On the other hand, the combination didn’t bring about any decrease in AKT phosphorylation in UNC10 cells (Figure 5D, lane 4), which also confirmed just additive rather than synergistic apoptosis. not reduced by the drug combination. Conclusion. Combined IGF-1R/HER family and IGF-1R/Src family inhibition may have therapeutic potential in HNSCC. AKT may be a node of convergence between IGF-1R signaling and pathways that compensate for IGF-1R inhibition. phenylmethylsulfonyl fluoride, 2 mpervanadate, 100 M benzamidine, 1 g/mL aprotinin, 2 M pepstatin, and 25 M leupeptin. The lysates were sonicated for 10 minutes in a Bioruptor ice-bath sonicator (Diagenode, Denville, NJ) at maximum output. The lysates were then centrifuged at 13,000 g for 15 minutes. Western blotting was carried out as previously explained.16 Immunoblots were analyzed using the Odyssey (LICOR Biosciences, Lincoln, Endoxifen E-isomer hydrochloride NE) imaging system. Drug screen Ninety-six-well plates (Corning Life Sciences/Costar, Corning, NY) were seeded with 3000 to 5000 cells/well in RPMI 1640 supplemented with 0.5% FBS using the FlexDrop Plus Reagent Dispenser (Perkin Elmer, Waltham, MA). The cells were incubated overnight to allow adherence. Ninety-six-well drug master plates were prepared by diluting drugs to 10 concentrations in RPMI 1640 + 0.5% FBS. The 10 drugs were transferred to cell plates at a final concentration of 1 1 using the Vprep Automated Liquid Handler (Agilent Technologies, Santa Clara, CA). Cells were incubated for 72 hours. alamarBlue was added using the Vprep to a final concentration of 10%, per the manufacturers instructions. Cells were incubated for 4 hours and fluorescence was go through at Ex lover560 nm/Em590 nm using a Molecular Devices M5 plate reader with Stakmax (Sunnyvale, CA). Statistics For each of the 9 cell lines and each of the 120 drug combinations, a 3-way analysis of variance was conducted around the log-transformed cell growth, with factors representing the dose of each drug in the combination and a factor to account for experiment-to-experiment variance in cell growth. The 3-way analysis of variance model included all main effects and 2-way interactions, but no 3-way interaction between experiment and the doses of Endoxifen E-isomer hydrochloride the 2 2 drugs in the combination. This model allows for an estimate, a standard error, and a test of statistical significance for the difference between the observed cytotoxicity and the Bliss predicted cytotoxicity. Within each cell collection, the combinations were ranked by the estimated degree of % synergy. Unsupervised hierarchal clustering of maximum synergies across drug combinations and cell lines using Euclidean distance as the similarity metric and average linkage within the R packages gplots17 and pvclust.18 RESULTS Combination screening identifies drug pairs that cause synergistic growth inhibition in head and neck squamous cell carcinoma To identify druggable targets that enhance the effects of IGF-1R pathway inhibition, we performed a combinatorial drug screen in 9 human papillomavirusCnegative HNSCC cell lines. As shown in Table 1, the inhibitor library was comprised of compounds that are Food & Drug Administration (FDA)-approved for clinical use (36%; 8 of 22 compounds), are currently in clinical trials (55%; 12 of 22 compounds), or have demonstrated antitumor efficacy in in vivo model systems (9%; 2 of 22 compounds). We screened 10 IGF-1R pathway inhibitors in combination with 12 inhibitors of other targets demonstrated to play important functions in the survival and proliferation of HNSCC cells for a total of 120 drug combinations. Drug concentrations were selected based on dose response experiments performed in all 9 cell lines. Doses that produced approximately 10%, 25%, and 40% growth inhibition were selected for screening. For each combination in each cell collection, we assessed the growth inhibition caused by each drug alone as well as 9 dose combinations (3 concentrations of drug A combined with 3 concentrations of drug B). These results were then compared to a predicted additive growth inhibition that was generated using the Bliss model of additivity19 to determine whether Rabbit polyclonal to PARP the drug conversation was synergistic, additive, or subadditive. From this analysis, we generated Endoxifen E-isomer hydrochloride what we refer to as the maximum % synergy value, which is the greatest difference between the actual observed growth inhibition and the Bliss predicted growth inhibition for a given drug combination in a given cell collection. We performed unsupervised hierarchical clustering using the maximum % synergy for each drug combination in each cell in order to observe patterns of response (Physique 1A). This clustering analysis revealed no 2 cell lines responded similarly to the combination panel ( .05), underscoring the diversity of possible adaptive responses Endoxifen E-isomer hydrochloride in HNSCC. Additionally, this analysis exhibited that strong synergies were rarely observed, implying that, when synergistic.