Caspase-3/7 Activation Assay for Apoptosis

Apoptosis was detected with the Apo-ONE^® homogeneous caspase-3/7 assay (Promega, Walldorf, Germany), according to the manufacturer’s instructions. It allows the detection of the active form of caspases 3 and 7—effector caspases in apoptosis. Cells were exposed to 10 µM of DDRI-18 or 5 µM of A12B4C3 before 24 h of treatment with CDDP/VP-16. The assay contains a profluorescent caspase-3/7 substrate, rhodamine 110 bis-(N-CBZ-l-aspartyl-l-glutamyl-l-valyl-aspartic acid amide) (Z-DEVD- R110), which becomes fluorescent after cleavage using caspase-3/7 enzymes. The amount of fluorescent product indicates the level of active caspase-3/7 in the sample. For each experiment, positive control cells were exposed to 100 µM VP-16, and negative control samples were performed. Cells were exposed to CDDP and VP-16 as single drugs or in combined treatment with or without DRI. Furthermore, we included the experimental scheme 20 µM pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) (Promega, Germany). It allowed confirming the apoptosis-mediated activation of caspases 3/7. The activity of 3/7 caspases was defined as a % activity of caspases 3/7 in the sample, compared to the untreated control.