AMP-activated protein kinase (AMPK)/Ulk1-dependent autophagic pathway contributes to C6 ceramide-induced cytotoxic effec
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AMP-activated protein kinase (AMPK)/Ulk1-dependent autophagic pathway contributes to C6 ceramide-induced cytotoxic effects in cultured colorectal cancer HT-29 cells Hai-zhong Huo • Bing Wang • Jian Qin • Shan-yu Guo • Wen-yong Liu • Yan Gu
Received: 12 December 2012 / Accepted: 23 February 2013 / Published online: 19 March 2013 Ó Springer Science+Business Media New York 2013
Abstract Colorectal cancer is the second leading cause of cancer-related deaths. Drug resistance and/or off-target toxicity against normal cells limit the effectiveness of current chemotherapies for the treatment of colorectal cancer. In the current study, we studied the potential cytotoxic effects of short-chain and cell-permeable C6 ceramide in cultured colorectal cancer HT-29 cells and focused on the underlying mechanisms. We observed that C6 ceramide-induced HT-29 cell death and growth inhibition in a dose- and time-dependent manner. However, no significant apoptosis was observed in C6 ceramide-treated HT-29 cells. Our data support that autophagy contributed to C6 ceramide-induced cytotoxic effects, as autophagy inhibitors, 3-methyladenine (3-MA) and hydroxychloroquine, inhibited C6 ceramide’s effect; however, autophagy activators, everolimus (RAD001) and temsirolimus, mimicked C6 ceramide effects and induced HT-29 cell death. Further, we indentified that AMP-activated protein kinase (AMPK)/Ulk1 signaling was required for autophagy induction by C6 ceramide, and AMPK silencing by a specific short hairpin RNA suppressed C6 ceramideinduced autophagy and cytotoxic effects. Reversely, forced activation of AMPK by its activator AICAR or by genetic manipulation caused autophagic death in HT-29 cells, which was inhibited by 3-MA. Our results suggest that autophagy, but not apoptosis, is a major contributor for C6 ceramide-induced cytotoxic effects in HT-29 cells, and activation of AMPK/Ulk1 is required for the process.
H. Huo B. Wang J. Qin S. Guo W. Liu Y. Gu (&) Department of General Surgery, The Ninth People’s Hospital Affiliated to Shanghai Jiao-tong University School of Medicine, 639 Zhizhaoju Road, Shanghai 200011, China e-mail: [email protected]
Keywords C6 ceramide AMPK/Ulk1 pathway Autophagy Colorectal cancer Abbreviations 3-MA 3-Methyladenine ACC Acetyl-CoA carboxylase AMPK AMP-activated protein kinase AICAR 5-Aminoimidazole-4-carboxamide ribotide JNK c-Jun N-terminal kinase LC3B Light chain 3B MTT 3-[4,5-Dimethylthylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide mTOR Mammalian target or rapamycin mTORC1 mTOR complex 1 sh-RNA Short hairpin RNA PI Propidium iodide
Introduction Colorectal cancer is the second leading cause of cancerrelated deaths in China and around the world [1, 2]. Drug resistance and/or off-target toxicity against normal cells limit the effectiveness of current chemotherapies for the treatment of colorectal cancer [3–5]; as such, the development of novel cancer chemotherapies is necessary and urgent [6, 7]. It has been well characterized that ceramide is a generalized mediator of cell death [8–13]. S
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