Components of cell-matrix linkage as potential new markers for prostate cancer

Prostate cancer is one of the most common tumor diseases worldwide. Often being non-aggressive, prostate tumors in these cases do not need immediate treatment. However, about 20% of diagnosed prostate cancers tend to metastasize and require treatment. Existing diagnostic methods may fail to accurately recognize the transition of a dormant, non-aggressive tumor into highly malignant prostate cancer. Therefore, new diagnostic tools are needed to improve diagnosis and therapy of prostate carcinoma. This review evaluates existing methods to diagnose prostate carcinoma, such as the biochemical marker prostate-specific antigen (PSA), but also discusses the possibility to use the altered expression of integrins and laminin-332 in prostate carcinomas as diagnostic tools and therapeutic targets of prostate cancer

Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation.

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways

Activation of sGC inhibits platelet aggregation induced by echicetin beads.

<p>Washed human platelets were preincubated with NO donor (SNP; 10 μM, 2 min) and/or sGC inhibitor (ODQ; 50 μM, 5 min) before EP0.1 or vWF/R stimulation. Representative aggregation curves of platelets stimulated with EP0.1 (A) or vWF/R (B) are shown. C, D. The graphs show corresponding average values of aggregation from A and B. Platelets stimulated with EP0.1 (E) or vWF/R (F) were lysed and the phosphorylation VASP was analyzed by Western blot. Total VASP served as loading control. Representative blots of three independent experiments are shown.⁺p<0.05 versus EP0.1.</p

Platelet adhesion and spreading on echicetin-coated surface.

<p>Cover slips were coated with various amounts of echicetin or BSA (control). Washed human platelets (1×10⁷/ml) were incubated on the coated cover slips for 15 min. Following incubation, cover slips were washed to remove unbound platelets and the adherent platelets were fixed with 1% formaldehyde. The fixed platelets were permeabilized with Triton X-100 and stained with phalloidin-Oregon Green (actin). The stained platelets were analyzed under the microscope (Zeiss, Axiovert 200) at 1000× magnification. The images were captured using a camera (Diagnostic Instruments, SpotPursuit 23) and VisiView software. A. Representative images of the bright-field (upper panel) and actin staining (lower panel) of the cover slips were shown for each sample. B. The number of adhered platelets was counted in 10 independent cover slips for each sample. C. Surface area of adhered and spread platelets was measured using VisiView software in 10 independent cover slips for each sample. Results are mean ± SEM from 3 independent experiments.</p

Src-kinases and SYK are downstream effectors of echicetin beads induced platelet activation.

<p>Washed human platelets were preincubated with Src-kinases inhibitor (PP2, 10 μM), an inactive analog (PP3, 10 μM) or Syk inhibitor (piceatannol, 10 μM) before stimulating with EP0.1 or vWF/R under stirring conditions. Representative aggregation curves of platelets stimulated with EP0.1 (A) or vWF/R (B) are shown. C, D. The graphs shows corresponding average values of aggregation from A and B. Platelets stimulated with EP0.1 (E) or vWF/R (F) were lysed at the indicated time and the phosphorylation of PKB, ERK and p38 was analyzed by Western blot. Total p38 served as loading control. Shown are representative blots of three independent experiments.⁺p<0.05 versus EP0.1.</p

ADP and TxA₂ play significant roles in echicetin bead induced platelet aggregation.

<p>Washed human platelets were preincubated with P2Y₁₂ antagonist (AR-C69931; 0.1 μM, 5 min), P2Y₁ antagonist (MRS2179; 1 μM, 5 min) or TxA₂ receptor antagonist (SQ-29548; 1 μM, 5 min) before EP0.1 or vWF/R stimulation. Representative aggregation curves of platelets stimulated with EP0.1 (A) or vWF/R (B) are shown. C, D. The graphs shows corresponding average values of aggregation from A and B. Platelets stimulated with EP0.1 (<b>E</b>) or vWF/R (<b>F</b>) were lysed on indicated time and the phosphorylation of PKB, ERK and p38 was analyzed by Western blot. Representative blots of three independent experiments are shown.⁺p<0.05 versus EP0.1.</p

The distance between echicetin molecules is critical for inducing platelet activation and aggregation.

<p>Washed human platelets were stimulated with EP under stirring conditions (1000 rpm). BSA coated polystyrene beads were used as negative control. Platelet aggregation stimulated by EP0.3 was considered as 100%. A. Representative aggregation curves of platelets stimulated with EP0.1, BSA-coated polystyrene beads, or echicetin-monomer are shown. B. Representative aggregation curves of platelets stimulated with EP0.3, EP0.2, EP0.1, EP0.05 or 5 times concentration of EP0.05 are shown. C. The graph shows the corresponding average values of aggregation from B. D. P-selectin surface expression on platelets was measured as Mean Fluorescence Intensity (MFI) using RPE-conjugated P-selectin antibody in flow cytometry. Thrombin (0.05 U/ml) and BSA-coated beads (BSA) were used as positive and negative controls, respectively. Results are mean±SD from 3 independent experiments. * p<0.05 versus EP0.3, and⁺p<0.05 versus EP0.1.</p

Echicetin beads but not vWF/R induces protein tyrosine phosphorylation.

<p>Washed human platelets were stimulated with EP0.1 (A) or vWF/R (B) after preincubation with aggrastat (1.25 μg/ml, 1 min) or echicetin monomer (25 μg/ml, 3 min). Protein tyrosine phosphorylation was analyzed by Western blot. Shown are representative blots of three independent experiments.</p

Echicetin coated polystyrene beads induce αIIbβ3–dependent aggregation in washed platelets.

<p>Representative aggregation curves of platelet rich plasma stimulated with ristocetin (A), and washed human platelets stimulated with EP0.1 in the absence of vWF or fibrinogen (B) or vWF-15 μg/ml and Ristocetin-1 mg/ml (C). Whenever indicated, platelets were preincubated with echicetin-monomer (25 μg/ml, 3 min) or aggrastat (1.25 μg/ml, 1 min). Shown are results of three independent experiments.</p