AIM Botanical preparations are widely used by patient with cancer in Korea, Japan and China.Rhus verniciflua Stokes (RVS) has traditionally been used as a medicinal ingredient for the therapy of stomach and uterine cancer.METHODS The AGS human gastric cancer cell line and normal rat intestinal epithelial (RIE1) cell lines were used and grown in RPMI 1640 or DMEM supplemented with 10% (V/V) heat-inactivated fetal bovine serum and 1% penicillin-streptomycin in a 37℃ humidified incubator with 5% CO2.Cell viability assay, Western blot and cytochrom c release analysis were conducted.To measure △Ψm, 1 × 106 cells treated with an ethanol extract of RVS for various amounts of time were stained with 5 rag· L-1 JC-1 (molecular probes, 5,5 ,6,6tetrachloro-1, 1 ,3,3-tetraethylbenzimidazol-carbocyanine iodide).For transient transfection, cells were seeded at a density of 1 × 105 cells per 60 mm dish in 1.5 ml of RPMI 1640 medium.Cells were then transfected with 2 μg of pCMV6.HA.Akt.K179M using LipofectAMINE 2000.The transfection was done as suggested by Qiagen with slight modifications.In brief, AGS ceils were plated at 1 × 105 per 60 mm dish.The next day, cells were transfected with 50 nmol-L-1 of siRNA-control and siRNA-Akt/PKB using Lipofe ctAMINE 2000.After transfection for 6 h, normal medium (RPMI 1640 medium containing 10% FBS) was added and cells were further incubatedfor 12 h prior to treatment with an ethanol extract of RVS.RESULTS The extract induces G1-ceil cycle arrest through the regulation of cyclins, the induction of p27Kipl, and decrease the CDK2 kinase activity.The upregulated p27Kipl level is caused by protein stability increment by the reduction of Skp2, a key molecule related with p27Kip1 ubiquitination and degradation, and de novo protein synthesis.RVS extract induces apoptosis through the expression of Bax, poly(ADP-ribose) polymerase (PARP) and activation of caspase 9 and caspase 3.It was mediated by the loss of mitochondrial membrane potential [MMP, Deltapsi(m)] and the release of cytochrome C from the mitochondrial intermembrane space.In addition, an ethanol extract of RVS inactivated PI3K-Akt/PKB kinase in a time-dependent manner.Moreover, combined treatment of an ethanol extract of RVS and LY294002 (a PI3K inhibitor)markedly increased apoptosis compared to treatment with an ethanol extract of RVS alone.The role of PI3K-Akt/PKB in this process was confirmed by constitutive expression of inactive mutants of this kinase in AGS cells.Finally, siRNA-mediated knockdown of Akt/PKB expression resulted in a significant reduction in AGS cell proliferation.CONCLUSION Taken together, these results suggest that an ethanol extract of RVS induces apoptosis via a mitochondrial death pathway in human gastric cancer cells, but not in normal cells, and inhibition of the PI3K-Akt/PKB pathway enhanced the mitochondrial death pathway, therefore we suggest that this extract could be a candidate medicine or compound for the development of novel class of anti-cancer drugs.