Chloroquine increases the anti‑cancer activity of epirubicin in A549 lung cancer cells

Liang,Ai‑Ling; Zhang,Jing; Du,Shen‑Lin; Zhang,Bin; Ma,Xuan; Wu,Cui‑Yun; Liu,Yong‑Jun

doi:10.3892/ol.2020.11567

Chloroquine increases the anti‑cancer activity of epirubicin in A549 lung cancer cells

Authors:
- Ai‑Ling Liang
- Jing Zhang
- Shen‑Lin Du
- Bin Zhang
- Xuan Ma
- Cui‑Yun Wu
- Yong‑Jun Liu
View Affiliations

Affiliations: Department of Clinical Biochemistry, Guangdong Medical University, Dongguan, Guangdong 523808, P.R. China, The Clinical Laboratory of Shunde Hospital, Southern Medical University, Foshan, Guangdong 528300, P.R. China, Blood Transfusion Department, Dongguan Tung Wah Hospital, Dongguan, Guangdong 523110, P.R. China, Guangdong Medical Molecular Diagnostic Key Laboratory, Guangdong Medical University, Dongguan, Guangdong 523808, P.R. China
Published online on: April 22, 2020 https://doi.org/10.3892/ol.2020.11567
Pages: 53-60
Copyright: © Liang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The present study investigated whether the autophagy inhibitor chloroquine (CQ) can improve the sensitivity of the A549 lung cancer cell line to epirubicin (EPI). The Cell Counting Kit 8 (CCK8) assay was used to determine the EPI IC50 in A549 cells treated for 72 h. A549 cells were treated with Western blot analysis was performed to detect the expression level of the autophagy‑associated protein, microtubule associated protein 1 light chain 3 β (LC3B), and apoptosis-associated proteins such as cleaved caspase‑9 and cleaved caspase‑3. CCK8, colony formation, wound healing and Transwell assays were performed to analyze cell proliferation, migration and invasion capacity. Reverse transcription-quantitative PCR (RT‑qPCR) was used to analyze the mRNA expression levels of LC3B and beclin‑1, and the apoptosis rate was analyzed by flow cytometry. The IC50 of EPI was 0.03 µg/ml. The CCK8 results demonstrated that the cell survival rate was lower in CQ + EPI‑treated cells when compared with the individual treatment groups. The colony formation results revealed that the number of clones in the EPI + CQ‑treated group was reduced compared with EPI or CQ treatment alone. The wound healing assay revealed that migration was reduced in the EPI + CQ‑treated group compared with the other treatment groups, and the Transwell results indicated that the number of cells passing through the Matrigel and membrane was lowest in the CQ + EPI treatment group. The mRNA expression levels of LC3B and beclin‑1 were increased in the CQ + EPI group by 51.5 and 61.2%, respectively, when compared with the control group. The results indicated that LC3B protein expression was enhanced by EPI in a concentration‑dependent manner, and the protein levels of cleaved caspase‑3 and cleaved caspase‑9 were higher in the combination group than in the EPI alone group. The flow cytometry results demonstrated that the apoptosis rate was highest in the EPI + CQ group. In conclusion, the autophagy inhibitor CQ increased the sensitivity of A549 cells to EPI, and the underlying mechanism of action may be associated with the activation of apoptosis.

