Figure 3

TRAIL and MG132 (or Bortezomib) combination induces a robust apoptosis in MPM cells. (A) The combinatorial treatment induces a robust apoptosis in NCI-H28 cells. A-1: Cell viability assay using WST-1 reagent indicates that 10 ng/ml TRAIL and 1 μM MG132 combination induces a dramatic cell death at 72 h after treatment. Values are expressed as the means ± SD of two experiments. *p < .05 versus control; A-2: Flowcytometry assay demonstrates that the two-agent combination induces a significant increase in DNA damage as indicated by the sub G0/G1 peak; A-3: Western blotting reveals that the two-agent combination induces a significantly increased protein cleavage for caspases 7, 9 and 10, Bid and PARP in NCI-H28 cells at 24 h after treatment. (B & C) Western blotting demonstrates that the two-agent combination significantly enhanced apoptosis in both NCI-H2452 and NCI-H2052 cells, as indicated by the enhanced protein cleavage for caspases 3, 9 and 10, Bid and PARP in NCI-H2452 cells at 36 h after treatment (B) and caspases 7, 9 and 10 and PARP in NCI-H2052 cells at 40 h after treatment (C). (D) The two-agent-induced-apoptosis is caspase-dependent. Specific inhibitor for the broad spectrum caspases (Z-VAD), caspase 3 (Z-DQMD), caspase 9 (Z-LEHD), caspase 8 (Z-IETD) or caspase 10 (Z-AEVD) reduces the two-agent-induced cell death (3D-1), as revealed by cell viability assay at 72 h after treatment, and protein cleavage for caspases 3 and 10, PARP, Mcl-1 and Akt, as demonstrated by Western blotting at 36 h after treatment. Z-FA is used as a negative control for all caspase inhibitors. *p < .05 versus control. Protein cleavage fragments in Western blots are indicated by arrows. (E) TRAIL and Bortezomib combination induces a robust apoptosis in NCI-H28 cells with a significant cleavage for both caspase 10 and Mcl-1 proteins.