Magnolol and Regorafenib Induce Apoptosis in Liver Cancer Cells

Hep3B and SK-Hep1 cells (5 × 10⁵) were seeded in 6-well plates and incubated with 50 μM magnolol, 10 μM regorafenib, and a combination for 48 h. ZVAD was added for 30 min before magnolol combined with regorafenib for 48 h. The cells were then harvested for the staining of different reagents, including Annexin-V/PI, cleaved-caspase-3 (1 μL, fluorescein isothiocyanate-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone (FITC-DEVD-FMK)), cleaved-PARP-1, FAS-FITC (1 μL), FAS-L-PE (1 μL), cleaved-caspase-8 (1 μL, sulforhodamine-Ile-Glu-Thr-Asp-fluoromethyl ketone (Red-IETDFMK), cleaved-caspase-9 (1 μL FITC-Leu-Glu-His-Asp-fluoromethyl ketone (FITC-LEHD-FMK)), DCFH-DA (500 μL at 10 μM) for ROS, DIOC6 (4 μmol/L) for Dy loss, and Fluo-3/AM (2.5 μg/mL) for Ca²⁺. For subG1 analysis, the harvested cells were fixed by 70% ethanol overnight at −20 °C and stained by a PI/RNase solution (cat: 550625, BD Biosciences). The fluoresce signal from the cells was detected and quantified by NovoCyte flow cytometry and the NovoExpress^® software (Agilent Technologies Inc., Santa Clara, CA, USA) [15 (link),17 (link)].