Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines
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Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines. / Crea, Francesco; Giovannetti, Elisa; Cortesi, Filippo; Mey, Valentina; Nannizzi, Sara; Gallegos Ruiz, Marielle I; Ricciardi, Simona; Del Tacca, Mario; Peters, Godefridus J; Danesi, Romano.
In: MOL CANCER THER, Vol. 8, No. 7, 07.2009, p. 1964-73.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines
AU - Crea, Francesco
AU - Giovannetti, Elisa
AU - Cortesi, Filippo
AU - Mey, Valentina
AU - Nannizzi, Sara
AU - Gallegos Ruiz, Marielle I
AU - Ricciardi, Simona
AU - Del Tacca, Mario
AU - Peters, Godefridus J
AU - Danesi, Romano
PY - 2009/7
Y1 - 2009/7
N2 - 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.
AB - 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.
KW - 14-3-3 Proteins/genetics
KW - Antimetabolites, Antineoplastic/pharmacology
KW - Antineoplastic Agents, Phytogenic/pharmacology
KW - Apoptosis
KW - Azacitidine/pharmacology
KW - Camptothecin/analogs & derivatives
KW - Cell Cycle/drug effects
KW - Cell Proliferation/drug effects
KW - Colorectal Neoplasms/drug therapy
KW - Cyclin-Dependent Kinase Inhibitor p16/genetics
KW - DNA Damage/drug effects
KW - DNA Methylation
KW - DNA Topoisomerases, Type I/genetics
KW - Drug Therapy, Combination
KW - Epigenesis, Genetic
KW - Humans
KW - Irinotecan
KW - RNA, Messenger/genetics
KW - Reverse Transcriptase Polymerase Chain Reaction
KW - Tumor Cells, Cultured
KW - Tumor Suppressor Protein p53/genetics
U2 - 10.1158/1535-7163.MCT-09-0027
DO - 10.1158/1535-7163.MCT-09-0027
M3 - SCORING: Journal article
C2 - 19531575
VL - 8
SP - 1964
EP - 1973
JO - MOL CANCER THER
JF - MOL CANCER THER
SN - 1535-7163
IS - 7
ER -
