Inhibition of autophagy by 3-MA potentiates cisplatin-induced apoptosis in esophageal squamous cell carcinoma cells
First Online: 30 December 2009 Received: 10 December 2009 Accepted: 15 December 2009 DOI:
Cite this article as: Liu, D., Yang, Y., Liu, Q. et al. Med Oncol (2011) 28: 105. doi:10.1007/s12032-009-9397-3 Abstract
Cisplatin (DDP)-based adjuvant chemotherapy is widely used for the treatment of esophageal cancer. However, DDP resistance has become more common and thus new approaches are required to be explored. Cisplatin was used to induce autophagy in the human esophageal cancer cell line, EC9706 cells, and the effect of autophagy on the survival of EC9706 cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by CCK8 assay. Apoptosis and cell cycle were detected by flow cytometry. Monodansylcadaverine (MDC) was used to detect autophagy. Western blotting assay was used to investigate the molecular changes that occurred in the course of treatment. DDP inhibited cell proliferation, induced cell death and cell cycle arrest at S phage. Moreover, autophagy was activated through class III PI3K pathway. The expression of autophagy-related Beclin1 and LC3-I was up-regulated and part of LC3-I was converted into LC3-II. However, after the combination treatment of 3-MA and DDP, the cell inhibitory rate increased; the apoptosis rate and the numbers of cells in S phase also increased. Furthermore, the accumulation of autophagic vacuoles was decreased; the expression of Beclin1 and LC3 was significantly down-regulated and the release of cytochrome c was decreased. DDP-induced apoptosis in EC9706 cells can be enhanced by the inhibitor of autophagy, 3-MA. Autophagy might play a role as a self-protective mechanism in DDP-treated esophageal cancer cells, and its inhibition could be a novel strategy for the adjuvant chemotherapy of esophageal cancer.
Keywords Autophagy Apoptosis Esophageal squamous cell carcinoma Cisplatin References
Pisani P, Parkin DM, Bray F, Ferlay J. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer. 1999;83:18–29.
Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med. 2003;349:2241–52.
Sun Y, Peng ZL. Programmed cell death and cancer. Postgrad Med J. 2009;85:134–40.
Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27–42.
Kroemer G, et al. Classification of cell death: recommendations of the nomenclature committee on cell death. Cell Death Differ. 2005;12:1463–7.
Li J, et al. Inhibition of autophagy by 3-MA enhances the effect of 5-FU-induced apoptosis in colon cancer cells. Ann Surg Oncol. 2009;16:761–71.
Lomonaco SL, et al. The induction of autophagy by gamma-radiation contributes to the radioresistance of glioma stem cells. Int J Cancer. 2009;125:717–22.
Chen Y, Lu Y, Lu C, Zhang L. Beclin-1 expression is a predictor of clinical outcome in patients with esophageal squamous cell carcinoma and correlated to hypoxia-inducible factor (HIF)-1alpha expression. Pathol Oncol Res. 2009. doi:
Munafo DB, Colombo MI. A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci. 2001;114:3619–29.
Biederbick A, Kern HF, Elsasser HP. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol. 1995;66:3–14.
Goodsell DS. The molecular perspective: cytochrome
and apoptosis. Stem Cells. 2004;22:428–9.
Liang XH, et al. Induction of autophagy and inhibition of tumorgenesis by Beclin1. Nature. 1999;402:672–6.
Kabeya Y, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000;19:5720–8.
Galluzzi L, et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryote. Cell Death Differ. 2009;16:1093–107.
Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science. 2004;306:990–5.
Rabik CA, Dolan ME. Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treat Rev. 2007;33:9–23.
Lefranc F, Kiss R. Autophagy, the Trojan horse to combat glioblastomas. Neurosurg Focus. 2006;20:E7. doi:
Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene. 2003;22:7265–79.
Kartalou M, Essigmann JM. Mechanisms of resistance to cisplatin. Mutat Res. 2001;478:23–43.
Seglen PO, Gordon PB. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA. 1982;79:1889–92.
Lum JJ, et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell. 2005;120:237–48.
Herman-Antosiewicz A, Johnson DE, Singh SV. Sulforaphane causes autophagy to inhibit release of cytochrome c and apoptosis in human prostate cancer cells. Cancer Res. 2006;66:5828–35.
Nishikawa T, et al. Inhibition of autophagy potentiates sulforaphane—induced apoptosis in human colon cancer cells. Ann Surg Oncol. 2009 [Epub ahead of print]. doi:
Degenhardt K, et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 2006;10:51–64.
Karantza-Wadsworth V, et al. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev. 2007;21:1621–35.
Katayama M, Kawaguchi T, Berger MS, Pieper RO. DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells. Cell Death Differ. 2007;14:548–58.
Pan J, et al. Autophagy induced by farnesyltransferase inhibitors in cancer cells. Cancer Biol Ther. 2008;7:1679–84.
Park MA, et al. Vorinostat and sorafenib increase ER stress, autophagy and apoptosis via ceramide-dependent CD95 and PERK activation. Cancer Biol Ther. 2008;7:1648–62.
de Bruin EC, Medema JP. Apoptosis and non-apoptotic death in cancer development and treatment response. Cancer Treat Rev. 2008;34:737–49.
Maiuri MC, et al. Control of autophagy by oncogenes and tumor suppressor genes. Cell Death Differ. 2009;16:87–93.
Ding WX, et al. Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J Biol Chem. 2007;282:4702–10.
Wu YC, et al. Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells. Biochem Biophys Res Commun. 2009;382:451–6.
Gozuacik D, Kimch A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene. 2004;23:2891–906.
Yang C, et al. Inhibition of autophagy induced by overexpression of mda-7/interleukin-24 strongly augments the antileukemia activity in vitro and in vivo. Cancer Gene Ther. 2009 [Epub ahead of print]. doi:
Chen N, Karantza-Wadsworth V. Role and regulation of autophagy in cancer. Biochim Biophys Acta. 2009;1793:1516–23.
Iwamaru A, et al. Silencing mammalian target of rapamycin signaling by small interfering RNA enhances rapamycin-induced autophagy in malignant glioma cells. Oncogene. 2007;26:1840–51.
Takeuchi H, et al. Synergistic augmentation of rapamycin-induced auto-phagy in malignant glioma cells by phosphatidylinositol 3-kinase protein kinase B inhibitors. Cancer Res. 2005;65:3336–46.
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