Expression and methylation status of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor that inhibits plasmin-dependent activation of several metalloproteinases. Downregulation of TFPI-2 could thus enhance the invasive potential of neoplastic cells in several cancers, including lung cancer. In this study, TFPI-2 mRNA was measured using a real-time PCR method in tumours of 59 patients with non-small-cell lung cancer (NSCLC). Tumour TFPI-2 mRNA levels appeared well correlated with protein expression evaluated by immunohistochemistry and were 4–120 times lower compared to those of nonaffected lung tissue in 22 cases (37%). Hypermethylation of the TFPI-2 gene promoter was demonstrated by restriction enzyme-polymerase chain reaction in 12 of 40 cases of NSCLC (30%), including nine of 17 for whom tumour TFPI-2 gene expression was lower than in noncancerous tissue. In contrast, this epigenetic modification was shown in only three of 23 tumours in which no decrease in TFPI-2 synthesis was found (P=0.016). Decreased TFPI-2 gene expression and hypermethylation were more frequently associated with stages III or IV NSCLC (eight out of 10, P=0.02) and the TFPI-2 gene promoter was more frequently hypermethylated in patients with lymph node metastases (eight out of 16, P=0.02). These results suggest that silencing of the TFPI-2 gene by hypermethylation might contribute to tumour progression in NSCLC

Reduced expression of tissue factor pathway inhibitor-2 contributes to apoptosis and angiogenesis in cervical cancer

<p>Abstract</p> <p>Background</p> <p>Tissue factor pathway inhibitor-2 (TFPI-2) is an extracellular matrix associated broad-spectrum Kunitz-type serine proteinase inhibitor. Recently, down regulation of TFPI-2 was suggested to be involved in tumor invasion and metastasis in some cancers.</p> <p>Methods</p> <p>This study involved 12 normal cervical squamous epithelia, 48 cervical intraepithelial neoplasia (CIN), and 68 cervical cancer. The expression of TFPI-2, Ki-67 and vascular endothelial growth factor (VEGF) were investigated by immunohistochemistry staining. The apoptolic index(AI) was determined with an in situ end-labeling assay(TUNEL). And the marker of CD34 staining was used as an indicator of microvessel density (MVD).</p> <p>Results</p> <p>TFPI-2 expression has a decreasing trend with the progression of cervical cancer and was significantly correlated with FIGO stage, lymph node metastasis and HPV infection. In addition, there were significant positive correlations between the grading of TFPI-2 expression and AI(P = 0.004). In contrast, the expression of TFPI-2 and VEGF or MVD was negatively correlated (both p < 0.001). However, we did not establish any significant correlation between Ki-67 and TFPI-2 expression in cervical cancer.</p> <p>Conclusions</p> <p>The results suggested that the expression of TFPI-2 had a decreasing trend with tumor progression of cervical cancer. There was a close association between the expression of TFPI-2 and tumor cell apoptosis and angiogenesis in patients with cervical cancer. TFPI-2 may play an inhibitive role during the development of cervical cancer.</p

Computer model of the interaction of human TFPI-2 Kunitz-type serine protease inhibitor with human plasmin

