Effect of combined therapy with low-dose 5-aza-2'-deoxycytidine and irinotecan on colon cancer cell line HCT-15.
BACKGROUND
Aberrant promoter hypermethylation is an epigenetic change that silences the expression of crucial genes, resulting in inactivation of the apoptotic pathway in various cancers. This hypermethylation can be restored by the demethylating agent 5-aza-2'-deoxycytidine (DAC). DAC might increase the tumor sensitivity to chemotherapy through demethylation and restoration of gene expression. We investigated the effect of combined therapy with DAC and irinotecan (CPT-11) on the human colon cancer cell line HCT-15.


METHODS
Human colon cancer cell line HCT-15 was treated with DAC and/or CPT-11 both in vitro and in vivo. The changes in mRNA expression of several apoptosis-related genes were investigated by reverse transcriptase-polymerase chain reaction (PCR). Promoter methylation was detected by methylation-specific PCR and combined bisulfite restriction analysis. Suppression of tumor growth was observed during the treatment with DAC and/or CPT-11 and apoptosis in the tumors was investigated by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay.


RESULTS
Promoter methylation of p14ARF, p16 INK4a, BNIP3, and XAF1 was confirmed, and DAC restored mRNA expression of these genes. Demethylation and restoration of gene expression was observed with low-dose DAC, and demethylation status was sustained for several weeks. Combined therapy with DAC and CPT-11 produced marked suppression in tumor growth compared with DAC or CPT-11 alone, both in vitro and in vivo.


CONCLUSIONS
Pretreatment with low-dose DAC may have the potential to be used as a "biosensitizer" of DNA-damaging agents such as CPT-11 when the apoptotic pathway is inactivated as a result of aberrant promoter methylation in the cancer.
Ishiguro
Megumi
M
Department of Surgical Oncology, Tokyo Medical and Dental University, Graduate School, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. ishiguro@ndmc.ac.jp
Iida
Satoru
S
Uetake
Hiroyuki
H
Morita
Shinji
S
Makino
Hiroshi
H
Kato
Keiji
K
Takagi
Yoko
Y
Enomoto
Masayuki
M
Sugihara
Kenichi
K
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D002166:Camptothecin / Q000008:administration & dosage / Q000031:analogs & derivatives; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D000077209:Decitabine; D004359:Drug Therapy, Combination; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020287:In Situ Nick-End Labeling; D047908:Intracellular Signaling Peptides and Proteins; D000077146:Irinotecan; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015996:Survival Rate; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C090121:BNIP3 protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D025803:Tumor Suppressor Protein p14ARF; C417551:XAF1 protein, human; D000077146:Irinotecan; D000077209:Decitabine; D001374:Azacitidine; D002166:Camptothecin