C Polymorphism of Interleukin 6 Gene Promoter Affects Interleukin 6 Serum Level in Patients with Colorectal Cancer

Purpose: Experimental data suggest that interleukin 6 (IL-6) plays an important role in the development and progression of metastasis from colorectal cancer (CRC), and 174 G>C polymorphism has been identified recently in the IL-6 gene promoter. Therefore, the aim of the present study was to investigate the significance of this type of polymorphism in patients with CRC. Experimental Design: Using enzyme immunoassay, IL-6 concentrations were measured in preoperative serum samples from 65 stage I-IV CRC patients. DNA was extracted from peripheral blood mononuclear cells, and 174 G>C polymorphism detected using PCR, followed by NlaIII restriction enzyme digestion and electrophoresis. Results: The median IL-6 serum level was 0.14 pg/ml in patients with stage I-III disease versus 0.41 pg/ml in patients with stage IV disease (P < 0.001). DNA amplification was possible in 62 cases. On grouping genotypes at the 174 G>C locus as C (CC and CG) and C (GG), a significant association was observed between the type of polymorphism and IL-6 serum level: the median value for IL-6 was 0.14 pg/ml in C patients (n 32) and 0.32 pg/ml in C patients (n 30; P 0.034). Moreover, in patients with hepatic metastasis the median level of IL-6 was 0.23 pg/ml in C patients (n 9) and 0.96 pg/ml in C patients (n 9; P 0.004). Conclusions: In patients with CRC, the 174 G>C polymorphism status of the IL-6 gene promoter affects the IL-6 serum level, particularly in the presence of hepatic metastasis. INTRODUCTION IL-6, a Mr 25,000 glycoprotein growth factor, has multiple stimulatory effects on inflammation and cell growth (1). Experimental findings suggest that IL-6 acts as a potent stimulator of metastasis by up-regulating the expression on endothelial cells of adhesion receptors, such as intercellular adhesion molecule-1 and leukocyte adhesion molecule-1, and by stimulating the production of growth factors such as hepatocyte growth factor and vascular endothelial growth factor (2–4). Several clinical studies have reported that in different tumor types, a high IL-6 serum level is associated with advanced stage disease (5–8) and a worse outcome (5, 9, 10). In particular, we reported elsewhere that a high preoperative IL-6 serum level is associated with the presence of metastasis and is a negative prognostic factor in patients with CRC (11). The status of a common functional single G C base exchange polymorphism in the human IL-6 gene promoter (chromosome 7p21) located at 174 bp, upstream from the start site of transcription ( 174 G C locus) (12), has been reported to influence IL-6 levels in vitro and in vivo (13, 14). The G allele increases IL-6 expression, both in basal and stimulated conditions, the highest IL-6 levels in plasma and serum being found in subjects homozygous for the G allele (13, 14). Moreover, it has been reported that 174 G C polymorphism is involved in several chronic conditions, such as insulin and noninsulin-dependent diabetes mellitus, juvenile chronic arthritis, coronary heart disease, dementia, peripheral artery occlusive disease, and postmenopausal osteoporosis (14–21). However, to our knowledge, no studies have been conducted to evaluate the effect of 174 G C polymorphism on IL-6 serum levels in patients with solid tumors. Therefore, the aim of the present study was to investigate the influence of 174 G C polymorphism on IL-6 serum levels in patients with CRC, particularly those with hepatic metastasis. PATIENTS AND METHODS Patients. The study population consisted of 65 fully informed consent patients (42 men and 23 women; mean age 66 years; range, 43–83 years), who underwent surgery for colorectal adenocarcinoma at our institution between January 2001 and December 2001. Patients with hereditary CRC, inflammatory bowel disease, and obstructing or perforated tumor were excluded from the study. None of the patients underwent preoperative chemoradiotherapy. The tumor was located in the colon in 47 cases (72%) and in the rectum in 18 cases (28%). The histological grade was assessed according to WHO criteria (22): 13 tumors (20%) were well differentiated, 34 (53%) modReceived 9/30/02; revised 12/16/02; accepted 12/19/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Department of Oncological and Surgical Sciences, University of Padova, Via Giustiniani, 2, 35128 Padova, Italy. Phone: 390498212056; Fax: 39049651891; E-mail: claudio.belluco@unipd.it. 2 The abbreviations used are: IL, interleukin; CRC, colorectal cancer; C, cytosine; G, guanine; TNM, tumor-node-metastasis; NF, nuclear factor. 2173 Vol. 9, 2173–2176, June 2003 Clinical Cancer Research Research. on October 2, 2017. © 2003 American Association for Cancer clincancerres.aacrjournals.org Downloaded from erately differentiated, and 18 (27%) poorly differentiated. Following the International Union Against Cancer classification and TNM staging system (23), 20 of the tumors (31%) were stage I, 14 (21%) stage II, 13 (20%) stage III, and 18 (28%) stage IV. All of the patients with stage IV disease had hepatic metastasis, which involved 50% of the liver in 10 patients (56%) and 50% of the liver in 8 patients (44%). Four of the patients with hepatic metastasis (22%) underwent resection, 8 (45%) systemic chemotherapy, 2 (11%) locoregional treatment, and 4 (22%) were treated by supportive care only. Blood samples were obtained from fasting patients immediately before anesthesia and surgery. Serum IL-6 Levels. Blood, collected in pyrogen-free glass tubes, was centrifuged at 600 rpm for 10 min. The serum was removed and stored at 80°C in pyrogen-free plastic tubes until analysis. Serum IL-6 levels were measured using a commercially available dual antibody sandwich enzyme-linked immunoassay (BioSource Cytoscreen human IL-6 UltraSensitive kit; Biosource International, Camarillo, CA). Using this kit, which has been used in previous studies (13, 24), the minimum detectable dose of IL-6 was 0.10 pg/ml, and no cross-detection of other cytokines was observed. All of the samples were thawed only once and assayed in duplicate. The intra-assay coefficient of variation was 10%. Determination of 174 G>C Polymorphism. DNA, extracted from peripheral blood mononuclear cells using phenol-chloroform following standard procedures, was amplified by primers designed for the promoter region of the IL-6 gene, as described previously (12). The primers used were: TTG TCA AGA CAT GCC AAG TGC T (forward) and GCC TCA GAG ACA TCT CCA GTC C (reverse). PCR products were digested with the NlaIII restriction enzyme overnight and electrophoresed on 2% agarose gel. The presence of a cytosine (C allele) at nucleotide 174 was revealed by the presence of the NlaIII cutting site. The genotypes identified at the 174 G C locus were classified as G/G (homozygotes for the NlaIII cutting site absence), G/C (heterozygotes for the NlaIII cutting site absence and presence), and C/C (homozygotes for the NlaIII cutting site presence). On the basis of our previous experience (13), genotypes were subsequently grouped as C (CC and CG) and C (GG). Statistical Analysis. Because of the great departure from the normality of log-transformed data, a comparison was made between serum IL-6 levels in relation to genotype and clinicopathologic variables using the Mann-Whitney and KruskallWallis nonparametric rank tests (25). Ps 0.05 were considered significant. The data are reported as medians with the first and the third quartiles, the latter representing variability within each group. All of the analyses were computed with S-Plus software (StatInc., Seattle, WA).