ANTICANCER RESEARCH 26: 4213-4216 (2006)

Expression of -17 in Human Colorectal Cancer

DICK WÅGSÄTER1, STURE LÖFGREN2, ANDERS HUGANDER3 and JAN DIMBERG4

1Atherosclerosis Research Unit, King Gustav V Research Institute, Department of Medicine, Karolinska Institute, SE-171 76 Stockholm; 2Department of Clinical Microbiology and 3Department of Surgery, Ryhov County Hospital, SE-551 11 Jönköping; 4Department of Natural Science and Biomedicine, University College of Health Sciences, SE-551 11 Jönköping, Sweden

Abstract. The proinflammatory IL-17 plays a intestinal epithelial cells (10). IL-17 promotes tumorigenicity potential role in T-cell mediated angiogenesis and promotes of human cervical cancer and is associated with an increased tumourigenicity of human cervical cancer. The objective of this level of IL-6 which correlates well with the invasiveness of study was to determine whether IL-17 level is altered cervical tumours (11, 12). Moreover, IL-17 has been shown to in colorectal tumours (n=74) compared with paired normal be expressed in a considerable proportion of ovarian cancers mucosa and in plasma from patients (n=61) with colorectal and promotes tumour angiogenesis (13). Numasaki et al., cancer (CRC) compared with a healthy group (n=78). reported that IL-17 plays a crucial role in T-cell mediated Analyses by ELISA showed that IL-17 protein was angiogenesis (14) and it has recently been suggested that IL- undetectable in 48.6% of the patients with cancer, as well as 17 may promote angiogenesis by enhancing vascular corresponding normal tissue which may in part reflect an endothelial (VEGF) (15). A correlation individual difference. No significant difference was observed between vascularity and invasive behaviour in CRC has also regarding IL-17 protein levels between cancer and matched been demonstrated (16). Furthermore, it has been shown that normal tissue or in plasma between patients and the healthy VEGF expression in the early premalignant stage of colorectal group. Immunohistochemistry (n=20) revealed heterogenous tumour progression is significantly up-regulated compared to immunoreactivity in 65% of the cases. The results of this study normal colorectal tissue (17). These reports indicate a role of suggest that IL-17 plays a minor or partial role in CRC. IL-17 in the promotion of tumour progression. However, to our knowledge little is known about the expression and the Infiltration of inflammatory cells into colorectal cancer (CRC) pathophysiological role of IL-17 in human CRC. tissue is considered of importance for tumour progression (1). In this study IL-17 protein expression in specimens of cancer There is evidence that proinflammatory , which can and adjacent non-cancer tissues and levels of IL-17 protein in be produced by various tumour cells and tumour-associated plasma from colorectal cancer patients were investigated. leukocytes, play an important antitumourigenic role, but have also been shown to contribute to growth and spread of the Materials and Methods malignancy (2, 3). Examples are interleukin-1 (IL-1) and IL-6 which induce proliferation and prolonged cell survival (4-6). Patients and tissue sampling. This study utilized tissue samples, which The proinflammatory cytokine IL-17 is predominantly were obtained from 74 patients who underwent surgical resections produced and secreted by activated CD4 T-cells (7) but data for primary colorectal adenocarcinomas diagnosed at the in humans have subsequently demonstrated that CD8 T-cells Department of Surgery, Ryhov County Hospital, Jönköping, Sweden. Sporadic tumours from 40 male and 34 female subjects with a mean can also produce IL-17 (8). In addition to the age of 70 years (range 36-93) were collected and classified according proinflammatory response, IL-17 is able to regulate tight to Dukes’ classification system: stage A (n=11), stage B (n=30), junction formation (9) and has an anti-mitotic effect on the stage C (n=29) and stage D (n=4). The tumours were localized in the colon (n=34) and rectum (n=40). From each patient tumour tissue and adjacent normal mucosa (about 5 cm from the tumour) were excised and immediately frozen at –70ÆC until analysis. Correspondence to: Dr. Jan Dimberg, Department of Natural The study was approved by the Ethics Committee of our Science and Biomedicine, University College of Health Sciences, hospital and all patients gave informed consent. Box 1026, SE-551 11 Jönköping, Sweden. Tel: +46-36-101288, e- mail: [email protected] Tissue protein preparation and ELISA. Frozen tumour tissue and normal mucosa were thawed, homogenised in ice cold lysis buffer Key Words: Interleukin-17, protein expression, colorectal cancer. containing PBS (9.1 mM dibasic sodium phosphate, 1.7 mM

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Figure 1. The difference of IL-17 protein level between human colorectal cancer (grey spots) and matched normal tissue (black spots) was not significant.

