Nutrition Research and Practice

The Korean Nutrition Society (한국영양학회)
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Induction of cytoprotective autophagy by morusin via AMP-activated protein kinase activation in human non-small cell lung cancer cells

  • Park, Hyun-Ji (Department of Pathology, College of Korean Medicine, Dong-Eui University) ;
  • Park, Shin-Hyung (Department of Pathology, College of Korean Medicine, Dong-Eui University)
  • Received : 2020.03.24
  • Accepted : 2020.06.04
  • Published : 2020.10.01
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Abstract

BACKGROUND/OBJECTIVES: Morusin, a marker component of Morus alba L., possesses anti-cancer activity. The objective of this study was to determine autophagy-inducing effect of morusin in non-small cell lung cancer (NSCLC) cells and investigate the underlying mechanism. SUBJECTS/METHODS: Autophagy induction and the expression of autophagy-related proteins were analyzed by LC3 immunofluorescence and western blot, respectively. The role of autophagy and AMP-activated protein kinase (AMPK) was determined by treating NSCLC cells with bafilomycin A1, an autophagy inhibitor, and compound C, an AMPK inhibitor. Cytotoxicity and apoptosis induction were determined by MTT assay, trypan blue exclusion assay, annexin V-propidium iodide (PI) double staining assay, and cell cycle analysis. RESULTS: Morusin increased the formation of LC3 puncta in the cytoplasm and upregulated the expression of autophagy-related 5 (Atg5), Atg12, beclin-1, and LC3II in NSCLC cells, demonstrating that morusin could induce autophagy. Treatment with bafilomycin A1 markedly reduced cell viability but increased proportions of sub-G1 phase cells and annexin V-positive cells in H460 cells. These results indicate that morusin can trigger autophagy in NSCLC cells as a defense mechanism against morusin-induced apoptosis. Furthermore, we found that AMPK and its downstream acetyl-CoA carboxylase (ACC) were phosphorylated, while mammalian target of rapamycin (mTOR) and its downstream p70S6 kinase (p70S6K) were dephosphorylated by morusin. Morusin-induced apoptosis was significantly increased by treatment with compound C in H460 cells. These results suggest that morusin-induced AMPK activation could protect NSCLC cells from apoptosis probably by inducing autophagy. CONCLUSIONS: Our findings suggest that combination treatment with morusin and autophagy inhibitor or AMPK inhibitor might enhance the clinical efficacy of morusin for NSCLC.

