The apoptotic activity of evodiamine was shown to be due to selleck chem Erlotinib its inhibition of NF-��B activation via suppression of I��B�� kinase activity, which leads to inhibition of NF-��B-regulated gene products such as XIAP, Bcl-2, and Bcl-xL 8. Previous study has also revealed the molecular mechanism by which evodiamine increases the expression of proapoptotic Bax and decreases that of anti-apoptotic Bcl-2, which amplified the activation of the caspase cascade, triggering consequent responses and cell death 21-23. PI3K was also found to exhibit essential regulatory effects on functions of SIRT1, p53 and other signaling proteins involved in evodiamine-induced A375-S2 cell death 24, 25. Based on these reports, we hypothesized that evodiamine may augment the gemcitabine-induced anti-tumor effect on pancreatic cancer via direct or possibly indirect inhibition of the PI3K/Akt pathway targeting NF-��B.

This study showed that evodiamine inhibited the spontaneous and gemcitabine-induced NF-��B activation, and the expression of NF-��B-regulated proteins in SW1990 cells. Importantly, evodiamine inhibited the activation of PI3K/Akt pathway, phosphorylation of PTEN and mammalian target of rapamycin (mTOR), and activity of cAMP-dependent protein kinase A (PKA) that was not influenced by gemcitabine. Our data suggest that evodiamine may augment the therapeutic effect of gemcitabine in pancreatic cancer through direct or indirect negative regulation of the PI3K/Akt pathway that targets NF-��B. Materials and Methods Reagents Evodiamine (purity: >99%) was purchased from Sigma (St.

Louis, MO, USA), and dissolved in dimethylsulfoxide (DMSO) at 0.2 mmol/L to make the stock solution. Gemcitabine was purchased from Ely Lilly (Bad Homburg, Germany) and dissolved in sterile saline at 50 g/L for stock solution, with a final concentration of DMSO at <0.1%. Rabbit polyclonal antibodies against phospho-PTEN(Ser380/Thr382/383) and PI3K(Tyr458) were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal antibodies against Bax and Bcl-2, phospho-mTOR(Ser2448), Rictor-mTOR, phospho-Akt(Ser473), rabbit monoclonal antibody against NF-��B(p65), survivin and active caspase 3 were purchased from Abcam (Cambridge, UK). The in Situ Cell Death Detection Kit was purchased from Roche (Basel, Switzerland).

Cell culture Human pancreatic tumor cell line, SW1990, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 supplemented with 10% heat-incubated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 units/mL of penicillin, and 100 ��g/mL of streptomycin and incubated Brefeldin_A at 37?C in a humidified 5% CO2 atmosphere. Some SW1990 cells were stably transfected with luciferase, as previously described for Panc-1 cells 26. Luciferase-transfected SW1990 cells were routinely cultured in the same condition as SW1990 cells.