Abstract
Autophagy is linked to multiple cancer-related signaling pathways, and represents a defense mechanism for cancer cells under therapeutic stress. The crosstalk between apoptosis and autophagy is essential for both tumorigenesis and embryonic development. We studied the influence of autophagy on cell survival in pro-apoptotic conditions induced by anticancer drugs in three model systems: human cancer cells (NCI-H460, COR-L23 and U87), human normal cells (HaCaT and MRC-5) and zebrafish embryos (Danio rerio). Autophagy induction with AZD2014 and tamoxifen antagonized the pro-apoptotic effect of chemotherapeutics doxorubicin and cisplatin in cell lines, while autophagy inhibition by wortmannin and chloroquine synergized the action of both anticancer agents. This effect was further verified by assessing cleaved caspase-3 and PARP-1 levels. Autophagy inhibitors significantly increased both apoptotic markers when applied in combination with doxorubicin while autophagy inducers had the opposite effect. In a similar manner, autophagy induction in zebrafish embryos prevented cisplatin-induced apoptosis in the tail region while autophagy inhibition increased cell death in the tail and retina of cisplatin-treated animals. Autophagy modulation with direct inhibitors of the PI3kinase/Akt/mTOR pathway (AZD2014 and wortmannin) triggered the cellular response to anticancer drugs more effectively in NCI-H460 and zebrafish embryonic models compared to HaCaT suggesting that these modulators are selective towards rapidly proliferating cells. Therefore, evaluating the autophagic properties of chemotherapeutics could help determine more accurately the fate of different cell types under treatment. Our study underlines the importance of testing autophagic activity of potential anticancer agents in a comparative approach to develop more rational anticancer therapeutic strategies.
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References
Aburto MR, Hurle JM, Varela-Nieto I, Magarinos M (2012) Autophagy during vertebrate development. Cells 1(3):428–448. doi:10.3390/cells1030428
Aredia F, Guaman Ortiz LM, Giansanti V, Scovassi AI (2012) Autophagy and cancer. Cells 1(3):520–534. doi:10.3390/cells1030520
Assinder SJ, Dong Q, Kovacevic Z, Richardson DR (2009) The TGF-beta, PI3 K/Akt and PTEN pathways: established and proposed biochemical integration in prostate cancer. Biochem J 417(2):411–421. doi:10.1042/BJ20081610
Avalos Y, Canales J, Bravo-Sagua R, Criollo A, Lavandero S, Quest AF (2014) Tumor suppression and promotion by autophagy. Biomed Res Int 2014:603980. doi:10.1155/2014/603980
Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelarova H, Meijer AJ (1997) The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243(1–2):240–246
Brand M, Heisenberg CP, Warga RM, Pelegri F, Karlstrom RO, Beuchle D, Picker A, Jiang YJ, Furutani-Seiki M, van Eeden FJ, Granato M, Haffter P, Hammerschmidt M, Kane DA, Kelsh RN, Mullins MC, Odenthal J, Nusslein-Volhard C (1996) Mutations affecting development of the midline and general body shape during zebrafish embryogenesis. Development 123:129–142
Chakrabarti G (2015) Mutant KRAS associated malic enzyme 1 expression is a predictive marker for radiation therapy response in non-small cell lung cancer. Radiat Oncol 10:145. doi:10.1186/s13014-015-0457-x
Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27–55
Cole LK, Ross LS (2001) Apoptosis in the developing zebrafish embryo. Dev Biol 240(1):123–142. doi:10.1006/dbio.2001.0432
D’Amours D, Sallmann FR, Dixit VM, Poirier GG (2001) Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis. J Cell Sci 114(Pt 20):3771–3778
Detrich HW 3rd, Westerfield M, Zon LI (2010) The zebrafish: cellular and developmental biology, part A. Preface. Methods Cell Biol. doi:10.1016/B978-0-12-384892-5.00018-9
Dietze EC, Troch MM, Bean GR, Heffner JB, Bowie ML, Rosenberg P, Ratliff B, Seewaldt VL (2004) Tamoxifen and tamoxifen ethyl bromide induce apoptosis in acutely damaged mammary epithelial cells through modulation of AKT activity. Oncogene 23(21):3851–3862. doi:10.1038/sj.onc.1207480
