P2Y12 receptor antagonists and AR receptor agonists regulates Protein Disulfide Isomerase secretion from platelets and endothelial cells.

Secretion of PDI from platelets and endothelial cells is an important step of all thrombotic events. In the absence of extracellular PDI thrombus formation and fibrin generation may be impaired. Thrombin-mediated PDI secretion is regulated by the stimulation of P2Y12 receptors. This paper provides evidences that P2Y12 antagonists or AR agonists may modulate release of PDI molecules from platelets and with less efficiency from endothelial cells. Moreover P2Y12 antagonization or AR agonization modulates platelet-endothelial interaction. We prove that combinations of P2Y12 antagonists and AR agonists inhibit platelet-dependent adhesion of cancer cells to endothelium and attenuate cancer cell invasiveness, but longer exposition to AR agonists may stimulate migration of invasive breast cancer cells through endothelium thus leading to increased metastasis.

Functional inhibition of F11 receptor (F11R/junctional adhesion molecule-A/JAM-A) activity by a F11R-derived peptide in breast cancer and its microenvironment.

PURPOSE: To examine the involvement of the F11R/JAM-A protein in breast cancer metastasis, we utilized the F11R/JAM-A antagonistic peptide 4D (P4D) in experiments of transendothelial migration (TEM) of breast cancer cells. METHODS: Experiments were conducted in the mouse 4T1 breast cancer model utilizing the human mammary epithelial cell and endothelial cell lines. The levels of soluble F11R/JAM-A (sJAM-A) in the murine plasmas were measured by ELISA. Levels of F11R/JAM-A mRNA and protein in cell lines were assessed by qRT-PCR and Western blot, respectively. Cell surface expression of F11R/JAM-A was demonstrated by flow cytometry. Functional tests included the TEM of breast cancer cells and adhesion of breast cancer cells to the endothelium. The endothelial permeability was studied by fluorescent tracer assay and by the Real-Time Cell Analysis (RTCA). RESULTS: The tumor inducers Tß4 and TGF-ß1 reduced the levels of sJAM-A in murine plasma, and reduced the F11R/JAM-A protein levels in the human microvascular endothelial cell line HMEC-1. The adhesion and TEM measured between breast cancer cells and inflamed or Tß4-treated endothelium were inhibited by P4D. The presence of P4D did not destabilize the pre-existing tight junctions in the endothelial monolayer. The barrier-protecting effect of P4D was stronger than that of forskolin, when a booster dose of P4D was applied to the inflamed endothelium. CONCLUSIONS: F11R/JAM-A protein can be considered as a novel target in the treatment of breast cancer metastasis. In vivo and clinical studies are needed to further investigate the effectiveness of F11R/JAM-A-derived peptide as a possible anti-metastatic drug.

Modifications of disulfide bonds in breast cancer cell migration and invasiveness.

Cancer metastasis involves the adhesion of cancer cells to the endothelium. This process can be mediated by integrins which are surface receptors responsible for interactions with ECM proteins. Integrins ß1 and αVß3 represent factors are involved in cancer progression and metastasis. Activation of integrins can be promoted by thiol-disulfide exchanges initiated by Protein Disulfide Isomerase (PDI). The purpose of this study was to prove the involvement of disulfide rearrangements in the molecules of integrins in the course of cancer cell adhesion and migration through the endothelium. We present the evidence which proves that highly metastatic MDA-MB-231 breast cancer cell lines adhere to endothelial cells are more effective than non-invasive MCF-10A and MCF-7 cell lines and that the attachment of MDA-MB-231 to the endothelium can be attenuated either by the agents blocking free thiol groups (DTNB, cystamine or PCMBS) or by PDI inhibitors (Q3Rut, 16F16 or PACMA-31). Furthermore, we prove that the transendothelial migration of MDA-MB-231 cells and contraction of collagen can be blocked by thiol blockers or PDI inhibitors and that these factors affect exposition of free thiols on integrin molecules.

