We demonstrated that both Rg3 and DSL have effects on these processes but the latter selleck chem inhibitor is more powerful, and cannot be substituted by Rg3. DSL blocked the cells in the S phase, while GEM arrested the cells in the G0/G1 phase. Consequently, DSL combined with GEM could strengthen its capability against cancer. Moreover, molecular analysis of the apoptosis pathway revealed that the expression of dr5, pro-caspase and caspase proteins increased, while the level of bcl-2 and survivin decreased. These observations indicated that DSL induces apoptosis most likely via both mitochondrial and death receptor pathways. MATERIALS AND METHODS The human hepatoma cell line HepG2 was cultured in RPMI 1640 with 10% fetal bovine serum and 2 mM L-glutamine (Gibco/BRL). The effects of DSL (Zhengbang Pharmaceutical Company, Beijing, P.

R. China), Rg3 (Fusheng Nature Pharmaceutical Company, Dalian, P.R. China) and GEM (Haosen Pharmaceutical Company, Jiangsu, P.R. China) were evaluated according to previously described method[10]. The IC50 was calculated using the SPSS 13.0 software to standardise drug administration in the following experiments. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma, St Louise, MO, USA. Mouse monoclonal antibodies against human ES, VEGFR-2, caspase-3, caspase-8, caspase-9, dr5, bcl-2, and surviving were purchased from Santa Cruz Biotechnology Inc., Texas, USA. MTT cell viability assay: Cells were seeded in 96 well plates at 1000 cells per well in 200 ��l of medium the day before the experiment.

DSL, Rg3 and GEM were added to the cells at the indicated concentrations (DSL: 3.125, 6.25, 12.5, 25, 50 and 100 ��g/ml; Rg3: 5, 10, 20, 40, 80 and 160 ��g/ml; GEM: 0.158, 0.316, 0.625, 1.25, 2.5 and 5 ��g/ml). Cell growth was assayed at 24, 48 and 72 h after drug treatment, by aspiring half of the medium (100 ��l) and adding an equal volume of fresh medium containing MTT (1 mg/ml). Cells were incubated further at 37��, 5% CO2 for 4 h, and 150 ��l of dimethyl sulphoxide (DMSO, Sigma, USA) was added to each well, followed by mixing at room temperature for 10 min. The absorbance of the supernatant was measured at 570 nm using a Multifunction microplate reader (POLARstarOPTIMA, BMG, Germany). The inhibition of cell viability was calculated by the formula, %inhibition=[1-(A570 nm/A��570 nm)]��100, where A570 nm is from the treated cells and A��570 nm is from the untreated cells. Each experiment was repeated at least three times. Cell cycle analysis: Cells were treated by drugs (DSL 25 ��g/ml, Rg3 80 ��g/ml and GEM 2.5 ��g/ml) for 24 h. After 24 h intervention by drugs, treated and Entinostat untreated cells (1��106) were collected and washed with PBS, then fixed in 75% alcohol for 30 min at room temperature.