title: Molecular mechanisms involved in the synergistic interaction of the EZH2 inhibitor 3-deazaneplanocin A with gemcitabine in pancreatic cancer cells.
abstract: 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 (H3K27me3). 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 PCR in 15 PDAC cells, including seven primary cell cultures, showing that expression values correlated with their originator tumors (Spearman R(2) = 0.89, P = 0.01). EZH2 expression in cancer cells was significantly higher than in normal ductal pancreatic cells and fibroblasts. The 3-deazaneplanocin A (DZNeP; 5 μmol/L, 72-hour exposure) modulated EZH2 and H3K27me3 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 G(2)-M phase (e.g., from 27% to 19% in PANC-1, P < 0.05) and significantly increased apoptosis compared with gemcitabine alone. Moreover, DZNeP enhanced the mRNA and protein expression of the nucleoside transporters hENT1/hCNT1, possibly because of the significant reduction of deoxynucleotide content (e.g., 25% reduction of deoxycytidine nucleotides in PANC-1), as detected by liquid chromatography/tandem mass spectrometry. 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 PDACs.
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Amir
authors.initials: A
authors.lastname: Avan
authors.forename: Francesco
authors.initials: F
authors.lastname: Crea
authors.forename: Elisa
authors.initials: E
authors.lastname: Paolicchi
authors.forename: Niccola
authors.initials: N
authors.lastname: Funel
authors.forename: Elena
authors.initials: E
authors.lastname: Galvani
authors.forename: Victor E
authors.initials: VE
authors.lastname: Marquez
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Honeywell
authors.forename: Romano
authors.initials: R
authors.lastname: Danesi
authors.forename: Godefridus J
authors.initials: GJ
authors.lastname: Peters
authors.forename: Elisa
authors.initials: E
authors.lastname: Giovannetti
mesh_terms: D000071916:AC133 Antigen; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015820:Cadherins / Q000235:genetics; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004357:Drug Synergism; D000071221:Enhancer of Zeste Homolog 2 Protein; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D063151:Polycomb Repressive Complex 2 / Q000037:antagonists & inhibitors / Q000235:genetics*; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured; D000093542:Gemcitabine
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D000964:Antimetabolites, Antineoplastic; D015820:Cadherins; D033721:Equilibrative Nucleoside Transporter 1; D006023:Glycoproteins; D026901:Membrane Transport Proteins; C000606023:PROM1 protein, human; D010455:Peptides; C065171:cif nucleoside transporter; D003841:Deoxycytidine; C048460:3-deazaneplanocin; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; D000241:Adenosine; D000093542:Gemcitabine