Role of Interleukin-6 (IL-6) in Cell Invasion of Cholangiocarcinoma Cell Lines บทบาทของอ นเตอร ล วค น-6 ในการบ กร กเซลล เน อเย อของเซลล มะเร งท อน าด

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1 BMP4-1 Role of Interleukin-6 (IL-6) in Cell Invasion of Cholangiocarcinoma Cell Lines บทบาทของอ นเตอร ล วค น-6 ในการบ กร กเซลล เน อเย อของเซลล มะเร งท อน าด Kattaleeya Jindapornprasert (แคทล ยา จ นดาพรประเสร ฐ)* Dr.Waraporn Komyod (ดร.วราภรณ คายอด)** ABSTRACT Interleukin-6 (IL-6) is a pleiotropic cytokine that has been implicated in the progression of cholangiocarcinoma (CCA), a bile duct cancer with high prevalence in the northeastern Thailand. In the present study, we aimed to investigate the role of IL-6 in cell invasion of human cholangiocarcinoma cell lines. The results showed that exogenous IL-6 induced cell invasion of KKU-M213 cells. To investigate the mechanisms underlying IL-6-induced cell invasion, the effect of IL-6 on secretion of MMP-2 and MMP-9 was measured by gelatin zymography. Zymograms from conditioned media of IL- 6-stimulated KKU-M213 cells showed a clear band indicating MMP 9 activity. In conclusion, IL-6 promotes cell invasion of CCA by up-regulating the MMP-9 secretion and IL-6 signaling may be a potential anti-invasion target for the treatment of cholangiocarcinoma. บทค ดย อ อ นเตอร ล วค น-6 เป นไซโตไคน ท ม หน าท หลากหลายท ม ผลต อการพ ฒนาของมะเร งท อน าด ซ งเป นมะเร งท พบมากใน ภาคตะว นออกเฉ ยงเหน อของไทย ในงานว จ ยน เราต องการศ กษาบทบาทของอ นเตอร ล วค น-6 ในการบ กร กเซลล เน อเย อของ เซลล มะเร งท อน าด ของคน ผลการว จ ยแสดงให เห นว าอ นเตอร ล วค น-6 ท ได ร บจากภายนอกกระต นให เก ดการบ กร กของ เซลล ในเซลล KKU-M213 เพ อท ต องการศ กษากลไกการทางานของอ นเตอร ล วค น-6 ท กระต นให เก ดการบ กร กของเซลล จ ง ทาการศ กษาผลของอ นเตอร ล วค น-6 ต อการหล งของเอ มเอ มพ -2 และ เอ มเอ มพ -9 โดยการว ดเจลาต นไซโมกราฟ ผลไซโม แกรมส ของอ นเตอร ล วค น-6 ท กระต นเซลล KKU-M213 ปรากฎให เห นแถบสว างอย างช ดเจนบ งบอกถ งการทางานของเอ ม เอ มพ -9 โดยสร ป อ นเตอร ล วค น-6 ส งเสร มการบ กร กของเซลล ของมะเร งท อน าด โดยเพ มการหล งเอ มเอ มพ -9 และการย บย ง ส ญญาณของอ นเตอร ล วค น-6 อาจเป นเป าหมายท เป นไปได ในการต านการบ กร กของเซลล เพ อการร กษามะเร งท อน าด Key Words: Interleukin-6, Cholangiocarcinoma, Cell invasion คาส าค ญ: อ นเตอร ล วค น-6 มะเร งท อน าด การบ กร กเซลล เน อเย อ * Student, Master of Science Program in Biochemistry, Faculty of Science, Mahidol University ** Lecturer, Department of Biochemistry, Faculty of Science, Mahidol University 518

