Division of Hematology/Oncology, Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
Pediatr Blood Cancer. 2013 Feb;60(2):248-53. doi: 10.1002/pbc.24211. Epub 2012 May 30.
Expression of toll-like receptor-4 (TLR4) on tumor cells is known to mediate innate immune responses that influence tumor cell growth and migration. This study aimed to characterize TLR4 expression and elucidate its functional significance in human hepatoblastoma (HB) cells.
Immunohistochemistry (IHC) was used to determine TLR4 expression level and its distribution pattern in HB liver tissues. Transcripts of tumor necrosis factor (TNF)-α, interleukin (IL)-8, matrix metalloproteinase (MMP)-2, MMP-13, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 in HB HepG2 cells with lipopolysacharide (LPS) treatment were measured by quantitative PCR. Soluble cytokines and peptides in conditioned media were measured by ELISA. MMP-2 activity was determined by using gelatin zymography. Cell motility and invasiveness was determined using wound healing migration and Matrigel invasion assays, respectively.
TLR4 IHC staining demonstrated that TLR4 overexpression in HB liver tissues dramatically vanished after chemotherapy. In vitro study using an HB cell line, HepG2, showed that TLR4 agonist, LPS, significantly decreased transcripts of IL-8 and TNF-α, but did not affect MMP-13 mRNA level. By contrast, LPS only down-regulated IL-8 production and MMP-2 gelatinolytic activity. The latter might be in part due to the increased levels of MMP-2/TIMP-2 complex in conditioned media, thus leading to the decreased motility and invasiveness of HepG2 cells.
HB cells overexpress TLR4, whereas TLR4 agonistic treatment inhibits migration and invasion of HB HepG2 cells. These findings suggest that TLR4 signaling pathway is a potential therapeutic target for control of HB tumor progression.
肿瘤细胞上 Toll 样受体 4（TLR4）的表达被认为介导了影响肿瘤细胞生长和迁移的固有免疫反应。本研究旨在表征 TLR4 的表达，并阐明其在人肝癌（HB）细胞中的功能意义。
采用免疫组织化学（IHC）检测 TLR4 在 HB 肝组织中的表达水平及其分布模式。用定量 PCR 检测 HB HepG2 细胞中转录因子 TNF-α、IL-8、MMP-2、MMP-13、金属蛋白酶组织抑制剂（TIMP）-1 和 TIMP-2 的表达，用 LPS 处理。用 ELISA 法检测条件培养基中可溶性细胞因子和肽的水平。用明胶酶谱法测定 MMP-2 活性。用划痕愈合迁移和 Matrigel 侵袭试验分别测定细胞迁移和侵袭能力。
TLR4 IHC 染色表明，化疗后 HB 肝组织中 TLR4 过度表达明显消失。体外研究使用 HB 细胞系 HepG2 表明，TLR4 激动剂 LPS 显著降低了 IL-8 和 TNF-α 的转录，但不影响 MMP-13 mRNA 水平。相反，LPS 仅下调 IL-8 的产生和 MMP-2 的明胶酶活性。后者可能部分归因于条件培养基中 MMP-2/TIMP-2 复合物水平的增加，从而导致 HepG2 细胞的迁移和侵袭能力降低。
HB 细胞过度表达 TLR4，而 TLR4 激动剂治疗抑制 HB HepG2 细胞的迁移和侵袭。这些发现表明 TLR4 信号通路是控制 HB 肿瘤进展的潜在治疗靶点。
