Fig. 3.

High levels of NT5C1A increase chemotherapeutic resistance towards gemcitabine in pancreatic cancer cells. A) Pharmacokinetic analysis of gemcitabine metabolites in murine PDAC cells. Murine KPC-BL6 cells (A) and human L3.6pl cells (B) (+NT5C1A) and respective control cells (+vector) were simultaneously treated with 1 μM gemcitabine-hydrochloride for 2 h. The concentration of the active gemcitabine metabolite dFdCTP (KPC-BL6: 27.3 vs. 9.4 pM per 1*10⁶ cells, p = .0008 and L3.6pl: 67.3 vs. 30.8 pM per 1*10⁶ cells, p = .021) and of native gemcitabine dFdC (C) were determined in homogenates of cell pellets using LC-MS/MS-analysis. Three biological replicates and the mean value are shown. Each dot represents the mean of three (KPC-BL6) or two (L3.6pl) technical replicates, respectively. All dFdC measurements of KPC-BL6 control cells were below the level of quantification, thus the values were set to zero. D) Murine and human pancreatic cancer cell lines were incubated with different concentrations of gemcitabine for six days. Crystal violet staining was more pronounced in cell lines with high NT5C1A expression. Three independent experiments, with each two technical replicates, were performed for all cell lines shown. E) Quantification of crystal violet assays for KPC1 cells using 10% acetic acid to solubilize crystal violet stain and photometric measurements at 595 nm. Results were normalized to untreated control cells. Two-way ANOVA with Sidak's multiple comparisons test was performed (12 nM: p = .0007, 20 nM: p < .0001). Graph shows mean ± SEM. F) Western blot analysis revealed reduced cleaved caspase-3 (CC3) protein levels in NT5C1A-expressing cells following gemcitabine treatment for 24 h. Strong NT5C1A and HA-tag expression was demonstrated in the +NT5C1A-cell line. Three independent experiments were performed. G) Quantification of CC3 protein expression in these three Western blot analyses. Two-way ANOVA with Sidak's multiple comparisons test was performed (30 nM: p = .249, 50 nM: p = .002). Graph shows mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)