kjpp The Korean Journal of Physiology & Pharmacology Korean J Physiol Pharmacol 1226-4512 2093-3827 Korean J Physiol Pharmacol 10.4196/kjpp.2011.15.1.1 kjpp-15-1 Original Article Involvement of ROS in Curcumin-induced Autophagic Cell Death LeeYoun Ju 1 KimNam-Yi 2 SuhYoung-Ah 3 LeeChuHee 2 Department of Pharmacology, College of Medicine, Catholic University of Daegu, Daegu 705-718, Korea Department of Biochemistry and Molecular Biology, School of Medicine, Yeungnam University, Daegu 705-717, Korea Institute for Innovative Cancer Research, Asan Medical Center, Seoul 138-736, Korea Corresponding to: ChuHee Lee, Department of Biochemistry & Molecular Biology and Aging-Associated Vascular Disease Research Center, School of Medicine, Yeungnam University, 317-1 Daemyung-5-dong, Daegu 705-717, Korea. (Tel) 82-53-620-4522, (Fax) 82-53-654-6651, (E-mail) chlee2@ynu.ac.kr 022011 18022011 15 1 1 7 11102010 07022011 07022011 Copyright © 2011 Korean J Physiol Pharmacol 2011 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract

Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosomelysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 1/2 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell. However, ROS-dependent activation of ERK1/2 and p38 MAPK, but not JNK, might not be involved in the curcumin-induced autophagy.

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Curcumin causes conversion of LC3 protein and SQSTM1 degradation. HCT116 cells were treated with the indicated concentrations of curcumin for 20 h. (A) LC3-I and LC3-II protein levels were determined by Western blot analysis. Equal amounts of proteins were loaded and immunoblot of GAPDH was used as the loading control. (B) LC3 trunover was calculated based on the relative amount of LC3-I or LC3-II protein measured by the software Image Gauge 3.01 (Fujifilm). Data are expressed as the mean±SD of three independent experiments (∗p<0.05). (C) Total RNAs were isolated and subjected to RT-PCR. LC3 mRNA expression was determined and normalized to that of GAPDH. (D) SQSTM1 protein levels in cell lysates were determined by Western blot analysis. Equal amounts of proteins were loaded and immunoblot of GAPDH was used as the loading control. The data shown are representative of three independent experiments.

Curcumin induces GFP-LC3 puncta formation and endogenous LC3 conversion, and degradation of autophagy substrates. GFP-LC3 expressing HCT116 cells were treated with the indicated concentrations of curcumin for 20 h. (A) GFP-LC3 puncta was observed under fluorescence microscope. (B) Total cell lysates were prepared and subjected to Western blot analysis to detect the level of GFP-LC3, endogenous LC3-II, and SQSTM1 proteins. GAPDH was used as a loading control. The data shown are representative of at least three independent experiments.

Curcumin-generated ROS production is involved in autophagy induction. (A) HCT116 cells were treated as in Fig. 1 and stained with DCF-DA to detect intracellular ROS. ROS generation was determined using flow cytometry. The data shown are representative of three independent experiments. (B) GFP-LC3 expressing HCT116 cells were treated with indicated concentrations of hydrogen peroxide for 20 h. The level of GFP-LC3, endogenous LC3-II, and SQSTM1 protein was determined by Western blot analysis and GAPDH was used as a loading control. (C) Cells were exposed to 20μM curcumin with or without pretreatment of 10 mM NAC and stained with DCF-DA. Intracellular ROS level was determine using flow cytometry. The data shown are representative of three independent experiments. (D) HCT116 cells were treated with indicated concentrations of NAC for 2 h and subsequently exposed to a range of curcumin for 20 h. Western blot analysis was conducted to detect GFP-LC3, endogenous LC3, and SQSTM1 protein. GAPDH was used as a loading control. (E) LC3 mRNA expression was determined with total RNAs isolated from cells after treatment and normalized to the level of GAPDH. Results are from three independent experiments. The data shown are representative of three independent experiments.

Bafilomycin A, an autophagy inhibitor, diminishes curcumin-induced cytotoxicity. Cells were treated with a range of curcumin with or without NAC or bafilomycin A. (A) Cell viability was determined by WST-8 assay. Data are expressed as the mean± SD of three independent experiments conducted in triplicate (∗p <0.05 control vs 10 mM NAC, and control vs 50μM bafilomycin A). (B) Western blot analysis was conducted to detect LC3 and SQSTM1 protein. GAPDH was used as a loading control. The data shown are representative of three independent experiments.

Curcumin-induced ROS production and activation of ERK1/2 and p38 MAPK, but not Jun JNK causes induction and processing of LC3 proteins. (A) HCT116 cells were treated with indicated concentrations of NAC for 2 h and subsequently stimulated with 20μM curcumin for 20 h. Phosphorylation of ERK1/2, p38 MAPK, and JNK was determined by Western blot analysis. Phosphorylation of MAPKs was determined with Western blot analysis and GAPDH was used as a loading control. Results are from three independent experiments. (B∼D) Cells were pre-treated with MAPK inhibitors and then exposed to 20μM curcumin for 20 h. Total cell lysates were applied to Western blot analysis to detect GFP-LC3, endogenous LC3-II, and SQSTM1 protein levels. GAPDH was used as a loading control. The data shown are representative of at least three independent experiments.