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Cannabinoid Receptor Activation Induces Apoptosis through Tumor Necrosis Factor –Mediated Ceramide De novo Synthesis in Colon Cancer Cells

Authors' Affiliations: Departments of ¹ Medical and Surgical Critical Care, ² Experimental Pathology and Oncology, ³ Preclinical and Clinical Pharmacology, ⁴ Human Pathology and Oncology, and ⁵ Excellence Center for Research, Transfer and High Education De Novo Therapies, University of Florence, Florence, Italy; and ⁶ Department of Pharmaceutical Sciences, University of Pisa, Pisa, Italy

Requests for reprints: Sergio Capaccioli, Dipartimento Patologia e Oncologia Sperimentale Medical School, University of Florence, Viale G.B. Morgagni 85, Firenze 50134, Italy. Phone: 3955-4598208; Fax: 3955-4598900; E-mail: sergio{at}unifi.it.

Purpose: Cannabinoids have been recently proposed as a new family of potential antitumor agents. The present study was undertaken to investigate the expression of the two cannabinoid receptors, CB₁ and CB₂, in colorectal cancer and to provide new insight into the molecular pathways underlying the apoptotic activity induced by their activation.

Experimental Design: Cannabinoid receptor expression was investigated in both human cancer specimens and in the DLD-1 and HT29 colon cancer cell lines. The effects of the CB₁ agonist arachinodyl-2'-chloroethylamide and the CB₂ agonist N-cyclopentyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (CB13) on tumor cell apoptosis and ceramide and tumor necrosis factor (TNF)- production were evaluated. The knockdown of TNF- mRNA was obtained with the use of selective small interfering RNA.

Results: We show that the CB₁ receptor was mainly expressed in human normal colonic epithelium whereas tumor tissue was strongly positive for the CB₂ receptor. The activation of the CB₁ and, more efficiently, of the CB₂ receptors induced apoptosis and increased ceramide levels in the DLD-1 and HT29 cells. Apoptosis was prevented by the pharmacologic inhibition of ceramide de novo synthesis. The CB₂ agonist CB13 also reduced the growth of DLD-1 cells in a mouse model of colon cancer. The knockdown of TNF- mRNA abrogated the ceramide increase and, therefore, the apoptotic effect induced by cannabinoid receptor activation.

Conclusions: The present study shows that either CB₁ or CB₂ receptor activation induces apoptosis through ceramide de novo synthesis in colon cancer cells. Our data unveiled, for the first time, that TNF- acts as a link between cannabinoid receptor activation and ceramide production.