Flavonol isolated from ethanolic leaf extract of Thuja occidentalis arrests the cell cycle at G2-M and induces ROS-independent apoptosis in A549 cells, targeting nuclear DNA.

ABSTRACT

OBJECTIVES: The K-ras gene mutation commonly found in lung adenocarcinomas contributes to their non-invasive expansion. Our main objective here was to develop a chemopreventive agent against K-ras-mutated lung adenocarcinoma cell line like-A549. MATERIALS AND METHODS: We isolated flavonol from ethanolic leaf extract of Thuja occidentalis, and evaluated its apoptotic potentials on A549 cells. They were treated with 1-10 µg/ml of flavonol and viability was tested retaining normal lung cells L-132 as control. We performed assays such as TUNEL, annexin V, cell-cycle and mitochondrial membrane potentials, by FACS analysis. ROS-mediated oxidative stress and drug-DNA interactions were analysed along with gene expression studies for p53, Bax-Bcl2, cytochrome c, the caspase cascade genes and PARP. RESULTS: Flavonol reduced A549 cell viability in a dose- and time-dependent manner (IC50 value = 7.6 ± 0.05 µg/ml following 48 h incubation) sparing normal L-132 cells. It effected G2-M phase cell cycle arrest and apoptosis, as indicated by progressive increase in the sub-G1, annexin V and TUNEL-positive cell populations. Apoptotic effects appeared to be mitochondria-dependent, caspase-3-mediated, but ROS-independent. Analysis of circular dichroism data revealed that flavonol intercalated with nuclear DNA. In vivo studies on non small cell lung carcinoma (NSCLC)-induced mice confirmed anti-cancer potential of flavonol. CONCLUSION: Flavonol-induced apoptosis apparently resulted from intercalation of cells' nuclear DNA. Flavonol inhibited growth of induced lung tumours in the mice, indicating its potential as an effective agent against NSCLC.