Epigenetic changes induced by reactive oxygen species in hepatocellular carcinoma: methylation of the E-cadherin promoter.
OBJECTIVE
In addition to genetic alterations, epigenetic changes underlie tumor progression and metastasis. Promoter methylation can silence tumor suppressor genes, and reactive oxygen species (ROS) promote DNA damage, although the relationship between ROS and epigenetic changes in cancer cells is not clear. We sought to determine whether ROS promote hypermethylation of the promoter region of E-cadherin, a regulator of the epithelial-to-mesenchymal transition, in hepatocellular carcinoma (HCC) cells.


METHODS
HCC cells were exposed to H(2)O(2) or stably transfected to express Snail, a transcription factor that down-regulates E-cadherin expression. E-cadherin and Snail expression levels were examined by real-time reverse-transcriptase polymerase chain reaction and immunoblot analyses. The methylation status of E-cadherin was examined by methyl-specific polymerase chain reaction, bisulfite sequencing, and chromatin immunoprecipitation. The interactions between Snail, histone deacetylase 1, and DNA methyltransferase 1 were assessed by immunoprecipitation/immunoblot and immunofluorescence analyses. ROS-induced stress, E-cadherin expression, Snail expression, and E-cadherin promoter methylation were confirmed in HCC tissues by immunoblot, immunohistochemistry, and methyl-specific polymerase chain reaction analyses.


RESULTS
We demonstrated that ROS induce hypermethylation of the E-cadherin promoter by increasing Snail expression. Snail induced DNA methylation of the E-cadherin promoter by recruiting histone deacetylase 1 and DNA methyltransferase 1. In human HCC tissues, we observed a correlation among ROS induction, E-cadherin down-regulation, Snail up-regulation, and E-cadherin promoter methylation.


CONCLUSIONS
These findings provide novel mechanistic insights into epigenetic modulations induced by ROS in the process of carcinogenesis. They are potentially relevant to understanding the activity of ROS in silencing various tumor suppressor genes and in subsequent tumor progression and metastasis.
Lim
Seung-Oe
SO
Department of Biological Sciences, Seoul National University, Seoul, Korea.
Gu
Jin-Mo
JM
Kim
Min Sook
MS
Kim
Hyun-Soo
HS
Park
Young Nyun
YN
Park
Cheol Keun
CK
Cho
Jae Won
JW
Park
Young Min
YM
Jung
Guhung
G
D000328:Adult; D000368:Aged; D015820:Cadherins / Q000235:genetics* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D047369:Chromatin Immunoprecipitation; D004273:DNA, Neoplasm / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008745:Methylation / Q000187:drug effects; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000235:genetics; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D015820:Cadherins; D004273:DNA, Neoplasm; D017382:Reactive Oxygen Species