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Open Access 01-12-2017 | Research

Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells

Authors: Hyo-Jin Kim, Ki-Eun Hwang, Do-Sim Park, Seon-Hee Oh, Hong Young Jun, Kwon-Ha Yoon, Eun-Taik Jeong, Hak-Ryul Kim, Young-Suk Kim

Published in: Journal of Translational Medicine | Issue 1/2017

Abstract

Background

Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action.

Methods

Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo.

Results

Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin.

Conclusions

Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.

Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74. doi:10.1016/j.cell.2011.02.013.CrossRefPubMed

Laubenbacher R, Hower V, Jarrah A, Torti SV, Shulaev V, Mendes P, et al. A systems biology view of cancer. Biochim Biophys Acta. 2009;1796(2):129–39. doi:10.1016/j.bbcan.2009.06.001.PubMedPubMedCentral

Levine B, Yuan J. Autophagy in cell death: an innocent convict? J Clin Investig. 2005;115(10):2679–88.CrossRefPubMedPubMedCentral

Martinet W, Agostinis P, Vanhoecke B, Dewaele M, De Meyer GR. Autophagy in disease: a double-edged sword with therapeutic potential. Clin Sci. 2009;116(9):697–712. doi:10.1042/CS20080508.CrossRefPubMed

Levine B, Deretic V. Unveiling the roles of autophagy in innate and adaptive immunity. Nat Rev Immunol. 2007;7(10):767–77.CrossRefPubMed

Cecconi F, Levine B. The role of autophagy in mammalian development: cell makeover rather than cell death. Dev Cell. 2008;15(3):344–57. doi:10.1016/j.devcel.2008.08.012.CrossRefPubMedPubMedCentral

Kroemer G, Levine B. Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol. 2008;9(12):1004–10. doi:10.1038/nrm2529.CrossRefPubMedPubMedCentral

Shen HM, Codogno P. Autophagic cell death: Loch Ness monster or endangered species? Autophagy. 2011;7(5):457–65.CrossRefPubMed

Edinger AL, Thompson CB. Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol. 2004;16(6):663–9.CrossRefPubMed

10.

Christofferson DE, Yuan J. Necroptosis as an alternative form of programmed cell death. Curr Opin Cell Biol. 2010;22(2):263–8. doi:10.1016/j.ceb.2009.12.003.CrossRefPubMedPubMedCentral

11.

Degterev A, Yuan J. Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol. 2008;5:378–90. doi:10.1038/nrm2393.CrossRef

12.

Papathanasiou K, Papageorgiou C, Panidis D, Mantalenakis S. Our experience in laparoscopic diagnosis and management in women with chronic pelvic pain. Clin Exp Obstet Gynecol. 1999;26(3–4):190–2.PubMed

13.

Chen X, Yang L, Oppenheim JJ, Howard MZ. Cellular pharmacology studies of shikonin derivatives. Phytother Res. 2002;16(3):199–209.CrossRefPubMed

14.

Zhao Q, Assimopoulou AN, Klauck SM, Damianakos H, Chinou I, Kretschmer N, et al. Inhibition of c-MYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells. Oncotarget. 2015;6(36):38934–51. doi:10.18632/oncotarget.5380.PubMedPubMedCentral

15.

Song J, Zhao Z, Fan X, Chen M, Cheng X, Zhang D, et al. Shikonin potentiates the effect of arsenic trioxide against human hepatocellular carcinoma in vitro and in vivo. Oncotarget. 2016;7(43):70504–15. doi:10.18632/oncotarget.12041.PubMedPubMedCentral

16.

He S, Liao TT, Chen YT, Kuo HM, Lin YL. Glutathione-S-transferase enhances proliferation-migration and protects against shikonin-induced cell death in breast cancer cells. Kaohsiung J Med Sci. 2011;27(11):477–84. doi:10.1016/j.kjms.2011.06.010.CrossRefPubMed

17.

Huang C, Luo Y, Zhao J, Yang F, Zhao H, Fan W, et al. Shikonin kills glioma cells through necroptosis mediated by RIP-1. PLoS ONE. 2013;8(6):e66326. doi:10.1371/journal.pone.0066326.CrossRefPubMedPubMedCentral

18.

Kanzawa T, Bedwell J, Kondo Y, Kondo S, Germano IM. Inhibition of DNA repair for sensitizing resistant glioma cells to temozolomide. J Neurosurg. 2003;99(6):1047–52.CrossRefPubMed

19.

Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell. 2009;137(6):1112–23. doi:10.1016/j.cell.2009.05.037.CrossRefPubMedPubMedCentral

20.

