Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines.
Irinotecan is a topoisomerase-I (Top-I) inhibitor used for the treatment of colorectal cancer. DNA demethylating agents, including 5-azacytidine (5-aza), display synergistic antitumor activity with several chemotherapy drugs. 5-Aza may enhance irinotecan cytotoxicity by at least one of the following mechanisms: (a) Top-I promoter demethylation, (b) activation of genes involved in Top-I transcriptional regulation (p16 or Sp1), and (c) modulation of the cell cycle and apoptosis after DNA damage. The growth-inhibitory effects of SN38, the active metabolite of irinotecan, 5-aza, and their combinations, were studied in four colorectal cancer cell lines. The effects of treatments on cell cycle were analyzed by flow cytometry, and apoptosis was measured by fluorescence microscopy. Top-I, Sp1, and p53 expression modulated by 5-aza were measured by real-time PCR. Methylation of Top-I, p16, 14-3-3sigma, and hMLH1 promoters before and after 5-aza treatment were measured by MethyLight PCR and DNA bisulfite sequencing. Low-dose 5-aza significantly enhanced the apoptotic effect of irinotecan in all colorectal cancer cells, whereas a synergistic cytotoxic effect was observed only in p53-mutated cells (HT29, SW620, and WiDr). This synergistic effect was significantly correlated with Top-I up-regulation by 5-aza, and coupled to p16 demethylation and Sp1 up-regulation. p16 demethylation was also associated with enhanced cell cycle arrest after irinotecan treatment. In contrast, 5-aza down-regulated Top-I expression in the p53 wild-type LS174T cells in a p53-dependent manner, thereby reducing SN38 cytotoxicity. In conclusion, 5-aza modulates Top-I expression by several mechanisms involving Sp1, p16, and p53. If confirmed in other models, these results suggest that p16 and p53 status affects the 5-aza-irinotecan interaction.
Crea
Francesco
F
Scuola Superiore Sant'Anna, Pisa, Italy. fcrea@sssup.it
Giovannetti
Elisa
E
Cortesi
Filippo
F
Mey
Valentina
V
Nannizzi
Sara
S
Gallegos Ruiz
Marielle I
MI
Ricciardi
Simona
S
Del Tacca
Mario
M
Peters
Godefridus J
GJ
Danesi
Romano
R
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis; D001374:Azacitidine / Q000494:pharmacology; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D019175:DNA Methylation; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D004359:Drug Therapy, Combination; D044127:Epigenesis, Genetic; D006801:Humans; D000077146:Irinotecan; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D048948:14-3-3 Proteins; D000964:Antimetabolites, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D000077146:Irinotecan; D004264:DNA Topoisomerases, Type I; D001374:Azacitidine; D002166:Camptothecin