Nutr Res Pract.  2020 Oct;14(5):478-489. 10.4162/nrp.2020.14.5.478.

Induction of cytoprotective autophagy by morusin via AMP-activated protein kinase activation in human non-small cell lung cancer cells

Affiliations
  • 1Department of Pathology, College of Korean Medicine, Dong-Eui University, Busan 47227, Korea

Abstract

BACKGROUND/OBJECTIVES
Morusin, a marker component of Morus alba L., possesses anticancer 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.

Keyword

Non-small cell lung cancer; autophagy; AMP-activated protein kinases; apoptosis

Figure

  • Fig. 1 Induction of autophagy by morusin in non-small cell lung cancer cells. Cells were treated with morusin (20 μM) for indicated time periods. (A) Cellular morphology was photographed with a microscope (×200 magnification). (B) Cells were subjected to immunostaining with anti-LC3 antibody and fluorescent-dye conjugated secondary antibody to observe LC3 puncta. DAPI staining was performed to visualize nuclei. LC3 puncta (green) and nuclei (blue) were photographed and merged using a fluorescence microscope (×200 magnification). (C and D) Expression of indicated protein was detected by western blot. Actin was a loading control. ImageJ software was used for densitometric analysis of immunoblots.*P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated control group.

  • Fig. 2 Inhibition of autophagy enhances morusin-induced apoptosis in non-small cell lung cancer cells. H460 cells were treated with indicated concentration of morusin for 12 h (A) or 72 h (B-E) w/ or w/o bafilomycin A1 (1 nM), an autophagy inhibitor. (A) The expression of LC3II was detected by western blot analysis. Actin was used as an internal control. (B) Cell viability was measured by MTT assay. (C) Percentage of survived cells was evaluated by trypan blue exclusion assay. (D) Cell cycle analysis was performed by flow cytometry to measure the percentage of sub-G1 phase cells (apoptotic cells). (E) Annexin V/PI double staining assay was conducted to detect apoptosis. Annexin V(+) cells were determined as apoptotic cells.Mo, morusin; Baf, bafilomycin A1.***P < 0.001 vs. respective controls.

  • Fig. 3 Role of AMPK/mTOR pathway in morusin-induced autophagy in non-small cell lung cancer cells. Cells were treated with morusin (20 μM) at various time periods. (A) The expression of indicated protein was detected by western blot. Actin was a loading control. (B) The ratio between phosphorylated proteins and total proteins was calculated after densitometric analysis of immunoblots using Image J software.AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; mTOR, mammalian target of rapamycin; p70S6K, phospho-p70S6 kinase.***P < 0.001 vs. untreated controls.

  • Fig. 4 Inhibition of AMPK enhances morusin-induced apoptosis in non-small cell lung cancer cells. H460 cells were incubated with morusin (5 μM) for 24 h (A) or 72 h (B-D) w/ or w/o compound C (1 μM), an AMPK inhibitor. (A) Expression of indicated protein was detected by western blot analysis. Actin was used as an internal control. (B) Cell viability was examined by MTT assay. (C) The percentage of survived cells was evaluated by trypan blue exclusion assay. (D) The percentage of sub-G1 phase cells (apoptotic cells) was measured by flow cytometry.AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase.***P < 0.001 vs. respective controls.


Reference

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018; 68:7–30. PMID: 29313949.
Article
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; 83:584–594. PMID: 18452692.
Article
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; 28:5311–5320. PMID: 21079145.
Article
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; 18:147–156. PMID: 30247518.
Article
5. Feng Y, He D, Yao Z, Klionsky DJ. The machinery of macroautophagy. Cell Res. 2014; 24:24–41. PMID: 24366339.
Article
6. Kroemer G, Mariño G, Levine B. Autophagy and the integrated stress response. Mol Cell. 2010; 40:280–293. PMID: 20965422.
Article
7. Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science. 2004; 306:990–995. PMID: 15528435.
Article
8. White E. Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer. 2012; 12:401–410. PMID: 22534666.
Article
9. Helgason GV, Holyoake TL, Ryan KM. Role of autophagy in cancer prevention, development and therapy. Essays Biochem. 2013; 55:133–151. PMID: 24070477.
Article
10. Levy JM, Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer. 2017; 17:528–542. PMID: 28751651.
Article
11. Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol. 2011; 13:1016–1023. PMID: 21892142.
Article
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; 101:3329–3335. PMID: 14985505.
Article
13. Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011; 13:132–141. PMID: 21258367.
Article
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; 6:91–99. PMID: 15261145.
Article
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; 30:214–226. PMID: 18439900.
Article
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. PMID: 21624888.
Article
17. Lin WL, Lai DY, Lee YJ, Chen NF, Tseng TH. Antitumor progression potential of morusin suppressing STAT3 and NFκB in human hepatoma SK-Hep1 cells. Toxicol Lett. 2015; 232:490–498. PMID: 25476160.
Article
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; 6:17396–17404.
Article
19. Zoofishan Z, Hohmann J, Hunyadi A. Phenolic antioxidants of Morus nigra roots, and antitumor potential of morusin. Phytochem Rev. 2018; 17:1031–1045.
Article
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. PMID: 30243717.
Article
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; 7:518–530. PMID: 28401008.
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; 40:1929–1940. PMID: 22367066.
Article
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; 41:1431–1442. PMID: 22858649.
Article
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; 42:2069–2077. PMID: 23591552.
Article
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. PMID: 9639028.
Article
26. Li W, Saud SM, Young MR, Chen G, Hua B. Targeting AMPK for cancer prevention and treatment. Oncotarget. 2015; 6:7365–7378. PMID: 25812084.
Article
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; 34:625–635. PMID: 23524572.
Article
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; 455:1069–1075. PMID: 18948947.
Article
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; 68:580–588. PMID: 18199555.
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; 26:1616–1625. PMID: 16953221.
Article
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. PMID: 21430184.
Article
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; 1866:232–251. PMID: 27681874.
Article
33. Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky DJ. Potential therapeutic applications of autophagy. Nat Rev Drug Discov. 2007; 6:304–312. PMID: 17396135.
Article
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. PMID: 31067694.
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. PMID: 28407774.
Article
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.
Article
37. Duffy A, Le J, Sausville E, Emadi A. Autophagy modulation: a target for cancer treatment development. Cancer Chemother Pharmacol. 2015; 75:439–447. PMID: 25422156.
Article
Full Text Links
  • NRP
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr