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: 5 0 Biotin-CGTCATACCGCATGTGCA CATGGAC-Biotin 3 0 ) 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.

Protein Z/protein Z-dependent protease inhibitor system in loco in human gastric cancer

“…Of note, our study demonstrated mRNAs encoding PZ and ZPI synthesis (apart from respective proteins expression) in gastric cancer cells, pointing to the constitutive expression of PZ and ZPI in gastric cancer cells. Similar findings were observed in human breast and colon cancer tissues [22, 23, 27]. Pancreatic endocrine cancers as well as enterochromaffin and neuroendocrine carcinoma cells were characterized by overexpression of ZPI mRNA in both primary and metastatic tumors [35, 36].…”

“…However, in lymphoblasts obtained from children suffering from acute lymphoblastic leukemia, no staining of PZ mRNA was observed, although some levels of ZPI mRNA were detected [17]. Noteworthy information was reported concerning the role of PZ and ZPI in obstetrics and inflammation, which suggests the potential function of the above inhibitors not only in the regulation of blood coagulation but also in local biological processes [17, 22, 23, 37]. Of interest is also the presence of the examined inhibitors in TAMs, which is in accordance with our previous findings in breast and colon cancer as well as in NSCLC tissues [22, 23, 27, 28].…”

“…Immunoreactive score values ranging between 1 and 4 were interpreted as weak, 5 and 8 medium, and 9 and12 strong protein expression, respectively [20, 21]. A monospecific polyclonal antibody against homogeneous plasma-derived human PZ was prepared in rabbits, and then purified from immune sera by protein A-Sepharose chromatography [22]. Specific mouse monoclonal anti-human ZPI IgM (4249.2) was a generous gift from Dr. George J. Broze Jr (Division of Hematology, Barnes-Jewish Hospital, St. Louis, MO, USA) [23].…”

“…The in situ hybridization (ISH) protocol employed biotin-labeled 25-nucleotide single-stranded DNA probes: (probe sequence: 5′ biotin-CGTCATACCGCATGTGCACATGGAC-biotin 3′) specific for PZ mRNA [22], (probe sequence: 5′ biotin-GCCATCGTGCCTCATGGAGATCTTT -biotin 3′) specific for ZPI mRNA [23]. The probes were synthesized by Sigma-Aldrich, Poznan, Poland, and the ISH protocol of R&D Systems (R&D Systems, Minneapolis, MN, USA) was followed.…”

Novel risk models for early detection and screening of ovarian cancer

“…Haemostatic system inhibitors are known to play a role in determining the malignancy of cancers [31]. Protein Z has previously been found down regulated in patients diagnosed with acute leukaemia compared with controls [32], but has also been found to be upregulated in tissues of gastric [33], breast [34] and colon [35] cancers. It is possible the down regulation of serum Protein Z reflects sequestration to the tumour site where it may prevent coagulation.…”