The Roles Of Mitochondria In Radiation-induced Autophagic Cell Death In Human Cancer Cells

Autophagy, also known as "self-eating," is a degradation process, by which the damaged cellular proteins and organelles can be embraced and digested by double membrane vesicles namely autophagosome. Then the fusion between autophagosome and lysosome contributes to the formation of autophagy-lysosome. Hence, autophagy plays an important role in satisfying the stability of cell metabolism, updating corresponding organelles and recycling basic metabolic units. Traditionally, autophagy acts as a pro-survival process. However, a growing number of studies found that autophagy also shows a pro-death consequence, thought of as autophagic cell death or type Ⅱprogrammed cell death.Mitochondria are the "power industry" in eukaryotic cells and critical for cell fate. Firstly, numbers of proteins containing in mitochondria play important role in regulating apopotosis. Secondly, mitochondria are main sources of(Reactive oxygen species, ROS) which show crucial role in autophagy. In this study, we focused on the roles of mitochondria in radiation-induced autophagic cell death in human cancer cells. ObjectiveIn order to explore the effects of mitochondria on the radiation-induced autophagic cell death, different human cancer cells such as cervical cancer He La cells and breast cancer MCF 7 cells, MDA 231 were selected in this study. Methods(1) cell lines: Human cervical cancer cell line He La, human breast cancer cell line MCF 7 and MDA 231 were used in the study;(2) irradiation condition: X-320 ix RAD irradiator was used and the irradiation conditions were as follows: voltage 180 k V, current 20.0 m A, filtration plate 2.0 mm Al, distance between target and X source 70 cm, dose rate 1.0 Gy/min;(3) detection index: MTT assays was used to evaluate cell viability; protein expression was detected by western blot; Mito-Tracker Green staining was used to detect mitochondrial integrity; the level of ROS, autophagy and MMP were analyzed by flow cytometry.(4) Data Analysis: SPSS software was used for statistical analysis; the experimental data were showed by`x±s; p<0.05 was considered the difference was statistically significant; Origin8.0 software was used to make statistical figures. Results1. IR induced autophagic cell death in He La, MCF 7 and MDA 231 cellsHe La cells, MCF 7 cells and MDA 231 cells were exposed to 8 Gy radiation, MTT results showed that the viability decreased in a time-dependent manner. After the treatment with 3-MA and CQ, the decrease of viability was reversed, indicating that 3-MA and CQ could antagonize the IR-induced cell death. The ratio of MAPLC3/LC3Ⅰwas higher than 0 Gy group at 12 h, 24 h and 48 h after IR treatment in He La, MCF 7 and MDA 231 cells. The level of Beclin 1 expression obviously increased in the three cell lines, and the decreasing of p62 expression was detected in breast cancer cells. The above results suggested that IR could induce autophagic cell death in He La, MCF 7 and MDA 231 cells.2. ROS participated in the IR-induced autophagic cell deathCells were exposed to 4- or 8-Gy radiation, ROS generation increased 12 h and 24 h after radiation. NAC as an antioxidant plays a key role in eliminating ROS. In our results, after the treatment with NAC, IR-induced autophagy was significantly decreased. In addition, SOD2-Ri cell model were established in He La cells. 8 Gy radiation increased ROS, autophagy and cell viability as compared with the sham-irradiation group, while in SOD2-Ri cells, the IR-induced ROS and autophagy cell death was enhanced, indicating that ROS participated in the IR-induced cell death via the regulation of autophagy.3. Antioxidants protected mitochondria from IR-induced damageIn He La cells, 4G and 8 Gy radiation significantly reduced the mitochondrial membrane potential(MMP). Moreover, we found the same results that the decrease of MMP was detected after 8 Gy radiation in breast cancer cells. The Mito Tracker Green staining also showed that mitochondrial integrity was obviously decreased after exposure to 8 Gy. However, NAC protected mitochondria from IR-induced impairment.4. Mitochondria participated in IR-induced autophagic cell deathIn this study, we used TTFA(an inhibitor of mitochondrial electron-transport chain complex Ⅱ) to induce mitochondrial damage, and Cs A(an inhibitor of the mitochondrial permeability transition pore) was used as a protector of mitochondria. Compared with the control group, TTFA significantly enhanced the rate of mitochondria damage, the level of ROS, autophagy rate and cell death induced by IR. Interestingly, Cs A showed adverse results, demonstrating that mitochondria participate in the IR-induced autophagic cell death in human cancer cells. Conclusion1. Ionizing radiation induces autophagic cell death in He La, MCF 7 and MDA 231 cells.2. Ionizing radiation leads to ROS production and mitochondrial damage which play an important role in autophagic cell death in He La, MCF 7 and MDA 231 cells.