Investigation of ADAM17 and ADAMTS-1, -4 and -5 in liver carcinoma.

Background: Proteolytic enzymes are important mediators of cellular proliferation, angiogenesis and remodelling of the extracellular matrix (ECM); all processes required for tumour growth and metastasis. However, the studies of proteolytic enzymes in hepatic tumours, both primary and metastatic, have largely been limited to specific matrix metalloproteinases e.g. MMP-2, -7 and -9, and urokinase-type plasminogen activator. ADAM17 (a disintegrin and metalloproteinase 17), a membrane-bound sheddase, releases membrane-bound proteins including growth factors, which could contribute to liver tumour growth. Fractalkine is also shed by ADAM 17, and can act as an angiogenic mediator, potentially aiding the development of tumour neovasculature. ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) -1, -4 and -5 are secreted enzymes, which primarily degrade ECM components, and may participate in the remodelling of liver ECM during invasive processes. TIMP3 (tissue inhibitor of metalloproteinases) is the major, endogenous inhibitor of ADAM17, ADAMTS-1, -4 and -5, and its dysregulation in cancer could aid tumour progression.Methods: Three liver derived cell lines were utilised throughout this investigation, namely HepG2, HuH-7 (well-differentiated hepatocellular carcinoma cell lines), and LX-2 (an activated hepatic stellate cell line). ADAM17, ADAMTS-1, -4, -5 and TIMP3 mRNA expression was investigated by quantitative real-time RT-PCR using the SYBR green method, their protein expression by western blotting using the SDS-PAGE Laemmli system, and their cellular distribution by immunocytochemistry with confocal laser scanning microscopy. The modulation of each of these characteristics by cytokines (IL-1beta, IL-6 and TNF-alpha) was also investigated, and MTT assays performed to determine the proliferative effect of these treatments. ADAM 17 activity was studied using a fractalkine ELISA, as was the effect of ADAM17 down-regulation by siRNA. Furthermore, cell surface and intracellular ADAM 17 protein levels were quantified using flow cytometry and related to shed fractalkine levels following cytokine treatments.Results: This investigation established the presence of ADAM17, ADAMTS-1, 4,-5 and TIMP3 at the mRNA level in foetal and adult human liver, and confirmed the presence of ADAM17, ADAMTS-1 and TIMP3 at the mRNA and protein level in liver derived cells (HepG2, HuH-7 & LX-2 cell lines). Furthermore, the expression of ADAMTS-4 and -5 at the mRNA and protein level was demonstrated for the first time in liver cell lines. Their expression in these cells was differentially modulated at the mRNA and protein level by pro-inflammatory cytokines elevated during liver tumour development (IL-1beta, IL-6 and TNF-alpha). The same cytokines also increased the cellular proliferation of hepatoma cells (HepG2 & HuH-7), but not activated hepatic stellate cells (LX-2). Fractalkine shedding was significantly increased following IL-1beta and TNF-alpha treatments of HepG2 cells, although this did not correlate with the relatively weak up-regulation of ADAM 17 protein following the same treatments, and was not reduced by the down-regulation of ADAM17 with specific siRNA, indicating the involvement of other proteinases in this process.Conclusions: The modulation of these enzymes and their endogenous inhibitor in normal or transformed hepatic cells may provide a microenvironment that facilitates ECM remodelling to allow cancer cell invasion, and subsequent growth and development of tumours into the liver parenchyma.