Distinct Radiosensitivity of Lung Carcinoma Stem-Like Side Population and Main Population Cells

Abstract

Lung cancer is a leading cause of cancer death worldwide. Efficacy of radiation therapy on lung cancer is hindered by many factors. Among these, both cancer stem-like side population (SP) and main population (MP) cells may contribute to tumorigenesis and resistance to radiation therapy. However, the detailed mechanism responsible for this effect remains unknown. The SP and MP cells were obtained from lewis lung carcinoma cells and analyzed the DNA dye (Hoechst 33342) method and flow cytometry. The levels of ABCG2 and CD133 markers were examined by reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. The effects of ionizing radiation (IR) on the growth and apoptosis of SP and MP cells were determined by 3-(4, 5-dimethylthiazol-2-y)-2, 5-diphenylterazolium bromide (MTT), colony formation, and apoptosis assays. Mitochondrial membrane potential and intracellular reactive oxygen species production were measured by flow cytometry. Finally, the expression of Bax, Bcl-xL, Bcl-2, activated caspase-3 and caspase-9 proteins were examined by Western Blot. IR decreased proliferation, increased apoptosis and mitochondria damage in MP, but not in SP cells. Protein levels of Bcl-2 and Bcl-xl were decreased, while Bax expression was increased in MP cells following IR exposure. In addition, increased activation of caspase-3 and caspase-9 were detected after IR exposure in MP cells, but not in SP cells. Our results show that IR decreases proliferation, increases apoptosis, and induces mitochondria damage in MP cells, but not in SP cells, through increased Bax and decreased Bcl-2 and Bcl-xl protein expression. This protein expression pattern induces activation of caspase-3 and caspase-9. This study suggests that IR exposure targets MP cells through a mitochondrial apoptosis pathway. However, more work is required to further confirm these results using in vivo xenograft models. More importantly, further studies are warranted to elucidate the radioresistant mechanisms of SP cells.