Figure 6

Overexpression of FOXO3a and RUNX3 restored cell growth and attenuated apoptosis affected by baicalein. A, H1650 cells were transfected with control or FOXO3a siRNA for 30 h, followed by control or FOXO3a expression vectors for up to 24 h before exposure of the cells to baicalein for an additional 24 h. Afterwards, cell growth was determined by MTT assays. The upper insert panel represents blots of expression of FOXO3a protein detected by Western blot. B-C, H1650 cells were transfected with control or FOXO3a, or RUNX3 expression vectors for 24 h before exposing the cells to baicalein for an additional 24 h. Afterwards, cell viability were detected by MTT assays. Insert blots were FOXO3a and RUNX3 protein expression. D, H1650 cells were transfected with control or RUNX3 siRNA for 30 h before exposing the cells to baicalein for an additional 24 h. Afterwards, the cells were processed for analysis of apoptosis as determined by caspase 3/7 activity assays. Data are expressed as a percentage of total cells. Values in bar graphs were given as the mean ± SD from three independent experiments. *indicates significant difference as compared to the untreated control group (P<0.05). **indicates significant difference from baicalein treated alone (P<0.05). E, H1650 cells were transfected with control or FOXO3a, or RUNX3 expression vectors for 24 h before exposing the cells to baicalein for an additional 2 h. Afterwards, The expression of FOXO3a and RUNX3 protein, phosphorylation of AMPKα and ERK1/2 were determined by Western blot. F, The graph shows that baicalein inhibits growth and induces apoptosis of lung cancer cells through AMPKα- and ERK1/2-mediated increase in RUNX3 and FOXO3a protein expression. Overexpression of RUNX3 strengthens baicalein-induced phosphorylation of ERK1/2 and induces expression of FOXO3a. The crosstalk between AMPKα and ERK1/2, and the reciprocal incorporation of FOXO3a and RUNX3 converge on the overall anti-cancer responses of baicalein.