LatB induces caspase-dependent endothelial apoptosis. (A,G) Isolates of HBMECs from 4 (A) or 2 (G) different donors, or tumor cells (4 × 105/well) were allowed to spread on VN-coated/HD-BSA–blocked 6-well plates and then incubated for 24 hours with 0 to 1μM (A) or 0.2μM (G) LatB in 0.4% BSA/RPMI 1640. Whole-cell lysates were resolved on 10% SDS-PAGE and analyzed by Western blotting (PARP cleavage and c-Abl). (A) The blot of PARP cleavage for HBMECs-3 and HBMECs-4 is shown at longer exposure than for HBMECs-1 and HBMECs-2 resulting from lower PARP expression in HBMECs-3 and HBMECs-4. GAPDH or ERK 1/2 and β-tubulin served as loading controls. (B) HBMECs (2 × 105 cells/well) seeded on VN-coated/HD-BSA–blocked 6-well plates in 0.4% BSA/RPMI 1640 were treated with vehicle or LatB (0.5μM) for 48 hours. Apoptosis was assessed by flow cytometry using the Apo-Direct kit, measuring FITC-dUTP and PI content. Percentage of apoptotic cells (FITC-dUTP+ cells in top quadrants) is indicated for each condition. Shown is a representative panel from 1 experiment of 12 with similar results. (C) HBMECs (2 × 105 cells/well) were allowed to spread on VN-coated/HD-BSA–blocked 6-well plates and then incubated for 24 hours with 0.5μM LatB or 5 μg/mL RGDfV in 0.4% BSA/RPMI 1640. Whole-cell lysates were resolved on 12.5% SDS-PAGE and analyzed by Western blotting for caspase-8 cleavage. GAPDH served as loading control. (D) HBMECs treated as in panel C were analyzed by flow cytometry for effect on mitochondrial polarization. Loss of mitochondrial membrane potential (ΔΨm) was measured by flow cytometry using the JC-1 mitochondrial probe. The transition of red fluorescence to green indicates mitochondrial membrane depolarization by the drug(s). Indicated are the percentages of mitochondrial membrane-depolarized cells. (E) HBMECs (5 × 104 cells/mL) were cultured in 3D collagen as described in supplemental Data and treated with LatB (0.5μM) for 18 hours. Cells were stained with Hoechst and annexin V–FITC and photographed by light microscopy or fluorescence. Shown are representative fields from each condition (original magnification ×400). (F) HBMECs spread on VN in serum-free RPMI 1640 were preincubated with the caspase inhibitors zVAD-FMK or zBOC-FMK (25μM or 50μM) or vehicle control for 2 hours, and then LatB (0.05μM) or RGDfV (5 μg/mL) was added for another 24 hours. Whole-cell lysates were resolved on 10% SDS-PAGE and analyzed by Western blotting (PARP cleavage, ERK, β-tubulin). (H) UW228-2 medulloblastoma cells (2 × 105 cells/well) seeded on 6-well plates were treated for 48 hours with LatB (0.5μM) as in panel A, and apoptosis was analyzed by flow cytometry as in panel B. (I) HBMECs (4 × 105/well) were cultured in 6-well plates in RPMI 1640 without additions, or in the presence of 10% FBS or 0.4% FAF-BSA. After cells were spread, 0 to 2μM LatB was added for 24 hours as indicated, and lysates were processed as indicated in panel A.