International audienceComputer model of the interaction of human TFPI-2 Kunitz-type serine protease inhibitor with human plasmin Dear Sir, Tissue factor pathway inhibitor-2 (TFPI-2) is a 32-kDa serine proteinase inhibitor that strongly inhibits trypsin, plasmin, factor XIa and plasma kallikrein [1], and is mainly associated with extracellular matrices [2]. By inhibiting plasmin, TFPI-2 thus efficiently decreases activation of several metalloproteinases [2] and reduces tumor invasion and metastasis [3,4]. In addition, TFPI-2 can also regulate plasmin in atherosclerotic plaques by modulating extracel-lular proteolytic mechanisms [5]. TFPI-2, which is a member of the Kunitz-type serine proteinase inhibitor family, consists of three tandemly repeated Kunitz-type domains, the first of which (K1) exhibits similarities in amino acid sequence to TFPI-1 and bovine pancreatic trypsin inhibitor (BPTI) (45% and 40%, respectively). The inhibition of serine proteinases by Kunitz-type inhibitors involves their binding to K1 and P1 residue that interacts with the proteinase pocket previously identified to be an arginine or a lysine residue [6-9]. Using site-directed mutagenesis experiments, Kamei et al. [10] reported that a TFPI-2 mutant with glutamine substituted for arginine lost its inhibitory activity towards trypsin and plasmin. Several structures showing the interaction of one serine proteinase with a Kunitz-type inhibitor, i.e. thrombin with BPTI [6], factor Xa/VIIa with TFPI-1 [7,8] and alpha-chymotrypsin with BPTI [9], have been determined crystallographically. However , TFPI-2 structure and plasmin complexed with a Kunitz-type inhibitor have not to date been described. This report is the first to address the structural basis for molecular recognition of plasmin by the K1 domain of TFPI-2 (TFPI-2/K1). The 3D models of TFPI-2/K1 was built using the automatic comparative modeling server swissPDB (http:// www.expasy.org). The structure of TFPI-1/K2 (44% identity with TFPI-2/K1 in 55 residues) was used as template to determine TFPI-2/K1. The resulting model was further energy-minimized using the steepest descent algorithm (SYBYL 6.9, Tripos, http://www.tripos.com), the atoms of the backbone being constrained during this step, and un-constrained using the conjugate gradient algorithm until the maximum derivative was < 0.1 kcal/mol/A ˚. After checking the stereochemical quality (WHATIF, http://www.cmbi. kun.nl/), all parameters were good for ensemble structure. The TFPI-2/K1 model and plasmin (coordinates extracted from the PDB file 1 bml) were superimposed on the Ca atoms of the TFPI-1/trypsin complex (coordinates extracted from the PDB file 1tfx). Finally, the few atoms that overlapped at the complex interface were relieved by energy minimization. The electrostatic potentials of TFPI-2/K1 and plasmin were calculated with swissPDB viewer 3.7b software and using formal charges at pH 7.4 (arginine, lysine, N-terminus, + 1; glutamate, aspartate and the C-terminus, À 1; and histidine, neutral), ionic strength in the aqueous environment of 0.15 M and dielectric constant of 80. The three-dimensional structure of TFPI-2/K1 exhibited the typical Kunitz-type proteinase inhibitor folding, i.e. a double stranded anti-parallel h-sheet from Arg20 to Phe33, and an a-helix from Trp48 to Ala54. The core of the domain, comprising the secondary structure elements and the three disulfide bonds, was highly conserved. TFPI-2/K1 exhibited the same overall fold as BPTI [6] and TFPI-1/K2 [7], with rms deviations (root-mean-square) for all 55 Ca atom positions of 0.70 and 1.09 A ˚ , respectively. These values are in agreement with the 40% identity with BPTI and 45% identity with TFPI-1/K2. The binding loop of TFPI-2/K1 (Leu19-Tyr33) was in direct contact with the active site of plasmin involving the characteristic main-main conforma-tional and intermolecular hydrogen bond interactions of canonical binding proteinase inhibitors [7-9]. Indeed, as shown in Table 1, TFPI-2/K1 residues positioned close to the active site of plasmin (PV2, PV1, P1 and P3 residues) were involved in hydrogen bonds. As compared with the TFPI-1/trypsin complex [7], two additional hydrogen bonds were found at the interface between TFPI-2/K1 and plasmin, probably improving the complementarity between the two molecules. The side-chain of TFPI-2 P1 residues (Arg15) extended into the plasmin-specific pocket, with its guanidi-nium group forming a salt bridge with the carboxylate group 0049-3848/$-see front matter

Improved PCR Method for Amplification of GC-Rich DNA Sequences

Most housekeeping genes, tumor-suppressor genes, and approx 40 % of tissue-specific genes contain CpG islands in their promoter region that are limited regions of high-density CpG residues

Human Small Breast Epithelial Mucin: The Promise of a New Breast Tumor Biomarker

International audienceBreast cancer remains one of the most frequently diagnosed cancers today. In developed countries, one in eight women is expected to present with breast cancer within her lifetime and an estimated 1,000,000 cases are detected each year worldwide. 1 (Canadian Cancer Statistics, http://www.cancer.ca/vgn/images/ portal/cit_86751114/14/33/1959864 11niw_stats2004_en.pdf). For women with recurrent disease, the median time of survival is about 2 years. Despite optimal surgery, adjuvant irradiation, hormonal treatment, and chemotherapy, approximately 30% of patients with localized breast cancer finally develop distant metastases. Early detection, which enables intervention at a localized and potentially curable stage, remains a central goal in breast cancer treatment. Indeed, the 5-year survival rate for women with breast cancer has been shown to increase dramatically when the disease is diagnosed at an early stage: from less than 25% in women with disseminated cancer to about 75% in patients with regional disease and over 95% in women with a localized tumor (Breast Cancer Facts and Figures, 2001-2002, http://www.cancer.org/downloads/STT/BrCaFF 2001.pdf). Unfortunately, only 60% of all breast cancers are diagnosed at a local stage. Any improvement in early detection through identification of tumor biomarkers would have a significant impact on reducing overall breast cancer mortality