Effects Of Sodium Butyrate On Radiosensitivity Of NSCLC A549 Cells And Its Mechanisms

Background:Lung cancer is the most common malignant tumor and the leading cause of cancer death.About 70%of non-small cell lung cancer（NSCLC）cases are diagnosed as advanced.Most patients are difficult to resect when diagnosed,and their treatment mainly depends on the combination of chemotherapy and radiotherapy.However,the radiation resistance of tumor and the side effects of radiation therapy limit its therapeutic effect and application.Combined use of radiosensitizer can improve its therapeutic potential.Sodium butyrate（NaBt）is a histone deacetylase inhibitor（HDACi）,which has been shown to inhibit the proliferation of tumor cells,promote apoptosis,and delay the invasion and metastasis of tumor cells in a variety of cancers.However,its exact role and mechanism in NSCLC have not been clarified.Purpose:To investigate the effect of NaBt on the radiosensitivity of NSCLC A549 cells and its mechanism,and to clarify the role of MDM2/p53/p21 signaling pathway.Methods:1.Interfering plasmid construction and transfection expression:p53 siRNA interfering plasmid was constructed and transfected into A549（p53 wild-type）cells to obtain A549（p53 KD）transient transfected cell line.2.CCk-8 method:The effect of NaBt combined with ionizing radiation on the proliferation activity of A549 cells was detected to determine the optimal concentration of NaBt and the time of administration.3.Clone formation experiment:Before and after p53 silencing,the effects of NaBt combined with ionizing radiation on colony formation ability of A549 cells were detected.4.Flow cytometry:Apoptosis（Annexinv-FITC staining）,Ca²⁺（Fluo-3AM probe）,ROS（DCFH-DA probe）,mitochondrial membrane potential（Rhodamine 123 staining）and cell cycle distribution（PI staining）of A549 cells were detected after treatment with NaBt and ionizing radiation.5.qRT-PCR method:After NaBt and ionizing radiation,the expression changes of apoptosis and cell cycle related genes in A549 cells were detected.6.Western Blot method:Apoptosis of A549 cells and expression changes of cell cycle-related proteins were detected after NaBt and ionizing radiation,and silencing was verified after transfection with siRNA-p53.7.Immunofluorescence method:cell mitochondrial membrane potential was detected.8.Statistical analysis:Graphpad prism 7 statistical software was used,and data were expressed as mean±standard deviation（（?）±s）.One-way ANOVA was used for comparison of sample data between multiple groups,and independent sample T test was used for comparison of sample mean between two groups.Results:1.Effects of NaBt combined with ionizing radiation on proliferation of A549 cellsNaBt inhibited the proliferation of A549（p53 wild-type）cells in a concentration and time dependent manner（P<0.05 or P<0.01）,and combined with ionizing radiation further inhibited the proliferation of A549（p53 wild-type）cells（P<0.05 or P<0.01）.After A549（p53 KD）cells were treated with NaBt alone or in combination with 4GyX-ray for 24 h,the proliferation rate of A549（p53 KD）cells in the irradiation group after p53 silencing was increased compared with that before silencing（P<0.05）.In the irradiation combined with NaBt group,the cell proliferation rate was also increased after p53 silencing compared with before silencing（P<0.05）.2.Effects of NaBt combined with ionizing radiation on colony formation ability of A549 cells.A549（p53 wild-type）cells were irradiated with 2,4,6 and 8 Gy,and the number of colony formation was reduced compared with the non-irradiated group,and the decrease was more obvious with the increase of dose.After combined with NaBt,the number of colony formation also showed a significant decreasing trend compared with the irradiation group.The number of colony formation in A549（p53 KD）cells was significantly increased after 2,4,6,8 Gy irradiation after p53 silencing compared with 2,4,6,8 Gy irradiation without p53 silencing.After p53 silencing,the number of colony formation in the irradiation combined with NaBt group also showed an obvious increase trend compared with the non-silencing p53 irradiation combined with NaBt group.3.Effects of NaBt combined with ionizing radiation on apoptosis of A549 cellsThe apoptosis rate of A549 cells（p53 wild-type）treated with NaBt alone or in combination with 4 Gy X-ray for 24 and 48 h was significantly lower than that of the control group,and the apoptosis rate of NaBt combined with NaBt was significantly higher than that of the control group alone（P<0.01）.A549 cells（p53 KD）were treated by NaBt alone or in combination with 4 GyX ray for 24 h,the apoptosis rate of the control group,the 4 Gy X ray irradiation group and the 10 m M NaBt combined with 4 Gy X ray irradiation group after p53 silencing was decreased compared with that before silencing（P<0.05）.4.Effects of NaBt on ROS induced by ionizing radiation in A549 cellsNaBt alone or combined with 4 GyX-ray irradiation increased ROS level in A549cells（P<0.01）,and the increase was more obvious in the combined group.5.Effects of NaBt combined with ionizing radiation on mitochondrial membrane potential of A549 cellsNaBt alone or combined with 4 GyX-ray irradiation caused a decrease in mitochondrial membrane potential of A549 cells（P<0.05 or P<0.01）,and the decrease was more obvious in the combined group.6.Effects of NaBt combined with ionizing radiation on Ca²⁺generation in A549cellsNaBt alone or combined with 4 GyX-ray irradiation increased Ca²⁺in A549 cells（P<0.01）,and the increase was more obvious in the combined group（P<0.01）.7.Effects of NaBt combined with ionizing radiation on cell cycle of A549NaBt alone induced G₁phase arrest of A549 cells,and ionizing radiation alone or combined with NaBt induced G₂ phase arrest,which was more obvious in combined group（P<0.01）.NaBt alone or in combination with ionizing radiation can reduce the gene and protein expression levels of Cyclin B,and the decrease is more obvious in the combined group.8.The role of MDM2/p53/p21 signaling pathway in the effect of NaBt on the radiosensitivity of A549 cells.NaBt alone or in combination with 4 GyX-ray irradiation can effectively activate p53 in A549 cells and affect the transcription of p53 regulatory genes,including increasing the expression of cell cycle regulator p21 and decreasing the expression of MDM2,ultimately leading to cell cycle arrest and cell death,thereby improving the radiosensitivity of NSCLC A549 cells.Conclusions1.NaBt alone can inhibit the proliferation activity of A549 cells,increase ROS level,decrease mitochondrial membrane potential,increase Ca²⁺concentration,induce G₁ phase arrest and induce apoptosis.2.NaBt may enhance the radiosensitivity of A549 cells by apoptotic pathway.3.MDM2/p53/p21 pathway plays an important role in the radio-sensitization of NaBt.