| Background and purposeAt present,cancer is one of the major diseases that seriously threaten human health,and the death caused by lung cancer ranks first among cancer-related death.The risk factors of lung cancer include smoking,working environment,environmental pollution,ionizing radiation and previous pulmonary infection.At present,the treatment methods for cancer include surgery,radiotherapy,chemotherapy,targeted therapy,immunotherapy and so on.However,the 5-year survival rate of patients is still low,and the problems such as tumor recurrence,serious adverse reactions and drug resistance cannot be effectively solved.In recent years,with the internationalization of Chinese medicine and its unique advantages in modern diseases,Chinese medicine has been more and more recognized at home and abroad.Many natural ingredients derived from traditional Chinese medicine exhibit antitumor activities by inhibiting proliferation,inducing apoptosis,inhibiting metastasis,angiogenesis,regulating autophagy,reversing drug resistance,balancing immunity,and enhancing chemotherapy.Dioscin is derived from Dioscoreaceae plants,including Dioscorea,Dioscorea nipponica,etc.It has been reported that dioscin has therapeutic effect on lung cancer,but its mechanism has not been further studied.In this study,lung adenocarcinoma cell lines and mouse lung adenocarcinoma model were used to explore the anti-tumor effect of dioscin and its mechanism,and provide some clues and basis for the study of dioscin as an anti-tumor drug.Methods1.Evaluation of anti-tumor activity of dioscin in vivoThe mouse lung adenocarcinoma model was induced by cigarette smoke(twice a day)and urethane(600 mg/kg,once a week)for 8 weeks.At the same time,control group and model group were given 0.5%CMCC-Na,25 mg/kg group,50 mg/kg group,100 mg/kg group were given the corresponding concentration of dioscin suspension,continuous administration for 12 weeks,weighing once a week.After the experiment,the lung tissue was carefully removed and photographed,and the number of lung tumors in mice was counted.Part of the lung tissue was cut with a scalpel and stored in a refrigerator at-80℃ for protein extraction.The protein expression of E-cadherin,MMP2,active-caspase3 and PCNA was detected by Western blot.The remaining lung tissue was fixed in 4%paraformaldehyde for H&E staining,IF staining(Ki67),TUNEL staining,IHC(PCNA,active-caspase3).In addition,liver and kidney were collected and weighed to calculate organ index.2.Evaluation of anti-tumor activity of dioscin in vitro(1)MTT assay was used to explore the effect of dioscin on cell viabilityLung adenocarcinoma cells A549 and PC-9 were treated with different concentrations of dioscin for 24 hours,48 hours and 72 hours,respectively.MTT assay was used to detect the effect of dioscin on the viability of lung adenocarcinoma cells and IC50 was calculated.At the same time,normal lung epithelial cells BEAS-2B and colon epithelial cells NCM460 were treated with dioscin for 24 hours.MTT method was also used to detect cell viability and compared with lung adenocarcinoma cells to explore whether dioscin had a stronger inhibitory effect on lung adenocarcinoma cells.(2)Laser holographic imaging experiment was used to explore the effect of dioscin on the morphology and volume of A549 and PC-9 cellsAfter A549 and PC-9 were treated with dioscin for 24 hours,the effects on cell morphology and volume were investigated by laser holographic imaging and analysis system.(3)EdU experiment and clone formation experiment were used to explore the effect of dioscin on the proliferation of A549 and PC-9 cellsAfter A549 and PC-9 cells were treated with dioscin for 24 hours,100 μL containing 50μM EdU medium was added to each well for 2 hours,and then 4%paraformaldehyde was added to fix the cells.Finally,Apollo staining and Hoechst 33342 staining were performed.The images were collected under fluorescence microscope,and the effect of dioscin on the proliferation of lung adenocarcinoma cells was analyzed by EdU positive rate.At the same time,clone formation assay was used to further explore the effect of dioscin on the proliferation of lung adenocarcinoma cells.(4)AO/EB and Annexin V/PI double staining experiments were used to explore the effect of dioscin on apoptosis of A549 and PC-9 cellsAfter the A549 and PC-9 cells were treated with dioscin for 24 hours,the effects of dioscin on the apoptosis of A549 and PC-9 cells were detected by AO/EB double staining and Annexin V/PI double staining.