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Pancreatic ductal adenocarcinoma (PDAC) is characterized by overexpression of Enhancer-of Zeste-Homolog-2 (EZH2), which plays a pivotal role in cancer-stem-cell (CSC) self-renewal through methylation of histone-H3-lysine-27 (H3K27m3). Against this background, EZH2 was identified as an attractive target and we investigated the interaction of the EZH2-inhibitor DZNeP with gemcitabine. EZH2 expression was detected by quantitative-RT-PCR in 15 PDAC cells, including 7 primary cell cultures, showing expression values correlated with their originator tumors (Spearman-R2=0.89, P=0.01). EZH2 expression in cancer cells was significantly higher than in normal ductal pancreatic cells and fibroblasts. DZNeP (5 μM, 72-hour-exposure) modulated EZH2 and H3K27m3 protein expression, and synergistically enhanced the antiproliferative activity of gemcitabine, with combination index values of 0.2 (PANC-1), 0.3 (MIA-PaCa-2) and 0.7 (LPC006). The drug combination reduced the percentages of cells in G2/M phase (e.g., from 27 to 19% in PANC-1, P<0.05), and significantly increased apoptosis compared to gemcitabine-alone. Moreover, DZNeP enhanced the mRNA and protein expression of the nucleoside transporters hENT1/hCNT1, possibly because of the significant reduction of deoxynucleotides content (e.g., 25% reduction of deoxycytidine-nucleotides in PANC-1), as detected by LC-MS/MS. DZNeP decreased cell migration, which was additionally reduced by DZNeP/gemcitabine combination (-20% in LPc006, after 8-hour exposure, P<0.05), and associated with increased E-cadherin mRNA and protein expression. Furthermore, DZNeP and DZNeP/gemcitabine combination significantly reduced the volume of PDAC spheroids growing in CSC-selective-medium, and decreased the proportion of CD133+ cells. All these molecular mechanisms underlying the synergism of DZNeP/gemcitabine combination support further studies on this novel therapeutic approach for treatment of PDAC.

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