hirudin and Prostatic-Neoplasms

The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G(0) and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G. Cell cycle kinases and inhibitors in synchronized tumor cells revealed high levels of p27(Kip1) and low levels of Skp2 and cyclins D1 and A. Addition of thrombin, TFLLRN, or serum down-regulated p27(Kip1) with concomitant induction of Skp2, Cyclin D1, and Cyclin A with similar kinetics. LNCaP p27(Kip1)-transfected cells or Skp2 knockdown cells were refractory to thrombin-induced cell cycle activation. MicroRNA 222, an inhibitor of p27(Kip1), was robustly up-regulated by thrombin. The in vitro observations were tested in vivo with transgenic TRAMP mice. Repetitive thrombin injection enhanced prostate tumor volume 6- to 8-fold (P < 0.04). Repetitive hirudin, a specific potent antithrombin, decreased tumor volume 13- to 24-fold (P < 0.04). Thus, thrombin stimulates tumor cell growth in vivo by down-regulation of p27(Kip1).

Topics: Animals; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Female; Glioblastoma; Hirudins; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; MicroRNAs; Prostatic Neoplasms; S-Phase Kinase-Associated Proteins; Thrombin; Up-Regulation

PC-3 cells, a metastatic human prostate adenocarcinoma line, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma (PRP). PC-3 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) and limited by increasing concentrations of apyrase. This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2. PC-3 cell suspension caused marked, dose-dependent decreases in plasma recalcification times using normal, Factor VIII-deficient and Factor IX-deficient, but not Factor VII-deficient, human plasma. This effect was potentiated in cell lysates, but was inhibited in intact cells preincubated with sphingosine. Overall, these data suggest that PC-3 TCIPA arises from PC-3 tissue factor activity expression. Trigramin and rhodostomin, RGD-containing snake venom peptides which antagonise the binding of fibrinogen to platelet membrane glycoprotein IIb-IIIa, prevented PC-3 TCIPA. Similarly, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoproteins IIb-IIIa and Ib prevented PC-3 TCIPA, which was unaffected by control peptide GRGDS. On a molar basis, trigramin (IC50, 0.11 microM) and rhodostomin (IC50, 0.03 microM) were approximately 5000 and 18000 times, respectively, more potent than GRGDS (IC50, 0.56 mM).

Topics: Adenocarcinoma; Antibodies, Monoclonal; Apyrase; Cysteine Proteinase Inhibitors; Fibrinogen; Glycoproteins; Hirudins; Humans; Intercellular Signaling Peptides and Proteins; Male; Peptides; Phospholipases A; Phospholipases A2; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostatic Neoplasms; Thrombin; Tumor Cells, Cultured