Figure 5

TRAIL inhibited P-gp efflux function in MDR cells by P-gp cleavage via caspase-3 activation. (A) The cell lysates obtained from CEM/VLB₁₀₀ cells treated with or without TRAIL (10 ng/ml) for 3 ~ 9 h (left) and the cell lysates of the MDR cells treated with TRAIL (5 ng/ml) for 6 h or pretreated with 50 μM Z-DEVD-FMK, a specific caspase-3 inhibitor, for 3 h and then with TRAIL for 6 h (right) were subjected to western blot analysis to monitor levels of P-gp, DNA-PKcs, caspase-3 and PARP and their its cleavage fragment (CF). (B) flow cytometric assay of P-gp efflux activity in TRAIL-treated MDR cells was based on extrusion of the fluorescent P-gp substrate, rhodamine123 (Rho123). The efflux activity of P-gp is highly temperature sensitive because functions optimally 37°C but is inactive at 4°C. Cell suspension from CEM and CEM/VLB₁₀₀ cells treated with or without 10 ng/ml TRAIL for 6 h was incubated with Rho 123 and further incubated at 37°C for 3 h (TREATED 37°C as TRAIL-treated cells or CTRL37°C as TRAIL-untreated control) to allow P-gp-mediated drug efflux or on ice as control (CTRL 4°C).