Mechanisms Of Mitochondrial F1Fo ATPase Inhibitors In Regulating Radiation Sensitivity Of Non-small Cell Lung Cancer

Lung cancer is a high-risk type of cancer worldwide.Deaths caused by lung cancer have become the leading cause of cancer death.In recent years,the incidence of lung cancer is becoming more and more common in developing countries.The number of lung cancer patients has increased significantly in female and is still the most common cancer in men.According to the histological characteristics and clinical features,lung cancer has been majorly subdivided into two main groups: small cell lung cancer and non-small cell lung cancer,and non-small cell lung cancer accounts for more than 75% of all.Radiation therapy is currently recognized as an effective treatment for malignant tumors.Local advanced lung cancer is very suitable for radiation therapy.However,long-term survival rate is still poor with a 5-year survival in 5-25%.With the development of basic research and the advancement of radiology technology,more and more methods have been used to improve the effect of radiotherapy.Among them,drugs combined with radiotherapy have achieved radiological synergistic effects.Multiple mechanisms,such as inhibiting the repair of radiation-induced damage,inhibiting proliferation,enhancing apoptosis and oxidation,make tumor more sensitive to radiation.F1Fo ATPase catalyzes the activity of ATP synthesis depend on mitochondrial transmembrane potential under normal physiology.Under special conditions(hypoxia or ischemia),it can play the revese role of ATP hydrolase and restore mitochondrial membrane potential.The small molecule BTB and the endogenous small molecule protein ATP inhibitor factor 1(IF1)are inhibitors that selectively inhibit the F1 Fo ATPase from hydrolyzing ATP without affecting the F1 Fo ATPase synthesis of ATP.This paper mainly studies the effects and mechanisms of these two inhibitors on radiation sensitivity.Firstly,pre-experiment was carried out in the model animal zebrafish to clarify the radiation response of F1 FoATPase,and the toxicity of BTB was determined.It was preliminarily determined that BTB had the protective effect of increasing ATP content after irradiation in zebrafish embryo.Then,the effect and mechanism of BTB to increase radiation sensitivity were determined in non-small cell lung cancer A549 cells.Finally,the regulation mechanism of IF1 on A549 radiation sensitivity was studied.This research found that:(1)under normal physiological conditions,F1 Fo ATPase has both ATP synthesis and hydrolysis activity in zebrafish embryo.Three different types of ionizing radiation(carbon ion beam,proton beam,X-ray)induce dose-dependent embryonic death and malformation in zebrafish embryos.After non-lethal doses of radiation,there was no significant change in the expression of many complexes in the mitochondrial respiratory chain,ATP levels were reduced,and expression of the respiratory chain complex V(F1Fo ATPase)was elevated.BTB treatment increased the ATP level of irradiated embryos,and oligomycin had no significant effect on ATP levels of irradiated embryos,indicating that ionizing radiation can induce F1 Fo ATPase hydrolysis activity.(2)Non-small cell lung cancer has both aerobic glycolysis and oxidative phosphorylation pathways for producing ATP.After ionizing radiation,the expression of F1 Fo ATPase is increased,the ability to hydrolyze ATP is enhanced,and the mitochondrial membrane potential level is reduced first and then restored.After BTB combined with radiation treatment,the mitochondrial membrane potential remained low during the detection period,the cell proliferation rate decreased,and the mitochondria-dependent apoptotic pathway was activated,which significantly increased the radiation-induced apoptosis and increased the radio-sensitivity.(3)IF1 also responds to the stimulation of ionizing radiation.IF1 expression level increases with the increase of radiation dose,and the dynamic change gradually increases and then decreases after irradiation,which result in the decrease of membrane potential in the beginning and restored later after radiation.The study found that the high expression of IF1 causes mitochondrial depolarization,activating the mitochondrial autophagy through the PINK1-Parkin pathway,thereby playing a role in clearing damaged mitochondria and retaining healthy mitochondria,preventing cells from mitochondria-dependent apoptosis.Subsequently,decreased IF1 expression ensures that the F1 FoATPase functions under physiological function.In this paper,it was confirmed that ionizing radiation can induce the ATP hydrolysis of F1 FoATPase in two types of energy metabolism system in zebrafish embryo and tumor cell.In zebrafish embryos,BTB play a protective role for increasing the ATP level after irradiation.Both BTB treatment and knockdown of IF1 protein have enhanced radiation sensitivity.The former induces apoptosis through a sustained decrease in membrane potential.The latter inhibits mitochondrial autophagy and disrupts mitochondrial quality control mechanisms.Therefore,targeting hydrolysis function of F1 FoATPase is a potential pathway for radiation sensitization,which has positive significance for improving the effectiveness of radiation therapy.