The HCC cell lines tested in this study included PLC/PRF/5 (PLC5), Hep3B (from American Type Culture Collection), and Huh7 (from Health Science Research Resources Bank, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL). Primary human umbilical venous endothelial cells (HUVEC), which were used to test the anti-angiogenic activity of the drug combinations, were cultured as previously described [
20]. These cells were maintained in a humidified incubator under 5% CO
2 at 37°C.
The molecular targeted agents (MTAs) tested in this study included NVP-AEW541 (IGFR inhibitor, Novartis), MK2206 (Akt inhibitor, MSD), BEZ235 (PI3K/mTOR inhibitor, Novartis), and RAD001 (mTOR inhibitor, Novartis). For in vitro experiments, the drugs were dissolved in DMSO and the final DMSO concentration was kept below 0.1%. For in vivo experiments, the following the optimal dosage were used: NVP-AEW541 (dissolved in 25 mmol/L tartaric acid, 30 mg/kg, gavage) once per day for 25 days based on previous studies [
22,
23], BEZ235 (dissolved in 10% NMP (1-methyl-2-pyrrolidone)/PEG300 90%, 25 mg/kg, gavage) once per day for 25 days based on previous studies [
24,
25], and MK2206 (dissolved in 30% captisol, 100 mg/kg, i.p.) once per week for 4 weeks based on previous studies [
26,
27]. The antibodies used for Western blotting, and immunohistochemical staining included anti-Myc Tag (Origene, Rockville, MD), p-Akt, caspase 3, p-GSK3β, GSK3β, p-P70S6K, P70S6K, p-4EBP-1, Mcl-1, Bim, DR-5, FADD (Cell Signaling Technology, Danvers, MA), Bcl-2, Bad, Bax, survivin, GAPDH, and actin (Santa Cruz Biotechnology, Santa Cruz, CA).