(5)Single cell gel electrophoresis experiment(comet assay)was used to explore the effect of dioscin on DNA damage in A549 and PC-9 cellsAfter A549 and PC-9 cells were treated with dioscin for 24 hours,the cells were digested and collected,and the cells were resuspended with an appropriate amount of PBS solution.Comet assay was used to explore the effect of dioscin on DNA damage of A549 and PC-9 cells.(6)Mitochondrial membrane potential detection kit was used to explore the effect of dioscin on membrane potential in A549 and PC-9 cellsAfter A549 and PC-9 cells were treated with dioscin for 24 hours,1 mL JC-1 staining solution was added to each well and incubated at 37℃ for 20 minutes.Finally,the changes of mitochondrial membrane potential were detected by fluorescence microscope.(7)Reactive oxygen species detection kit was used to explore the effect of dioscin on reactive oxygen in A549 and PC-9 cellsAfter A549 and PC-9 cells were treated with dioscin for 24 hours,1 mL medium containing DCF-DA fluorescent probe was added to each well,and incubated at 37℃ for 20 minutes.Finally,the fluorescence changes of DCF were detected by fluorescence microscope,which indirectly reflected the changes of intracellular reactive oxygen.(8)Transwell experiment was used to explore the effect of dioscin on the invasion and migration of A549 and PC-9 cellsMigration assay:A549 and PC-9 cells were treated with dioscin for 24 hours,then the cells were digested and collected,washed with PBS for 3 times.The cells were resuspended in serum-free medium,200 μL cell suspension was added in the upper chamber,and 10%FBS complete medium was added in the lower chamber.After conventional culture for 24 hours,the medium was discarded,and 1 mL 4%paraformaldehyde was added to each well for 30 minutes.Using cotton swabs to wipe the cells from the upper chamber and stain cells with 0.1%crystal violet solution.The crystal violet solution was washed by tap water,and the images were collected by microscope after drying.Invasion experiment and migration experiment steps are basically same,except in the small room using matrix gel(1:8 dilution)simulated extracellular matrix environment.(9)The effects of dioscin on E-cadherin,MMP2,active-caspase3 and PCNA in A549 and PC-9 cells were detected by Western blot.The cells were digested and collected after A549 and PC-9 cells were treated with dioscin for 24 hours.Each sample was treated with an appropriate amount of RIPA lysate(containing 1%protease inhibitor and 1%phosphatase inhibitor)for 30 minutes.12000g,centrifuged at 4℃ for 10 minutes.Carefully absorbing the supernatant to 500 μL EP tube,using BCA kit for protein quantification.Adding the corresponding volume of 5 × Loading buffer and boiling the samples for 5 minutes.Finally,Western blot was used to detect the changes of E-cadherin,MMP2,active-caspase3,PCNA in A549 and PC-9 cells after dioscin treatment.3.Study on anti-tumor mechanism of dioscin(1)Network pharmacology analysisBased on the research strategy of network pharmacology,the potential targets of dioscin were collected from HERB database,and the targets of lung adenocarcinoma was acquired from the Genecard database.The ’dioscin-target network’ was constructed by Cytoscape software.At the same time,using an online tool to get dioscin and lung adenocarcinoma intersection targets.The intersection targets were import into the STRING database to acquire the protein-protein interaction network(PPI network).The PPI network was analyzed by the plug-in in Cytoscape software and the top five core targets were determined according to the degree value.Gene ontology analysis(cellular component,molecular function,biological process)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed by David database.(2)Molecular docking of dioscin with core targets2D structure of dioscin was download from PubChem database and the crystal structures of core targets were acquired from Protein Data Bank(PDB)database.After pretreatment of protein and ligand,Autodock Vina software was used for molecular docking of dioscin with core targets.