View Figures

View References

1	Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA and Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 68:394–424. 2018. View Article : Google Scholar : PubMed/NCBI
2	Sa H, Song P, Ma K, Gao Y, Zhang L and Wang D: Perioperative targeted therapy or immunotherapy in non-small-cell lung cancer. OncoTargets Ther. 12:8151–8159. 2019. View Article : Google Scholar
3	Jacot W, Pujol JL, Chakra M, Molinier O, Bozonnat MC, Gervais R and Quantin X: Epirubicin and ifosfamide in relapsed or refractory small cell lung cancer patients. Lung Cancer. 75:213–216. 2012. View Article : Google Scholar : PubMed/NCBI
4	Chen W, Yang Z, Deng G and Ye H: High-dose epirubicin combination therapy in the treatment of 48 cases with advanced chest malignant tumors. China Oncol. 11:355–356. 2001.(In Chinese).
5	Plosker GL and Faulds D: Epirubicin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in cancer chemotherapy. Drugs. 45:788–856. 1993. View Article : Google Scholar : PubMed/NCBI
6	Liu F, Jin H, Shen J, Wu D, Tian Y and Huang C: Gp130 degradation induced by epirubicin contributes to chemotherapy efficacy. Biochem Biophys Res Commun. 519:572–578. 2019. View Article : Google Scholar : PubMed/NCBI
7	Wachters FM, Van Putten JW, Kramer H, Erjavec Z, Eppinga P, Strijbos JH, de Leede GP, Boezen HM, de Vries EG and Groen HJ: First-line gemcitabine with cisplatin or epirubicin in advanced non-small-cell lung cancer: A phase III trial. Br J Cancer. 89:1192–1199. 2003. View Article : Google Scholar : PubMed/NCBI
8	Owonikoko TK, Ramalingam SS and Belani CP: Maintenance therapy for advanced non-small cell lung cancer: Current status, controversies, and emerging consensus. Clin Cancer Res. 16:2496–2504. 2010. View Article : Google Scholar : PubMed/NCBI
9	Cianfanelli V, Fuoco C, Lorente M, Salazar M, Quondamatteo F, Gherardini PF, De Zio D, Nazio F, Antonioli M, D'Orazio M, et al: AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation. Nat Cell Biol. 17:20–30. 2015. View Article : Google Scholar : PubMed/NCBI
10	Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, Mizushima N, Iwata J, Ezaki J, Murata S, et al: Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell. 131:1149–1163. 2007. View Article : Google Scholar : PubMed/NCBI
11	Liu JT, Li WC, Gao S, Wang F, Li XQ, Yu HQ, Fan LL, Wei W, Wang H and Sun GP: Autophagy inhibition overcomes the antagonistic effect between gefitinib and cisplatin in epidermal growth factor receptor mutant non-small-cell lung cancer cells. Clin Lung Cancer. 16:e55–e66. 2015. View Article : Google Scholar : PubMed/NCBI
12	Galluzzi L, Kepp O, Vander Heiden MG and Kroemer G: Metabolic targets for cancer therapy. Nat Rev Drug Discov. 12:829–846. 2013. View Article : Google Scholar : PubMed/NCBI
13	Hale AN, Ledbetter DJ, Gawriluk TR and Rucker EB III: Autophagy: Regulation and role in development. Autophagy. 9:951–972. 2013. View Article : Google Scholar : PubMed/NCBI
14	Liu R, Chen Z, Yi X, Huang F, Hu G, Liu D, Li X, Zhou H and Liu Z: 9za plays cytotoxic and proapoptotic roles and induces cytoprotective autophagy through the PDK1/Akt/mTOR axis in non-small-cell lung cancer. J Cell Physiol. 234:20728–20741. 2019. View Article : Google Scholar : PubMed/NCBI
15	Mizushima N: Autophagy: Process and function. Genes Dev. 21:2861–2873. 2007. View Article : Google Scholar : PubMed/NCBI
16	Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, Thomas-Tikhonenko A and Thompson CB: Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 117:326–336. 2007. View Article : Google Scholar : PubMed/NCBI
17	Chou HL, Lin YH, Liu W, Wu CY, Li RN, Huang HW, Chou CH, Chiou SJ and Chiu CC: Combination therapy of chloroquine and C(2)-ceramide enhances cytotoxicity in lung cancer H460 and H1299 cells. Cancers (Basel). 11:E3702019. View Article : Google Scholar : PubMed/NCBI
18	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
19	Pan Y, Qian JX, Lu SQ, Chen JW, Zhao XD, Jiang Y, Wang LH and Zhang GX: Protective effects of tanshinone IIA sodium sulfonate on ischemia-reperfusion-induced myocardial injury in rats. Iran J Basic Med Sci. 20:308–315. 2017.PubMed/NCBI
