The Response Mechanism Of DNA Damage And Mitochondrial Dysfunction Induced By Ionizing Radiation In Non-small Cell Lung Cancer

As the largest cancer in the world,lung cancer is a dangerous type of cancer that threatens human health.Radiotherapy has been used in every stage of lung cancer treatment as an effective treatment for lung cancer.Radiation effects of radiotherapy mainly cause damage to the nuclear genome and cytoplasm such as mitochondrial dysfunction.As an advanced radiotherapy technology,heavy ion therapy can induce significant biological effects and significantly improve the cure rate of lung cancer patients due to its unique physical advantages,but its mechanism is still unclear.In this study,we first investigated the dynamic response mechanism of complex DNA damage induced by X-rays,carbon ions and their combination with bleomycin in non-small cell lung cancer.Secondly,studies were carried out on the mitochondrial dysfunction induced by carbon ions alone or in collaboration with tigecycline.The interaction mechanism between DNA damage response and mitochondrial dysfunction was preliminarily explored.The purpose of this study is to highlight the biological effect mechanism of heavy ions in cancer therapy,so as to give full play to their advantages in cancer therapy.First,compared with X-ray,we found that lung cancer cells were more sensitive to carbon ion irradiation,and the frequency of complex DSB damage induced by carbon ions was increased compared with that of X-ray.The two types of irradiations were further combined with bleomycin and the damage frequency of complex DSB was in the order of X-ray<X-ray combined with bleomycin<carbon ion<carbon ion combined with bleomycin.With the increase of damage frequency,the recognition factors?H2AX and 53BP1 were more difficult to recruit to the damage site,and the recruitment lagged.The differential expression analysis of repair proteins showed that the expression of non-homologous end joining(NHEJ)repair factor Ku70 was up-regulated in all groups except for X-ray irradiation,while the expression of homologous recombination(HR)repair factor Rad51 was not significantly different,suggesting that the repair of complex DSB might be dominated by NHEJ.The expression of single-strand damage recognition protein XRCC1 was down-regulated in both the carbon ion group and the carbon ion combined bleomycin group,indicating that the single-strand DNA breaks(SSB)were gradually repaired.Next,compared with the anti-tumor drug ticecycline,it was found that carbon ions,ticecycline and their combination could inhibit the proliferation of A549 and H1299 cells and induce mitochondrial dysfunction.The toxic effect and mitochondrial dysfunction induced by the combination of carbon ions and ticecycline were the most significant.Mitochondrial dysfunction were shown as decreased ATP production,decreased mitochondrial membrane potential and increased mitochondrial Ca²⁺intake.The results further showed that in A549 cells,carbon ions,tigacycline and their combination induced apoptosis.However,in H1299 cells,carbon ions and their combined effects induced apoptosis and autophagy.At the molecular level,compared with the response pathway AMPK/mTOR of tigecycline,carbon ions and its combination with tigecycline changed mitochondrial translation proteins through the Akt/AMPK/mTOR pathway to affect mitochondrial translation,thereby controlling mitochondrial function.P53deficient H1299 cells were sensitive to mitochondrial translation inhibition.Finally,our preliminary investigation found that ATM/p53/mTOR signaling pathway is the common regulatory pathway of DNA damage and mitochondrial dysfunction.Inhibition of ATM increased the radiossensitivity of A549 cells to carbon ions.It mainly showed that supression of ATM could down-regulate the expression of DSB repair factors Ku70 and?H2AX,weaken the ability of DNA damage repair,and increase the uptake of Ca²⁺of mitochondria,leading to mitochondrial dysfunction.At the same time,inhibition of ATM could decrease the phosphorylation of p53 and increase the phosphorylation of mTOR after irradiation.It was further found that block of p53 phosphorylation could up-regulate mTOR activity by reducing AMPK phosphorylation and activating Akt.These results suggest that ATM not only participates in DSB repair,but also regulates mTOR pathway through p53,thereby regulating mitochondrial function.Meanwhile,after24 hours of carbon ion irradiation,LC3-II/I ratio decreased and caspase-3 activity increased,and inhibition of ATM could decrease LC3-II/I ratio.Therefore,the decrease of autophagy level and the increase of apoptosis jointly determined the fate of cells.Inhibition of ATM aggravated this phenomenon.To sum up,this study compared the dynamic response of DNA damage induced by heavy ions with that of X-ray,and provided new insights into mitochondrial dysfunction induced by heavy ions.In addition,we also explored the common regulatory mechanism of DNA damage response and mitochondrial dysfunction induced by carbon ion and connected the two-way signal between nucleus and mitochondria.This study provides the direct biological basis for the heavy ion cancer therapy.