application of siRNA complexed with lipids for the knock-down of Src, whose role in the maintenance of the complex phenotype of cancer is not clearly understood. It also seemed relevant to examine the effect of inhibiting more than a single signal transducer in the Src signaling pathway, or interacting pathways, through the use 
of other siRNAs in combination with Src siRNA. Direct application of siRNA avoids the use of viral-based vectors whose safety when used in humans is often currently under question. In this report, Src knock-down in combination with the knock-down of the downstream molecules STAT3 or cMyc is shown to result in a strong inhibition of the anchorage-independent growth, tumor growth, and metastasis of a human cancer cell line. Results siRNA targeting Src knocks down protein expression and affects the anchorage-independent growth of MDA-MB435S cells Of the several siRNAs targeting Src that were designed and tested, the most effective one was used to knock down Src in the highly metastatic human breast cancer cell line MDA-MB-435S, a cell line in which Src activity is elevated and potentially playing an important role in maintaining its neoplastic phenotype. Src siRNA caused a 62 or 67% reduction, respectively, in Src protein levels relative to either untransfected cells or cells transfected with a non-targeting control siRNA . The nontargeting control siRNA caused no significant change in Src levels relative to untrasnfected cells. This reduction by Src siRNA was accompanied by a corresponding reduction in Src kinase activity. The inhibitory effects typically lasted 4 days in rapidly dividing cells in culture, and this duration was sufficient to carry out cell culture growth experiments. We noted that siRNAs targeting other gene products often resulted in 905% decreases in protein levels, suggesting that the relatively long half-life of the cellular Src protein may play a role in the effectiveness of the several Src siRNA we tested, which typically gave reductions of 500%. In slowly growing cells and ��in vivo”, siRNA silencing can April 2011 | Volume 6 | Issue 4 | e19309 Inhibition of Tumor Growth Using siRNA be effective for greater than 21 days. Src siRNA was examined for its ability to alter certain growth characteristics of these tumor cells. The ability to suppress anchorage-independent growth in soft agar, a characteristic of transformed or cancer cells that is highly correlated with tumorigenicity, was initially examined. The Src siRNA was able to reduce the number of colonies in soft agar by 61% and 53% relative to either oligofectamine alone or negative controls siRNA treated cells, respectively. Treatment with Src siRNA also affected anchorage-dependent growth and resulted in a 50% reduction in cell number in comparison to untreated cells 5 days post transfection. Src knock-down combined with knock-down of STAT3 or cMyc augments the effects on anchorage-independent and -dependent growth The cell growth effects of Src siRNA alone were further examined to see if they could be RGFA-8 augmented by combining the Src siRNA with siRNAs that target signaling pathways that are 19176528 either downstream of 11423396 Src or that interact with the Src signal transduction cascade. siRNAs targeting proteins in several signaling pathways including MAPK1, Akt1, cMyc, and STAT3 were examined for their ability to modify cell growth. These siRNAs were initially examined for their ability to reduce their corresponding target proteins. Western blots de