(3)Drug affinity target stability experiment(DARTS)was used to explore the binding of dioscin to core targetsA549 cells were lysed with M-PER lysate containing 1%protease inhibitor and 1%phosphatase inhibitor for 30 minutes to extract total protein.After protein quantification,300μg total protein solution and corresponding final concentration of dioscin(0 μM,10 μM,100μM,1000μM)were added to each tube,and incubated at 4℃ for 12 hours.After incubation,1.25 μg streptomycin was added to each tube and incubated for 20 minutes at room temperature.Finally,13μL 5× loading buffer was added to terminate the reaction,and the prepared samples was used for further Western blot analysis.(4)Cellular Thermal Shift Assay(CETSA)was used to explore the binding of dioscin to AKT1A549 cells were collected and the total protein was extracted by RIPA lysate.After incubation with dioscin or solvent,the soluble components were extracted after heating at different temperatures.The expression of AKT1 protein was investigated by western blot.(5)Effect of dioscin on AKT1 protein stability was detected by protein half-life experimentA549 cells were treated with 0.1%DMSO or 5.75 μM dioscin,and then treated with 20μg/mL cycloheximide for 0 minute,30 minutes,60 minutes,90 minutes and 120 minutes,respectively.Finally,the total cell protein was extracted with RIPA lysate,and the expression of AKT1 was detected by Western blot.(6)Effect of dioscin on AKT1 mRNA level in A549 cells detected by qRT-PCRAfter A549 cells were treated with dioscin for 24 hours,the cells were digested and collected.Total mRNA was extracted by Trizol lysis chloroform extraction.After quantification,reverse transcription was performed using a kit.Finally,qRT-PCR was used to detect the change of AKT1 mRNA level in A549 cells after dioscin treatment.(7)The protein expression of AKT1,p-AKT1,mTOR,p-mTOR in lung adenocarcinoma cells and mouse lung tumors tissues after dioscin treatment were detected by Western blotAfter A549 and PC-9 cells were treated with dioscin for 24 hours,the total protein was extracted and the protein levels of AKT1,p-AKT1,mTOR and p-mTOR were detected by Western blot.Similarly,the protein levels of AKT1,p-AKT1,mTOR and p-mTOR in mice lung tumor tissues were detected by Western blot.(8)Effects of dioscin on proliferation,apoptosis,invasion and migration of A549 cells after silencing AKT1A549 cells were treated with siRNA for 48 hours,and the total protein were extracted to verify the knock-down efficiency.At the same time,EdU experiment,Transwell experiment and Annexin V/PI double staining experiment were used to explore the effects of dioscin on the proliferation,apoptosis,invasion and migration of A549 cells after AKT1 silencing.Results1.Dioscin inhibited the development of mouse lung adenocarcinoma(1)The mouse lung adenocarcinoma animal model was successfully constructed using urethane and cigarette smoking.Compared with the model group,25 mg/kg group,50 mg/kg group and 100 mg/kg group could significantly reduce the number of lung tumors in mice.In terms of body weight,except for the control group,there was no significant difference in body weight between the model group and the 25 mg/kg group,50 mg/kg group and 100 mg/kg group.Further H&E experiments showed that compared with the model group,the 25 mg/kg group,50 mg/kg group,and 100 mg/kg group could reduce lung tissue damage in mice.(2)Compared with the control group,the lung index of the model group was significantly increased.Compared with the model group,the 25 mg/kg group,50 mg/kg group and 100 mg/kg group significantly reduced the lung index,and the lung index gradually returned to normal.Similarly,the kidney index and liver index statistics found that there was no significant difference between the kidney index and liver index.(3)IF detection of Ki67 expression in lung tumor tissue of mice found that compared with the model group,the 25 mg/kg group,50 mg/kg group and 100 mg/kg group could significantly reduce Ki67 expression.At the same time,the expression of PCNA in lung tumor tissue of mice was detected by IHC and Western blot.Compared with the model group,the expression of PCNA was significantly decreased in the 25 mg/kg group,50 mg/kg group and 100 mg/kg group.Therefore,the expression of Ki67 and PCNA in lung tumor tissue of mice showed that dioscin could inhibited the proliferation of tumor cells.