Resistance mechanism to cisplatin in NCI-H460 non-small cell lung cancer cell line: investigating apoptosis, autophagy, and cytogenetic damage

Abstract

Aim: To investigate the effects of cisplatin on the human non-small cell lung carcinoma (NCI-H460) cell line regarding cytotoxicity, genotoxicity, and expression of genes associated with apoptosis (BIRC5) and autophagy (BECN1).

Methods: Cell cultures were treated with cisplatin concentrations (0.16-33.3 μmol/L) for 48 h. Mutagenicity and acute and chronic cytotoxicities were assessed using the MTT, clonogenic, and cytokinesis-block micronucleus assays. Gene expression of BIRC5 and BECN1 was evaluated by reverse transcription-polymerase chain reaction.

Results: Cisplatin IC₅₀ (0.33 μmol/L) increased micronucleus frequency 2.50 times. Cisplatin was also cytotoxic in the 0.6-33.3 μmol/L range, with reduced expression of the BIRC5 gene, suggesting induction of apoptosis. Besides reducing the expression of the BIRC5 gene, 33.3 μmol/L cisplatin increased the expression of the BECN1 gene, suggesting that autophagy can be related to cisplatin resistance.

Conclusion: Cisplatin inhibited NCI-H460 growth, and cisplatin IC₅₀ induced genotoxic damage. When higher cisplatin concentrations are used, the expression of genes associated with apoptosis and autophagy was changed. This results points to a further investigation of the role of autophagy in cisplatin resistance.

Keywords

BECN1 / BIRC5 / cisplatin / cytotoxicity / lung cancer

References

[1] World Health Organization. Cancer. February 2018. Available from: http://www.who.int/mediacentre/factsheets/fs297/en/ [Last accessed on 5 Mar 2018]

[2] Addonisio GLung cancer screening: still not the definitive answer..J Radiol Radiat Ther2014;2:1-3

[3] Karczmarek-Borowska BRabiej EThe quality of life of non-small cell lung cancer patients treated with chemotherapy..Pneumonol Alergol Pol2014;82:349-57

[4] Zhong CJiang LYao FChemotherapy plus best supportive care versus best supportive care in patients with non-small cell lung cancer: a meta-analysis of randomized controlled trials..PLoS One2013;8:e58466PMC3603584

[5] Raparia KDeCamp MMMehta MPMolecular profiling in non-small cell lung cancer..Arch Pathol Lab Med2013;137:481-91

[6] Langer CJPostmus PETargeted in the third-/fourth-line treatment of patients with advanced (stage III/IV) non-small-cell lung cancer (NSCLC)..Cancer Treat Rev2013;39:252-60

[7] Cosaert JPlatinum drugs in the treatment of non-small-cell lung cancer..Br J Cancer2002;87:825-33PMC2376170

[8] Nagasaka MRole of chemotherapy and targeted therapy in early-stage non-small cell lung cancer..Expert Rev Anticancer Ther2018;18:63-70

[9] Spinello AAn omics perspective to the molecular mechanisms of anticancer metallo-drugs in the computational microscope era..Expert Opin Drug Discov2017;12:813-25

[10] Gebel TPlessow KGenotoxicity of platinum and palladium compounds in human and bacterial cells..Mut Res1997;389:183-90

[11] Rjiba-Touati KSkhiri HZellema DBacha HInduction of DNA fragmentation, chromosome aberrations and micronuclei by cisplatin in rat bone-marrow cells: protective effect of recombinant human erythropoietin..Mut Res2012;747:202-6

[12] Bowden NANucleotide excision repair: why is it not used to predict response to platinum-based chemotherapy?.Cancer Lett2014;346:163-71

[13] Rabik CAMolecular mechanisms of resistance and toxicity associated with platinating agents..Cancer Treat Rev2007;33:9-23PMC1855222

