[Membrane cholesterol mediates the endocannabinoids-anandamide affection on HepG2 cells].
OBJECTIVE
To study the effect of anandamide (AEA) on necrosis in HepG2 cells and to explore the role of AEA in progression of liver cancer.


METHODS
Localization of the fatty acid hydrolytic enzyme (FAAH), cannabinoid receptors 1(CB1) and cannabinoid receptors 2 (CB2) proteins was detected in L02 and HepG2 cells using immunofluorescence. L02 and HepG2 cells were treated with different concentrations of AEA and methyl-beta-cyclodextrin, and the rates of cells necrosis were examined by PI stain. Meanwhile, the expression levels of FAAH, CB1 and CB2 receptor proteins, as well as P38 mitogen-activated protein kinase (p-P38 MAPK) and c-Jun-NH2-terminal kinase (p-JNK) proteins, were analyzed by Western blot.


RESULTS
The FAAH, CB1 and CB2 receptor proteins were observed both in cytoplasm and on membrane in L02 and HepG2 cells. The expression level of FAAH protein was higher in HepG2 than in L02 cells. The expression level of CB1 receptor protein was very low in both L02 and HepG2 cells. The expression level of CB2 receptor protein was high in both L02 and HepG2 cells. AEA treatment induced necrosis in HepG2 cells but not in L02 cells. Methyl-beta-cyclodextrin treatment prevented necrosis in HepG2 cells (t = 3.702; 5.274; 3.503, P less than 0.05). The expression patterns of FAAH, CB1 and CB2 receptor protein in L02 and HepG2 cells were confirmed by western blot, which were consistent with the immunofluorescence results. AEA treatment increased the levels of p-P38MAPK and p-JNK proteins in a dose-dependent manner in HepG2 cells (F = 11.908; 26.054, P less than 0.05) and the increase can be partially by prevented by MCD (t = 2.801; t = 12.829, P less than 0.05).


CONCLUSIONS
AEA treatment induces necrosis in HepG2 cells via CB1 and CB2 receptors and lipid rafts.
Wu
Wen-Jie
WJ
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Yang
Qiao
Q
Cao
Qin-Fang
QF
Zhang
Yao-Wen
YW
Xia
Yu-Jia
YJ
Hu
Xiao-Wen
XW
Tang
Wang-Xian
WX
D000581:Amidohydrolases / Q000378:metabolism*; D001095:Arachidonic Acids / Q000494:pharmacology*; D063385:Cannabinoid Receptor Modulators / Q000494:pharmacology*; D002784:Cholesterol / Q000378:metabolism; D063388:Endocannabinoids; D056945:Hep G2 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D009336:Necrosis; D053284:Polyunsaturated Alkamides / Q000494:pharmacology*; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism*; D043885:Receptor, Cannabinoid, CB2 / Q000378:metabolism*; D015398:Signal Transduction; D047392:beta-Cyclodextrins / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D002784:Cholesterol; D063388:Endocannabinoids; D048031:JNK Mitogen-Activated Protein Kinases; D053284:Polyunsaturated Alkamides; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D047392:beta-Cyclodextrins; D048051:p38 Mitogen-Activated Protein Kinases; D000099252:Fatty Acid Amide Hydrolases; C108732:methyl-beta-cyclodextrin; C078814:anandamide