Role Of Mitochondrial Damage In Cell Malignant Transformation Induced By Alpha Particles

Objective: To explore the role of mitochondrial damage in the carcinogenic process of lung cancer induced by alpha particles.Methods: (1) To establish the malignantly transformed human small airway epithelial (SAEC) cell model induced byαparticles. SAEC cells were exposed to different doses (0.2,0.4,0.8,1.0,2.0Gy) ofαparticles by either single or multiple treatment. Survival fraction was detected using plating efficiency immediately after the irradiation. Cells were cultured in SAGM at 37℃in a 5% CO2 incubator for a period of several months to measure changes in transformed phenotypes including resistance to serum and PALA, cell proliferation, anchorage independent growth in soft agar and invasion ability. C-myc and p53 expression were measured using western blot. The transformed cells were inoculated subcutaneously into nude mice to observe tumor growth in vivo. (2) To test the mitochondrial function and mt DNA mutation in transformed SAEC cells. Mitochondrial copy number was analyzed by real-time PCR with 12S rRNA/18S rRNA and 12S rRNA /GAPDH. Common deletion rate was tested with nested PCR combined with real-time PCR. Cytochrome C Oxidase activity and expression were measured with biochemical kit and western blot. Membrane potential was detected by JC-1 fluorescence with flow cytometry. Oxygen consumption was measured by oxygen electrode. Correlation between mitochondrion and transformation was analyzed by SPSS17.0 software. (3) Wistar rats were exposed to radon and its progeny with a multifunction ecological radon room at a constant radiation level of 100,000Bq/KL. The low, middle and high dose groups were exposed up to a cumulative dose of 100, 200 and 400WLM respectively. Pathology of lung tissue of rats was examined using HE stain and masson collagen fiber stain. Mitochondria of lung tissue were extracted and COX activity was measured with biochemical kit, and the correlation between COX activity and pathology was analyzed.Results: (1) Survival fraction of SAEC exposed to graded doses ofαparticles showed a dose-dependent manner in cytotoxicity. Serum resistance and PALA resistance were observed in the process of transformation. Proliferation and saturation density increased significantly 4 months after first irradiation, indicating an overcome of growth contact inhibition of cells. All the cells in the irradiated groups formed colonies in soft agar, of which the highest rate and biggest size of colony was detected in 0.8 Gy group, with an increased invasion ability in the transformed cells. Expression of c-myc increased and p53 decreased significantly among the transformed cells. Transformed SAEC cells formed tumors in three out of the five nude mice in the 0.8 Gy multiple treatment group 4 months after subcultanously injection and the tumors were of human epithelial in origin. (2) Real-time PCR showed a 1.4-2.9 fold increase in mitochondrial DNA copy number and a 2.4-4.9 fold increase in common deletion of the transformed cells, which might contribute to the dysfunction of mitochondria and initiation of transformation. Transformed cells showed a reduced COX activity and expression, dissipated mitochondrial membrane potential, and decreased oxygen consumption in each mitochondrion. Correlation analysis showed a linear correlation between the copy number, common deletion, COX activity and the soft agar colony formation/c-myc, indicating a correlation of mitochondrial function with cell transformation. (3) Pathological observation revealed congestion and edema of the lung interstitial, thickening of alveolar septum with infiltration of inflammatory cells such as neutrophilic leukocyte, lymphocyte, macrophage and collagen fiber, with the alveolar space gradually shrinked or diminished in size. The thickness of alveolar septum increased with a corresponding decrease in the area of alveolar space in the high dose group. The COX activity also decreased with a negative correlation with the thickness of alveolar septum and a positive correlation to the alveolar space.Conclusions: (1) Human SAEC cells could be malignantly transformed in vitro byαparticles and formed tumor upon subcultanouly injection in nude mice. (2) Alteration in mitochondrial function and mutations in mitochondrial DNA correlated with the process of cell transformation, indicating an important role of mitochondria in the carcinogenic process ofαirradiation. (3) Experiment of rats exposed to radon revealed a correlation between pathological changes of the lung tissue and the COX activity of mitochondria, and confirmed role of mitochondrial damage in tumorgenesis of lung cancer induced by radon exposure.