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Ischemia dysregulates DNA methyltransferases and p16INK4a methylation in human colorectal cancer cells.

ABSTRACT: Epigenetic modifications are involved in the initiation and progression of cancer. Expression patterns and activity of DNA methyltransferases (DNMTs) are strictly controlled in normal cells, however, regulation of these enzymes is lost in cancer cells due to unknown reasons. Cancer therapies which target DNMTs are promising treatments of hematologic cancers, but they lack effectiveness in solid tumors. Solid tumors exhibit areas of hypoxia and hypoglycaemia due to their irregular and dysfunctional vasculature, and we previously showed that hypoxia reduces global DNA methylation. Colorectal carcinoma (CRC) cells (HCT116 and 379.2; p53+/+ and p53-/-, respectively) were subjected to ischemia (hypoxia and hypoglycaemia) in vitro, and levels of DNMTs were assessed. We found a significant decrease in mRNA for DNMT1, DNMT3a and DNMT3b, and similar reductions in DNMT1 and DNMT3a protein levels were detected by western blotting. In addition, total activity levels of DNMTs (as measured by an ELISA-based DNMT activity assay) were reduced in cells exposed to hypoxic and hypoglycaemic conditions. Immunofluorescence of HCT116 tumor xenografts demonstrated an inverse relationship between ischemia (as revealed by carbonic anhydrase IX staining) and DNMT1 protein. Bisulfite sequencing of the proximal promoter region of p16INK4a showed a decrease in 5-methylcytosine following in vitro exposure to ischemia. These studies provide evidence for the down-regulation of DNMTs and modulation of methylation patterns by hypoxia and hypoglycaemia in human CRC cells, both in vitro and in vivo. Our findings suggest that ischemia, either intrinsic or induced through the use of anti-angiogenic drugs, may influence epigenetic patterning and hence tumor progression.

SUBMITTER: Skowronski K

PROVIDER: S-EPMC3322492 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Ischemia dysregulates DNA methyltransferases and p16INK4a methylation in human colorectal cancer cells.

Skowronski Karolina K Dubey Sonam S Rodenhiser David D Coomber Brenda B

Epigenetics 20100816 6

Epigenetic modifications are involved in the initiation and progression of cancer. Expression patterns and activity of DNA methyltransferases (DNMTs) are strictly controlled in normal cells, however, regulation of these enzymes is lost in cancer cells due to unknown reasons. Cancer therapies which target DNMTs are promising treatments of hematologic cancers, but they lack effectiveness in solid tumors. Solid tumors exhibit areas of hypoxia and hypoglycaemia due to their irregular and dysfunction ...[more]

PMID: 20543577

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Project description:AimRecent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells.Materials and methodsTo evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR.ResultsAs expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean ?-values of 0.12), however, non-significant changes in mean ?-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines.ConclusionsOur data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.

Dataset Information

Ischemia dysregulates DNA methyltransferases and p16INK4a methylation in human colorectal cancer cells.

Publications

Ischemia dysregulates DNA methyltransferases and p16INK4a methylation in human colorectal cancer cells.

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