Induction of cytoprotective autophagy by morusin via AMP-activated protein kinase activation in human non-small cell lung cancer cells
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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.
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.
참고문헌 (37)
- Siegel RL, "Cancer statistics, 2018", CA Cancer J Clin, 68, pp.7~30 (2018)
- Molina JR, "Non-small cell lung cancer : epidemiology, risk factors, treatment, and survivorship", Mayo Clin Proc, 83, pp.584~594 (2008)
- Langer CJ, "The evolving role of histology in the management of advanced non-small-cell lung cancer", J Clin Oncol, 28, pp.5311~5320 (2010)
- Yan YF, "Immune checkpoint inhibitors in non-small-cell lung cancer : current status and future directions", Brief Funct Genomics, 18, pp.147~156 (2019)
- Feng Y, "The machinery of macroautophagy", Cell Res, 24, pp.24~41 (2014)
- Kroemer G, "Autophagy and the integrated stress response", Mol Cell, 40, pp.280~293 (2010)
- Shintani T, "Autophagy in health and disease : a double-edged sword", Science, 306, pp.990~995 (2004)
- White E, "Deconvoluting the context-dependent role for autophagy in cancer", Nat Rev Cancer, 12, pp.401~410 (2012)
- Helgason GV, "Role of autophagy in cancer prevention, development and therapy", Essays Biochem, 55, pp.133~151 (2013)
- Levy JM, "Targeting autophagy in cancer", Nat Rev Cancer, 17, pp.528~542 (2017)
- Mihaylova MM, "The AMPK signalling pathway coordinates cell growth, autophagy and metabolism", Nat Cell Biol, 13, pp.1016~1023 (2011)
- Shaw RJ, "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, 101, pp.3329~3335 (2004)
- Kim J, "AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1", Nat Cell Biol, 13, pp.132~141 (2011)
- Shaw RJ, "The LKB1 tumor suppressor negatively regulates mTOR signaling", Cancer Cell, 6, pp.91~99 (2004)
- Gwinn DM, "AMPK phosphorylation of raptor mediates a metabolic checkpoint", Mol Cell, 30, pp.214~226 (2008)
- Grosdidier A, "SwissDock, a protein-small molecule docking web service based on EADock DSS", Nucleic Acids Res, 39, pp.W270~W277 (2011)
- Lin WL, "Antitumor progression potential of morusin suppressing STAT3 and NFκB in human hepatoma SK-Hep1 cells", Toxicol Lett, 232, pp.490~498 (2015)
- Ding B, "Anti-tumor effect of morusin from the branch bark of cultivated mulberry in Bel-7402 cells via the MAPK pathway", RSC Advances, 6, pp.17396~17404 (2016)
- Zoofishan Z, "Phenolic antioxidants of Morus nigra roots, and antitumor potential of morusin", Phytochem Rev, 17, pp.1031~1045 (2018)
- Park HJ, "Induction of apoptosis by morusin in human non-small cell lung cancer cells by suppression of EGFR", STAT3 activation, 2018, pp.505, 194~200
- Cho SW, "Autophagy inhibits cell death induced by the anti-cancer drug morusin", Am J Cancer Res, 7, pp.518~530 (2017)
- Park SH, "Role of autophagy in apoptosis induction by methylene chloride extracts of Mori cortex in NCI-H460 human lung carcinoma cells", Int J Oncol, 40, pp.1929~1940 (2012)
- Tsai SC, "Bufalin increases sensitivity to AKT", mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells, 2012, pp.41, 1431~1442
- Huang WW, "Kaempferol induces autophagy through AMPK and AKT signaling molecules and causes G2", M arrest via downregulation of CDK1, Int J Oncol, pp.2013, 42[DOI]
- Yamamoto A, "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, 23, pp.33~42 (1998)
- Li W, "Targeting AMPK for cancer prevention and treatment", Oncotarget, 6, pp.7365~7378 (2015)
- Yang YP, "Application and interpretation of current autophagy inhibitors and activators", Acta Pharmacol Sin, 34, pp.625~635 (2013)
- Ding L, "Somatic mutations affect key pathways in lung adenocarcinoma", Nature, 455, pp.1069~1075 (2008)
- Memmott RM, "Phosphatidylinositol ether lipid analogues induce AMP-activated protein kinase-dependent death in LKB1-mutant non small cell lung cancer cells", Cancer Res, 68, pp.580~588 (2008)
- Carretero J, "Dysfunctional AMPK activity, signalling through mTOR and survival in response to energetic stress in LKB1-deficient lung cancer", Oncogene, 26, pp.1616~1625 (2007)
- William WN, "The impact of phosphorylated AMP-activated protein kinase expression on lung cancer survival", Ann Oncol, 23, pp.78~85 (2012)
- Cheng J, "Functional characterization of AMP-activated protein kinase signaling in tumorigenesis", Biochim Biophys Acta, 1866, pp.232~251 (2016)
- Rubinsztein DC, "Potential therapeutic applications of autophagy", Nat Rev Drug Discov, 6, pp.304~312 (2007)
- Min TR, "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, 20, pp.E2244~ (2019)
- Zhao Y, "ROS signaling under metabolic stress : cross-talk between AMPK and AKT pathway", Mol Cancer, 16, pp.79~ (2017)
- Kwon Y, "Targeting autophagy for overcoming resistance to anti-EGFR treatments", Cancers(Basel), 11, pp.1374~ (2019)
- Duffy A, "Autophagy modulation : a target for cancer treatment development", Cancer Chemother Pharmacol, 75, pp.439~447 (2015)