The Endocannabinoid Anandamide Selectively Induces Cell Death in Hepatoma Cell Lines

Background: Endocannabinoids mediate antineoplastic effects in several cancers including breast and prostate cancer. We have shown that the endocannabinoid anandamide (AEA) does not induce cell death in primary hepatocytes due to high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH). Aim: To determine whether the endocannabinoid AEA induces growth arrest and cell death in hepatoma cell lines. Methods: Rat and human hepatoma cell lines (FaO, McA-RH7777, HepG2, Hep3B, PCL/PRF/5) and primary rat hepatocytes were treated with varying concentrations of AEA. Cell death was determined by LDH release, western blot for caspase 3 and PARP cleavage and propidium iodide (PI) uptake. Proliferation was analyzed by ³H-thymidine uptake assay. Reactive oxygen species (ROS) formation was monitored by DCFDA fluorescence. FAAH expression was analyzed by western blot and real time PCR. FAAH overexpression was achieved by adenoviral infection. FAAH activity was measured colorimetrically and inhibited with the specific inhibitor URB597. Glutathione was depleted by BSO. Results: AEA induced dose-dependent cell death in all human and rat hepatoma cell lines, but not in primary rat hepatocytes. The rat hepatoma cell line FaO expressed almost no FAAH and was highly sensitive to AEA-induced cell death (60% at 50μM after 4h), whereas the rat hepatoma McA-RH cell line expressed higher levels and activity of FAAH and required longer AEA treatment to efficiently induce cell death (50% at 50μM after 24h). Overexpression of FAAH rendered FaO cells resistant to AEA whereas inhibition of FAAH activity strongly increased AEA sensitivity in McA-RH cells. Accordingly, FAAH overexpression inhibited, and FAAH inhibition increased AEA-induced cell death in human hepatoma cells. AEA markedly induced ROS formation in hepatoma cells. Blocking AEA-induced ROS formation by GSH pretreatment reduced AEA-induced cell death whereas GSH-depletion further increased AEA sensitivity of all hepatoma cells. Sublethal concentrations of AEA inhibited FCS-induced ³H-thymidine uptake (p<0.05). Conclusion: Anandamide induces ROS-dependent cell death in all investigated hepatoma cell lines, but not in primary hepatocytes. This higher sensitivity of hepatoma cells is due to the lower expression of FAAH. In addition, sublethal doses of AEA block proliferation in hepatoma cells. Modulation of the endocannabinoid system may represent a new option for the treatment of primary hepatocellular cancer.

Key words

Anandamide - Anti-cancer Therapy - Cell death - Endocannabinoids - Hepatocytes - Hepatoma