The Effect And Potential Mechanism Of Ethacrynic Acid On Diminishing A549 Sphere

Objective:To study the effect and mechanism of ethacrynic acid (EA) on A549 spheres.Methods:1.A549 spheres were cultured in serum-free medium, and the protein expression of CD133, SOX2, EpCAM and ABCG2 was detected by western blot.2. MTT was used to evaluate the cell vability of A549 spheres and A549 cells after they were treated by 1, 2, 5, 10 and 20 mg/ml DDP for 48h.3.The activity of GST was measured by colorimetric method after A549 spheres were treated with 10uM, 50uM, 100uM and 200uM EA, respectively.4.The sphere form assay,flow cytometry(FCM), western blot,qPCR and luciferase assay had been used to analyze the levels of cellular ROS, the ability of formed sphere, the apoptosis rate and cell growth inhibition of A549 sphere , The mRNA and protein expression of β-catenin, Sox2 and ABCG2 and the promoter activity of β-catenin upon 200uM EA treated cells for 48h.5.A549 sphere was infected with β-catenin adenovirus for 24h,then cells was treated with 200uM EA (and or no 5mg/ml DDP) for 24h, The mRNA and protein expression of β-catenin, Sox2 and ABCG2 was detected by qPCR and western blot,and cell growth inhibition of A549 sphere was evaluated by MTT.Results:1.The expression of stem cells marker CD 133, SOX2, EpCAM and drug resistance related gene ABCG2 is higher in A549 sphere than in A549 cell lines, and A549 sphere was resisted the killing effect of difference dose DDP. The activity of GST of A549 spheres was inhibited by 10uM, 50uM, 100uM and 200uM EA.2. It was dose-dependent manner. After 200uM EA had treated A549 sphere for 48h, the level of ROS was significantly increased (p <0.05), and the mRNA and protein expression of P-catenin, Sox2 and ABCG2, and promoter activity of β-catenin was notably decreased (p <0.05), compared to control group. Furthermore, the formed-sphere ability of A549 sphere was inhibited after cells was treated with 200uM EA. 200uM EA could suppressed the proliferation of A549 sphere and induced cells apoptosis. The proliferation of A549 cells was significantly inhibited and cell apoptosis rate was increased by 200uM EA combined with 5mg/ml DDP than by 200uM EA alone.3.Up-regulation β-catenin by adenovirus partly reversed the effect of EA inhibition the expression of β-catenin, Sox2 and ABCG2, compared to empty vector adenovirus group.4.Additionaly, over expression β-catenin could partly remitted the proliferation inhibition effect of 200uM EA and 5mg/ml DDP in A549 sphere.Conclusion:1.EA inhibited the cell proliferation and stem cells trait,and induced apoptosis in A549 sphere by the inhibition of GST activity and β-catenin expression.2.EA is a novel drug for lung cancer and cancer stem cells treatment.