Based On Wnt/β-catenin Signaling To Investigate The Mechanism Underlying β-Asarone-induced Apoptosis In Lung Cancer Cells

Objective:Lung cancer is the leading cause of cancer death worldwide.Chemotherapy is one of the most effective strategies for lung cancer treatment.However,the side effects of chemotherapy limit the application of chemotherapeutic agents.The β-Asarone,a low-toxicity natural compound from a traditional Chinese medicinal herb,has been demonstrated to display anticancer activities in multiple cancer types.However,the anticancer activities ofβ-Asarone in lung cancer have not been shown,and the underlying molecular mechanisms are still unclear.The current study aims to evaluate the anticancer activities of β-Asarone in lung cancer cells,and demonstrate the potential underlying molecular mechanism.Methods:1.To evaluate the anticancer effects of β-Asarone in lung cancer cel 1s,we treated cells(A549,NCI-H1299,NCI-H1650 and HCC827)with different c oncentrations(0 μM,200 μM,400 μM and 800 μM)of β-Asarone,and performe d MTT assay at various time points(24h,48h,72h and 96h)to evaluate cell viability.2.To measure the effect of β-Asarone on cell migration,invasion and adhesion,we performed in vitro migration and invasion assays using Transw ell chambers and performed in vitro cell adhesion assays-3.To further demonstrate the anticancer effects of β-Asarone,we exam ined whether β-Asarone exerts pro-apoptotic effects in lung cancer cell s.To this end,A549 cells were treated with β-Asarone at various concentr ations(0 μ M,100 μ M,200 μM and 400μM)for 48h,and the apoptotic cells wa s evaluated by Annexin V/PI staining and flow cytometry.4.The possible mechanism underlying β-Asarone-induced apoptosis.(1)Based on the pro-apoptotic effect of β-Asarone elicited in lung cancer c ells,we further examined the possible mechanism underlying β-Asarone-in duced apoptosis.Since caspase activation is critical in apoptosis mediate d by various stimuli,we first evaluated the proteolytic processing of ca spases.A549 cells were treated with the indicated concentration of β-Asa rone for 48h.Cells were subjected to western blot analysis.(2)We further confirmed this mechanism by utilizing different caspase inhibitors,incl uding LEHD(for the inactivation of caspase-9 or caspase-3),DEVD(for the i nactivation of caspase-3)and IETD(for the inactivation of caspase-8).A549 cells were treated with the indicated concentration of β-Asarone(400 uM)and(or)LEHD/DEVD/IETD for 48h.Cell apoptosis was evaluated by Anne xin V-FITC Apoptosis Detection Kit and flow cytometry.(3)To evaluate the apoptosis related proteins,we examined antiapoptotic(XIAP,Bcl-2,Survivin and Bcl-XL)and pro-apoptotic(XAF1,Bax,Bad,Bak and Puma)proteins by wes tern blotting.(4)A549 cells were treated with the indicated concentration of β-Asarone for 48h.Cells were subjected to mitochondrial protein sepa ration and the expression of Bax,Bad,phospho-Bax(Ser184)and phospho-Bad(Ser112)were evaluated by western blot analysis.(5)Mitochondrial membran e potential was evaluated by JC-1 staining.Representative images were acq uired by fluorescence microscopy.5.The infuence of β-Asarone on Wnt/β-catenin signaling in lung canc er cells.(1)A549 cells were treated with the indicated concentration ofβ-Asarone for 48h.Total protein was extracted,the protein expression of p-GSK-3β(Ser9,inactivated form),DVL2,β-catenin,Cyclin D1 and GSK-3βwere analyzed by western blotting.(2)A549,NCI-H1299 and NCI-H1650 cells w ere transiently cotransfected with TOPflash or FOPflash and Renilla pRL-T K reporter plasmids by Lipofectamine 2000.Six hours after transfection,th e cells were treated with the indicated concentration of β-Asarone for 48 h.Relative luciferase activity was normalized by transfection efficacy according to Renilla luciferase activity.(3)A549 cells were treated with the indicated concentration of β-Asarone for 48h.The mRNA was isolated and the expression of downstream target genes of the Wnt/β-catenin pathw ay,including c-Myc,Cyclin D1 and MMP-7 were evaluated by real-time PCR.6.The infuence of the activation of Wnt/β-catenin signaling on β-As arone induced anticancer effects.(1)We first assessed whether Wnt3a,a lig and of the Wnt/β-catenin pathway,stimulated the activation of the Wnt/β-catenin pathway.A549 cells were transiently cotransfected with TOPflash or FOPflash and Renilla pRL-TK reporter plasmids by Lipofectamine 2000.Si x hours after transfection,cells were treated with indicated concentratio n of Wnt3a for 48 h.Relative luciferase activity was normalized by trans fection efficiency as determined by Renilla luciferase activity.A549 Cel Is were treated with indicated concentration of Wnt3a for 48h.The mRNA w as isolated and the expression of downstream target genes of the Wnt/β-c atenin pathway,including c-Myc,CyclinDl and MMP-7 were evaluated by real-time PCR.(2)CelIs were treated with indicated concentration of Wnt3a for 48h.A549 cells were treated with 400 uM of β-Asarone and(or)300 ng/ml Wnt3a for the indicated time points.Cell viability was evaluated by MTT a ssay.