Enzastaurin blocks TPA-induced endothelial-cell signaling pathways and MM-cell growth triggered by endothelial-cell adhesion. (A) Dose-related effects of enzastaurin on endothelial-cell proliferation. HUVECs were cultured with indicated concentrations of enzastaurin. Proliferation was measured using [³H]-thymidine uptake during the last 8 hours of 24-hour and 48-hour cultures. (B) Enzastaurin inhibits endothelial tubule formation. Endothelial-cell suspensions were premixed with different concentrations of enzastaurin in EGM-2 and added on top of the ECMatrix. Tubule formation was assessed using an inverted light microscope at ×4 and ×10 magnification. Photographs are representative of each group and 3 independent experiments. (C) Enzastaurin specifically inhibits homologous Thr-514 and Ser-660 residue phosphorylation of PKC isoforms and downstream signaling molecules in endothelial cells. HUVECs were treated with enzastaurin (3 hours, 5 μM) or bisindolylmaleimide (BIM I) or left untreated. After stimulation with TPA (200 nM, 20 minutes), immunoblot analysis was performed with indicated antibodies. (D-E) Enzastaurin inhibits proliferation of MM cells adherent to endothelial cells. MM.1S cells (MM; D or CD138⁺ MM cells (pat. MM) isolated from a multidrug-resistant patient with clinically progressive disease (E) were cultured with or without HUVECs. Enzastaurin was added in the indicated concentrations, and proliferation was measured using [³H]-thymidine uptake. Data shown are the mean ±SD of experiments performed in quadruplicate.