Mechanisms of induction of cell cycle arrest and cell death by cryptolepine in human lung adenocarcinoma a549 cells.

We investigated p53-dependent and -independent molecular events associated with cell cycle alteration and cell death in human lung adenocarcinoma A549 cells using cryptolepine, a DNA-damaging agent. After a 24-h treatment, cryptolepine caused an accumulation of p53 at concentrations of 1.25-10 microM and induction of p21(Cip1/WAF1) but only at concentrations up to 5muM. p21(Cip1/WAF1) was also strongly induced by cryptolepine (2.5-5 microM) in cells with p53 largely ablated via small interfering RNA-mediated gene silencing. Cryptolepine induced G1-phase block at 1.25-2.5 microM, S-phase and G2/M-phase block at 2.5-5 microM, and cell death at 10 microM. The dead cells displayed condensed and fragmented nuclei, features of apoptosis. Wortmannin, an inhibitor of ataxia telangiectasia-mutated and DNA-dependent protein kinase (DNA-PK), caused cell cycle arrest at G1 phase without inducing p53 and p21(Cip1/WAF1) expression and cell death. The addition of wortmannin partially prevented cryptolepine-induced expression of p53 and p21(Cip1/WAF1) together with the S-phase block and sensitized cells to induction of cell death. NU7026, a DNA-PK-specific inhibitor, showed neither induction of cell cycle arrest and apoptosis nor the expression of p53 and p21(Cip1/WAF1). The presence of NU7026 caused further reduction of cells in G1 phase induced by cryptolepine at 5 microM without affecting the induction of p53 and p21(Cip1/WAF1) and cell death. This study using the A549 cell as a model demonstrated that cryptolepine selects different molecular pathways to cell cycle checkpoint activation in a dose-specific manner and evokes a wortmannin-sensitive antiapoptosis response.