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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

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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 ceramide-induced 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.

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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

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Acknowledgments

Funding/Support: This study was supported in part by the National Natural Science Foundation of China.

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Authors and Affiliations

  1. Department of General Surgery, The Ninth People’s Hospital Affiliated to Shanghai Jiao-tong University School of Medicine, 639 Zhizhaoju Road, Shanghai, 200011, China

    Hai-zhong Huo, Bing Wang, Jian Qin, Shan-yu Guo, Wen-yong Liu & Yan Gu

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  1. Hai-zhong Huo
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  2. Bing Wang
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Correspondence to Yan Gu.

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Huo, Hz., Wang, B., Qin, J. et al. AMP-activated protein kinase (AMPK)/Ulk1-dependent autophagic pathway contributes to C6 ceramide-induced cytotoxic effects in cultured colorectal cancer HT-29 cells. Mol Cell Biochem 378, 171–181 (2013). https://doi.org/10.1007/s11010-013-1608-8

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