| At present,cancer is still one of the major diseases endangering public health.According to the data of the International Cancer Agency in 2020,China ranks first in the list of newly diagnosed tumors,which seriously affect people’s quality of life and health.Cancer treatment has always been a social concern.Radiotherapy,the cornerstone of many cancer treatments,uses high-energy ionizing radiation（IR）to produce DNA double-strand breaks,induce cell cycle arrest,senescence,and various cell death patterns,including apoptosis,necrosis,autophagy,and mitotic catastrophes,ultimately to kill tumor cells.A large number of literatures have reported that the combination of traditional Chinese medicine with radiotherapy,chemotherapy or targeting therapy can improve the efficacy of radiotherapy,reduce the occurrence of related adverse reactions and complications,improve the quality of life and prolong the survival time of patients.Gallic Acid（GA）is widely found in gallnut and other plants,and has a variety of biological activities,including anti-cancer,anti-inflammatory,antiviral,anti-infection,cardiovascular protection,liver protection and enhancement of biofilm performance.Gallic Acid/Lecithin Complex（GA/LC）is composed of GA and lecithin molar ratio of 1:1.The synthesized nanomaterial has low toxicity of phospholipid,safe and simple synthesis method,and is combined with the parent drug GA to improve the biological activity,bioavailability and pharmacological activity of GA,and the prepared GA/LC can protect the liver and inhibit the growth of tumor cells,which is related to its participation in the antioxidant/oxidation effect.Ferroptosis is a kind of iron-dependent cell programmed death mode,and is different from cell apoptosis,cell necrosis and autophagy.Main mechanism is the polyunsaturated fatty acids containing phos-pholipids in cell membrane.PUFA-PLs is prone to peroxidation under the condition rich in iron and reactive oxygen species（ROS）,named lipid peroxidation.The accumulation of lipid peroxides in the cell membrane will eventually destroy the integrity of the membrane and thus induce cell death.This study mainly investigated the effects and molecular mechanisms of GA/LC combined with ionizing radiation on inducing ferroptosis by lipid peroxidation in non-small cell lung cancer cells,thereby enhancing the radiosensitivity of tumor cells.Objective:To explore the effects and molecular mechanisms of GA/LC mediated-ferroptosis to enhance the radiosensitivity of lung cancer cells,and to provide a new sensitizer for radiation killing of tumor cells.Methods:1.The optimal preparation conditions of GA/LC were determined by the combination of mathematical model and experimental conditions.Finally,GA/LC was synthesized by selecting 15 m L ethanol as the reaction solvent（mass concentration of GA was 12.5 mg/m L）,1:1 as the molar ratio of GA to phospholipid,50℃as the reaction temperature,and 3 h as the reaction time.2.X-rad 320i X deep irradiator was used for irradiation,irradiation conditions were as follows:voltage is 180 k V,current is 20 m A,target distance is 70 cm,dose rate is 1.02 Gy/min,dose is 6 Gy.3.The effects of GA/LC and irradiation on the proliferation ability of A549 and H1299 cells were detected by CCK-8 detection kit,and the optimal dose,treatment time and irradiation dose were selected.4.After A549 and H1299 cells were pretreated by Ferrostatin-1（Fer-1）,a ferroptosis inhibitor,cells were treated by GA/LC and irradiation,and then cell proliferation were detected by 5-ethynyl-2’-deoxyuridine（Ed U）staining.5.The changes of mitochondrial membrane potential and ROS in A549 and H1299 cells after GA/LC and irradiation treatment were detected by flow cytometry.6.The changes of colony formation ability of A549 and H1299 cells treated with GA/LC and radiation were detected by clonal formation assay.7.DCFH-DA probe was used to detect ROS production in A549 and H1299 cells after GA/LC and irradiation treatment,and fluorescence intensity was observed with Cytation 3 cell imaging multifunctional detection system.8.The changes of iron content in A549 and H1299 cells after GA/LC and irradiation treatment were detected by tissue iron（Fe）assay kit.9.Malondialdehyde（MDA）detection kit was used to detect the changes of MDA content in A549 and H1299 cells after GA/LC and irradiation treatment.10.The expressions of Nrf2,GPX4,ACSL4 and SLC7A11 were detected by Western blot after GA/LC,radiation,NAC and Fer-1 pretreatment.11.The effects of Erastin,a SLC7A11 inhibitor,on the expression of the proteins described above were detected by Western blot.12.SPSS 24.0 was used for statistical analysis.Cell survival rate,percentage of cell cycle distribution,cell apoptosis rate,intracellular iron content and MDA content were all expressed（x±s）,which were consistent with normal distribution by normality test.Analysis of variance was used for sample mean comparison among multiple groups,and independent sample t test was used for pair comparison between groups.P<0.05was considered statistically significant.Plot the resulting graph using Graph Pad Prism8 software.Results:1.Screening of GA/LC concentration,treatment time and ionizing radiation doseThe inhibitory effect of GA/LC on the proliferation of A549 and H1299 cells increased with the increase of drug concentration and treatment time,and the inhibitory effects of radiation on the proliferation in these two cells increased with the increase of radiation dose.After IC50 calculation and combined with references,the optimal concentration of GA/LC was determined to be 0.16 mg/m L in A549 cells,0.14 mg/m L in H1299 cells,the optimal treatmeng time was 24 h,and the optimal radiation dose was 6 Gy.2.Sensitizing effect of GA/LC on A549 and H1299 cells（1）GA/LC enhanced radiation induced-proliferation inhibition of A549 and H1299 cellsGA/LC and 6 Gy irradiation alone could inhibit the proliferation of A549 and H1299 cells（P<0.05）,and when GA/LC and 6 Gy irradiation was combined,the proliferation inhibition effect on the two cells was more obvious（P<0.05）.