Objective To investigate the effect of recombinant soluble CD40 ligand (rsCD40L) on Wogonin mediated antitumor activity in cancer cells and the underlying molecular mechanisms. Methods Cell death was detected based on the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit. For morphological study of cell death, cells were stained with 50 μg/mL of acridine orange and 50 μg/mL of ethidium bromide and observed and photographed under a fluorescence microscope. Activation of apoptosis pathway was evaluated by Western blot. The effects of pan-caspase inhibitor Z-VAD-FMK and tumor necrosis factor α (TNF-α) neutralizing antibody on cell death induced by rsCD40L and Wogonin co-treatment were also investigated. Results rsCD40L significantly enhanced Wogonin-induced cell death of ovarian cancer cells SKOV3. A dose-dependent synergism was found with a fixed rsCD40L dose (1 μg/mL) and increased concentrations of Wogonin (5 μmol/L-15 μmol/L). rsCD40L and Wogonin co-treated cells showed typical apoptotic morphologies and enhanced activation of caspases pathway. As expected, the pan-caspase inhibitor Z-VAD-FMK inhibited synergistic cell death of rsCD40L and Wogonin co-treated SKOV3 cells. Interestingly, the TNF-α neutralizing antibody that blocks TNF-α binding to its receptor also significantly suppressed the cell death enhancing effect, indicating that autocrine TNF-α played a role of sensitization. Conclusion rscCD40L sensitizes cancer cells to wogonin-mediated apoptosis, which may involve autocrine of TNF-α, and the combination of rsCD40L and Wogonin may have a potential for cancer therapy.
