HepG2 Hepatocellular Carcinoma Cells Express a High Density of Cannabinoid Receptors that Exhibit Atypical Binding and Functional Properties: A Novel Therapeutic Target? Amal M. Shoeib1, Lance N. Benson1, Shengyu Mu1, and Paul L. Prather1 1University of Arkansas for Medical Sciences, Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR Hepatocellular carcinoma is an aggressive form of liver malignancy with poor prognosis and few treatment options. Preclinical reports show that cannabinoids decrease cell viability in models of hepatocellular carcinoma; however, the exact mechanisms underlying these anti-tumor effects remain elusive. We recently found that cell lines derived from a variety of cancer types express cannabinoid receptors (CBRs) exhibiting atypical ligand binding and signaling characteristics. Most importantly, cannabinoids induce cytotoxicity and reduce cell viability in these cancer cell types possibly through CBRs. Here, we tested the hypothesis that the hepatocellular carcinoma cell line HEPG2 similarly expresses atypical CBRs. We demonstrate that HEPG2 cells express high levels of mRNA for both CB1 and CB2 receptors when compared to normal liver tissue. However, radioligand binding studies indicate that these receptors exhibit a distinct ligand binding profile relative to canonical cannabinoid receptors. For example, only 5 out of 15 well-characterized cannabinoid ligands known to exhibit high affinity for canonical CB1 and/or CB2 receptors are able to displace the non-selective cannabinoid [3H]WIN-55,212-2 from HEPG2 membrane homogenates. HEPG2 cells contain a remarkably high density of these atypical CBRs relative to that expressed in normal brain and other cancerous tissues, to which [3H]WIN-55,212-2 binds with a high affinity of 7.7 nM. Finally, similar to receptor binding studies, functional studies examining G-protein modulation indicate that CBRs expressed in HEPG2 cells exhibit a distinct functional profile relative to canonical CBRs as well. Collectively, these results suggest that similar to data determined in our laboratory for several other cancer cell lines, HEPG2 cells also express a high density of atypical CBRs with unique receptor ligand binding and signaling properties. These findings might have clinical significance, because such atypical CBRs expressed in HEPG2 cells could be unique to cancer cells and hold promise for the development of efficacious drugs to treat hepatocellular carcinoma with reduced adverse effects.