**8. References**

168 Hepatocellular Carcinoma – Basic Research

We went further to examine the effect of TFPI-2 expression on the invasion of HepG2 cells. Based on invasion and migration assays, we counted the cells that passed through the membranes (Table 1). The results show that the number of cells passing through the membranes was significantly lower in the HepG2-TFPI-2 group than the other two groups ( P<0.05), indicating that TFPI-2 suppresses the invasive potential of hepatocarcinoma cells. While no significant difference in migration ability was observed in the three groups (Table 1).

All data were presented as mean±SD. Stastical analysis was performed with SPSS statistical software. The Student two-tailed *t* test was used to compare the difference between groups,

TFPI-2 is a serine proteinase inhibitor which is frequently downregulated in malignant tumors (18). Previous studies have demonstrated that silencing of TFPI-2 by either histone deacetylation (19) or promoter hypermethylation contributes to its inactivation and tumor progression in several cancers including glioma (18), choricarcinoma (20), pancreatic carcinoma (17), lung carcinoma (21), breast cancer (22), melanoma (23) and hepatocarcinoma (24). In addition, the aberrant splicing form of TFPI-2 was detected during cancer progression (25), which represents an untranslated form providing another mechanism by

In this study, we investigated the expression and function of TFPI-2 in HCC. We first applied the *in situ* hybridization and immunohistochemistry methods to evaluate the expression of TFPI-2 mRNA and protein in hepatocarcinoma tissues and tumor-adjacent normal hepatic tissues. Consistent with previous studies, our results showed that TFPI-2 expression at both mRNA and protein levels was low in hepatocarcinoma tissues compared to adjacent normal hepatic tissues.

To find the mechanism by which TFPI-2 loss contributes to HCC, we employed HepG2 cells as a model. Our results demonstrate that reconstitution of TFPI-2 into HepG2 cells could inhibit the proliferation and invasion of HepG2 cells. Although the details for TFPI-2-mediated growth suppression are unknown, a previous study suggested that TFPI-2 induces apoptosis in glioma cells (26). Further studies are necessary to examine whether TFPI-2 promotes apoptosis of HepG2 cells. In agreement with previous reports that overexpression of TFPI-2 reduced the invasion of cancer cell lines derived from melanoma (27), prostate cancer (28), choriocarcinoma (29), glioblastoma (30) or meningiomas (31), our results showed that restoration of TFPI-2 was associated with a twofold decrease in invasive ability of HepG2 cells. In fact, TFPI-2 is thought to play a pivotal role in the regulation of plasmin-mediated ECM proteolysis during tumor invasion and metastasis (14). TFPI-2 inhibits the release of plasminor trypsin-dependent activation of pro-matrix metalloproteinase (MMP)-1 and pro-MMP-3, which leads to diminished ECM degradation and decreased invasion of HT-1080 fibrosarcoma cell lines (32, 33). In addition, TFPI-2 can inhibit MMP-2 activation in HT-1080 cells (34) and inhibit MMP-1, MMP-13, MMP-2 and MMP-9 in experimental models (35). Thus we assume that TFPI-2 inhibits HCC invasion and metastasis through modulating the activity of MMPs. In summary, we reported that TFPI-2 expression is lost in HCC. The results of our *in vitro* studies confirm that restoration of TFPI-2 caused decreased proliferative and invasive behaviors of HepG2 cells. Taken together, these data suggest that inactivation of TFPI-2 may contribute to the malignant behavior in hepatocarcinoma. Additional in vivo studies will

These results indicated that a decreased expression of TFPI-2 is implicated in HCC.

**7. Statistical analysis** 

*p*<0.05 was considered to be statistically significant.

which TFPI-2 is downregulated in tumor cells.


**8** 

**Proteoglycans in Chronic Liver Disease and** 

The last four decades witnessed a brilliant career of proteoglycans (PGs). Once regarded as mere space-fillers or passive structural components of matrices and charge-selective barriers, these fascinating molecules have been increasingly acknowledged as key players in cell-cell and cell-matrix communication, and have become recognized as modulators of most, if not all, aspects of cell behavior including survival, proliferation, and migration. Simultaneously, the range of disease processes with known involvement of PGs has steadily expanded, now covering areas as diverse as host-pathogen interactions, regulation of pathologic fibrogenesis, and tumor progression. Characteristic alterations of PGs in various human malignant tumors, including HCC, were first described more than 20 years ago (for

PGs, glycanated proteins with extensive posttranslational modifications, consist of a protein core and one or more long, linear, sulfated polysaccharide chains, called glycosaminoglycans (GAGs). GAGs are ligated to the protein core at specific serine, threonine, or asparagine residues, although the exact signal sequences that designate the position of attachment are mostly unknown. The multifunctionality of PGs arises from their inherently complex structure: some functions are assigned to the core protein, while others

The synthesis of each GAG chain (recently reviewed by Ly et al., 2010) is introduced by the attachment of a short linkage region to Ser in the case of heparan sulfate (HS)/heparin and chondroitin sulfate/dermatan sulfate (CS/DS), and either Asn or Ser/Thr in the case of keratan sulfate (KS) type I and type II, respectively. During the elongation phase of GAG synthesis, acetylated hexosamine and hexuronic acid or galactose residues are added in an alternating fashion to the growing polysaccharide chain. GAGs are classified by their disaccharide composition: the dimeric building block is N-acetyl-glucosamine / glucoronic acid in HS and heparin; N-acetyl-galactosamine / glucoronic acid in CS and DS; and Nacetlyl-glucosamine / galactose in KS. Completed GAG chains then undergo various chemical modifications including *N*-deacetylation, *N*- and *O*-sulfation, and epimerization of the hexuronic acid. Heparin, for example, differs from HS in the extent of sulfation (heparin is sulfated uniformly and nearly exhaustively, whereas HS is sulfated only partially and in a

**1. Introduction** 

an early review, see Tímár & Kovalszky, 1995).

are fulfilled by the GAG chains.

**Hepatocellular Carcinoma: An Update** 

Péter Tátrai and Ilona Kovalszky

*Semmelweis University* 

*Hungary* 

