Toll-like receptor 4 signaling promotes epithelial-mesenchymal transition in human hepatocellular carcinoma induced by lipopolysaccharide

Jing et al. BMC Medicine 2012, 10:98 http://www.biomedcentral.com/1741-7015/10/98 RESEARCH ARTICLE Open Access Toll-like receptor 4 signaling promotes epithelialmesenchymal transition in human hepatocellular carcinoma induced by lipopolysaccharide Ying-Ying Jing†, Zhi-Peng Han†, Kai Sun†, Shan-Shan Zhang, Jing Hou, Yan Liu, Rong Li, Lu Gao, Xue Zhao, Qiu-Dong Zhao, Meng-Chao Wu and Li-Xin Wei* Abstract Background: The endotoxin level in the portal and peripheral veins of hepatocellular carcinoma (HCC) patients is higher and lipopolysaccharide (LPS), a cell wall constituent of gram-negative bacteria, has been reported to inhibit tumor growth. However, in this study, we found that LPS-induced toll-like receptor 4 (TLR4) signaling was involved in tumor invasion and survival, and the molecular mechanism was investigated, Methods: Four HCC cell lines and a splenic vein metastasis of the nude mouse model were used to study the invasion ability of LPS-induced HCC cells and the epithelia-mesenchymal transition (EMT) in vitro and in vivo. A total of 106 clinical samples from HCC patients were used to evaluate TLR4 expression and analyze its association with clinicopathological characteristics Results: The in vitro and in vivo experiments demonstrated that LPS could significantly enhance the invasive potential and induce EMT in HCC cells with TLR4 dependent. Further studies showed that LPS could directly activate nuclear factor kappa B (NF-B) signaling through TLR4 in HCC cells. Interestingly, blocking NF-B signaling significantly inhibited transcription factor Snail expression and thereby inhibited EMT occurrence. High expression of TLR4 in HCC tissues was strongly associated with both poor cancer-free survival and overall survival in patients. Conclusions: Our results indicate that TLR4 signaling is required for LPS-induced EMT, tumor cell invasion and metastasis, which provide molecular insights for LPS-related pathogenesis and a basis for developing new strategies against metastasis in HCC. Keywords: Toll-like receptor 4, Epithelial-mesenchymal transition, Lipopolysaccharide, Human hepatocellular carcinoma Background Persistent inflammatory conditions can induce tumorigenesis [1]. Hepatocellular carcinoma (HCC) is closely associated with chronic inflammatory liver diseases and the endotoxin level in the portal and peripheral veins of those patients is higher owing to changes in the intestinal mucosal permeability and increased bacterial infection [2-4]. Lipopolysaccharide (LPS), a cell wall constituent of gramnegative bacteria, is released during lysis of bacteria. It has been reported that LPS can induce cytokines from * Correspondence: † Contributed equally Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, PR China immune cells and inhibit tumor growth [5,6], but recent studies have shown that LPS can alter cytokine levels in the tumor microenvironment and exert direct effects on tumor cell proliferation, invasion and metastasis in vitro and in vivo [7-10]. Toll-like receptor 4 (TLR4), the receptor for LPS, is not only important in the regulation of immune responses to infection [11], but also is involved in noninfectious inflammatory diseases, such as tumor invasion and survival [12]. TLR4 has been detected in many human cancer cell lines, including pancreatic, lung, breast, prostate, liver and colorectal cancer [10,12-16]. When silencing TLR4 expression, the invasion, survival, and tumorigenicity of human prostate cancer cells was inhibited, which © 2012 Jing et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jing et al. BMC Medicine 2012, 10:98 http://www.biomedcentral.com/1741-7015/10/98 indicates TLR4 plays a significant role in connecting inflammation and cancer invasion and progression [12]; however, the exact mechanism is still not clear. Epithelial to mesenchymal transition (EMT) is a process in which epithelial cell layers lose polarity together with cell to cell contacts which results in a dramatic remodeling of the cytoskeleton, and has an important role in tumor metastasis [17]. When human intrahepatic biliary epithelial cells (IBECs) were exposed to high levels of LPS, the IBECs could undergo EMT, potentially contributing to hepatic fibrosis or even hepatoma [18]. In HCC tissues, LPS was previously reported to promote adhesion and invasion in hepatoma cells [9]. These effects suggest that LPS-induced TLR4 signaling provides a survival benefit for metastatic tumors; however, whether TLR4 signaling can induce EMT in HCC cells and the mechanism involved remains unclear. In the present study, we provide evidence that LPSinduced TLR4 signaling promotes HCC cell invasion and EMT in vitro and in vivo, and a high expression of TLR4 in HCC tissues was strongly associated with both poor cancer-free survival and overall survival in patients, which indicates that LPS is closely related to tumor invasion and metastasis, rather than only anti-tumor effects. Methods Reagents and antibodies (Dulbecco’s) modified Eagle’s medium ((D)MEM), fetal bovine serum (FBS), penicillin, streptomycin sulfate, glutamine, and 0.05% trypsin/0.02% ethylenediamine tetraacetic acid (EDTA) solution were purchased from Invitrogen (Carlsbad, CA, USA). LPS derived from Escherichia coli strain 055:B5, Trizol, Lipofectamine 2000 and BAY11-7028, the inhibitor of nuclear factor kappa B (NF-B), were purchased from Sigma (St. Louis, MO, USA). A dual luciferase reporter gene assay kit to detect NF-B activity was purchased from Promega (Madison, WI, USA). Rabbit antihuman E-cadherin and Vimentin antibodies were obtained from Thermo (Fremont, CA, USA). Goat anti-human Snail antibody was purchased from R&D (Minneapolis, MN, USA). Rabbit anti-human TLR4 antibody was purchased from Bioworld Technology (St. Louis, MN, USA). Page 2 of 12 Patients and tissue specimens Specimens of HCC tissues were obtained from 106 HCC patients who underwent hepatic resection at the Eastern Hepatobiliary Surgery Hospital of the Second Military Medical University from October 2000 to November 2003. These patients included 88 men and 18 women with a median age of 49 years (range: 11 to 72), and all of the specimens were subjected to immunohistochemisty (IHC). Prior informed consent was obtained and the study protocol was approved by the Ethics Committee of the Eastern Hepatobiliary Surgery Hospital. Wound healing and Transwell assay The methods for wound healing and the Transwell assay have been described [19-21]. For the wound-healing assay, cells (5 × 104) were seeded on a 24-well dish and incubated for 24 hours, the monolayer was then disrupted with a cell scraper (1.2 mm width (...truncated)