Degterev A, Hitomi J, Germscheid M, Ch’en IL, Korkina O, Teng X, et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008;4(5):313–21. doi:10.1038/nchembio.83.CrossRefPubMedPubMedCentral

21.

Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000;1(6):489–95.CrossRefPubMed

22.

Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G. Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol. 2010;11(10):700–14. doi:10.1038/nrm2970.CrossRefPubMed

23.

Festjens N, Berghe TV, Cornelis S, Vandenabeele P. RIP1, a kinase on the crossroads of a cell’s decision to live or die. Cell Death Differ. 2007;14(4):400–10.CrossRefPubMed

24.

He S, Wang L, Miao L, Wang T, Du F, Zhao L, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α. Cell. 2009;137(6):1100–11. doi:10.1016/j.cell.2009.05.021.CrossRefPubMed

25.

Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science. 2009;325(5938):332–6. doi:10.1126/science.1172308.CrossRefPubMed

26.

Shen HM, Liu ZG. JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species. Free Radic Biol Med. 2006;40(6):928–39.CrossRefPubMed

27.

Lin Y, Devin A, Rodriquez Y, Liu ZG. Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 1999;13(19):2514–26.CrossRefPubMedPubMedCentral

28.

Feoktistova M, Geserick P, Kellert B, Dimitrova DP, Langlais C, Hupe M, et al. cIAPs block ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms. Mol Cell. 2011;43(3):449–63. doi:10.1016/j.molcel.2011.06.011.CrossRefPubMedPubMedCentral

29.

Bonapace L, Bornhauser BC, Schmitz M, Cario G, Ziegler U, Niggli FK, et al. Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance. J Clin Investig. 2010;120(4):1310–23. doi:10.1172/JCI39987.CrossRefPubMedPubMedCentral

30.

Farkas T, Daugaard M, Jaattela M. Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy. J Biol Chem. 2011;286(45):38904–12. doi:10.1074/jbc.M111.269134.CrossRefPubMedPubMedCentral

31.

Bell BD, Leverrier S, Weist BM, Newton RH, Arechiga AF, Luhrs KA, et al. FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells. Proc Natl Acad Sci USA. 2008;105(43):16677–82. doi:10.1073/pnas.0808597105.CrossRefPubMedPubMedCentral

32.

Osborn SL, Diehl G, Han SJ, Xue L, Kurd N, Hsieh K, et al. Fas-associated death domain (FADD) is a negative regulator of T-cell receptor-mediated necroptosis. Proc Natl Acad Sci USA. 2010;107(29):13034–9. doi:10.1073/pnas.1005997107.CrossRefPubMedPubMedCentral

33.

Oberst A. Autophagic cell death RIPs into tumor. Cell Death Differ. 2013;20(9):1131–2. doi:10.1038/cdd.2013.89.CrossRefPubMedPubMedCentral

34.

Ye YC, Yu L, Wang HJ, Tashiro S, Onodera S, Ikejima T. TNFα-induced necroptosis and autophagy via supression of the p38-NF-κB survival pathway in L929 cells. J Pharmacol Sci. 2011;117(3):160–9.CrossRefPubMed

35.

Ha HC, Snyder SH. Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc Natl Acad Sci USA. 1999;96(24):13978–82.CrossRefPubMedPubMedCentral

36.

Yu SW, Wang H, Poitras MF, Coombs C, Bowers WJ, Federoff HJ, et al. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science. 2002;297(5579):259–63.CrossRefPubMed

37.

Zong WX, Ditsworth D, Bauer DE, Wang ZQ, Thompson CB. Alkylating DNA damage stimulates a regulated form of necrotic cell death. Genes Dev. 2004;18(11):1272–82.CrossRefPubMedPubMedCentral

38.

Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem. 2007;282(33):24131–45.CrossRefPubMed

39.

Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell. 2009;137(6):1062–75. doi:10.1016/j.cell.2009.03.048.CrossRefPubMedPubMedCentral

Title: Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells
Authors: Hyo-Jin Kim
Ki-Eun Hwang
Do-Sim Park
Seon-Hee Oh
Hong Young Jun
Kwon-Ha Yoon
Eun-Taik Jeong
Hak-Ryul Kim
Young-Suk Kim
Publication date: 01-12-2017
Publisher: BioMed Central
Published in: Journal of Translational Medicine / Issue 1/2017
Electronic ISSN: 1479-5876
DOI: https://doi.org/10.1186/s12967-017-1223-7

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Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells

Abstract

Background

Methods

Results

Conclusions

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Abstract

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Conclusions

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