Targets And Mechanisms Of Glycyrrhetinic Acid Therapy For Lung Cancerbased On Quantitative Chemical Proteomics

Objective:Lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lung.With highly morbidity and mortality,it has become the leading cause of cancer death worldwide.As the progress of diagnosis and treatment technology,the treatment of lung cancer tends to be a precision model,among which various targeted therapies based on immunotherapy have made significant progress.Epidermal growth factor receptor（EGFR）inhibitors,including gefitinib,erlotinib,and osimertinib,have been shown to be effective in the treatment of non-small cell lung cancer,but their drug resistance limits their long-term efficacy.Therefore,it is very important to develop new lung cancer drugs to improve the therapeutic effect of lung cancer patients.Glycyrrhetinic acid is a natural pentacyclic triterpenoid compound isolated from glycyrrhiza licorice.It has obvious anticancer activity against lung cancer,but its target and mechanism remain unclear.Therefore,in this study,specific protein targets of glycyrrhetinic acid in the treatment of lung cancer were sought based on activity-based proteomics analysis（ABPP）,and the Mlecular mechanism of its pharmacological action was explored.It provides experimental reference for clinical application ofdrugs and new drug development.Methods:1.The effect of glycyrrhetinic acid on the proliferation and growth of different lung cancer cell lines was detected by CCK 8 method.2.Flow cytometry was usedto detect the effect of glycyrrhetinic acid on LLC cell apoptosis.3.Laser confocal fluorescence imaging and flow cytometry were used to detect the effect of glycyrrhetinicacid on the concentration of ROS and calcium ions in LLC cells.4.Click-chemistry reaction and gel electrophoresis were used to detect whether glycyrrhetinic acid could occupy the protein-binding siteof IAAprobe in LLC cells.5.Screen for the target proteins directly acted by glycyrrhetinic acid in LLC living cells through quantitative analysis and identification of enriched proteins via LC-MS/MS based on TMT,and high confidence target proteins of glycyrrhetinic acid in the treatment of lung cancer were explored via biogenicanalysis andbiological function analysis.6.The IAA probe and glycyrrhetinic acid labeling and competition experiments were conducted on recombinant mouse Casp3 and Prdx6 proteins using click chemistry.7.Fluorescence co-localization imaging was used to explore whether glycyrrhetinic acid could competewiththe protein sites occupied by the IAA?probe in LLC cells.8.Mlecular docking technology was used to explore the specific binding sites and binding stability of glycyrrhetinic acid with Casp3 and Prdx6 proteins.9.Flow cytometry and laser scanning confocal microscopy were used to detect the effects of glycyrrhetinic acid on LLC cell apoptosis,mitochondrial membrane potential and intracellular ROSafter theaddition of Casp3 inhibitor.10.Mouse in situ lung cancer model was established,and the effect of glycyrrhetinic acid on lungcancer was detected by invivo bioluminescenceimaging.11.The differential protein expression profiles of mouse lung cancer tissues in the administration group and the model group were analyzed by liquid chromatography-mass spectrometry（LC-MS/MS）.Results:1.Glycyrrhetinic acid significantly inhibited the growth of lung cancer cells.After treatment with glycyrrhetinic acid for 24 h,the IC₅₀value of glycyrrhetinic acid was 28.79μM for LLC cells,29.72μM for Calu-1 cells and30.10μM for A549 cells.2.Glycyrrhetinic acid could induce LLC cell apoptosis,and the apoptosis rates of LLC cells in the10μM group and 20μM group were19.38%and 86.76%,respectively（P<0.01）.3.Glycyrrhetinic acid could increase ROS and Ca²⁺level in LLC cells（P<0.05）.4.Glycyrrhetinic acid concentration-dependent occupied the binding sites of IAA probes in proteins in LLC cells.5.The target proteins Prdx6 and Casp3 directly affected by glycyrrhetinic acid were identified by ABPP technology and bioinformatics analysis.All the target proteins were mainly involved in endoplasmic reticulum stress,cell apoptosis and regulation of mitochondrial membrane permeability.6.Glycyrrhetinic acid concentration-dependent occupied the binding site of the IAA probe on Casp3 and Prdx6 proteins.7.Glycyrrhetinic acid can effectively compete for protein sites occupied by IAA?probes in LLC living cells.8.Glycyrrhetinic acid can form stable binding energy with Casp3 and Prdx6 proteins.9.The addition of Casp3 inhibitor slowed down the effect of glycyrrhetinic acid on LLC cell apoptosis,mitochondrial membrane potential decline and intracellular ROS accumulation（P<0.05）.10.Glycyrrhetinic acid could slow the growth rate of in situ lung cancer in mice（P<0.001）,and Casp3 inhibitor had certain antagonistic effect on the antitumor effect of glycyrrhetinic acid（P<0.05）.11.After the administration of glycyrrhetinic acid,the expression levels of different proteins were significantly changed,among which the metabolic pathways were concentrated in cellular oxidativestress,mitochondrial energy metabolism andcell apoptosis.Conclusion:Glycyrrhetinicacid up-regulated ROS level andpromoted oxidative damage by targeting Prdx6and Casp3 via mitochondrial apoptosis pathway,thus ledto apoptosis in NSCLC.