| Background: The number of cases and deaths of lung cancer is increasing year by year,so the lung cancer has become one of the main causes of cancer-related death worldwide.Malignant tumors,including lung cancer,bypass the cellular senescence process at the onset of the disease by epigenetic mutations or downregulation of relevant tumor suppression-related pathways,and gain the ability to replicate indefinitely.In senescent cells,DNA damage,cell cycle arrest,proliferation and migration ability will be weakened,and the senescent cells do not cause damage to normal tissue.Therefore,inducing lung cancer cells to enter the senescence state is a win-win treatment that can inhibit the proliferation of cancer cells and protect the function of normal cells to the greatest extent.Artemisinin has never been shown to have strong side effects since it was isolated as an antimalarial drug,so it has attracted much attention from researchers.There have studies show that artemisinin can increase the level of intracellular reactive oxygen species(ROS),damage the DNA of tumor cells,and induce tumor cells to enter G1 phase cell arrest,all of which are related to cell senescence.At present,there are no studies on the improvement of lung cancer by artemisinin induced senescence of tumor cells.Peptidyl arginine deiminase 4(PADI4)is a calcium-dependent protein that inhibits senescence cell production in model of Alzheimer’s disease,but its role in lung cancer is unclear.Purpose: This study is to investigate the relationship between artemisinin and PADI4 in lung cancer prevention and their underlying mechanism.Methods: In vitro studies,EDU experiment was used to explore the effect of artemisinin on DNA replication in lung cancer cells.Fluorescence probe DCFH-DA was used to detect the effect of artemisinin on ROS levels in lung cancer cells.DNA damage of lung cancer cells induced by artemisinin was detected by comet tail assay.The effects of artemisinin on the morphology,volume and surface area of lung cancer cells were detected by laser holography and cell morphology analysis.The effect of artemisinin on mitochondrial membrane potential in lung cancer cells was detected by JC-1 kit.Lysosome probe was used to detect the effect of artemisinin on the morphology and function of lysosome in lung cancer cells.The cell cycle arrest effect of artemisinin on lung cancer cells was tested by cell cycle kit.In vivo studies,the anti-tumor effect of artemisinin in the host versus graft model was detected.The direct antitumor effect of artemisinin was detected in tumor allograft model.The urethane and smokinginduced lung carcinogenic model was established to detect the ameliorative effect of artemisinin on lung cancer.The metabolism system was used to detected the protective effect of artemisinin on the lung function in lung cancer mice.The effect of artemisinin on the survival state of the mice with lung cancer was detected by the number of independent activities.The pathological changes of lung tissue of lung cancer mice were detected by HE staining.The inhibitory effect of artemisinin on tumour progression in lung cancer mice was detected by small animal in vivo imaging instrument.Transcriptome was used to analyze the differential genes in the solid tumor between host versus graft model and tumor allograft model.The results of transcriptome analysis were verified by Western blotting(WB).Molecular docking predicted the binding effect of artemisinin and the different expression gene that analyze by transcriptome.Immunohistochemistry was used to detected the effect of artemisinin on key proteins in lung tissue of mice with lung cancer.Mechanism study,PADI4 overexpression A549(A549O)cell line was established.A549 O cell line and PADI4 inhibitor GSK484 were used as controls to investigate the effects of PADI4 expression level on proliferation and migration of A549 cells.Network pharmacology was used to analyze search the downstream targets of artemisinin and the pathway that artemisinin involved in.Target affinity assay was used to detect the direct interaction between artemisinin and PADI4 and downstream targets.The proteinprotein interaction between PADI4 and downstream targets was analyzed and verified by protein-protein docking,GEO gene chip mining and co-immunoprecipitation(CO-IP)experiments.In the urethane and smoking-induced lung carcinogenic model,immunofluorescence was used to verify the effect of artemisinin on PADI4 and downstream targets,and whether the relative expression of PADI4 and downstream targets was consistent with the predicted trend.SA-β-gal staining was used to detect the effect of artemisinin on the senescence level of lung cancer cells in vitro and in vivo.WB verified the effect of artemisinin on PADI4 and P53 related senescence pathway proteins in vitro and in vivo.Result: In vitro studies showed that EDU inclusion in artemisinin administration groups were significantly reduced compared with control group in the EDU experiment(P < 0.05),indicating that artemisinin could reduce the proliferation ability of A549 cells in a dose-dependent manner.In ROS assay,compared with control group,the absorption peak at 525 nm in artemisinin administration groups were significantly increased(P < 0.05),indicating that artemisinin could increase ROS level in A549 cells in a dose-dependent manner.The comet trailing experiment,compared with control group,the tail of DNA in artemisinin administration groups were significantly longer(P < 0.01),indicating that artemisinin could damage the DNA of A549 cells in a dose-dependent way.The results of laser holographic detection showed that,compared