Cellular Distribution of Transcripts for Tissue Inhibitor of Metalloproteinases 1 and 2 in Human Hepatocellular Carcinomas

Abstract

HARUSHIGE NAKATSUKASA, KOUZOU ASHIDA, TOSHIHIRO HIGASHI, SOUHEI OHGUCHI, SO TSUBOI, NAOKI HINO, KAZUHIRO NOUSO, YOSHIAKI URABE, NOBUYUKI KINUGASA, KEIGO YOSHIDA, SHUJI UEMATSU, MASAHIKO ISHIZAKI, YOSHIYUKI KOBAYASHI, AND TAKAO TSUJI Hepatocellular carcinoma (HCC) is one of the most prevaThe cellular distribution of tissue inhibitor of melent malignancies in Japan and China, and frequently occurs talloproteinases (TIMP)-1, and TIMP-2 was studied by in hepatitis B or C virus-related chronic hepatitis or cirrhousing in situ hybridization in surgically removed husis. In particular, cirrhosis has been regarded as a high-risk man hepatocellular carcinomas (HCCs) and cholandisease state for developing HCCs. A unique feature of HCC giocellular carcinomas (CCCs). The purpose of this development is the occurrence of the tumor in fibrotic or cirstudy was to characterize the potential involvement rhotic liver that contains abundant extracellular matrices of TIMPs in the development of HCCs and CCCs. All (ECMs). Therefore, the capacity of HCC to degrade the surHCCs and CCCs expressed TIMPs. The distribution rounding ECMs may be an important trait for growth, invaof transcripts for TIMPs in the tumors was mostly sion, and metastasis. homogeneous. Expression of TIMPs in cancer cellsThe characteristic features of malignant tumors are the was more intense than that in the surrounding noninvasion to cross tissue boundaries and the metastasis to cancerous liver (either, cirrhosis, chronic hepatitis, distant organs. Many steps that occur during cancer invasion or normal), and expression of TIMP-1 was stronger and metastasis require specific interactions between maligthan that of TIMP-2. Expression of TIMPs varied nant cells and the ECMs, 1 particularly in regard to HCC and among HCC nodules, but there was no obvious associcirrhotic liver. Multiple humoral factors are involved in this ation between the expression level of TIMPs and difprocess: matrix metalloproteinases (MMPs), tissue inhibitor ferentiation stages or invasiveness of the HCCs. Tranof metalloproteinases (TIMPs), and various cytokines. An imscripts for TIMPs were clearly demonstrated in the balance between TIMPs and MMPs may be an important metastatic HCC nodules in the lung. Expression of factor in tumor invasion and metastasis. TIMP-1 in CCC was strong, and small nodules of CCC Three members of the TIMP family have been described. 2 were recognized in the liver. ImmunohistochemicalEach TIMP is the product of a separate gene. TIMP-1 is a 28-study for TIMP-1 revealed a consistent staining of the kd glycoprotein, whereas TIMP-2 is a 21-kd nonglycosylated TIMP-1 protein with the transcripts. In the perituprotein. Between TIMP-1 and TIMP-2, there is 37% amino moral histologically normal liver, which was not inacid identity and 65.6% overall homology. 3 TIMP-3 has been fected with either hepatitis B or C virus, expression identified as a 21-kd protein and shares an amino acid seof TIMP-1 was found in various cell types, but that of quence homology of 40% with TIMP-1 and 45%...

Pravastatin reduces lung metastasis of rat hepatocellular carcinoma via a coordinated decrease of MMP expression and activity

“…In addition, pravastatin also greatly decreased the expression of TIMP-1. TIMP-1 is not involved in pro-MMP-2 activation but is known to be overexpressed in HCC [28], especially in high-grade tumors [29]. The role of TIMP-1 in HCC is unclear but high levels of TIMP-1 have been related to a poor prognosis and/or to an increased metastasis risk in other cancers [30][31][32].…”

The formation of capsule and septum in human hepatocellular carcinoma

“…D In situ hybridization with sense probe as a negative control disclosed no significant staining (serial section of B and C and counterstained with hematoxylin) production of ECMs, particularly collagens, is essential. Matrix metalloproteinases [2,15] are the major degrading enzymes and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), also play an important role [13]. In this regulation network, the activation of α-SMA-positive cells (activated stellate cells) appears to be a pivotal step.…”

“…In situ hybridization was performed as described previously [13]. Briefly, tissue sections were deparaffinized, rehydrated, and pretreated with 0.2 N HCl and 100 mg/ml pepsin, 0.3% Triton X-100 in phosphate-buffered saline (PBS), 20 µg/ml proteinase K (Boehringer-Mannheim, Tokyo, Japan) in 0.1 M Tris HCl with 50 mmol/l ethylene diamine tetraacetic acid (EDTA), 4% paraformaldehyde in PBS, 0.2% glycine, and 0.2 mol/l Tris (pH 7.5) and with 0.25% acetic anhydride.…”

“…Northern-blot analyses using chemiluminescence was performed as was described previously [13]. Briefly, 20 µg of total RNA, extracted using the acid phenol-guanidinium thiocyanate-chloroform extraction method [20], was fractionated on a 1% agarose/formaldehyde gel and subsequently transferred onto a nylon membrane.…”

“…The membranes were washed once in 2×SSC and 0.1% SDS and twice in 0.1×SSC and 0.1% SDS at 70°C for 15 min. The visualization of the DIG using chemiluminescence was performed according to the manufacturer's protocol [13]. The luminescent light emission was recorded on X-ray films (Kodak, Tokyo, Japan).…”