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Ethacrynic acid promotes apoptosis in lung cancer A549 cells when combined with cisplatin chemotherapy



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Ethacrynic acid promotes apoptosis in lung cancer A549 cells when combined with cisplatin chemotherapy


HUANG Lingping XIE Lixia QIU Yuchao HU Ping YE Xiaoqun

Department of Respiratory Diseases, Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, 330006; Department of Internal Medicine Teaching, College of Clinical Medicine, Jinggangshan University, Ji’an, Jiangxi Province, 343000; Department of Respiratory Diseases, Xingang Central Hospital of Jiangxi Province, Xinyu, Jiangxi Province, 338001,  China


lung cancer stem cells ethacrynic acid glutathione S-transferase &beta-catenin

R73-361; R734.2; R979.1

Objective     To investigate the killing effect of ethacrynic acid (EA) on lung cancer A549 cells derived spheres and explore the underlying mechanism. Methods     A549 spheres were cultured in serum-free medium, and the protein expression of CD133, SOX2, EpCAM and ABCG2 was detected by Western blotting. MTT assay was used to evaluate the cell viability of A549 spheres and A549 cells after treated by 1, 2, 5, 10 and 20 mg/mL cisplatin (DDP) for 48 h. The activity of glutathione S-transferase (GST) was measured by colorimetric method after A549 spheres were treated with 10, 50, 100 and 200 μmol/L EA, respectively. Flow cytometry, Western blotting, real-time PCR and luciferase assay were used to analyze the levels of cellular reactive oxygen species (ROS), formation of A549 spheres, mRNA and protein expression levels of βcatenin, Sox2 and ABCG2, and promoter activity of β-catenin upon 200 μmol/L EA treated cells for 48 h. A549 sphere was infected with β-catenin adenovirus for 24 h, followed by 200 μmol/L EA treatment (in presence or absence of 5 mg/mL DDP) for 24 h. The expression of βcatenin, Sox2 and ABCG2 at mRNA and protein levels was detected by real-time PCR and Western blotting, and cell growth of A549 spheres was evaluated by MTT assay.  Results    The A549 spheres, with high expression of tumor stem cells markers CD133, SOX2, EpCAM and drug resistance related molecule ABCG2, and resistance to DDP at different doses, were successfully derived. After 200 μmol/L EA had treated A549 sphere for 48 h, the levels of ROS were significantly increased (P<0.05), and the mRNA and protein levels of β-catenin, Sox2 and ABCG2, and promoter activity of β-catenin were notably decreased (P<0.05). The treatment of 200 μmol/L EA enhanced the inhibitory effect on proliferation and the promoting effect on apoptosis in A549 spheres induced by 5 mg/mL DDP (P<0.05). Up-regulation of β-catenin by adenoviral infection partly reversed the effects of 200 μmol/L EA on suppressing the expression levels of β-catenin, Sox2 and ABCG2, compared to the spheres infected with blank adenovirus. Additionally, β-catenin over-expression significantly remitted the inhibitory effect of 200 μmol/L EA and 5 mg/mL DDP on the proliferation in A549 spheres. Conclusion    EA exerts inhibitory effect on the proliferation and stemness of A549 spheres through suppressing GST activity and βcatenin expression, and then promotes cell apoptosis. EA might be a novel drug in treatment of lung cancer and cancer stem cells.


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Last Update: 2017-09-04