Protein Z is present in human breast cancer tissue

Abstract

Thromboembolic complications are common findings in breast cancer patients [1, 2]. Hemostatic system components play a role in tumor progression, independent of their role in hemostasis [1, 2]. Since the report of multifactorial activation of factor X in cancer patients [1], inhibitors which control the activity of this factor have attracted attention. Protein Z (PZ), a vitamin K-dependent plasma nonpeptidase glycoprotein [3, 4], is involved in one mechanism responsible for the direct inhibition of factor Xa. PZ circulates in plasma complexed with PZ-dependent protease inhibitor (ZPI) and enhances the rate of ZPImediated factor Xa inhibition [5, 6]. The presence of ZPI in breast cancer tissue was previously reported [7], while information on PZ in tumor malignant tissue is lacking. The purpose of the present study was to evaluate the localization of PZ (both protein and mRNA) at the primary site of human breast cancer. Tumor fragments were obtained during the surgical treatment of 13 previously untreated breast cancer patients (clinical stage T1-2N0M0). Studies were performed on 4 cases of G2 and 9 cases of G3 invasive ductal carcinomas. Immunohistochemical studies (IHC) were performed (Vectastain Kits, Vector Laboratories, Burlingame, CA, USA) [7] employing a polyclonal antibody against homogeneous, plasma-derived human PZ (prepared in rabbits, and purified from immune sera by protein A-Sepharose chromatography). The antibody did not cross-react with all other purified human plasma vitamin K-dependent proteins including prothrombin, protein C, protein S, factor VII, factor X, and factor IX by ELISA. Controls consisted of omission of the primary antibody from the procedure. The control fragments of normal breast tissues obtained from neoplasm-free surgical margins were processed simultaneously as well. Antigen staining was detected by the dark brown reaction product. A semiquantitative analysis of PZ IHC expression in cancer cells (based on both the percentage of cancer cells with PZ positive staining and the intensity of staining) was performed [7]. Immunoreactive score (IRS) values of 1–4 were interpreted as weak, 5–8 as medium, and 9–12 as strong PZ expression. In situ hybridization (ISH) method employed a biotin-labeled 25-nucleotide single-stranded DNA probe (probe sequence: 50 Biotin-CGTCATACCGCATGTGCA CATGGAC-Biotin 30) specific for PZ mRNA. The probe was synthesized by Sigma-Aldrich, Poznan, Poland. The ISH protocol of R&D Systems (R&D Systems, Minneapolis, MN, USA) was followed. Hybrids were detected with rabbit anti-biotin monoclonal antibodies according to the IHC ABC technique associated with ImmunoMax amplification technique described in detail elsewhere [7]. Negative controls included hybridization without addition of the molecular probe and incubation of slides in a RNase A solution (R&D Systems, Minneapolis, MN, USA) before hybridization. Reaction results appeared as dark brown staining. E. Sierko M. Z. Wojtukiewicz (&) Department of Oncology, Medical University, 12 Ogrodowa St, Bialystok, Poland e-mail: mzwojtukiewicz@gmail.com