Cannabinoid receptor activation induces apoptosis through tumor necrosis factor alpha-mediated ceramide de novo synthesis in colon cancer cells.
OBJECTIVE
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.


METHODS
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)-alpha production were evaluated. The knockdown of TNF-alpha 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-alpha 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-alpha acts as a link between cannabinoid receptor activation and ceramide production.
Cianchi
Fabio
F
Department of Medical and Surgical Critical Care, University of Florence, Florence, Italy.
Papucci
Laura
L
Schiavone
Nicola
N
Lulli
Matteo
M
Magnelli
Lucia
L
Vinci
Maria Cristina
MC
Messerini
Luca
L
Manera
Clementina
C
Ronconi
Elisa
E
Romagnani
Paola
P
Donnini
Martino
M
Perigli
Giuliano
G
Trallori
Giacomo
G
Tanganelli
Elisabetta
E
Capaccioli
Sergio
S
Masini
Emanuela
E
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002518:Ceramides / Q000096:biosynthesis*; D003110:Colonic Neoplasms / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008819:Mice, Nude; D034741:RNA, Small Interfering; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism*; D043885:Receptor, Cannabinoid, CB2 / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D002518:Ceramides; D034741:RNA, Small Interfering; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D014409:Tumor Necrosis Factor-alpha