Combining RAD001 with MK-2206 synergistically suppresses proliferation of HCC cell lines. (A) HCC cell lines were treated with the indicated concentration of MK-2206 over 24 h, and changes in mTOR- and AKT-signaling were analyzed by Western Blot. HSC70 served as loading control. (B) HCC cells were seeded into 96 well plates and incubated with increasing concentrations of either RAD001 (triangle), MK2206 (box), or a combination (inverted triangle) of both with a fixed ratio of 1:5. Controls were treated with DMSO only. Proliferation was analyzed after 72 h BrdU-incorporation. Asterisks indicate a significantly stronger inhibition of the drug combination compared to each compound alone, * p < 0.05. (C) HCC cell lines were treated with RAD001 (triangle) or a combination of RAD001 and MK-2206 (inverted triangle) with a constant concentration of 1.7 μM MK2206 for 72 h. Cells treated with DMSO only served as control. Proliferation was analyzed as in (B). Each data point represents mean of at least three independent experiments, normalized to controls; bars, SD. Asterisks indicate a significantly stronger inhibition of the drug combination compared to each compound alone, * p < 0.05. (D) Fractional effect plot for the effect of RAD001 and MK2206 as seen in (B). (E) Cell cycle analysis of HCC cell lines after 24 h treatment with 100 nM RAD001, 1.7 μM MK-2206, or the combination of both, compared to DMSO treated controls. Colums: mean of one representative experiment, performed in triplicates; bars: SD. The drug combination resulted in a significant increase of cells in G0/G1 phase compared to each drug alone and compared to controls in all three cell lines (p < 0.05).