|Table of Contents|

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

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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

Issue:
2017年第17期
Page:
1720-1727
Research Field:
基础医学
Publishing date:

Info

Title:

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

Author(s):

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

Keywords:

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

PACS:
R73-361; R734.2; R979.1
DOI:
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Abstract:

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.

References:

[1]RAHMAN M, DELEYROLLE L, VEDAMMAi V, et al. The cancer stem cell hypothesis: failures and pitfalls[J]. Neurosurgery, 2011, 68(2): 531-545. DOI: 10.1227/NEU.0b013e3181ff9eb5.
[2]TYSNES B B, BJERKVIG R. Cancer initiation and progression: involvement of stem cells and the microenvironment[J]. Biochim Biophys Acta, 2007, 1775(2): 283-297. DOI: 10.1016/j.bbcan.2007.01.001.
[3]QI W, CHEN J, CHENG X, et al. Targeting the wnt-regulatory protein CTNNBIP1 by microRNA-214 enhances the stemness and self-renewal of cancer stem-like cells in lung adenocarcinomas[J]. Stem Cells, 2015, 33(12): 3423-3436. DOI: 10.1002/stem.2188.
[4]YE X Q, LI Q, WANG G H, et al. Mitochondrial and energy metabolismrelated properties as novel indicators of lung cancer stem cells[J]. Int J Cancer, 2011, 129(4): 820-831. DOI:10.1002/ijc.25944.
[5]SUN F F, HU Y H, XIONG L P, et al. Enhanced expression of stem cell markers and drug resistance in A549 sphere-forming non-small cell lung cancer cells[J]. Int J Clin Exp Pathol, 2015, 8(6): 6287-6300.
[6]LI F, TIEDE B, MASSAGU  J, et al. Beyond tumorigenesis: cancer stem cells in metastasis[J]. Cell Res, 2007, 17(1): 3-14. DOI:10.1038/sj.cr.7310118.
[7]WANG R, LI C, SONG D, et al. Ethacrynic acid butyl-ester induces apoptosis in leukemia cells through a hydrogen peroxide mediated pathway independent of glutathione S-transferase P1-1 inhibition[J]. Cancer Res, 2007, 67(16): 7856-7864. DOI:10.1158/0008-5472.CAN-07-0151.
[8]WANG R, LIU C, XIA L, et al. Ethacrynic acid and a derivative enhance apoptosis in arsenic trioxide-treated myeloid leukemia and lymphoma cells: the role of glutathione S-transferase p1-1[J]. Clin Cancer Res, 2012, 18(24): 6690-6701. DOI: 10.1158/1078-0432.CCR-12-0770.
[9]AIZAWA S, OOKAWA K, KUDO T, et al. Characterization of cell death induced by ethacrynic acid in a human colon cancer cell line DLD-1 and suppression by N-acetyl-L-cysteine[J]. Cancer Sci, 2003, 94(10): 886-893. DOI:10.1111/j.1349-7006.2003.tb01371.x.
[10]LIU B, HUANG X, HU Y, et al. Ethacrynic acid improves the antitumor effects of irreversible epidermal growth factor receptor tyrosine kinase inhibitors in breast cancer[J]. Oncotarget, 2016, 7(36): 58038-58050. DOI:10.18632/oncotarget.10846.
[11]DHANBHOORA C M, BABSON J R. Thiol depletion induces lethal cell injury in cultured cardiomyocytes[J]. Arch Biochem Biophys, 1992, 293(1): 130-139. DOI:10.1016/00039861(92)903757.
[12]PARODY J P, ALVAREZ M L, QUIROGA A, et al. Hepatocytes isolated from preneoplastic rat livers are resistant to ethacrynic acid cytotoxicity[J]. Arch Toxicol, 2007, 81(8): 565-573. DOI:10.1007/s0020400701838.
[13]BYUN S S, KIM S W, CHOI H, et al. Augmentation of cisplatin sensitivity in cisplatinresistant human bladder cancer cells by modulating glutathione concentrations and glutathione-related enzyme activities[J]. BJU Int, 2005, 95(7): 1086-1090. DOI:10.1111/j.1464410X.2005.05472.x.
[14]HO M M, NG A V, LAM S, et al. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells[J]. Cancer Res, 2007, 67(10): 4827-4833. DOI:10.1158/00085472.CAN063557.
