Smac inhibits IAPs and promotes caspase activation done and apoptosis. Recently, small molecule mimetics of Smac have been developed that can promote cancer cell apoptosis either alone or in combination with Inhibitors,Modulators,Libraries other proapoptotic agents. In fact the Smac mimetic JP1201 has recently been shown to augment the Gem response in PDAC MIA PaCa 2 cells. In the present study we evaluated the effect of JP on the in vitro and in vivo therapeutic efficacy of various cytotoxic chemotherapy agents in an effort to provide a more effective antitumor strategy for PDAC. Methods Cell culture and reagents Human PDAC cell lines, AsPC 1, Panc 1, BxPC 3 and MIA PaCa 2 were obtained from the American Type Culture Collection. Cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 Uml penicillinstreptomycin solu tion at 37 C in a humidified 5% CO2 atmo sphere.

JP was obtained from Joyant Pharmaceuticals, Gem was purchased from Eli Lilly Cor porations, doxorubicin was pur chased from Ben Venue Laboratories and docetaxel was purchased from Sanofi aventis. Inhibitors,Modulators,Libraries Cell viability assay In vitro cell viability of PDAC cell lines was evaluated by using Inhibitors,Modulators,Libraries the colorimetric WST 1 reagent as described earlier. The assay is based on the ability of viable cells to cleave the sulfonated tetrazolium salt WST 1 2 2H 5 tetrazolio 1,3 benzene disulfo nate by mitochondrial dehydrogenases. Briefly, PDAC cells were plated in a 96 well plate in regular growth medium and after 16 hours the med ium was replaced with Inhibitors,Modulators,Libraries 2% FBS containing medium. After 5 hours incubation the cells were treated with JP, Gem, Dox or DT, either alone or in combination.

After additional incubation of 72 hours, 10 ul WST 1 reagent was added in each well followed Inhibitors,Modulators,Libraries by incubation for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Western blot analysis A monolayer of cells at 75 to 80% confluence was placed in 2% FBS containing medium for at least 5 hour before treatment with JP, gemcitabine for 24 hours. Cells were lysed and equal amounts of total protein were separated by SDS PAGE and trans ferred to PVDF membranes. The membranes were blocked for 1 hour at room tem perature with gentle shaking in TBS T, 150 mM NaCl, 0. 05% Tween 20. The membranes were incubated overnight at 4 C with the following antibodies phospho JNK. poly poly merase 1. a tubulin or GAPDH.

The membranes were then incubated with corresponding HRP conju gated secondary antibodies for 1 hour at room temperature. Specific bands were detected using the enhanced chemilumines cence reagent on autoradiographic film and quantitated by densitometry. Apoptosis Axitinib VEGFR inhibitor assay by Annexin V FITC and propidium iodide staining Effect of JP and Gem on AsPC 1 cell apoptosis was detected by using the Annexin V FITC apoptosis detec tion kit as per manufacturers protocol.