Eskelinen EL, Saftig P (2009) Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta 1793(4):664–673. doi:10.1016/j.bbamcr.2008.07.014
Fimia GM, Piacentini M (2010) Regulation of autophagy in mammals and its interplay with apoptosis. Cell Mol Life Sci 67(10):1581–1588. doi:10.1007/s00018-010-0284-z
Furutani-Seiki M, Jiang YJ, Brand M, Heisenberg CP, Houart C, Beuchle D, van Eeden FJ, Granato M, Haffter P, Hammerschmidt M, Kane DA, Kelsh RN, Mullins MC, Odenthal J, Nusslein-Volhard C (1996) Neural degeneration mutants in the zebrafish, Danio rerio. Development 123:229–239
Guichard SM, Curwen J, Bihani T, D’Cruz CM, Yates JW, Grondine M, Howard Z, Davies BR, Bigley G, Klinowska T, Pike KG, Pass M, Chresta CM, Polanska UM, McEwen R, Delpuech O, Green S, Cosulich SC (2015) AZD2014, an inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer when administered using intermittent or continuous schedules. Mol Cancer Ther 14(11):2508–2518. doi:10.1158/1535-7163.MCT-15-0365
Guo JY, Chen HY, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G, Kamphorst JJ, Chen G, Lemons JM, Karantza V, Coller HA, Dipaola RS, Gelinas C, Rabinowitz JD, White E (2011) Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 25(5):460–470. doi:10.1101/gad.2016311
Halaby MJ, Kastein BK, Yang DQ (2013) Chloroquine stimulates glucose uptake and glycogen synthase in muscle cells through activation of Akt. Biochem Biophys Res Commun 435(4):708–713. doi:10.1016/j.bbrc.2013.05.047
Hippert MM, O’Toole PS, Thorburn A (2006) Autophagy in cancer: good, bad, or both? Cancer Res 66(19):9349–9351. doi:10.1158/0008-5472.CAN-06-1597
Inohara N, Nunez G (2000) Genes with homology to mammalian apoptosis regulators identified in zebrafish. Cell Death Differ 7(5):509–510. doi:10.1038/sj.cdd.4400679
Janji B, Viry E, Baginska J, Van Moer K, Berchem G (2013) Role of autophagy in cancer and tumor progression. In: Bailly Y (ed) Autophagy - a double-edged sword - cell survival or death? InTech. doi:10.5772/55388
Jeong EH, Choi HS, Lee TG, Kim HR, Kim CH (2012) Dual inhibition of PI3K/Akt/mTOR pathway and role of autophagy in non-small cell lung cancer cells. Tuberc Respir Dis (Seoul) 72(4):343–351
Lee E, Koo Y, Ng A, Wei Y, Luby-Phelps K, Juraszek A, Xavier RJ, Cleaver O, Levine B, Amatruda JF (2014) Autophagy is essential for cardiac morphogenesis during vertebrate development. Autophagy 10(4):572–587. doi:10.4161/auto.27649
Li J, Yuan J (2008) Caspases in apoptosis and beyond. Oncogene 27(48):6194–6206. doi:10.1038/onc.2008.297
Li J, Hou N, Faried A, Tsutsumi S, Kuwano H (2010) Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model. Eur J Cancer 46(10):1900–1909. doi:10.1016/j.ejca.2010.02.021
Liu D, Yang Y, Liu Q, Wang J (2011) Inhibition of autophagy by 3-MA potentiates cisplatin-induced apoptosis in esophageal squamous cell carcinoma cells. Med Oncol 28(1):105–111. doi:10.1007/s12032-009-9397-3
Livesey KM, Tang D, Zeh HJ, Lotze MT (2009) Autophagy inhibition in combination cancer treatment. Curr Opin Investig Drugs 10(12):1269–1279
Looyenga BD, Hutchings D, Cherni I, Kingsley C, Weiss GJ, Mackeigan JP (2012) STAT3 is activated by JAK2 independent of key oncogenic driver mutations in non-small cell lung carcinoma. PLoS One 7(2):e30820. doi:10.1371/journal.pone.0030820
Martinet W, Agostinis P, Vanhoecke B, Dewaele M, De Meyer GR (2009) Autophagy in disease: a double-edged sword with therapeutic potential. Clin Sci (Lond) 116(9):697–712. doi:10.1042/CS20080508
Mathew R, Karantza-Wadsworth V, White E (2007) Role of autophagy in cancer. Nat Rev Cancer 7(12):961–967. doi:10.1038/nrc2254
Matsushima H, Yonemura K, Ohishi K, Hishida A (1998) The role of oxygen free radicals in cisplatin-induced acute renal failure in rats. J Lab Clin Med 131(6):518–526
Meijer AJ, Codogno P (2004) Regulation and role of autophagy in mammalian cells. Int J Biochem Cell Biol 36(12):2445–2462. doi:10.1016/j.biocel.2004.02.002
Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140(3):313–326. doi:10.1016/j.cell.2010.01.028