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References

  1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin 2018;68:7-30. https://doi.org/10.3322/caac.21442
  2. Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA. Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 2008;83:584-94. https://doi.org/10.4065/83.5.584
  3. Langer CJ, Besse B, Gualberto A, Brambilla E, Soria JC. The evolving role of histology in the management of advanced non-small-cell lung cancer. J Clin Oncol 2010;28:5311-20. https://doi.org/10.1200/JCO.2010.28.8126
  4. Yan YF, Zheng YF, Ming PP, Deng XX, Ge W, Wu YG. Immune checkpoint inhibitors in non-small-cell lung cancer: current status and future directions. Brief Funct Genomics 2019;18:147-56. https://doi.org/10.1093/bfgp/ely029
  5. Feng Y, He D, Yao Z, Klionsky DJ. The machinery of macroautophagy. Cell Res 2014;24:24-41. https://doi.org/10.1038/cr.2013.168
  6. Kroemer G, Marino G, Levine B. Autophagy and the integrated stress response. Mol Cell 2010;40:280-93. https://doi.org/10.1016/j.molcel.2010.09.023
  7. Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science 2004;306:990-5. https://doi.org/10.1126/science.1099993
  8. White E. Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer 2012;12:401-10. https://doi.org/10.1038/nrc3262
  9. Helgason GV, Holyoake TL, Ryan KM. Role of autophagy in cancer prevention, development and therapy. Essays Biochem 2013;55:133-51. https://doi.org/10.1042/bse0550133
  10. Levy JM, Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer 2017;17:528-42. https://doi.org/10.1038/nrc.2017.53
  11. Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol 2011;13:1016-23. https://doi.org/10.1038/ncb2329
  12. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, Cantley LC. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 2004;101:3329-35. https://doi.org/10.1073/pnas.0308061100
  13. Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 2011;13:132-41. https://doi.org/10.1038/ncb2152
  14. Shaw RJ, Bardeesy N, Manning BD, Lopez L, Kosmatka M, DePinho RA, Cantley LC. The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 2004;6:91-9. https://doi.org/10.1016/j.ccr.2004.06.007
  15. Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE, Shaw RJ. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 2008;30:214-26. https://doi.org/10.1016/j.molcel.2008.03.003
  16. Grosdidier A, Zoete V, Michielin O. SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Res 2011;39:W270-7. https://doi.org/10.1093/nar/gkr366
  17. Lin WL, Lai DY, Lee YJ, Chen NF, Tseng TH. Antitumor progression potential of morusin suppressing STAT3 and $NF{\kappa}B$ in human hepatoma SK-Hep1 cells. Toxicol Lett 2015;232:490-8. https://doi.org/10.1016/j.toxlet.2014.11.031
  18. Ding B, Lv Y, Zhang YQ. Anti-tumor effect of morusin from the branch bark of cultivated mulberry in Bel-7402 cells via the MAPK pathway. RSC Advances 2016;6:17396-404. https://doi.org/10.1039/C5RA21321E
  19. Zoofishan Z, Hohmann J, Hunyadi A. Phenolic antioxidants of Morus nigra roots, and antitumor potential of morusin. Phytochem Rev 2018;17:1031-45. https://doi.org/10.1007/s11101-018-9565-1
  20. Park HJ, Min TR, Chi GY, Choi YH, Park SH. Induction of apoptosis by morusin in human non-small cell lung cancer cells by suppression of EGFR/STAT3 activation. Biochem Biophys Res Commun 2018;505:194-200. https://doi.org/10.1016/j.bbrc.2018.09.085
  21. Cho SW, Na W, Choi M, Kang SJ, Lee SG, Choi CY. Autophagy inhibits cell death induced by the anti-cancer drug morusin. Am J Cancer Res 2017;7:518-30.
  22. Park SH, Chi GY, Eom HS, Kim GY, Hyun JW, Kim WJ, Lee SJ, Yoo YH, Choi YH. Role of autophagy in apoptosis induction by methylene chloride extracts of Mori cortex in NCI-H460 human lung carcinoma cells. Int J Oncol 2012;40:1929-40. https://doi.org/10.3892/ijo.2012.1386
  23. Tsai SC, Yang JS, Peng SF, Lu CC, Chiang JH, Chung JG, Lin MW, Lin JK, Amagaya S, Wai-Shan Chung C, Tung TT, Huang WW, Tseng MT. Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells. Int J Oncol 2012;41:1431-42. https://doi.org/10.3892/ijo.2012.1579
