Inhibition of autophagy significantly enhances combination therapy with sorafenib and HDAC inhibitors for human hepatoma cells

Figure 1 Antiproliferative effects of sorafenib, vorinostat, or the drug combination on human hepatoma cell lines. HepG2 (A), Hep3B (B), and PLC/PRF/5 (C) cells were treated with various concentrations of sorafenib, vorinostat, or the drug combination. The results are expressed as the percentage of viable cells in the control group. The results are expressed as mean ± SD from three independent experiments. ^aP < 0.05 and ^bP < 0.01 vs control as evaluated by Student’s t test.

Figure 2 Sorafenib, vorinostat, or the drug combination induces cell cycle arrest and apoptosis in human hepatoma cell lines. HepG2, Hep3B and PLC/PRF/5 cells were treated with various concentrations of sorafenib (Sor), vorinostat (Vor), or the drug combination. A: Cell cycle distributions were analyzed by flow cytometry, and the percentage of cells in the G₀/G₁, S, or G₂/M phases of the cell cycle is indicated; B: The apoptosis-specific caspase-3 activity induced by various concentrations of sorafenib, vorinostat, or the drug combination. The results are expressed as mean ± SD from three independent experiments. ^bP < 0.01 using Student’s t test.

Figure 3 Sorafenib, vorinostat, or the drug combination induces the modulation of apoptosis-, cell cycle- and autophagy-related proteins. A: Representative images of Western blot showing the effect of treatment with siBeclin-1 and vorinostat/sorafenib on apoptosis-, cell cycle-, and autophagy-related proteins in HepG2 cells. siSCR, siRNA scramble; B: Representative images of Western blot showing the acetylated p53 level in HepG2 cells treated with or without siBeclin-1 or vorinostat/sorafenib. siSCR, siRNA scramble; C: Protein level of Beclin-1 in HepG2 cells infected with sip53 combined with or without vorinostat/sorafenib. All experiments were performed independently in triplicate. siSCR, siRNA scramble.

Figure 4 Knockdown of Beclin-1 enhances the synergistic effect of the combination of vorinostat with sorafenib. A: Knockdown of Beclin-1 enhanced the vorinostat/sorafenib drug combination-stimulated cell cycle alterations. siSCR, siRNA scramble; B: Knockdown of Beclin-1 enhanced the vorinostat/sorafenib drug combination-stimulated apoptosis. The results are expressed as mean ± SD from three independent experiments. ^aP < 0.05 and ^bP < 0.01 using Student’s t test. siSCR, siRNA scramble.

Figure 5 Knockdown of Beclin-1 or the autophagy inhibitor 3-MA enhances the growth inhibitory effects of the combination of vorinostat with sorafenib. A: HepG2; B: Hep3B; C: PLC/PRF/5. The results are expressed as mean ± SD from three independent experiments. ^aP < 0.05 and ^bP < 0.01 vs the siSCR DMSO group using Student’s t test.