Figure 4

Gossypol combined with phenformin enhances cell cycle arrest and induces cell death in non-small-cell lung cancer (NSCLC). (a) G2/M transition of synchronized cells (following the protocol of a single thymidine block as shown in the upper panel) was monitored via mitotic index assessment (middle panel) and western blot analysis (lower panel) in the presence or absence of the indicated chemicals. Cyclin B1 is a marker for mitotic exit. pY15 of Cdk1 is a marker for the G2/M transition. (b) Effect of gossypol (10 μM), phenformin (100 μM) or a combined treatment for 24 h on the mitotic index of asynchronous cells. *P<0.05, **P<0.01, ***P<0.001 compared with vehicle control. (c) G1/S transition of synchronized cells (following the protocol of a single thymidine block as shown in the upper panel) was monitored via DNA FACS analysis (lower panel) in the presence or absence of the indicated chemicals. Note that EKVX cells were mainly arrested at the early S phase, whereas A549 cells were mainly arrested at the G1 phase. (d) To measure cell death, A549 and EKVX cells were treated with gossypol (10 μM), phenformin (100 μM), combination for 13 and 10 h, respectively. Cell death was measured by flow cytometric analysis. Data are representative of the mean and s.d. of three independent experiments. *P<0.05 compared with control.