[14] Yang LWu LAn XCao SRen XCytokine-induced killer cells modulates resistance to cisplatin in the A549/DDP cell line..J Cancer2017;8:3287-95PMC5665046

[15] Galluzzi LVitale IMartins ICastedo MMolecular mechanisms of cisplatin resistance..Oncogene2012;31:1869-83

[16] Lin JFTsai TFChou KYCisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells..Drug Des Devel Ther2017;11:1517-33PMC5439993

[17] Scudiero DAPaull KDTierney SCurrens MJBoyd MREvaluation of asoluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines..Cancer Res1988;48:4827-33

[18] Riss TLNiles ALBenink HAMinor LCell Viability Assays..In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, Austin C, Baell J, Bejcek B, Chung TDY, Dahlin JL, Devanaryan V, Foley TL, Glicksman M, Hall MD, Hass JV, Inglese J, Iversen PW, Kahl SD, Kales SC, Lal-Nag M, Li Z, McGee J, McManus O, Riss T, Trask OJ Jr, Weidner JR, Xia M, Xu X, editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences;2004;

[19] Franken NAStap Jvan Bree CClonogenic assay of cells in vitro..Nat Protoc2006;1:2315-9

[20] Fenech MThe in vitro micronucleus technique..Mutat Res2000;455:81-95

[21] Boom RSalimans MMWertheim-van Dillen PMRapid and simple method for purification of nucleic acids..J Clin Microbiol1990;28:495-503PMC269651

[22] Livak KJAnalysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method..Methods2001;25:402-8

[23] Barr MPHoffmann ACThomale JFennell DAO'Leary JJGeneration and characterisation of cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature..PLoS One2013;8:e54193PMC3547914

[24] Ferri NMazzarella LLucchini GGandolfi RPellizzoni MCytotoxic effect of (1-methyl-1H-imidazol-2-yl)-methanamine and its derivatives in Pt(II) complexes on human carcinoma cell lines: a comparative study with cisplatin..Bioorg Med Chem2013;21:2379-86

[25] Luo SZhu HLiu MLiang SMolecular, biological characterization and drug sensitivity of chidamide-resistant non-small cell lung cancer cells..Oncol Lett2017;14:6869-75

[26] Lewis CWCancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA..Cell Cycle2016;15:3131-45PMC5134707

[27] Regenthal RKoeppel CDrug levels: therapeutic and toxic serum/plasma concentrations of common drugs..J Clin Monit1999;5:529-44

[28] Tegeder ISeegel MTurowski BKovács AFCisplatin tumor concentrations after intra-arterial cisplatin infusion or embolization in patients with oral cancer..Clin Pharmacol Ther2003;73:417-26

[29] Jafri SHMills GAdvance lung cancer inflammation index (ALI) at diagnosis is a prognostic marker in patients with metastatic non-small cell lung cancer (NSCLC): a retrospective review..BMC Cancer2013;13:1-7PMC3618002

[30] da Silva GNde Camargo EASakamoto-Hojo ETCell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine..Exp Biol Med2010;235:814-24

[31] Tian LRen XGao WSun YOverexpression of miR-26b decreases the cisplatin-resistance in laryngeal cancer by targeting ATF2..Oncotarget2017;8:79023-33PMC5668017

[32] Steen NVGiovannetti ERuijtenbeek RPauwels PMolecular mechanism underlying the pharmacological interactions of the protein kinase C-β inhibitor enzastaurin and erlotinib in non-small cell lung cancer cells..Am J Cancer Res2017;7:816-30PMC5411790

[33] Miyamoto MAoyama TIshibashi HIwahashi HKuwahara MSakamoto TFuruya KPhenoxodiol increases cisplatin sensitivity in ovarian clear cancer cells through XIAP down-regulation and autophagy inhibition..Anticancer Res2018;38:301-6

[34] Herzog ELyng FMByrne HJA new approach to the toxicity testing of carbon-based nanomaterials--the clonogenic assay..Toxicol Lett2007;174:49-60

