Data are the mean ± SD of triplicate determinations. Effect of gemcitabine, sorafenib and EMAP on EC and fibroblast proliferation Targeting endothelial cells and fibroblasts for solid tumor treatment has been shown to be potentially quite effective [34, 35]. In our study, analysis of in vitro HUVEC and WI-38 cell proliferation

in growth factor containing medium revealed that single agent gemcitabine, sorafenib and EMAP induced significant dose-dependent inhibitory effects. Importantly, combination of these agents had some additive effects on inhibition of cell proliferation of both cell lines. At an intermediate concentration of HSP inhibitor gemcitabine (1 μM), sorafenib (1 μM) and EMAP (1 μM), the percent inhibition Selonsertib concentration in HUVEC proliferation was 63, 69, 53, 79, 82, 72 and 79 in the Gem, So, EMAP, Gem+So, Gem+EMAP, So+EMAP and Gem+So+EMAP groups,

respectively. In fibroblast WI-38 cells at an intermediate concentration of gemcitabine (500 nM), sorafenib (500 nM) and EMAP (500 nM) the percent inhibition Tucidinostat order in WI-38 proliferation was 73, 66, 49, 80, 82, 77 and 83 in the Gem, So, EMAP, Gem+So, Gem+EMAP, So+EMAP and Gem+So+EMAP groups, respectively (Figure 3). Figure 3 Gemcitabine (Gem), sorafenib (So) and EMAP (E) inhibit in vitro cell proliferation of EC (HUVECs) and fibroblast cells (WI-38). Cells were plated on 96-well plate and treated with gemcitabine, sorafenib and EMAP. After 72 hours incubation, WST-1 reagent was added in each well and number of viable cells was calculated by measuring absorbance of color produced in each well. Data are representative of mean values ± SD of triplicate determinants. Symbols +, * and • represent p values of less than 0.05, 0.005 and 0.0005 compared to controls. Effect of gemcitabine, sorafenib and EMAP on apoptosis markers Western blot analysis to evaluate if inhibition in cell proliferation was due to the induction in apoptosis revealed that sorafenib treatment either alone or in combination with gemcitabine Cyclin-dependent kinase 3 and EMAP induced apoptosis as observed via PARP-1 cleavage and caspase-3 cleavage in HUVECs and WI-38 cells (Figure 4). Sorafenib-induced

expression of cleaved PARP-1 and cleaved caspase-3 was similar in HUVECs and WI-38 cells. Gemcitabine caused a significant increase in PARP-1 or caspase-3 cleavage in WI-38 fibroblast cells but no detectable change in HUVECs (Figure 4). EMAP treatment caused a small change in these apoptosis marker protein in HUVECs but not in WI-38 cells. In a parallel setting with AsPC-1 PDAC cells, no detectable change in apoptosis marker proteins was observed after gemcitabine, sorafenib or EMAP treatment (data not shown). Figure 4 Effects of gemcitabine (G), sorafenib (So) and EMAP (E) treatment on cleavage of PARP-1 and caspase-3 proteins. A sub-confluent cell monolayer was treated with gemcitabine (10 μM), sorafenib (10 μM) and EMAP (10 μM).