(3)Cells were treated with indicated concentration of Wnt3a for 48h.Cell migration,invasion,and adhesion were evaluated.Representative image s of migration and invasion and statistical results of migration and inva sion assay are shown.Statistical results of adhesion assay are also show n.(4)Cells were treated with indicated concentration of Wnt3a for 48 h.Ce 11s were subjected to apoptosis analysis by Annexin V/PI staining and flo w cytometry.(5)Cells were treated with indicated concentration of Wnt3a f or 48h.Total protein was extracted and the indicated proteins(caspase-9,caspase-3,caspase-8,pBax(S184),Puma,Bcl-2 and Survivin)were analyz ed by western blotting.(6)Cells were treated with indicated concentration of Wnt3a for 48h.Cells were subjected to mitochondrial protein separatio n and the indicated proteins(pBax(S184),Bax,Cytochrome C)were analyzed by western blotting.Results:1.β-Asarone decreases the viability of lung cancer cells.Cells displayed a mild decrease in cell viability at 24h of treatment with various concentrations of β-Asarone.However,the decrease in cell viability was significantly aggravated when the treatment time was longer than 24h.Moreover,β-Asarone induced the decrease in cell viability in a dose-dependent manner.2.β-Asarone inhibits the migration,invasion,and adhesion of lung cancer cells.Based on the migration assay,compared with the control,β-Asarone reduced migration in a dose-dependent manner.According to the invasion and adhesion assay,β-Asarone treatment resulted in a dose-dependent decrease in invasion and adhesion,respectively,in A549,NCI-H1299 and NCI-H1650 cells.3.β-Asarone induces apoptosis in lung cancer cells.Different concentrations of β-Asarone exerted apoptosis-inducing effects on A549 cells.We further confirmed the pro-apoptotic effects of β-Asarone in other lung cancer cells.Additionally,β-Asarone induced apoptosis in a dose-dependent manner in NCI-H1299 and NCI-H1650 lung cancer cells.4.β-Asarone promoted apoptosis through mitochondria-related mechanis m in lung cancer cells.(1)The activities of Caspase-9 and Caspase-3 signi ficantly increased with β-asarone treatment in a dose-dependent manner,c orrelating with increased PARP cleavage.However,the cleavage of procaspas e-8 was not significantly altered after β-Asarone treatment-(2)Our data showed that LEHD or DEVD effectively reversed β-Asarone mediated apopto sis,while IETD did not reverse β-Asarone-mediated apoptosis,indicating that Caspase-9 and Caspase-3 were associated with β-Asarone-induced apop tosis.(3)β-Asarone increased the expression of XAF1 and Puma but had lit tle effect on the expression of Bax,Bad,Bak and Bcl-XL.In contrast,β-A sarone significantly reduced the expression of XIAP,Bcl-2 and Survivin.(4)Cytochrome C released from the mitochondria to the cytosol was signifi cantly increased.Mitochondrial levels of Bax,Bad,phospho-Bax(Ser184)and phospho-Bad(Ser112)were increased in P-Asarone treated cells.(5)Consi stently,our data showed that JC-1 red fluorescence,due to an aggregated form of JC-1 generated during high mitochondrial membrane potential,gradu ally decreased increasing concentrations of β-Asarone.Concomitantly,JC-1 green fluorescence,due to a monomeric form of JC-1 generated during lo w mitochondrial membrane potential,gradually increased.5.β-Asarone suppresses Wnt/β-catenin signaling in lung cancer cells.(1)Indeed,β-Asarone significantly downregulated the protein expression o f p-GSK-3β,DVL2,β-catenin and Cyclin D1 in a dose-dependent manner in A 549 cells.Conversely,the protein expression of GSK-3β was gradually upr egulated with increasing concentrations of β-asarone.(2)β-Asarone reduc ed the Tcf dependent luciferase activity(TOPflash)in a dose-dependent m anner in all 3 lung cancer cells after 48h of treatment,while FOPflash a ctivity,a mutant of catenin/Tcf binding,remained unchanged.(3)Consistentl y,the expression of downstream target genes of the Wnt/β-catenin pathwa y,including c-Myc,Cyclin D1 and MMP-7,were reduced by β-Asarone in A549 cells in a dose-dependent manner.6.Stimulating the activation of Wnt/β-catenin signaling reverses β-Asarone-induced anticancer effects.(1)Wnt3a significantly activated Tcf-d ependent luciferase activity and promoted the expression of c-Myc,Cyclin D1 and MMP-7.(2)Mimicking the activation of Wnt/β-catenin pathway overc ame β-Asarone induced reduction in cell viability,migration,invasion and adhesion.(3)Similarly,β-Asarone-induced apoptosis was also reversed by Wnt3a treatment.(4)Accordingly,the activation of Caspase-9 and Caspase-3,the expression of pBax(S184)/Puma/Bc1-2/Survivin and the translocationof pBax(S184)/Bax/Cytochrome C were also rescued by Wnt3a treatment.Conclusions:(1)β-Asarone functioned as an anticancer reagent in lung cancer cells.(2)β-Asarone reduced the viability,migration,invasion and adhesion of lung cancer cells.(3)β-Asarone induced mitochondria-related apoptosis in lung cancer cells.(4)inhibition of the activation of the Wnt/β-catenin pathway was critical for β-Asarone-induced anticancer effects.