20	Lee JG, Shin JH, Shim HS, Lee CY, Kim DJ, Kim YS and Chung KY: Autophagy contributes to the chemo-resistance of non-small cell lung cancer in hypoxic conditions. Respir Res. 16:1382015. View Article : Google Scholar : PubMed/NCBI
21	Pedrosa P, Corvo ML, Ferreira-Silva M, Martins P, Carvalheiro MC, Costa PM, Martins C, Martins LMDRS, Baptista PV and Fernandes AR: Targeting cancer resistance via multifunctional gold nanoparticles. Int J Mol Sci. 20:E55102019. View Article : Google Scholar : PubMed/NCBI
22	Wu T, Wang MC, Jing L, Liu ZY, Guo H, Liu Y, Bai YY, Cheng YZ, Nan KJ and Liang X: Autophagy facilitates lung adenocarcinoma resistance to cisplatin treatment by activation of AMPK/mTOR signaling pathway. Drug Des Devel Ther. 9:6421–6431. 2015. View Article : Google Scholar : PubMed/NCBI
23	Sugita S, Ito K, Yamashiro Y, Moriya S, Che XF, Yokoyama T, Hiramoto M and Miyazawa K: EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells. Biochem Biophys Res Commun. 461:28–34. 2015. View Article : Google Scholar : PubMed/NCBI
24	Tang MC, Wu MY, Hwang MH, Chang YT, Huang HJ, Lin AM and Yang JC: Chloroquine enhances gefitinib cytotoxicity in gefitinib-resistant nonsmall cell lung cancer cells. PLoS One. 10:e01191352015. View Article : Google Scholar : PubMed/NCBI
25	Sui X, Kong N, Zhu M, Wang X, Lou F, Han W and Pan H: Cotargeting EGFR and autophagy signaling: A novel therapeutic strategy for non-small-cell lung cancer. Mol Clin Oncol. 2:8–12. 2014. View Article : Google Scholar : PubMed/NCBI
26	Chang CY, Kuan YH, Ou YC, Li JR, Wu CC, Pan PH, Chen WY, Huang HY and Chen CJ: Autophagy contributes to gefitinib-induced glioma cell growth inhibition. Exp Cell Res. 327:102–112. 2014. View Article : Google Scholar : PubMed/NCBI
27	Cheng Y, Li H, Ren X, Niu T, Hait WN and Yang J: Cytoprotective effect of the elongation factor-2 kinase-mediated autophagy in breast cancer cells subjected to growth factor inhibition. PLoS One. 5:e97152010. View Article : Google Scholar : PubMed/NCBI
28	Pietrocola F, Izzo V, Niso-Santano M, Vacchelli E, Galluzzi L, Maiuri MC and Kroemer G: Regulation of autophagy by stress-responsive transcription factors. Semin Cancer Biol. 23:310–322. 2013. View Article : Google Scholar : PubMed/NCBI
29	Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M, Baehrecke EH, Bahr BA, Ballabio A, et al: Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 4:151–175. 2008. View Article : Google Scholar : PubMed/NCBI
30	Varbiro G, Veres B, Gallyas F Jr and Sumegi B: Direct effect of Taxol on free radical formation and mitochondrial permeability transition. Free Radic Biol Med. 31:548–558. 2001. View Article : Google Scholar : PubMed/NCBI
31	Fulda S, Galluzzi L and Kroemer G: Targeting mitochondria for cancer therapy. Nat Rev Drug Discov. 9:447–464. 2010. View Article : Google Scholar : PubMed/NCBI
32	Namba T, Takabatake Y, Kimura T, Takahashi A, Yamamoto T, Matsuda J, Kitamura H, Niimura F, Matsusaka T, Iwatani H, et al: Autophagic clearance of mitochondria in the kidney copes with metabolic acidosis. J Am Soc Nephrol. 25:2254–2266. 2014. View Article : Google Scholar : PubMed/NCBI
33	Randow F and Youle RJ: Self and nonself: How autophagy targets mitochondria and bacteria. Cell Host Microbe. 15:403–411. 2014. View Article : Google Scholar : PubMed/NCBI
34	Boyer-Guittaut M, Poillet L, Liang Q, Bole-Richard E, Ouyang X, Benavides GA, Chakrama FZ, Fraichard A, Darley-Usmar VM, Despouy G, et al: The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells. Autophagy. 10:986–1003. 2014. View Article : Google Scholar : PubMed/NCBI
35	Karsli-Uzunbas G, Guo JY, Price S, Teng X, Laddha SV, Khor S, Kalaany NY, Jacks T, Chan CS, Rabinowitz JD and White E: Autophagy is required for glucose homeostasis and lung tumor maintenance. Cancer Discov. 4:914–927. 2014. View Article : Google Scholar : PubMed/NCBI
36	Guo JY, Xia B and White E: Autophagy-mediated tumor promotion. Cell. 155:1216–1219. 2013. View Article : Google Scholar : PubMed/NCBI