(San Diego, CA). agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective. 0.05 ** 0.01 when compared with the respective agonist control. The percentage inhibition of the light transmission when compared to the control is given in parentheses. 2CP inhibits PDPN-induced TCIPA C6/Lung cells were used as the cancer cell model to address whether 2CP is able to inhibit PDPN-induced TCIPA. This was based on the findings that C6/Lung cells expressed high levels Dynamin inhibitory peptide of PDPN and induced platelet aggregation (Figure ?(Figure2A)2A) and the formation of tumor cell-platelet aggregates (Figure ?(Figure2B).2B). In contrast, the parental C6/LG cells did not express PDPN and were not able to induce platelet aggregation and form tumor cell-platelet aggregates (Figure ?(Figure2A2A and ?and2B).2B). Expression of PDPN short hairpin RNA in C6/Lung cells abrogated C6/Lung cells-induced platelet aggregation (Figure ?(Figure2C),2C), implying that PDPN is the key molecule mediating TCIPA. Open in a separate Dynamin inhibitory peptide window Figure 2 2CP inhibits PDPN-mediated TCIPAA. The expression of PDPN proteins in the indicated cell lines was determined by Western blotting using the anti-PDPN antibody (inserted panel). The expression of -actin was used for the control of equal protein loading. The cells (1.5 106) from the indicated cell lines were added to the human washed platelet suspension (1 109/ml) to stimulate Dynamin inhibitory peptide platelet aggregation. Tumor cell-induced platelet aggregation was measured and recorded by using an aggregometer. Representative traces of platelet aggregation are shown. B. Calcein-AM green-labeled platelets (1 109/ml, green) were incubated with the calcein-AM orange/red-labeled cells (1.5 106, red) in an aggregometer. The reaction mixtures were then placed on a glass slide for fluorescence microscopy analysis. Representative fluorescent images are shown to demonstrate the interaction between tumor cells and platelets. Scale bar = 20 m. C. The expression of PDPN protein in the indicated cell lines was determined by Western blotting using the anti-PDPN antibody (left panel). The expression of -actin was used for the control of equal protein loading. The platelet aggregation-inducing activities of these sublines were evaluated by TCIPA assays. Representative traces of platelet aggregation are shown (right panel). Arrows indicate the point of cells being added. D. The cells from the indicated cell lines were added into the washed platelets with or without pre-incubation with 2CP (20 M). Representative traces of platelet aggregation (left panel) and the turbidity of the reactions (center panel) are shown. The time to reach 50% of the maximal aggregation was defined as the aggregation Rabbit polyclonal to PAX2 time that is shown as Box with whiskers (Min to Max) plot (right panel). The value is set to 1000 sec when no platelet aggregation was observed. ** 0.01 when compared with the vehicle treatment. ECF. Platelets and C6/Lung cells were treated with the indicated concentrations of 2CP and the LDH and caspase 3/7 activities were measured. The data represent the mean S.E of three to six independent experiments. TCIPA assays were performed by incubation of C6/Lung cells with human platelets in the presence or absence of 2CP. 2CP inhibited C6/Lung cells-induced platelet aggregation (Figure ?(Figure2D,2D, left panel) and caused the turbidity of the reaction mixture (Figure ?(Figure2D,2D, center panel). The time to reach 50% aggregation for the control and 2CP-treated group was 496.5 65.4 sec (= 41) and 910.6 123.1 sec (= 39), respectively (Figure ?(Figure2D,2D, right panel, 0.01). The lactose dehydrogenase (LDH) release and caspase 3/7 activity assays revealed that 2CP did not cause cytotoxic or apoptotic effects in platelets and tumor cells. This implies that cell stress or cell death does not account for the inhibitory activity of 2CP on TCIPA induced by C6/Lung cells (Figure ?(Figure2E2E and Figure ?Figure2F).2F). 2CP.