Contribution of activated beta3 integrin in the PDI release from endothelial cells.

Protein disulfide isomerase (PDI) is an abundant reticulum endoplasmic protein but also acts as an important functional regulator of some extracellular surface proteins. Recent studies suggest that PDI plays a role in integrin activation and thrombus formation. The aim of this study was to examine whether activation of integrin is the first stage leading to release of PDI from the subcellular compartments of endothelial cells to extracellular space. Our results show that endothelial cells which adhere to fibronectin or fibrinogen release significantly more PDI than those which adhere to poly-L-lysine. Cells treated with RGD peptide, Src and FAK kinase inhibitors and anti alphaVbeta3 antibody display lower PDI secretion. The destruction of the actin cytoskeleton of endothelial cells by cytochalasin D inhibits PDI release. When the endothelial cells adhere to fibrinogen or fibronectin, PDI and alphaVbeta3 gain free thiol groups. Our data suggest that upon activation of integrins, PDI is released from endothelial cells and forms a disulfide bond complex with alphaVbeta3 integrin.

The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11.

Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11ß1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11.

[Role of protein disulfide isomerase in activation of integrins]. / Rola izomerazy disiarczkowej w aktywacji integryn.

Integrins belong to a large family of transmembrane cell adhesion receptors that communicate biochemical and mechanical signals in a bidirectional manner across the plasma membrane. Integrins and their ligands play a crucial role in a number of physiological and pathological processes, including cell migration, cell differentiation, hemostasis, adhesion, angiogenesis, cancer, cell invasiveness and wound healing. Intracellular signals switch integrins into a ligand-competent state as a result of conformational changes within the integrin molecule. Binding of extracellular ligands induces structural changes that can transmit signals to the cell interior. Transition of integrins from an inactive to a ligand binding state involves rearrangement of the disulfide bonding pattern. The rearrangement of disulfide bonds is modulated by protein disulfide isomerase (PDI). PDI has been found on the surface of several types of cells, including endothelial cells, hepatocytes, cancer cells, pancreatic cells and B cells. PDI was identified on the platelet surface, where it plays an important role in platelet reactions such as adhesion, aggregation and secretion. PDI was found to directly interact with integrins. Disulfide-thiol exchange mediated by PDI appears to be involved in the conformational changes in integrin activation. In this report we describe the structure of integrin and the role of disulfide bond rearrangement in its activation.

BLM and RAD51 genes polymorphism and susceptibility to breast cancer.

DNA repair by homologous recombination is one of the main processes of DNA double strand breaks repair. In the present work we performed a case-control study (304 cases and 319 controls) to check an association between the genotypes of the c.-61 G>T and the g.38922 C>G polymorphisms of the RAD51 gene and the g.96267 A>C and the g.85394 A>G polymorphisms of the BLM gene and breast cancer occurrence. Genotypes were determined in DNA from peripheral blood by PCR-RLFP and by PCR-CTPP. We observed an association between breast cancer occurrence and the T/G genotype (OR 4.41) of the c.-61 G>T-RAD51 polymorphism, the A/A genotype (OR 1.69) of the g.85394 A>G-BLM polymorphism and the A/A genotype (OR 2.49) of the g.96267 A>C-BLM polymorphism. Moreover, we demonstrated a correlation between intra- and intergenes genotypes combinations and breast cancer occurrence. We found a correlation between progesterone receptor expression and the T/G genotype (OR 0.57) of the c.-61 G>T- RAD51 polymorphism. We also found a correlation between the T/G genotype (OR 1.86) and the T/T genotype (OR 0.56) of the c.-61 G>T- RAD51 polymorphism and the lymph node metastasis. We showed an association between the A/A genotype (OR 2.45) and the A/C genotype (OR 0.41) of the g.96267 A>C-BLM polymorphism and G3 grade of tumor. Our results suggest that the variability of the RAD51 and BLM genes may play a role in breast cancer occurrence. This role may be underlined by a common interaction between these genes.