Expression of ADAMTS-1, ADAMTS-4, ADAMTS-5 and TIMP3 by hepatocellular carcinoma cell lines

Abstract

Abstract.Little is known about the expression or role of ADAMTS-1, -4 and -5 and their endogenous inhibitor TIMP3 in the liver in physiological and pathological conditions. Their expression was, therefore, investigated in the hepatocellular carcinoma cell lines HepG2 and HuH-7 using qRT-PCR and western blotting, and their cellular localisation by immunocytochemistry. Cytokine treatments were used to assess mRNA and protein modulation. ADAMTS-1, -4, -5 and TIMP3 mRNA and protein were detected in both HepG2 and HuH-7 cells. IL-1β and IL-6 treatments significantly modulated ADAMTS-1 mRNA expression and IL-1β treatment ADAMTS-4 mRNA expression in HepG2 cells. Modulations of mRNA by ≥5-fold did not translate to increased protein expression. This study showed that ADAMTS-1, -4, -5 and TIMP3 were expressed at differential levels in hepatocellular carcinoma cell lines. The pro-inflammatory cytokines IL-1β, TNF-α or IL-6 induced changes in mRNA expression, although these did not translate to the protein level. IntroductionHepatocellular carcinoma (HCC) is the most common primary malignant tumour occurring in the liver which accounts for 80-90% of all primary liver cancers (1). In many cases, the neoplastic transformation of hepatocytes results from accumulation of genetic changes during enhanced cell proliferation in the injured liver in response to paracrine growth factor and cytokine stimulation (2). HCCs have a considerable capacity for vascular invasion, with metastasis and recurrence being the major factors associated with the poor prognosis of HCC (3).Proteolytic modification of cell surface proteins and extracellular matrix (ECM) plays a pivotal role in cancer development and metastasis. Proteolytic enzymes are involved in several processes, at the cellular and organism level, which are dysregulated in cancer, e.g. cell adhesion and migration, cell invasion and angiogenesis. In particular, they mediate tumour invasion at several stages, e.g. detachment of cells from the primary tumour, crossing vessel walls and extravasation into target organs. This involves attachment of oncogenically transformed cells to the ECM followed by its degradation and movement of invading cells through the damaged ECM (4,5).Expression and activity of several classes of proteolytic enzymes have been shown to be modified in cancer, including recently discovered a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs). There are 19 ADAMTSs which can be involved in pathological processes such as ECM breakdown and angiogenesis (6). They consist of several domains: the prodomain, metalloproteinase domain, disintegrin-like domain, cysteine-rich domain, thrombospondin type-I repeats (TSRs) and a spacer domain. Several ADAMTSs also contain an additional unique domain(s). They are synthesised as zymogens and after proteolytic processing at the N-terminus to remove the signal sequence and prodomain, they are secreted from cells. For most ADAMTSs this is an important step in their activation. ADAMTSs may also undergo C-terminal proc...

TIMP-3 Expression Associates with Malignant Behaviors and Predicts Favorable Survival in HCC

“…Furthermore, IHC analysis of an HCC TMA similarly showed a reduced cytoplasmic expression of TIMP-3 protein in cancer cells relative to normal non-cancerous cells. These results are similar to the studies that indicated that the expression of TIMP-3 was inhibited in malignant cancers [16], [18], [20], [38]. Moreover, positive TIMP-3 expression in HCC was negatively correlated with certain clinical pathologic items, including portal vein invasion and lymph node metastasis.…”

Prognostic Value of ADAMTS Proteases and Their Substrates in Epithelial Ovarian Cancer

“…TIMP-3 was localized in tumor cell cytoplasm, as previously described for renal, pancreatic, and esophageal tumors [22]. Despite its known inhibitory protease activity, increased TIMP-3 expression was associated with head-and-neck tumor development and progression [60].…”

“…Hyalectans have the ability to bind HA through their N-terminal globular domain (G1); the central domain carries most of the GAG chains and exhibits lectin-like activity located in the C-terminal globular domain (G3) [13,19]. The endogenous tissue inhibitor of metalloproteinase 3 (TIMP-3) regulates ADAMTS-1, -4, and -5 proteolytic activity [20,21,22]. …”

ADAMTS5 deficiency protects against non‐alcoholic steatohepatitis in obesity

“…We previously observed expression of several ADAMTS members in murine adipose tissue and found a marked upregulation of ADAMTS5 during the development of obesity . Furthermore, ADAMTS5 is expressed by hepatocellular carcinoma cell lines . ADAMTS5 is the most prominent enzyme in aggrecan degradation , although it also degrades other substratres including versican, biglycan, decorin and collagen .…”