- Authors: Tsai, J. J., Pan, P. J., Hsu, F. T.
- Year: 2017
- Journal: Oncol Rep 37 1036-1044
- Applications: in vitro / DNA / jetPEI
- Cell type: SK-HEP-1
Cells (1x10^6 ) were seeded into 10-cm dish and incubated overnight. DNA solution (10 µg NF-κB/luc2 plasmid dissolved in 250 µl of 150 mM NaCl) was mixed with 250 µl jetPEI solution (20 µl of jetPEI reagent diluted in 230 µl of 150 mM NaCl), and then incubated for 30 min at room temperature to make 500 µl DNA/JetPEI mixture. The DNA/JetPEI mixture was added to the SK-HEP-1 cells in a 10-cm diameter dish and incubated for 24 h.
The aim of the present study was to investigate the role of NF-kappaB inactivation in regorafenib-induced apoptosis in human hepatocellular carcinoma SK-HEP-1 cells. SK-HEP-1 cells were treated with different concentrations of the NF-kappaB inhibitor 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine (QNZ) or regorafenib for different periods. The effects of QNZ and regorafenib on cell viability, expression of NF-kappaB-modulated anti-apoptotic proteins and apoptotic pathways were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, DNA gel electrophoresis, flow cytometry and NF-kappaB reporter gene assay. Inhibitors of various kinases including AKT, c-Jun N-terminal kinase (JNK), P38 and extracellular signal-regulated kinase (ERK) were used to evaluate the mechanism of regorafenib-induced NF-kappaB inactivation. The results demonstrated that both QNZ and regorafenib significantly inhibited the expression of anti-apoptotic proteins and triggered extrinsic and intrinsic apoptosis. We also demonstrated that regorafenib inhibited NF-kappaB activation through ERK dephosphorylation. Taken all together, our findings indicate that regorafenib triggers extrinsic and intrinsic apoptosis through suppression of ERK/NF-kappaB activation in SK-HEP-1 cells.