4.11. Assay of Caspase-3 and -6 Activities

Cascade activation of caspases-3 and -6 in human TMZ-sensitive U87 MG and -resistant U87 MG-R glioblastoma cells was assayed using a fluorogenic substrate method as described previously [53]. After exposure to enzalutamide, the specific substrates, DEVD and VEID, were used to respectively measure activities of caspases-3 and -6. These fluorogenic substrates conjugated with 7-amino-4-trifluoromethyl coumarin for fluorescent detection were purchased from R&D Systems (Minneapolis, MN, USA). Cell lysates of human glioblastoma cells were prepared by lysing them in an ice-cold lysis buffer. Protein concentrations of cell lysates were measured using a bicinchonic acid protein assay kit (Thermo Fisher Scientific). Cell lysates at 25 mg total protein were mixed with 50 mM of the DEVD and VEID substrates in a cell-free system buffer at a volume of 20 μL. Activities of caspases-3 and -6 were measured with a fluorescent spectrophotometer.