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Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo

Abstract

Ceramidases (CDases) play a key role in cancer therapy through enhanced conversion of ceramide into sphingosine 1-phosphate (S1P), but their involvement in hepatocarcinogenesis is unknown. Here, we report that daunorubicin (DNR) activated acid CDase post-transcriptionally in established human (HepG2 cells) or mouse (Hepa1c1c7) hepatoma cell lines as well as in primary cells from murine liver tumors, but not in cultured mouse hepatocytes. Acid CDase silencing by small interfering RNA (siRNA) or pharmacological inhibition with N-oleoylethanolamine (NOE) enhanced the ceramide to S1P balance compared to DNR alone, sensitizing hepatoma cells (HepG2, Hep-3B, SK-Hep and Hepa1c1c7) to DNR-induced cell death. DNR plus NOE or acid CDase siRNA-induced cell death was preceded by ultrastructural changes in mitochondria, stimulation of reactive oxygen species generation, release of Smac/DIABLO and cytochrome c and caspase-3 activation. In addition, in vivo siRNA treatment targeting acid CDase reduced tumor growth in liver tumor xenografts of HepG2 cells and enhanced DNR therapy. Thus, acid CDase promotes hepatocarcinogenesis and its antagonism may be a promising strategy in the treatment of liver cancer.

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Abbreviations

Ac-DEVD-AMC:

Ac-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin

Cdases:

ceramidases

DCF:

2′,7′-dichlorofluorescin

DMS:

dimethyl sphingosine

GCS:

glucosylceramide synthase

D-MAPP:

D-erythro-2-tetradecanoylamino-1-phenyl-1-propanol

NOE:

N-oleylethanolamine

NSMase:

neutral sphingomyelinase

ROS:

reactive oxygen species

SK:

sphingosine kinase

S1P:

sphingosine-1-phosphate

TMRM:

tetramethylrhodamine methyl ester

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Acknowledgements

This work was supported in part by the Research Center for Liver and Pancreatic Diseases Grant P50-AA11999 and 1R21 AA014135-01 funded by the National Institute on Alcohol Abuse and Alcoholism, Plan Nacional de I+D Grants SAF2001-2118, SAF2002-3564, SAF2003-4974, Fondo de Investigaciones Sanitarias, FISS 02/3057 and FISS 03/0426, and Red Temática de Investigación Cooperativa G03/015 and Red de Centros C03/02 supported by Instituto de Salud Carlos III. The technical assistance of Susana Nuñez and the Servicio Cientificotecnico of IDIBAPS is highly appreciated. AM and AV are Ramón y Cajal Investigators.

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Correspondence to J C Fernández-Checa.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Morales, A., París, R., Villanueva, A. et al. Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo. Oncogene 26, 905–916 (2007). https://doi.org/10.1038/sj.onc.1209834

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