（2）GA/LC inhibited the colony forming ability of A549 and H1299 cells induced by radiationIt was also observed that the combined effect of GA/LC and 6 Gy irradiation on the colony formation ability of A549 and H1299 cells was greater than that of GA/LC and 6 Gy alone,suggesting that GA/LC had sensitizing effects on the two kinds of cells.（3）Effects of GA/LC on radiation-induced migration of A549 and H1299 cellsWhen GA/LC was treated on A549 and H1299 cells,the migration ability was significantly reduced compared with the Control group,and the scratch spacing was wider.When GA/LC was combined with 6 Gy irradiation,the migration ability was further weakened,and the scratch spacing was more obvious than that treated with GA/LC and IR alone.3.Effects of GA/LC on mitochondria and oxidative damage of lung cancer cells induced by radiation（1）GA/LC can cooperate with radiation-induced ROS production in lung cancer cellsAfter a certain time of GA/LC treatment,the ROS production in A549 and H1299cells increased compared with the Control group.When GA/LC combined with 6 Gy,ROS was significantly more than that of GA/LC alone（P<0.05）,and DCFH-DA probe staining showed increased fluorescence intensity.（2）MDA content in lung cancer cells was increased by GA/LC combined with radiationCompared with the Control group,GA/LC and 6 Gy irradiation alone increased the MDA content of A549 and H1299 cells,and the combined action of GA/LC and 6Gy increased the MDA content of the two cells（P<0.05）.After pretreatment with ROS scavenger NAC,the increase of intracellular MDA content was inhibited.（3）The effect of GA/LC on the expression of oxidative stress protein in lung cancer cells induced by radiationCompared with the Control group,GA/LC and 6 Gy treatment could reduce the expression of oxidative stress-related proteins Nrf2,GPX4 and SLC7A11 in A549 and H1299 cells,and the combined effect was more obvious.However,the expression of ACSL4 was increased after the treatment of GA/LC combined with 6 Gy.（4）The effect of GA/LC on ionizing radiation-induced mitochondrial damage in lung cancer cellsCompared with Control group,the mitochondrial membrane potential of A549 and H1299 cells treated with GA/LC for a certain time was decreased.When GA/LC combined with 6 Gy,the mitochondrial membrane potential was further decreased compared with that treated with GA/LC and IR alone（P<0.05）,and the mitochondrial damage was more serious.The results of Mi To-Tracker Green probe staining were consistent with the results of flow cytometry.In the Control group,the fluorescence intensity was high and evenly distributed in the cytoplasm.GA/LC and 6 Gy irradiation alone reduced the fluorescence intensity,damaged mitochondria and reduced the integrity of mitochondrial membrane.The combination causes the loss of cell membrane integrity and weak fluorescence.4.Effects and molecular mechanisms of GA/LC on enhancing ferroptosis and radiosensitivity of lung cancer cells（1）Ed U staining results showed that Fer-1,a ferroptosis inhibitor,could save the proliferative activity of A549 and H1299 cells induced by GA/LC and 6 Gy,and Ed U staining highlights were restored compared with GA/LC and 6 Gy treatment groups.（2）The results of colony formation experiment showed that Fer-1,a ferroptosis inhibitor,could reduce the effects of GA/LC and 6 Gy on the colony formation ability of A549 and H1299 cells.（3）The results of iron ion detection showed that Fer-1,a ferroptosis inhibitor,could recover the effects of GA/LC and 6 Gy on the content of iron in A549 and H1299cells（P<0.05）.（4）Compared with the Control group,GA/LC and 6 Gy treatment could reduce the expression of ferroptosis related proteins Nrf2,GPX4 and SLC7A11 in A549 and H1299 cells,and the combined effect was more obvious.The expression of ACSL4increased after GA/LC and 6 Gy,and the increase was more obvious.Moreover,pretreatment of Fer-1,a ferroptosis inhibitor,could reduce the effect of GA/LC and 6Gy irradiation on the expression of ACSL4.5.Inhibitory effects of SLC7A11 on ferroptosisAfter Erastin pretreatment,compared with the Control group,the SLC7A11protein of A549 and H1299 cells was decreased after Erastin combined with GA/LC and 6 Gy.Moreover,when Erastin,GA/LC and 6 Gy were treated with the two cells at the same time,the SLC7A11 protein in the two cells was decreased.The expression SLC7A11 shows to disappear.Nrf2 and GPX4 expressions showed strong decreasing effect after Erastin pretreatment.ACSL4 protein in A549 cells was enhanced after GA/LC,6 Gy and Erastin treatment.However,in H1299 cells,the inhibition of SLC7A11 decreased the sensitivity of radiation to ferroptosis,and the combined effect of GA/LC and 6 Gy was also lost.These results indicated that the susceptibility of A549and H1299 cells to ferroptosis was significantly affected when the level of SLC7A11was inhibited.Conclusions:1.GA/LC can increase the radiosensitivity of lung cancer cells A549 and H1299by enhancing the reduction of clonogenesis ability,migration ability and proliferation inhibition induced by radiation.2.GA/LC can enhance the level of lipid peroxidation induced by radiation,thus inducing iron death.3.Fer-1,an iron death inhibitor,can reduce the effect of GA/LC on enhancing radiosensitivity.4.Erastin,a SLC7A11 inhibitor,significantly altered the effect of GA/LC on the expression of Nrf2/SLC7A11/GPX4 pathway,which is involved in the molecular regulation of GA/LC-induced iron-sensitised lung cancer.In general,GA/LC increases radiosensitivity to A549 and H1299 cells by increasing intracellular lipid peroxidation levels and mediating ferroptosis. |
