Screening of new natural products that enhance sensitivity to TRAIL using the TRAIL-resistant ATLL cell line KOB. (A) TRAIL receptor expression on KOB and Jurkat cells. Cells were stained with antibodies to TRAIL receptors or with an isotype-matched control antibody and were analyzed by FCM. Shaded and unshaded peaks correspond to specific and control staining, respectively. RFI was determined as the ratio of mean fluorescence intensity for specific staining to that for control staining and is indicated in each panel. (B) Sensitivity of KOB (▪) and Jurkat (•) cells to TRAIL. KOB cells (5 × 10⁵/mL) and Jurkat cells (10⁶/mL) were cultured for 24 hours in the presence of the indicated concentrations of TRAIL, and cell proliferation was evaluated by MTS assay. Results are expressed relative to the values for the control cells cultured without TRAIL. Data are the mean ± SD of 3 independent experiments. (C) The structure of methyl dihydro quercetin extracted from B balsamifera (BB-1). (D) Evaluation of apoptosis. KOB cells (5 × 10⁵/mL) were incubated for 48 hours with 0.5 μg/mL TRAIL, 12.5 μg/mL BB-1, and a combination of both and apoptotic cells were evaluated by Annexin V binding and propidium iodide (PI) staining. Cells (10⁴) were counted and analyzed by FCM. Percentages of Annexin V⁺ /PI^– cells are shown in each dot-plot graph. (E) Synergism of TRAIL and BB-1. KOB cells (5 × 10⁵/mL) or PBMCs (10⁶/mL) were incubated for 48 hours with 0.5 μg/mL TRAIL (T), 12.5 μg/mL BB-1 (B), BB-1+TRAIL (simultaneous addition, B+T), or BB-1+TRAIL (0.5 μg/mL TRAIL was added after incubation with 12.5 μg/mL BB-1 for 24 hours, B→T). Cell proliferation was assessed by MTS assay, and results are expressed relative to the value for the control cells [C] cultured without inducers. Data are the mean ± SD of 3 independent experiments.