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

Lingping HUANG; Lixia XIE; Yuchao QIU; Ping HU; Xiaoqun YE

doi:10.16016/j.1000-5404.201701068

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

Basic Medicine | 更新时间：2026-01-09

- Ethacrynic acid promotes apoptosis in lung cancer A549 cells when combined with cisplatin chemotherapy
- Journal of Third Military Medical University Vol. 39, Issue 17, Pages: 1720-1727(2017)
- 作者机构：
  
  1.330006 南昌，南昌大学第二附属医院呼吸内科
  2.343000 江西吉安，江西省井冈山大学临床医学院内科教研室
  3.338001 江西新余，江西省新钢中心医院呼吸内科
- 作者简介：
- 基金信息：
  
  the Tackling Project of Social Development of Jiangxi Provincial Department of Science and Technology(20141BBG70035);the Major Science and Technology Project of Jiangxi Province(20143ACB20011)
- DOI：10.16016/j.1000-5404.201701068
  CLC： R73-361;R734.2;R979.1
- Received：11 January 2017，
  
  Revised：2017-03-11，
  
  Published：15 September 2017
- 稿件说明：
移动端阅览
Lingping HUANG, Lixia XIE, Yuchao QIU, et al. Ethacrynic acid promotes apoptosis in lung cancer A549 cells when combined with cisplatin chemotherapy[J]. Journal of Third Military Medical University, 2017, 39(17): 1720-1727.
DOI：

Lingping HUANG, Lixia XIE, Yuchao QIU, et al. Ethacrynic acid promotes apoptosis in lung cancer A549 cells when combined with cisplatin chemotherapy[J]. Journal of Third Military Medical University, 2017, 39(17): 1720-1727. DOI： 10.16016/j.1000-5404.201701068.

摘要

目的

探讨依他尼酸(ethacrynic acid，EA)杀伤肺癌A549细胞球的作用及机制研究。

方法

无血清培养基中培养A549细胞球，应用Western blot检测CD133、SOX2、EpCAM和ABCG2的蛋白表达水平。应用1、2、5、10、20 mg/mL的浓度顺铂(cisplatin，DDP)分别处理A549及细胞球48 h，用MTT检测48 h内细胞的存活率。应用比色法检测10、50、100、200 μmol/L EA对A549细胞球中谷胱甘肽S-转移酶(glutathione S-transferase，GST)活性的抑制作用。应用流式细胞术、Western blot、Real-time-PCR、相差显微镜观察检测200 μmol/L EA处理A549细胞球前后ROS水平、细胞成球能力、β-catenin、Sox2和ABCG2 mRNA和蛋白以及β-catenin启动子活性的变化情况。应用β-catenin腺病毒感染A549细胞球后，再用200 μmol/L EA的处理A549细胞球，利用Real-time PCR和Western blot检测β-catenin S、Sox2和ABCG2 mRNA和蛋白的变化，并用MTT检测A549细胞球增殖的抑制作用。

结果

成功培养出悬浮的A549细胞球，其高表达肿瘤干细胞标志物CD133、SOX2、EpCAM以及耐药相关蛋白ABCG2，并可耐受不同浓度DDP的杀伤作用。200 μmol/L EA处理A549细胞球后ROS水平明显升高，而GST活性、β-catenin、Sox2和ABCG2 mRNA和蛋白的表达、β-catenin的启动子活性、A549细胞球的成球能力显著下降，并且200 μmol/L EA联合5 mg/mL DDP可增强对抑制A549细胞球增殖的作用和增加细胞凋亡的水平(

0.05)。过表达β-catenin后，200 μmol/L EA对β-catenin、Sox2和ABCG2 mRNA和蛋白的抑制作用较空载体组显著减弱。此外，过表达β-catenin可显著缓解200 μmol/L EA联合5 mg/mL DDP对A549细胞球的增殖抑制作用。

结论

EA通过抑制GST活性和β-catenin水平，发挥抑制A549细胞球增殖和干性，促进其凋亡的作用。EA可望成为治疗肺癌及肺癌干细胞的药物。

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

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

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 (

0.05)

and the mRNA and protein levels of β-catenin

Sox2 and ABCG2

and promoter activity of β-catenin were notably decreased (

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 (

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

novel drug in treatment of lung cancer and cancer stem cells.

关键词

Keywords

references

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

BERTOLINI G, ROZ L, PEREGO P, et al. Highly tumorigenic lung cancer CD133+ cells display stem-likefeatures and are spared by cisplatin treatment[J]. Proc Natl Acad Sci U S A, 2009, 106(38): 16281-16286. DOI:10.1073/ pnas.0905653106.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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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