Ng SS, Tsao MS, Nicklee T, Hedley DW (2001) Wortmannin inhibits pkb/akt phosphorylation and promotes gemcitabine antitumor activity in orthotopic human pancreatic cancer xenografts in immunodeficient mice. Clin Cancer Res 7(10):3269–3275
Ou HC, Raible DW, Rubel EW (2007) Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line. Hear Res 233(1–2):46–53. doi:10.1016/j.heares.2007.07.003
Parng C (2005) In vivo zebrafish assays for toxicity testing. Curr Opin Drug Discov Devel 8(1):100–106
Pommier Y, Leo E, Zhang H, Marchand C (2010) DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol 17(5):421–433. doi:10.1016/j.chembiol.2010.04.012
Sasore T, Kennedy B (2014) Deciphering combinations of PI3K/AKT/mTOR pathway drugs augmenting anti-angiogenic efficacy in vivo. PLoS One 9(8):e105280. doi:10.1371/journal.pone.0105280
Scarlatti F, Bauvy C, Ventruti A, Sala G, Cluzeaud F, Vandewalle A, Ghidoni R, Codogno P (2004) Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1. J Biol Chem 279(18):18384–18391. doi:10.1074/jbc.M313561200
Smith DG, Sturmey RG (2013) Parallels between embryo and cancer cell metabolism. Biochem Soc Trans 41(2):664–669. doi:10.1042/BST20120352
Spangler JB, Manzari MT, Rosalia EK, Chen TF, Wittrup KD (2012) Triepitopic antibody fusions inhibit cetuximab-resistant BRAF and KRAS mutant tumors via EGFR signal repression. J Mol Biol 422(4):532–544. doi:10.1016/j.jmb.2012.06.014
Spears LD, Tran AV, Qin CY, Hobbs SB, Burns CA, Royer NK, Zhang Z, Ralston L, Fisher JS (2016) Chloroquine increases phosphorylation of AMPK and Akt in myotubes. Heliyon 2(3):e00083. doi:10.1016/j.heliyon.2016.e00083
Stern ST, Adiseshaiah PP, Crist RM (2012) Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part Fibre Toxicol 9:20. doi:10.1186/1743-8977-9-20
Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005) Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 1(2):84–91
Thompson CB (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267(5203):1456–1462
Uribe PM, Mueller MA, Gleichman JS, Kramer MD, Wang Q, Sibrian-Vazquez M, Strongin RM, Steyger PS, Cotanche DA, Matsui JI (2013) Dimethyl sulfoxide (DMSO) exacerbates cisplatin-induced sensory hair cell death in zebrafish (Danio rerio). PLoS One 8(2):e55359. doi:10.1371/journal.pone.0055359
Varga M, Sass M, Papp D, Takacs-Vellai K, Kobolak J, Dinnyes A, Klionsky DJ, Vellai T (2014) Autophagy is required for zebrafish caudal fin regeneration. Cell Death Differ 21(4):547–556. doi:10.1038/cdd.2013.175
Westerfield M (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press, Eugene
Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H, Bause A, Li Y, Stommel JM, Dell’antonio G, Mautner J, Tonon G, Haigis M, Shirihai OS, Doglioni C, Bardeesy N, Kimmelman AC (2011) Pancreatic cancers require autophagy for tumor growth. Genes Dev 25(7):717–729. doi:10.1101/gad.2016111
Yu SW, Wang H, Poitras MF, Coombs C, Bowers WJ, Federoff HJ, Poirier GG, Dawson TM, Dawson VL (2002) Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science 297(5579):259–263. doi:10.1126/science.1072221
Yu CC, Huang HB, Hung SK, Liao HF, Lee CC, Lin HY, Li SC, Ho HC, Hung CL, Su YC (2016) AZD2014 radio sensitizes oral squamous cell carcinoma by inhibiting AKT/mTOR axis and inducing G1/G2/M cell cycle arrest. PLoS One 11(3):e0151942. doi:10.1371/journal.pone.0151942
Zhang S, Wang C, Tang S, Deng S, Zhou Y, Dai C, Yang X, Xiao X (2014) Inhibition of autophagy promotes caspase-mediated apoptosis by tunicamycin in HepG2 cells. Toxicol Mech Methods 24(9):654–665. doi:10.3109/15376516.2014.956915
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This research was supported by Ministry of Education, Science and Technological Development of Serbia (Grant Nos. III41031 and 173008).
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Divac Rankov, A., Ljujić, M., Petrić, M. et al. Targeting autophagy to modulate cell survival: a comparative analysis in cancer, normal and embryonic cells. Histochem Cell Biol 148, 529–544 (2017). https://doi.org/10.1007/s00418-017-1590-4
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DOI: https://doi.org/10.1007/s00418-017-1590-4