In UK Pan 1 cells, a significant effect was observed for combined treatment when the highest concentration of AG1478 was used in combination and as seen with PANC 1 cells, the combination treatment caused only a marginal increase of growth suppression. These observa tions suggest, although the combined treatments increased growth inhibition, the effects were less than additive. STAT3Tyr705 selleck inhibitor phosphorylation was not inhibited by treating cells with either AG1478 or gemcitabine Inhibitors,Modulators,Libraries alone, except in BxPC3, where higher concentrations Inhibitors,Modulators,Libraries of AG1478 caused some inhibition. Similarly, combining both drugs had a minimal affect on the level of STAT3Tyr705 phosphorylation except for BxPC3 where higher doses of AG1478 resulted in some reduc tion of STAT3Tyr705 phosphorylation.

It should be noted that 10 uM concentration of AG1478 was suffi cient to inhibit phosphorylation of EGFR suggesting that molecular affects requiring concentrations of AG1478 greater than 10 uM may represent off target effects. Inhibition of STAT3 by shRNA sensitizes PDAC cells to gemcitabine in vitro Because STAT3Tyr705 phosphorylation was maintained in cells treated Inhibitors,Modulators,Libraries with AG1478 or gemcitabine, Inhibitors,Modulators,Libraries we hypothe sized that targeting STAT3 may serve as an independent therapeutic target or may cause PDAC cells to be more sensitive to gemcitabine. To inhibit STAT3, PDAC cells PANC 1, UK Pan 1, MIA PaCa 2 and BxPC3 were transfected with a vector that expresses a shRNA against STAT3 and individual stable clones were established after antibiotic selection. These clones were tested for the expression of STAT3 along with control cells that express the vector alone.

Control Inhibitors,Modulators,Libraries cells and isogenically matched cells that express STAT3 shRNA were treated with gemcitabine and were assessed for growth by MTT assays. As shown in Figure 4, cells that express shRNA against STAT3 were significantly more sensitive to gemcitabine treatment as compared to control cells. UK Pan 1 and PANC 1 cells showed a sig nificant dose dependent sensitivity to gemcitabine at doses of 6 and inhibitor licensed 4 ngml respectively and knockdown of STAT3 further increased their sensitivity as significant growth inhibition was observed from 0. 5 ngml and greater. MIA PaCa 2 and BxPC3 cells were more resis tant to gemcitabine compared to UK Pan 1 and PANC 1. Statistically significant growth inhibition was observed for doses of gemcitabine from 25 ngml and above for MIA PaCa 2 cells and 8 ngml and greater for BxPC3 cells. Interestingly, knockdown of STAT3 in creased their sensitivity to gemcitabine to a level similar to that seen for the more sensitive cell lines, UK Pan 1 and PANC 1. Significant growth inhibition was seen in STAT3 knock down cells at doses of 4 ng ml and 1 ngml for MIA PaCa 2 and BxPC3 cells re spectively.