2 BMP4-2 Introduction Cholangiocarcinoma (CCA) refers to all tumors arising from bile duct epithelium. Many risk factors that lead to CCA development have been reported, such as primary sclerosing cholangitis (PSC), specific carcinogens including nitrosamines, and liver fluke infestation (Clonorchis sinensis and Opisthorchis viverrini ) (Bartlett et al., 2011). CCA was observed in a high percentage of liver cancers from northeast Thailand, where the prevalence of O. viverrini infection is higher than elsewhere in this country. Khon Kaen province in northeast Thailand has the highest incidence of this type of cancer in the world (Sripa et al., 2007). Interleukin-6 (IL-6) is now known as a multifunctional cytokine that can be produced by various cell types, including tumor cells. IL-6 plays important roles with a wide range of biological activities in immune regulation, hematopoiesis, and oncogenesis. Most importantly, IL-6 is involved in the proliferation and differentiation of various malignant tumor cells (Guo et al., 2012). IL-6 signaling generally induces phosphorylation of STAT3 which allows for dimerization of STAT3. The dimer form of STAT3 translocates into the nucleus and binds to DNA at specific binding sites, thereby directing transcription of target genes (Huang et al., 2005). Recently, supporting report suggested that a single plasma IL-6 measurement has excellent positive predictive value for the detection of bile duct cancer in regions with high O. viverrini transmission (Sripa et al., 2012). Metastasis is the process whereby cancer cells spread throughout the body, establishing new colonies in organs at a distance from the one where the primary tumor originated (Bacac, Stamenkovic, 2008). In order to break away from the primary tumor and initiate the metastatic process, individual or small groups of cancer cells must acquire the ability to migrate and invade. These traits enable cells to degrade and move through the extracellular matrix of the surrounding tissue toward blood and lymphatic vessels, which in turn provide highways for their passage to distant secondary sites. Understanding the mechanisms of this process is likely to be important for preventing metastasis in patients who are diagnosed with early cancer lesions (Chaffer, Weinberg, 2011). However, mechanisms of cell invasion that are triggered by IL-6 in CCA still unknown. Objective of the study The objective of this study was to investigate whether IL-6 plays a role in cell invasion of human CCA cell lines. Methodology Cell culture Two human cholangiocarcinoma cell lines, KKU-M213 and KKU-100, were obtained from Thai patients and cultured in Ham's F12 culture medium (Invitrogen, New Zealand) supplemented with 10% fetal bovine serum (FBS), 100 U/ml antibioticantimycotic, 100 μg/ml (Gibco BRL, Paisley, Scotland) and 15 mm HEPES (Merck, Darmstadt, Germany). Cells were incubated at 37 C in a humidified atmosphere with 5% CO 2. In-vitro Transwell invasion assay Invasion assay was performed by Matrigel Transwell in vitro invasion assay. In brief, the upper chamber of a Transwell unit (6.5 mm diameter polycarbonate membrane with 8-µm-pore size) (Corning, United States) was coated with 30 µg of 519

3 BMP4-3 Matrigel (BD Biosciences, United States). Cells ( cells) in 200 µl of serum-free medium with 20 ng/ml recombinant human IL-6 were seeded to the upper chamber. The lower chamber was filled with 600 µl of media containing 1% FBS. After 12 hrs of incubation at 37 under CO 2 atmosphere, non invading cells in the upper chamber were removed and cells that invaded the Matrigel and had attached to the lower surface of the Transwell membrane were fixed with 25% methanol for 15 min and stained with 0.25% crystal violet. Invaded cells were counted in 5 random fields under light microscope at 10x magnification. Gelatin zymography To examine the expression and activation of matrix metalloproteinases (MMPs), cells were seeded on mm tissue culture dishes to reach 80% confluence for 24 hrs. Then, cells were treated with a recombinant human IL-6 (20 ng/ml) for 24 hrs at 37 C and conditioned media was collected by centrifugation. Samples were mixed with sample buffer in the absence of reducing agent and separated by 7.5% acrylamide gel containing 1% gelatin at 4 C. Then, the gel was washed in 2.5% Triton X-100 solution at room temperature with gentle agitation and soaked in incubation buffer (500 mmol/l Tris- HCl ph 8, 100 mmol/l CaCl 2, 1 mmol/l ZnCl 2 and 1% TritonX-100) at 37 C overnight. Afterwards, gel was stained with 0.25 % Coomassie blue for 2 hrs at room temperature, destained for 10 min and dried with cellophane paper. MMPs were detected as clear band against a blue background. Results Effect of exogenous IL-6 on cell invasion in CCA was determined in two CCA cell lines, KKU- M213 and KKU-100. As shown in Figure 1, KKU- M213 cell line displayed higher basal level of invasiveness than KKU-100 cell line. Moreover, IL-6 treatment (20 ng/ml) could increase cell invasion of KKU-M213 cells but not of KKU-100 cells. Figure 1 Matrigel-Transwell assay was used to determine cell invasion after KKU-M213 and KKU-100 cell lines were incubated without or with IL-6 (20 ng/ml) for 12 hrs. The proteolysis of basement membrane by MMPs is one of metastasis process that successfully promotes cell invasion. Thus, to investigate the mechanisms underlying IL-6-induced cell invasion, the effect of IL-6 on secretion of MMP-2 and MMP- 9 in KKU-M213 cells was measured by gelatin zymography. The results showed that IL-6 could significantly increase MMP-9 secretion but not MMP-2 in KKU-M213 cells (Figure 2). A clear band of MMP-9 indicated that MMP-9 (gelatinase enzyme) could digest gelatin substrate in gel. 520

