Luteolin acts as a radiosensitizer in non-small cell lung cancer cells by enhancing apoptotic cell death through activation of a p38/ROS/caspase cascade

Abstract

To improve radiation therapy, the development of effective radiosensitizer is required. Fifty percent inhibitory concentration (IC50) values of 3',4',5',7'‑tetrahydroxyflavone (luteolin) against NCI‑H460 and ‑H1299 non‑small cell lung cancer (NSCLC) cells were determined using 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) assays. Radiosensitizing activity was assessed in vitro by treating cells with luteolin prior to irradiation of γ‑ionizing radiation (IR), and performing cell count and clonogenic assays. Cell signaling pathways involved in the radiosensitizing effects of luteolin were examined using propidium iodide (PI) uptake, reactive oxygen species (ROS) detection and immunoblot assays, with or without specific chemical inhibitors. Apoptotic cell death was confirmed by PI uptake and immunoblot assays. In vivo radiosensitizing activity was tested using an NCI‑H460 cell xenograft model in nude mice. Tumor size was measured and apoptosis was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in animals in four treatment groups: mock‑treated control, luteolin only, IR only, and luteolin plus IR. Treatment with luteolin or IR induced NSCLC cell death in vitro, but the combination of luteolin pre‑treatment and IR was more effective than either agent alone, yielding dose enhancement ratios (DERs) of 1.22 and 1.35 for NCI‑H460 and ‑H1299 cells, respectively. Combined treatment with luteolin and IR enhanced apoptotic cell death in association with downregulation of B‑cell lymphoma 2 (Bcl‑2) and activation of caspase‑3, ‑8, and ‑9; it also induced phosphorylation of p38 mitogen‑activated protein kinase (MAPK) and ROS accumulation. Inhibition of p38 MAPK decreased ROS production, and inhibition of either p38 MAPK or ROS production attenuated apoptotic cell death and activation of caspase‑8 and ‑9. In a xenograft model, tumor growth was delayed by 21.8 days in the luteolin/IR combination group compared with controls, and apoptotic cell death was increased. The enhancement factor of the luteolin and IR combination was 1.83. Collectively, these findings indicate that luteolin acts as a radiosensitizer by enhancing apoptotic cell death through activation of a p38/ROS/caspase cascade.