Fig. 5

Bicyclol suppressed the PI3K/AKT and the Ras/Raf/MEK/ERK pathways. a Dose-dependent effects of bicyclol on the PI3K/AKT and the Ras/Raf/MEK/ERK pathway-related proteins. The cells were disrupted after treatment with various concentrations (0, 50, 100, 200 and 500 μM) of bicyclol for 6 h. The proteins were collected, and cellular β-actin, p-ERK1/2 (Thr202 and Tyr 204), total ERK, Ras, p-AKT (Thr 450), p-AKT (Ser 473), total AKT, p-mTOR (Ser 2448), and total mTOR were analyzed by western blotting. b The AKT cDNA rescued HepG2 cells from bicyclol-induced cell anti-proliferation. The cells were transiently transfected with the AKT cDNA expression vector using Lipofectamine 2000 and incubated for 48 h. Then, the transfected cells were treated with 200 μM bicyclol for 48 h. The A₅₇₀ was measured after the MTT incubation. c The bicyclol-induced G1 arrest was reduced by the AKT cDNA. The cells were transfected with the AKT cDNA expression vector and then treated with 200 μM bicyclol for 24 h. The phase distribution was determined by flow cytometry. d The AKT cDNA suppressed the effect of bicyclol on the PI3K/AKT and the Ras/Raf/MEK/ERK pathways. The cells were transfected with the AKT cDNA expression vector and then treated with 200 μM bicyclol for 6 h. The cells were disrupted, and cellular β-actin, p-AKT (Thr 450), p-AKT (Ser 473), total AKT, p-ERK1/2 (Thr202 and Tyr 204), total ERK, p-Rb (Ser 807) and LC3 I and II were analyzed by western blotting. e The number of autophagosomes and autolysosomes were reduced by the AKT cDNA. Bar graphs represent the means ± SD from three independent experiments. (*p < 0.05 versus bicyclol treatment)