Mechanism of anti-cancer activity of andrographolide and DRF 3188 – In vitro studies: Western blot analysis of cell cycle proteins A. Western blot analysis of cells treated with Andrographolide (25 μM) or DRF 3188 (5 μM) for 24 hours. An induction of the cell cycle inhibitor p27 and reduction in the levels of Cdk4 is observed. No change is observed in the levels of Cyclin D1, Cdk1 or Cyclin B1 in DRF 3188 treated cells. A similar effect is seen in the andrographolide treated cells, but a slight decrease in the level of Cdk 1 is observed. Lovastatin treated cells show decreased levels of Cdk1 and cyclin B1. Actin is used as the internal loading control. B) Densitometric analysis of the western blot with p27 antibody. An induction of p27 is seen in MCF7 cells treated with 25 uM Lovastatin (4.5 fold), 25 uM Andrographolide (3 fold) and 5 uM DRF3188 (2 fold). C) Densitometric analysis of the western blot with Cdk4 antibody. A decrease in the levels of Cdk4 is seen when MCF7 cells are treated with 25 uM Lovastatin (0.4 fold), 25 uM Andrographolide (0.8 fold) and 5 uM DRF3188 (0.4 fold).