Honokiol induces caspase-dependent apoptosis of B-CLL cells. (A-B) PBMCs from B-CLL patients were pretreated with or without 50 μM z-VAD-fmk for 30 minutes and then incubated in the presence or absence of 40 μM honokiol for 16 hours. Apoptosis was determined by annexin V-FITC/PI double staining. The numbers in each quadrant indicate the percentage of cells labeled with annexin V-FITC (bottom right), PI (top left), or annexin V-FITC and PI (top right), or the percent unlabeled (bottom left). A representative example is shown in panel A, and panel B represents the means of PI-negative cells ± standard deviation from 5 patients' cells. Results were normalized to cells incubated without drug. ^*P < .001 when cells treated with honokiol were compared with cells incubated in medium alone. †P < .05 when cells treated with honokiol were compared with cells pretreated with z-VAD-fmk and honokiol. (C) B-CLL cells were incubated with or without 40 μM honokiol for 16 hours. Cells were analyzed for caspase-3 activity using a fluorogenic peptide substrate. The histogram is representative of 4 experiments. The gate indicates the percentage of cells containing active caspase 3. (D-E) B-CLL cells were incubated with or without 40 μM honokiol for the indicated times. Cells were lysed, and Western analysis was performed using antibodies that recognize (D) the proform of caspase-8 as well as caspase-8 cleavage products or the proform of caspase-9 and (E) Bcl-2 or Bax. Blots were stripped and reprobed for β-actin as a loading control. One representative example of 4 is shown. (F) CLL cells were incubated for 4 hours with 20, 40, 60, and 80 μM honokiol or left untreated. Cells were lysed, and Western analysis was performed using antibodies that recognize the caspase-3 substrate, PARP. The proform of PARP is 115 kDa, and the cleaved, activated form is 80 kDa. The blot was stripped and reprobed for STAT3 as a loading control. Two representative examples of 6 are shown.