Glioblastoma multiforme (GBM) are among the most aggressive of all known human tumors and the median survival time after GBM diagnosis remains in the 12-15 months range.The main pathologic characteristics of GBM are unregulated cell proliferation, necrosis, and neovascularization, which are influenced by the phosphatidylinositol 3-kinase (PI3K) pathway and more specifically by the activity of its downstream target AKT.Integrin-linked kinase (ILK) has been proposed to be involved in AKT activation.Our research team is pursuing the evaluation of treatment strategies involving ILK as a therapeutic target.One therapeutic strategy involves the use of short interfering RNA (siRNA) targeting ILK expression.The technology of RNA interference has proven to be one of the most potent, robust and easy-to-use gene expression inhibitors, and has been extensively explored in the context of anti-cancer therapy development.However, in this case, delivery of the siRNA inside target cell populations limits the technologys therapeutic potential.Delivery and stability issues can be effectively addressed through use of lipidbased nano-delivery systems.The vast majority of these lipid based delivery systems use cationic lipids to complex the polyanionic siRNA.However, it is becoming increasingly apparent that the cationic lipid influences more then just siRNA delivery.In this research, we used the glioblastoma cell line U251 to assess the relevance of siRNAmediated inhibition of ILK in GBM treatment.Efficacy and specificity of a commercially available cationic liposome formulation (LipofectamineTM 2000) was compared to nucleofection-mediated transfection.Methods to assess delivery included fluorescence microscopy and flow cytometry analysis, as well as single timepoint and five days tirnecourse western immunoblot assays of ILK expression.It is demonstrated that the molecular signaling consequences associated with siRNA mediated ILK suppression changes over extended time courses; ILK suppression-induced decreases in phosphorylated-AKT which was followed be a re-phosphorylation event that may be independent of ILK.We also report that the cationic liposome formulation used for siRNA delivery engendered non-specific side effects on mTOR/PDK1/AKT pathways which had an impact on our interpretation of effects do to ILK silencing.