(A) Chemical structure of noscapine. (B) Noscapine potentiates cytotoxicity induced by TNF and chemotherapeutic agents. KBM-5 cells were pretreated with 25 μM noscapine for 12 h and then incubated with 1 nM TNF, 10 μg/mL thalidomide, 5 nM paclitaxel, and 20 nM bortezomib for 24 h. Cell viability was then analyzed by MTT assay. (C, left) Noscapine potentiates TNF-induced apoptosis. KBM-5 or U266 cells (1 × 106) were pretreated with 25 μM noscapine for 12 h and then incubated with 1 nM TNF for 24 h. Cells were stained with live/dead assay reagent for 30 min and then analyzed under a fluorescence microscope. (right) Cells were pretreated with 25 μM noscapine for 12 h and then incubated with 1 nM TNF for 16 h. Cells were incubated with FITC-conjugated antibody to annexin V, or with TUNEL assay reagent, and then analyzed by flow cytometry for early apoptotic effects. (D, left) Noscapine enhances TNF-induced caspase activation. Cells were pretreated with 25 μM noscapine for 12 h and then incubated with 1 nM TNF for 16 h. Whole-cell extract were prepared and analyzed by western blotting using the indicated antibodies. (right) Cells were incubated with 1 nM TNF, alone or with 25 μM noscapine, for the indicated times. PARP cleavage was determined by western blot analysis.