[Effects of 5-Aza-2'-deoxycytidine and trichostatin A on DNA methylation and expression of hMLH1 in ovarian cancer cell line COC1/DDP].
OBJECTIVE
Cisplatin (DDP) can cause DNA damage in cells. DNA mismatch repair proteins serve to detect DDP-caused DNA damage and generate an injury signal that eventually contributes to the triggering of tumor cell apoptosis. As a member of the mismatch repair system, the absence of hMLH1 expression contributes to the resistance of tumor cells to DDP. This study was to explore the role of hMLH1 expression and DNA methylation in DDP-resistance of human ovarian cancer, and to evaluate the reversal effects of 5-aza-2'-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) on DDP-resistance.


METHODS
We cultured human ovarian cancer cell line COC1 and its DDP-resistant subline, COC1/DDP. We treated the two cell lines with 5-Aza-dC and TSA. DNA methylation at hMLH1 gene promoter was detected by methylation-specific polymerase chain reaction (MSP). The expression of hMLH1 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The inhibition rate of cell proliferation was detected by MTT assay.


RESULTS
In COC1 cells, both hMLH1 mRNA and protein were detected, while no DNA methylation of hMLH1 gene was detected. 5-Aza-dC and TSA used alone or in combination had no effects on DNA methylation, hMLH1 mRNA or protein expression (P>0.05), and cell proliferation. In COC1/DDP cells, DNA hypermethylation of hMLH1 gene was detected, while no hMLH1 mRNA or protein was detected. 5-Aza-dC resulted in DNA demethylation and restoration of hMLH1 expression. TSA had no effect on DNA demethylation or restoration of hMLH1 expression. 5-Aza-dC plus TSA also resulted in DNA demethylation, restored hMLH1 expression more obviously than 5-Aza-dC did (P<0.05), and restricted the proliferation of COC1/DDP cells.


CONCLUSIONS
Hypermethylation of DNA promoter is related to the silencing of hMLH1 in ovarian cancer COC1/DDP cells. 5-Aza-dC alone or in combination with TSA results in DNA demethylation of hMLH1 gene, restoration of hMLH1 expression and reversal of DDP-resistance of COC1/DDP cells.
Meng
Chun-Feng
CF
Department of Surgical Oncology, Department of General Surgery, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning, 110001, P. R. China.
Dai
Dong-Qiu
DQ
Guo
Ke-Jun
KJ
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D019008:Drug Resistance, Neoplasm; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000378:metabolism* / Q000473:pathology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000964:Antimetabolites, Antineoplastic; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; C012589:trichostatin A; D000077209:Decitabine; D000070957:MutL Protein Homolog 1; D001374:Azacitidine; D002945:Cisplatin