4 BMP ng/ml IL-6 KKU-M MMP-9 (92 kda) MMP-2 (73 kda) Figure 2 MMP-2 (73 kda) and MMP-9 (92 kda) secretion were determined by gelatin zymography after KKU-M213 cells were treated without or with IL-6 (20 ng/ml). Discussion and Conclusions IL-6 is a multifunctional cytokine which plays an important role in a wide range of biological activities in different types of cells including tumor cells. IL-6 is involved in the host immune defense mechanism as well as the modulation of growth and differentiation in various malignancies. Clinical studies have revealed that increased serum IL-6 concentrations in patients are associated with advanced tumor stages of various cancers and short survival in patients. Therefore, blocking IL-6 signaling is a potential therapeutic strategy for cancer. Several investigators have reported an aberrant IL-6 pathway activation in a variety of human cancer cell lines and solid tumors and IL-6 has been found to play an important role in various tumor behaviors including cell invasion. In this study, we used two different CCA cell lines, KKU-M213 and KKU-100, as models to study in vitro cell invasion of CCA. The results showed that IL-6 could increase cell invasion through MMP-9 secretion in KKU-M213 cells. Our results are consistent with the previous studies showing the effect of IL-6 in human malignant non- Hodgkin s lymphomas (Kossakowska et al., 1999) and human cerebral endothelial cell (Yao et al., 2006). However, IL-6 had no effect on cell invasion in KKU-100 cells. The lack of IL-6 effect may be due to lack of IL-6 receptors expression in this cell line. In conclusion, IL-6 can promote cell invasion by up-regulating MMP-9 secretion in CCA and IL-6 signaling may be a potential target for the treatment of cholangiocarcinoma. Acknowledgements This work was supported by research grant from the Faculty of Science, Mahidol University. References Bacac M, Stamenkovic I. Metastatic Cancer Cell. Annu Rev Pathol Mech Dis 2008; 3: Bartlett DL, Ramanathan RK, Edgar Ben-Josef. Cancer of the Biliary Tree. In: Vincent T. DeVita J, Theodore S. Lawrence, Steven A. Rosenberg, editors. Devita, Hellman, and Rosenberg's Cancer: Principles & Practice of Oncology. 9 th ed. Philadelphia, USA: Two Commerce Square; Chaffer CL, Weinberg RA. A Perspective on Cancer Cell Metastasis. Science 2011; 331(6024): Guo Y, Xu F, Lu T, Duan Z, Zhang Z. Interleukin-6 signaling pathway in targeted therapy for cancer. Cancer Treatment Reviews 2012; 38(7):

5 BMP4-5 Huang HF, Murphy TF, Shu P, Barton AB, Barton BE. Stable expression of constitutivelyactivated STAT3 in benign prostatic epithelial cells changes their phenotype to that resembling malignant cells. Molecular Cancer 2005; 4(2): doi: / Kossakowska AE, Edwards DR, Prusinkiewicz C, Zhang MC, Guo D, Urbanski SJ, et al. Interleukin-6 Regulation of Matrix Metalloproteinase (MMP-2 and MMP-9) and Tissue Inhibitor of Metalloproteinase (TIMP-1) Expression in Malignant Non- Hodgkin s Lymphomas. Blood 1999; 94(6): Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, Smout M, et al. Liver fluke induce cholangiocarcinoma. PLoS Med 2007; 4(7): e201. doi: /journal.pmed Sripa B, Thinkhamrop B, Mairiang E, Laha T, Kaewkes S, Sithithaworn P, et al. Elevated Plasma IL-6 Associates with Increased Risk of Advanced Fibrosis and Cholangiocarcinoma in Individuals Infected by Opisthorchis viverrini. PLoS Negl Trop Dis 2012; 6(5): e1654. Yao JS, Zhai W, Young WL, Yang GY. Interleukin-6 triggers human cerebral endothelial cells proliferation and migration: the role for KDR and MMP-9. Biochem Biophys Res Commun 2006; 342(4):