained with Crystal violet. Invasive cells from three random microscope fields per filter were selected for cell counting. Flow cytometry

The effect of IL17A on AGS and SGC7901 cell viability were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay. Briefly, cells grown in 96 well were exposed to various concentrations of IL17A dissolved in DMSO (final concentration, 10, 50, and 100 ng/ml) for different time intervals (24, 48, and 72 h). Controls were treated with DMSO vehicle at equal concentrations. Then, 20 μl of MTT (5 mg/ml) was added to each well and cells were incubated continuously at 37 °C for 4 h. After removal of medium, the crystals were dissolved in DMSO and absorbance was assessed at 492 nm with a microplate reader (Bio-Tek, USA).

To determine the presence of early apoptosis, cell death was analyzed by staining cells with Annexin V and propidium iodide (PI). Cells were pretreated with H2O2 (0.25 mmol/l) for 2 h. Before staining, cells were exposed to various concentrations of IL17A (final concentration, 0, 10, and 100 ng/ml) for 12 h. Cells were washed with PBS, centrifuged, and suspended in annexin V binding buffer (HEPES, 10 mM; NaCl, 140 mM; CaCl2, 2.5 mM; and pH,7.4) containing 5 μl/100 μl of annexin V and PI (final concentration, 5 μg/ml) as provided by the manufacturer. Cells were incubated at 5 % CO2 at 37 °C for 15 min and analyzed using flow cytometry (BD Biosciences Pharmingen, San Diego, CA, USA). The fluorochrome was excited using the 488-nm line of argon ion laser, and annexin V and PI emissions were monitored at 525 and 620 nm, respectively. A total of at least 1×105cells were analyzed per sample.

Monolayer wound healing assay

Statistical analysis

Six-well plates with markings on the outer bottom were prepared to be used as reference points during image acquisition. Then, seed the cells in six-well plates and culture until confluent. At the day of analysis, using a yellow pipette tip, a straight scratch was made simulating a wound. In preparation for making the wound, the growth medium is aspirated and replaced by calcium-free PBS to prevent killing of cells at the edge of the wound by exposure to high-calcium concentrations. The wounds are observed using phase contrast microscopy on an inverted microscope. Then, cells were exposed to various concentrations of IL17A (final concentration, 0, 10,

All statistical analyses were performed using SPSS 13.0 software package (SPSS Inc., Chicago, IL, USA). All quantitative data were expressed as mean±SD. Independent-sample t test was used for comparing between two groups, while multiple samples were compared using one-way ANOVA and the LSD method. Immunohistochemical results were analyzed using nonparametric test. Mann–Whitney U test was used to compare the staining results between two independent samples in two groups. Kruskal–Wallis test was used to compare the staining results of independent multiple samples. Spearman test was used for correlation analysis. These are P