[35] Toulany MHoller MRodemann HPCisplatin-mediated radiosensitization of non-small cell lung cancer cells is stimulated by ATM inhibition..Radiother Oncol2014;111:228-36

[36] Liang HLiu HZZhou QLThe effects of combined treatment with sevoflurane and cisplatin on growth and invasion of human adenocarcinoma cell line A549..Biomed Pharmacother2013;67:503-9

[37] OECD Guidelines for the Testing of Chemicals. In vitro mammalian cell micronucleus test. Adopted: 26 Sep 2014. Available from: https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecd-tg487-2014-508.pdf [Last accessed on 6 Mar 2018]

[38] Jamieson ERStructure, recognition, and processing of cisplatin-DNA adducts..Chem Rev1999;99:2467-98

[39] Siddik ZHCisplatin: mode of cytotoxic action and molecular basis of resistance..Oncogene2003;22:7265-79

[40] Li FChu EYTognin SAltieri DCControl of apoptosis and mitotic spindle checkpoint by survivin..Nature1998;396:580-4

[41] Wang HQWang JArctigenin enhances chemosensitivity to cisplatin in human nonsmall lung cancer H460 cells through downregulation of survivin expression..J Biochem Mol Toxicol2014;28:39-45

[42] Wong JCFiscus RRCyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells..J Cell Biochem2012;113:3587-98

[43] Ueno TNakahata KSurvivin selective inhibitor YM155 promotes cisplatin induced apoptosis in embryonal rhabdomyosarcoma..Int J Oncol2016;48:1847-54

[44] Alotaibi AAADavies JDAnderson DInhibition of survivin expression after using oxaliplatin and vinflunine to induce cytogenetic damage in vitro in lymphocytes from colon cancer patients and healthy individuals..Mutagenesis2017;32:517-24

[45] Wu XZhao XLiu NLi CZhang BRole of Beclin-1-mediated autophagy in the survival of pediatric leukemia cells..Cell Physiol Biochem2016;39:1827-36

[46] Zhou ZMesenchymal stem cells alleviate LPS-induced acute lung injury in mice by miR-142a-5p- controlled pulmonary endothelial cell autophagy..Cell Physiol Biochem2016;38:258-66

[47] Duffy ASausville EAutophagy modulation: a target for cancer treatment development..Cancer Chemother Pharmacol2015;75:439-47

[48] Li CLiu HShen CChen XBi YYang ZWang KZhang JZou HZhao SImpact of autophagy inhibition at different stages on cytotoxic effect of autophagy inducer in glioblastoma cells..Cell Physiol Biochem2015;35:1303-16

[49] Martins WKCruz MCMiotto RBaptista MSParallel damage in mitochondrial and lysosomal compartments promotes efficient cell death with autophagy: the case of the pentacyclic triterpenoids..Sci Rep2015;5:12425PMC4515638

[50] Maiuri MCCriollo AVicencio JMKroemer GControl of autophagy by oncogenes and tumor suppressor genes..Cell Death Differ2009;16:87-93

[51] Zou ZDing HZhang YChen XZhang QMicroRNA-30a sensitizes tumor cells to cis-platinum via suppressing beclin 1-mediated autophagy..J Biol Chem2012;287:4148-56PMC3281695

[52] Yang XXu YChen LIntensified Beclin-1 mediated by low expression of miR-30a-5p promotes chemoresistance in human small cell lung cancer..Cell Physiol Biochem2017;43:1126-39

[53] Degenhardt KBeaudoin BAnderson DMukherjee CGélinas CNelson DAWhite EAutophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis..Cancer Cell2006;10:51-64PMC2857533

[54] Zhang LWang YFZhang WFKulkarni ABDual induction of apoptotic and autophagic cell death by targeting survivin in head neck squamous cell carcinoma..Cell Death Dis2015;6:e1771PMC4669714