[15]BERTOLINI G, ROZ L, PEREGO P, et al. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment[J]. Proc Natl Acad Sci U S A, 2009, 106(38): 16281-16286. DOI:10.1073/pnas.0905653106.
[16]SALCIDO C D, LAROCHELLE A, TAYLOR B J, et al. Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer[J]. Br J Cancer, 2010, 102(11): 1636-1644. DOI:10.1038/sj.bjc.6605668.
[17]LI H, CHEN X, CALHOUN-DAVIS T, et al. PC3 human prostate carcinoma cell holoclones contain self-renewing tumor-initiating cells[J]. Cancer Res, 2008, 68(6): 1820-1825. DOI: 10.1158/0008-5472.CAN-07-5878.
[18]ZHOU Z H, PING Y F, YU SC, et al. A novel approach to the identification and enrichment of cancer stem cells from a cultured human glioma cell line[J]. Cancer Lett, 2009, 281(1): 92-99. DOI: 10.1016/j.canlet.2009.02.033.
 [19]YAN F, YANG W K, LI X Y, et al. A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity[J]. Biochim Biophys Acta, 2008, 1780(6): 869-872. DOI: 10.1016/j.bbagen.2008.03.003.
[20]ITO K, SUDA T. Metabolic requirements for the maintenance of selfrenewing stem cells[J]. Nat Rev Mol Cell Biol, 2014, 15(4): 243-256. DOI:10.1038/nrm3772.
[21]LIM E J, HEO J, KIM Y H. Tunicamycin promotes apoptosis in leukemia cells through ROS generation and downregulation of survivin expression[J]. Apoptosis, 2015, 20(8): 1087-1098. DOI: 10.1007/s10495-015-1135-z.
[22]ZHANG Y, HAN L, QI W, et al. Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROSCa(2+)JNK mitochondrial pathways[J]. Biochem Biophys Res Commun, 2015, 456(4): 926-932. DOI: 10.1016/j.bbrc.2014.12.036.
[23]LU D, LIU J X, ENDO T, et al. Ethacrynic acid exhibits selective toxicity to chronic lymphocytic leukemia cells by inhibition of the Wnt/betacatenin pathway[J]. PLoS ONE, 2009, 4(12): e8294. DOI: 10.1371/journal.pone.0008294.
[24]XI Y, CHEN Y. Wnt signaling pathway: implications for therapy in lung cancer and bone metastasis[J]. Cancer Lett, 2014, 353(1): 8-16. DOI: 10.1016/j.canlet.2014.07.010.
[25]DUCHARTRE Y, KIM YM, KAHN M. The Wnt signaling pathway in cancer[J]. Crit Rev Oncol Hematol, 2016, 99: 141-149. DOI: 10.1016/j.critrevonc.2015.12.005.
[26]WU G, LIU A, ZHU J, et al. MiR-1207 overexpression promotes cancer stem cell-like traits in ovarian cancer by activating the Wnt/β-catenin signaling pathway[J]. Oncotarget, 2015, 6(30): 28882-28894. DOI:10.18632/oncotarget.4921.
[27]GE C, WU S, WANG W, et al. miR-942 promotes cancer stem cell-like traits in esophageal squamous cell carcinoma through activation of Wnt/β-catenin signalling pathway[J]. Oncotarget, 2015, 6(13): 10964-10977. DOI:10.18632/oncotarget.3696.
[28]YONG X, TANG B, XIAO Y F, et al. Helicobacter pylori upregulates nanog and oct4 via Wnt/βcatenin signaling pathway to promote cancer stem cell-like properties in human gastric cancer[J]. Cancer Lett, 2016, 374(2): 292-303. DOI:10.1016/j.canlet.2016.02.032.
[29]YANG N, HUI L, WANG Y, et al. Overexpression of SOX2 promotes migration, invasion, and epithelial-mesenchymal transition through the Wnt/β-catenin pathway in laryngeal cancer Hep-2 cells[J]. Tumour Biol, 2014, 35(8): 7965-7973. DOI:10.1007/s13277-014-2045-3.
[30]JIN G, LU D, YAO S, et al. Amide derivatives of ethacrynic acid: synthesis and evaluation as antagonists of Wnt/beta-catenin signaling and CLL cell survival[J]. Bioorg Med Chem Lett, 2009, 19(3): 606-609. DOI:10.1016/j.bmcl.2008.12.067.
[31]KIM Y, GAST S M, ENDO T, et al. In vivo efficacy of the diuretic agent ethacrynic acid against multiple myeloma[J]. Leuk Res, 2012, 36(5): 598-600. DOI:10.1016/j.leukres.2012.01.025.

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Last Update: 2017-09-04