(4)TUNEL detection of apoptosis in mouse lung tumor tissue showed that compared with the model group,25 mg/kg group,50 mg/kg group and 100 mg/kg group could significantly increase the number of TUNEL positive cells.At the same time,the expression of active-caspase3 in lung tumor tissue of mice was detected by IHC and Western blot.Compared with the model group,25 mg/kg group,50 mg/kg group and 100 mg/kg group could significantly increase the expression of active-caspase3.Therefore,the expression of active-caspase3 detected by TUNEL assay,IHC and Western blot showed that dioscin could induce apoptosis of tumor cells.(5)The expression of E-cadherin and MMP2 in lung tumor tissue of mice was detected by Western blot.Compared with model group,the expression of E-cadherin was significantly increased and the expression of MMP2 was significantly decreased in 25 mg/kg group,50 mg/kg group and 100 mg/kg group.Therefore,the expression of Ecadherin and MMP2 detected by Western blot showed that dioscin could inhibited the metastasis of tumor cells.2.Dioscin inhibited proliferation,invasion,migration,and induced apoptosis of A549 and PC-9 cells(1)MTT assay showed that dioscin inhibited the cell viability of A549 and PC-9 cells in a concentration-dependent and time-dependent manner.Compared with lung epithelial cells BEAS-2B and colon epithelial cells NCM460,dioscin has stronger inhibitory effect on lung adenocarcinoma cells.(2)After dioscin treatment,laser holographic detection showed that the volume and number of A549 and PC-9 cells decreased,and the cell morphology shrank and became round.The EdU experiment and clone formation experiment showed that dioscin could significantly inhibit the proliferation and clone formation of A549 and PC-9 in a concentration-dependent manner.(3)AO/EB and Annexin V/PI experiments showed that the number of apoptotic cells in A549 and PC-9 cells significantly increased after dioscin treatment.(4)Wound healing experiment showed that the migration ability of A549 and PC-9 cells significantly reduced after dioscin treatment.(5)Transwell chamber experiment showed that the number of invasion and migration cells of A549 and PC-9 cells significantly decreased after dioscin treatment.(6)In addition,the mitochondrial membrane potential of A549 and PC-9 cells significantly decreased,and intracellular ROS production significantly increased after dioscin treatment.Comet assay showed that A549 and PC-9 cells showed obvious tailing phenomenon and induced DNA damage after dioscin treatment.3.Dioscin exerts anti-tumor effect by targeting AKT1(1)98 potential targets of dioscin were found from HERB database,and 2120 lung adenocarcinoma-related targets were found from GeneCard database(correlation score>10).A total of 76 intersection targets of dioscin and lung cancer were obtained from an online tool.In the PPI network,AKT1,CASP3,IL6,JUN and TP53 are the top five targets according to topological parameters.Gene enrichment analysis was done through the David database,including MAPK signaling pathway,PI3K-AKT signaling pathway,apoptosis,pertussis,insulin resistance.(2)Dioscin was virtually docked with the core target by Autodock VinaAfter molecular docking,the binding energy was less than 4.5 kcal/mol,suggesting that dioscin might have good binding ability with AKT1,CASP3,IL6,JUN and TP53.(3)DARTS experiments showed that dioscin could bind to AKT1 and slow down the degradation of Streptomyces protease,while dioscin had no binding effect with P53,C-JUN,IL-6 and CASP3.At the same time,CETSA experiments further demonstrated that dioscin binds to AKT1 and increases the thermal stability of AKT1.(4)Further protein half-life experiment and qRT-PCR experiment showed that dioscin could promote AKT1 degradation without affecting its transcription level.(5)Western blot experiments showed that dioscin could reduce the protein expression of AKT1,p-AKT1 and p-mTOR in vitro and in vivo.(6)After silencing AKT1 with siRNA,the anti-proliferation,inhibition of invasion and migration,and induction of apoptosis of dioscin on A549 were blocked.ConclusionIn summary,this study concluded that dioscin has a significant therapeutic effect on lung adenocarcinoma in vivo and in vitro.It may promote the degradation of AKT1 and inhibit the activation of AKT1 pathway by binding to AKT1,thereby inhibiting the proliferation,invasion,migration and inducing apoptosis of lung adenocarcinoma. |
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