Screening of natural compounds using the TRAIL-resistant cell line KOB. (A) TRAIL receptor expression was analyzed by FCM. Shaded and unshaded peaks correspond to specific and control staining, respectively. RFI (the ratio of mean fluorescence intensity for specific staining to that for control staining) is indicated in each panel. (B) The structure of the cycloanthranilylproline-derivative, Fuligocandin B (FCB). (C) Inhibition of cell proliferation. KOB cells (3.5 × 10⁵/mL) were cultured for 48 hours with TRAIL and/or sFCB and cell proliferation was evaluated by MTS assay. The sensitivity of Jurkat cells to TRAIL is also indicated. (D) Isobolographic analysis. The fractional inhibitory concentrations were determined by using the IC₅₀ of either agent alone or in combination. Sums of less than 1, 1, and greater than 1 indicate synergy, additivity, and antagonism, respectively. Four experimental points were found to be significantly below the theoretical additive line (dotted line), indicating a synergistic effect. (E-G) Combined treatment of ATLL cell lines, primary ATLL cells, and normal PBMCs. Cells (3.5-5.0 × 10⁵/mL) were incubated for 48 hours with 500 ng/mL of TRAIL, 2.5 μg/mL of sFCB, sFCB→TRAIL (TRAIL was added after incubation with sFCB for 24 hours), or without reagents. (E) Annexin-V/PI staining of KOB. Percentages of intact cells, and early and late apoptotic cells are indicated in the bottom panels. (F) Annexin-V-positive cells in various ATLL cell lines are indicated. (G) Results of MTS assays of primary ATLL cells and normal PBMCs. Values are expressed as mean plus or minus SD.