  24. Huang WW, Tsai SC, Peng SF, Lin MW, Chiang JH, Chiu YJ, Fushiya S, Tseng MT, Yang JS. Kaempferol induces autophagy through AMPK and AKT signaling molecules and causes G2/M arrest via downregulation of CDK1/cyclin B in SK-HEP-1 human hepatic cancer cells. Int J Oncol 2013;42:2069-77. https://doi.org/10.3892/ijo.2013.1909
  25. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 1998;23:33-42. https://doi.org/10.1247/csf.23.33
  26. Li W, Saud SM, Young MR, Chen G, Hua B. Targeting AMPK for cancer prevention and treatment. Oncotarget 2015;6:7365-78. https://doi.org/10.18632/oncotarget.3629
  27. Yang YP, Hu LF, Zheng HF, Mao CJ, Hu WD, Xiong KP, Wang F, Liu CF. Application and interpretation of current autophagy inhibitors and activators. Acta Pharmacol Sin 2013;34:625-35. https://doi.org/10.1038/aps.2013.5
  28. Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, Sougnez C, Greulich H, Muzny DM, Morgan MB, Fulton L, Fulton RS, Zhang Q, Wendl MC, Lawrence MS, Larson DE, Chen K, Dooling DJ, Sabo A, Hawes AC, Shen H, Jhangiani SN, Lewis LR, Hall O, Zhu Y, Mathew T, Ren Y, Yao J, Scherer SE, Clerc K, Metcalf GA, Ng B, Milosavljevic A, Gonzalez-Garay ML, Osborne JR, Meyer R, Shi X, Tang Y, Koboldt DC, Lin L, Abbott R, Miner TL, Pohl C, Fewell G, Haipek C, Schmidt H, Dunford-Shore BH, Kraja A, Crosby SD, Sawyer CS, Vickery T, Sander S, Robinson J, Winckler W, Baldwin J, Chirieac LR, Dutt A, Fennell T, Hanna M, Johnson BE, Onofrio RC, Thomas RK, Tonon G, Weir BA, Zhao X, Ziaugra L, Zody MC, Giordano T, Orringer MB, Roth JA, Spitz MR, Wistuba II, Ozenberger B, Good PJ, Chang AC, Beer DG, Watson MA, Ladanyi M, Broderick S, Yoshizawa A, Travis WD, Pao W, Province MA, Weinstock GM, Varmus HE, Gabriel SB, Lander ES, Gibbs RA, Meyerson M, Wilson RK. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 2008;455:1069-75. https://doi.org/10.1038/nature07423
  29. Memmott RM, Gills JJ, Hollingshead M, Powers MC, Chen Z, Kemp B, Kozikowski A, Dennis PA. Phosphatidylinositol ether lipid analogues induce AMP-activated protein kinase-dependent death in LKB1-mutant non small cell lung cancer cells. Cancer Res 2008;68:580-8. https://doi.org/10.1158/0008-5472.CAN-07-3091
  30. Carretero J, Medina PP, Blanco R, Smit L, Tang M, Roncador G, Maestre L, Conde E, Lopez-Rios F, Clevers HC, Sanchez-Cespedes M. Dysfunctional AMPK activity, signalling through mTOR and survival in response to energetic stress in LKB1-deficient lung cancer. Oncogene 2007;26:1616-25. https://doi.org/10.1038/sj.onc.1209951
  31. William WN, Kim JS, Liu DD, Solis L, Behrens C, Lee JJ, Lippman SM, Kim ES, Hong WK, Wistuba II, Lee HY. The impact of phosphorylated AMP-activated protein kinase expression on lung cancer survival. Ann Oncol 2012;23:78-85. https://doi.org/10.1093/annonc/mdr036
  32. Cheng J, Zhang T, Ji H, Tao K, Guo J, Wei W. Functional characterization of AMP-activated protein kinase signaling in tumorigenesis. Biochim Biophys Acta 2016;1866:232-51.
  33. Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky DJ. Potential therapeutic applications of autophagy. Nat Rev Drug Discov 2007;6:304-12. https://doi.org/10.1038/nrd2272
  34. Min TR, Park HJ, Park MN, Kim B, Park SH. The root bark of Morus alba L. suppressed the migration of human non-small-cell lung cancer cells through inhibition of epithelial-mesenchymal transition mediated by STAT3 and Src. Int J Mol Sci 2019;20:E2244.
  35. Zhao Y, Hu X, Liu Y, Dong S, Wen Z, He W, Zhang S, Huang Q, Shi M. ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway. Mol Cancer 2017;16:79. https://doi.org/10.1186/s12943-017-0648-1
  36. Kwon Y, Kim M, Jung HS, Kim Y, Jeoung D. Targeting autophagy for overcoming resistance to anti-EGFR treatments. Cancers (Basel) 2019;11:1374. https://doi.org/10.3390/cancers11091374
  37. Duffy A, Le J, Sausville E, Emadi A. Autophagy modulation: a target for cancer treatment development. Cancer Chemother Pharmacol 2015;75:439-47. https://doi.org/10.1007/s00280-014-2637-z

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