with the control group,the cells in the artemisinin administration groups lost the spindle structure,the cell body appeared flat shape,and the cell volume and surface area increased significantly(P< 0.01),indicating that A549 cells stimulated by artemisinin showed the morphological characteristics of cell senescence.After lysosome probe incubation,the lysosome fluorescence probe response was enhanced in the artemisinin administration group compared with the control group,indicating that artemisinin could increase the lysosome function of A549 cells in a dose-dependent manner.After JL-1 probe incubation,the red-light intensity of cells in artemisinin administration groups were significantly decreased and the green light intensity was significantly increased compared with the control group(P < 0.01),indicating that artemisinin could disrupt the mitochondrial membrane potential of A549 cells in a dose-dependent way.In the cell cycle detection,compared with the control group,the proportion of G1 phase cells in artemisinin administration groups were significantly increased(P < 0.01),indicating that artemisinin could arrest A549 cell cycle in G1 phase in a dose-dependent manner.In vivo studies showed that the tumor mass and tumor growth rate were significantly decreased in the artemisinin administration groups compared with the control group in the host versus graft model(P <0.05),indicating that artemisinin could inhibit solid tumor growth in the host versus graft model in a dosedependent manner.In the tumor allograft model,the tumor inhibition rate in the artemisinin administration group was 13.6% compared with the control group,which was much lower than it in the host versus graft model.In the urethane and smoking-induced lung carcinogenic model,compared with the model group,the respiratory metabolism,voluntary activity and survival rate of mice in the artemisinin administration group were significantly increased(P < 0.05),and the size and number of lung tumor nodules were significantly decreased(P < 0.05),indicating that artemisinin can protect lung function of lung cancer mice in a dosedependent manner,improve the survival state and survival rate of lung cancer mice,and improve the lung cancer progression in lung cancer mice.Transcriptome results showed that PADI4 was the most significant differential gene between host versus graft model and tumor allograft model by log FC and P-value sequencing.Molecular docking results showed that artemisinin had a good docking fraction with PADI4,and PADI4 may be the key target of artemisinin in improving lung cancer.Immunohistochemical results showed that PADI4 expression was significantly increased in the model group compared with the normal group(P < 0.01),the expression of PADI4 in artemisinin administration group was significantly lower than that in model group,suggesting that artemisinin could reduce PADI4 expression in lung tissue of lung cancer mice in a dose-dependent manner.The mechanism study showed that,in the Plate cloning assay and Transwell assay,compared with A549 group,the number of clones and migrating cells in A549 O group was increased significantly(P<0.05),but the number of clones and migrating cells in GSK484 group and artemisinin administration groups were significantly decreased(P<0.05),indicating that the expression level of PADI4 was positively correlated with the proliferation and migration capacity of A549 cells,and artemisinin could inhibit the proliferation and migration capacity of A549 cell.Network pharmacological analysis showed that only P53 and PADI4 had protein interaction in PPI network,and it had the highest degree value,which may be the downstream target of PADI4.Artemisinin is mainly involved in four pathways: "P53 signaling pathway","reactive oxygen species","cell cycle" and "senescence pathway".Target affinity experiment showed that artemisinin had direct interaction with PADI4,but not with P53.COIP experiment showed that there was an interaction between PADI4 and P53.Protein-protein docking and GEO gene chip mining results showed that the low expression level of PADI4 is more conducive to the expression of P53,which was further verified by tissue immunofluorescence.In the SA-β-gal staining assay,compared with A549 group,the percentage of senescent cells in the A549 O group was significantly decreased(P<0.05),the percentage of senescent cells in GSK484 group and artemisinin administration groups was significantly increased(P<0.05),indicating that the expression level of PADI4 was inversely correlated with the senescence level of A549 cells,and artemisinin could induce the senescence of A549 cells in a dose-dependent manner.In the SA-β-gal staining experiment of lung tissue sections of lung cancer mice,the percentage of senescent cells in the sections of artemisinin administration groups was significantly increased compared with the model group,suggesting that artemisinin could induce senescence of tumor cells in lung tissue of lung cancer mice in a dose dependent manner.The results of WB showed that artemisinin significantly decreased the level of PADI4 and increased the expression levels of P53 and P53-related senescence pathway proteins in vitro and in vivo(P<0.05).Conclusions: In conclusion,artemisinin has a significant improvement effect on lung cancer.By reducing the level of PADI4 and increasing the protein expression levels of P53,P21 and P16 in the p53-related senescence pathway,artemisinin can induce lung cancer cells to enter the senescence state,which may be an important mechanism for artemisinin to improve lung cancer. |
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