0 software (Chicago, IL, USA). A P-value of less than 0.05 was considered statistically significant. Results Cytotoxicity of evodiamine plus gemcitabine against SW1990 cells in vitro As shown by MTT assays (Fig. (Fig.2A),2A), treatment with evodiamine or gemcitabine alone reduced the cell viability by nearly 42% and 57%, respectively. Treatment sellckchem with combination of evodiamine and gemcitabine significantly reduced the cell viability by 80%. Further analysis by cell apoptosis assay indicated that single evodiamine increased the apoptotic rate from 12.6% to 28.6% and single gemcitabine increased the apoptotic rate from 12.6% to 34.2%, while treatment with evodiamine plus gemcitabine induced nearly 57.6% of cell apoptosis (Fig. (Fig.2B2B and C).

These data are consistent with results from cell growth inhibition studies using MTT, suggesting that the loss of viable cells by evodiamine and/or gemcitabine is partly due to the induction of cell apoptosis. Figure 2 Treatment with evodiamine plus gemcitabine inhibits the proliferation and induces apoptosis of SW1990 cells in vitro. SW1990 cells at 2��105/mL were cultured overnight in six-well plates and treated in triplicate with 20 ��mol/L of gemcitabine … Effect of evodiamine on the gemcitabine-induced NF-��B activity in vitro SW1990 cells were treated with 20 ��M of gemcitabine for varying periods and the DNA binding activity of NF-��B in the nuclear extracts was characterized by EMSA. As shown in Fig. Fig.3A,3A, SW1990 cells in the absence of gemcitabine displayed low levels of DNA binding activity, indicating low levels of spontaneous NF-��B activation.

Treatment of SW1990 cells with gemcitabine for 24 h increased the DNA binding activity of NF-��B by 436% in vitro, then activity of NF-��B gradually decreased to nearly a normal level at 72 h after treatment (Fig. (Fig.3A).3A). Treatment with different doses of evodiamine inhibited the DNA binding activity of NF-��B in SW1990 cells (Fig. (Fig.3B).3B). Batimastat More importantly, treatment with gemcitabine plus evodiamine significantly reduced the DNA binding of NF-��B, as compared with that of gemcitabine treatment or control cells (Fig. (Fig.3C).3C). Therefore, treatment with evodiamine effectively inhibited the gemcitabine-induced NF-��B activation in vitro, which may contribute to inducing more SW1990 cells apoptosis. Figure 3 Effect of evodiamine on the spontaneous and gemcitabine-induced NF-��B activation in vitro. (A) Gemcitabine (20 ��mol/L) induces NF-��B activation in SW1990 cells, as evidenced by EMSA supershift experiment. (B) Treatment with evodiamine …