FIGURE 4. Suppression of TRAIL-induced apoptosis by 1,25-(OH)₂D₃ in OVCAR3 cells.

A, cells were pretreated with 1,25-(OH)₂D₃ (VD) or vehicle (EtOH) for 3 days. Equal numbers of treated cells were plated into 96-well plates and further treated with the indicated concentrations of TRAIL for 24 h. MTT assays were performed and absorption at 595 nm (A_{595 nm}) was determined. Eight samples were analyzed in parallel for each data point and the experiments were reproduced twice. Percentages of viable cells were calculated relative to the A₅₉₅ values of cells before TRAIL treatment (pretreated cells), which was set as 100%. *, p < 0.01 when compared with the corresponding values from cells pretreated with vehicle. B, cells were pretreated similarly as in A and subsequently treated with or without 100 ng/ml TRAIL for the indicated times before MTT assays were performed. Percentages of viable cells were calculated by dividing A₅₉₅ values with the corresponding value before treatment for each group. *, p < 0.01 when compared with the group treated with TRAIL following vehicle. C and D, cells were pretreated similarly as in A followed by secondary treatment with 100 ng/ml TRAIL for the indicated times. Apoptosis of treated cells was determined by flow cytometry after Annexin V staining. A representative profile of the flow cytometry analysis is shown in C. The apoptotic index from two independent analyses is shown in D. E, effect of 1,25-(OH)₂D₃ on TRAIL-induced caspase-7 activation. Cells were treated with 10⁻⁷ m 1,25-(OH)₂D₃ or EtOH for 0, 1, 3, or 6 days followed by treatment with 100 ng/ml TRAIL for 4 h. Capase-7 activity was determined as described under “Materials and Methods.” F, effect of 1,25-(OH)₂D₃ on TRAIL-induced caspase-3 activation. Cells were treated as in E and caspase-3 activity was measured. FITC, fluorescein isothiocyanate.