Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines

Francesco Crea1, Elisa Giovannetti2, Valentina Mey2, Sara Nannizzi2, Simona Ricciardi2, Mario Del Tacca2, Romani Danesi2
1Scuola Superiore Sant'Anna, Pisa, Italy, 2Department of Internal Medicine, Division of Pharmacology, University of Pisa, Pisa, Italy

Irinotecan is a topoisomerase-I (Top-I) targeting drug employed for colorectal cancer treatment. DNA demethylating agents, including 5-azacytidine, displayed synergistic antitumor activity with several chemotherapy drugs. The aim of the present study was to investigate whether 5-azacytidine may enhance irinotecan cytotoxicity by at least one of the following mechanisms: 1) Top-I promoter demethylation 2) activation of genes involved in Top-I transcriptional regulation (p16, Sp1, p53) 3) modulation of cell cycle and apoptosis after DNA damage. Growth inhibitory effect of irinotecan active metabolite (SN38), 5-azacytidine and their combination was studied in four colorectal cancer cell lines (HT29, SW620, WiDr, LS174T). The effects of these treatments on the cell cycle was analyzed by flow cytometry, and apoptosis was measured by fluorescence microscopy. Top-I and Sp1 mRNA modulation by 5-azacytidine were measured by real-time PCR. Methylation of Top-I, p16, 14-3-3Σ, hMLH1 promoters before and after 5-azacytidine treatment was measured through Methylight PCR. Results demonstrated that low dose 5-azacytidine significantly enhances the apoptotic effect of irinotecan in all colorectal cancer cells. However, 5-azacytidine enhanced irinotecan growth inhibitory effect only in p53-mutated cells, with a 22.1-fold (HT29), 10.3-fold (SW620) and 2.9-fold (WiDr) IC50 reduction. Top-I up-regulation by 5-azacytidine was significantly correlated with this effect, and was coupled to p16 demethylation and Sp1 up-regulation. P16 demethylation by 5-azacytidine was also associated with an enhanced cell cycle arrest after irinotecan treatment. In p53-wt LS174T cells, 5-azacytidine downregulated Top-I expression, thereby reducing SN38 cytotoxicity. In conclusion, 5-azacytidine modulates Top-I expression in a Sp1-, p16- and p53-dependent manner. Based on these findings, 5-azacytidine-irinotecan combination appears to be particularly effective against p53-mutated, p16-methylated colorectal cancers.