monobasic sodium phosphate, 150 mM NaCl, pH=7.4) and 1% For negative control, non-immune rabbit isotypic IgG (Santa Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl Cruz Biotechnology) was added instead of the primary antibody. sulphate (SDS), 100 mg/ml phenylmethylsulphonyl flouride, 2 Ìg/ml aprotinin, 1 mM sodium orthovanadate and 1 Ìg/ml leupeptin. The Statistical analysis. Data are presented as mean values ± standard lysate was placed on ice for 30 minutes and then centrifuged at error of the mean (SEM). Differences of IL-17 protein expression 13,000 xg for 10 minutes. Protein content of the supernatant fluid between tumour and paired normal tissues were examined by the was determined for each sample using the Lowry assay (18). Wilcoxon signed test. Statistical analysis was performed using The IL-17 protein levels of cancer and paired normal tissues the SPSS for Windows computer package (Rel. 11.5, 2002, Chicago: from 74 patients were measured using established commercially SPSS Inc.). Results were considered significant at a level of p<0.05. available enzyme-linked immunosorbent assay (ELISA) (R&D Systems Europe, UK). The IL-17 protein level was expressed as Results picograms per milligram of protein (pg/mg). All analyses were performed in duplicate and the mean values were used for Levels of IL-17 in tissue and plasma. IL-17 protein statistical calculations. concentration was measured by ELISA in protein-lysates of Plasma samples and ELISA. Sixty-one of the patients (31 rectal CRC tissues and matched normal tissues from 74 patients. cancers and 30 colon cancers) were available for plasma collection. IL-17 protein expression was undetectable in 48.6% (36/74) Venous blood was collected before surgery and separated by of the CRC cases, as well as in corresponding normal tissue. centrifugation within 1 hour. Evaluation of the relative expression (tumour versus normal In order to establish the IL-17 level among healthy individuals 78 tissue) showed 31.1% (23/74) up-regulation and 20.3% control subjects were selected from volunteer blood donors at Ryhov (15/74) suppression. The levels of IL-17 protein in cancer County Hospital. The mean age was 60 (55-67) years and included 41 tissue (6.9±1.35 pg/mg) showed no significant difference in males and 37 females. All plasma from patients and control subjects was stored at –70ÆC until IL-17 was measured by ELISA (R&D comparison with normal tissue (7.1±1.93 pg/mg), (p=0.421) Systems Europe, UK) following the manufacturer’s instructions. and are shown in Figure 1 which is given as scatter graph. IL-17 in plasma was detected in 12 out of 61 in the CRC Immunohistochemistry. Twenty out of the 74 tumour samples were group and 8 out of 78 in the healthy donor group and the available for immunohistochemical staining to study the cell type origin range was 2.1-73.8 pg/ml and 8.8-54.1 pg/ml, respectively. of the IL-17 expression. Staining was performed using a standard There was no statistical difference in IL-17 levels between protocol on 4 Ìm sections from formalin-fixed paraffin-embedded these groups and no relation between IL-17 levels in tissue tissue blocks. Endogenous peroxidase activity was quenched by treatment with 3% hydrogen peroxide for 5 min followed by incubation and plasma (data not shown). with 5% blocking bovine serum albumin solution. Sections were subsequently incubated with a primary rabbit anti-human polyclonal Location of IL-17 expression in CRC tissue. To identify the antibody (Santa Cruz Biotechnology, USA) in appropriate dilution localization of IL-17 protein in CRC tissue (1:100) overnight at 4ÆC. After rinsing in tris-buffered saline, sections immunohistochemical analysis was performed in 20 CRC were incubated with secondary biotinylated bovine anti-rabbit IgG cases. The resection border reflects normal tissue and (Santa Cruz Biotechnology). Avidin-biotin peroxidase complexes haematoxylin staining revealed stromal cells which were (Dako Cytomation, Denmark) were added followed by visualization with 3,3’-diaminobenzidine tetrahydrochloride (Dako Cytomation). All identified by their morphological appearance. Immuno- sections were counterstained with Mayer’s haematoxylin (Histolab reactivity was detected in 13 out of 20 (65%) cases and Products AB, Sweden). showed heterogenous and focal staining of IL-17. IL-17

4214 Wågsäter et al: Interleukin-17 in Human Colorectal Cancer

Figure 2. Images of immunohistochemical staining of IL-17 in cancer (A) and normal (C) tissue from patients with colorectal cancer. No staining was observed when rabbit isotypic IgG antibody was used as negative control in corresponding cancer (B) or normal (D) tissue. immunoreactivity was localised in cancer and normal In the current study an ELISA assay was used to epithelial cells but also in stromal cells with the determine the level of IL-17 protein in CRC and paired morphological characteristics of lymphocytes and normal tissue. No significant difference regarding IL-17 (Figure 2). Overall, an intensity of cytoplasmic staining protein expression was observed between cancer and varying from none to weak and occasionally strong was matched normal tissue in the patient samples. Moreover, noted. Negative controls had no immunoreactivity. IL-17 protein could not be detected in 48.6% of the patients The levels of IL-17 protein in all the analyzed tissue and in either cancer or paired normal tissue. The discrepancy plasma samples from the CRC patients did not correlate may in part reflect individual differences. To examine with clinical characteristics such as age, gender, location and whether IL-17 protein was expressed in tumour and normal Dukes’ stage (data not shown). epithelial or stromal cells in CRC immunohistochemistry was carried out. Staining revealed heterogenous Discussion immunoreactivity with no specific difference in cancer and normal epithelial or stromal cells. Elevation of proinflammatory cytokines, such as IL-1‚ and Circulating cytokines such as IL-1 and IL-6 are associated IL-6 is considered to be associated with colorectal cancer (4, with CRC (6, 25, 26) as is the angiogenetic factor VEGF (27). 19). In in vitro assay, IL-17 induces IL-1‚ from While searching for tumour markers, the levels of IL-17 in and has been shown to stimulate epithelial and fibroblastic cells plasma from CRC patients were also studied. However, the to secrete IL-6 (20, 21). Recently, IL-17 has been demonstrated levels of IL-17 in plasma from the patients were not to increase the production of active metalloproteinase MMP-9 significantly different than those in the healthy control group. (22). The role of metalloproteinases in the progression of Until today there has been little information available human malignancies has been well documented in numerous about IL-17 in cancer, especially in human CRC. Our reports (23, 24). Moreover, IL-17 promotes angiogenesis and results may suggest that IL-17 plays a minor or partial role tumour growth (14). These circumstances may implicate a role in CRC for instance in connection with T-cell mediated for IL-17 in colorectal carcinogenesis. angiogenesis and IL1-‚/IL-6 derived tumour growth.

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