Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G2/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G2/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.

Abstract. Torilis japonica is a wild biennial herb and has been used as a traditional medicine for the treatment of inflammation, skin disease and impotence. Here, we studied the effects of a T. japonica extract on the proliferation of the U87MG human glioblastoma cell line. The extract inhibited cell proliferation in a dose-and time-dependent manner, as determined using the MTT assay. We next investigated the molecular mechanisms underlying its anti-proliferation properties by examining cell cycle progression and cell death. T. japonica extract induced S-phase cell cycle arrest and inhibited the expression of cell cycle-regulatory proteins, including cyclin A, cyclin-dependent protein kinase 2 and E2F1. The extract also induced apoptotic cell death as evaluated by nuclear morphology and flow cytometry using Annexin-V/ PI dual staining. Furthermore, Western blot analysis showed that apoptotic cell death was mediated by both mitochondriaindependent and caspase-dependent pathways. Together, our findings indicate that the T. japonica extract contains bioactive compounds with anti-cancer effects. These materials may be useful in the chemotherapy of human glioblastoma.