Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors still associated with poor prognosis in advanced stages. Gemcitabine is one of the standard agents for the treatment of PDAC, without having a major impact on the clinical outcome. Combining two compounds acting via different ways of action may result in a better efficacy. Methods:We investigated the effects of gemcitabine in combination with the dual PI3k/mTOR inhibitor BEZ235 in four human pancreatic cancer cell lines (Panc-1, BxPC-3, MiaPaCa-2 and AsPC-1). The cells were analysed with MTT assay for cell viability, FACS-analysis for cell cycle distribution. Real-time RT-PCR and Western blot for survivin/ BIRC5, STAT3, BCl-xL and WNT16 mRNA and protein expression and γH2AX.Results: Application of NVP-BEZ235 or gemcitabine inhibited cell viability of AsPC-1 and BxPC-3 cells while Panc-1 and MiaPaCa-2 remained nearly unaffected. Combined treatment of gemcitabine and BEZ235, however, enhanced the inhibitory effect on cell viability of Panc-1 and MiaPaCa-2 cells of about 80% compared to control cells. This effect was boosted by time-delayed application of the two compounds. The biggest impact on cell growth, viability and downstream gene regulations were achieved by a sequential incubation with gemcitabine followed by BEZ235 24 hours later. Conclusions:Combining gemcitabine with dual PI3K/mTOR inhibitors like NVP-BEZ2235 improved the efficacy on growth inhibition in human pancreatic cell lines especially by sequential application of both agents.