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S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression.

ABSTRACT: Global DNA hypomethylation is a characteristic feature of colorectal carcinoma (CRC). The tumor inhibitory effect of S-adenosylmethionine (SAM) methyl donor has been described in certain cancers including CRC. However, the molecular impact of SAM treatment on CRC cell lines with distinct genetic features has not been evaluated comprehensively. HT-29 and SW480 cells were treated with 0.5 and 1 mmol/L SAM for 48 h followed by cell proliferation measurements, whole-genome transcriptome and methylome analyses, DNA stability assessments and exome sequencing. SAM reduced cell number and increased senescence by causing S phase arrest, besides, multiple EMT-related genes (e.g., TGFB1) were downregulated in both cell lines. Alteration in the global DNA methylation level was not observed, but certain methylation changes in gene promoters were detected. SAM-induced ?-H2AX elevation could be associated with activated DNA repair pathway showing upregulated gene expression (e.g., HUS1). Remarkable genomic stability elevation, namely, decreased micronucleus number and comet tail length was observed only in SW480 after treatment. SAM has the potential to induce senescence, DNA repair, genome stability and to reduce CRC progression. However, the different therapeutic responses of HT-29 and SW480 to SAM emphasize the importance of the molecular characterization of CRC cases prior to methyl donor supplementation.

SUBMITTER: Zsigrai S

PROVIDER: S-EPMC7464653 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression.

Zsigrai Sára S Kalmár Alexandra A Nagy Zsófia B ZB Barták Barbara K BK Valcz Gábor G Szigeti Krisztina A KA Galamb Orsolya O Dankó Titanilla T Sebestyén Anna A Barna Gábor G Szabó Vanessza V Pipek Orsolya O Medgyes-Horváth Anna A Csabai István I Tulassay Zsolt Z Igaz Péter P Takács István I Molnár Béla B

Cells 20200809 8

Global DNA hypomethylation is a characteristic feature of colorectal carcinoma (CRC). The tumor inhibitory effect of S-adenosylmethionine (SAM) methyl donor has been described in certain cancers including CRC. However, the molecular impact of SAM treatment on CRC cell lines with distinct genetic features has not been evaluated comprehensively. HT-29 and SW480 cells were treated with 0.5 and 1 mmol/L SAM for 48 h followed by cell proliferation measurements, whole-genome transcriptome and methylom ...[more]

PMID: 32784836

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Project description:Background & aimsElevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is the most common DNA mismatch repair defect in colorectal cancers, observed in approximately 60% of specimens. This acquired genotype correlates with metastasis and poor outcomes for patients, and is associated with intra-epithelial inflammation and heterogeneous nuclear levels of the mismatch repair protein hMSH3. Inflammation and accompanying oxidative stress can cause hMSH3 to change its intracellular location, but little is known about the source of oxidative stress in cancer cells. We investigated whether cytokines mediate this process.MethodsWe analyzed levels of interleukin 6 (IL6) and its receptor (IL6R) in human colon and lung cancer cell lines by flow cytometry and enzyme-linked immunosorbent assay; proteins were localized by immunofluorescence and immunoblot analyses. IL6 signaling was blocked with antibodies against IL6, soluble glycoprotein 130 Fc fragments, and the signal transducers and activators of transcription 3 inhibitor NSC74859; a constitutively active form of STAT3 was expressed in colon and lung cancer cell lines to replicate IL6R signaling. EMAST was detected by DNA fragment analysis. Immunohistochemistry was used to examine levels of IL6 in 20 colorectal tumor and adjacent nontumor tissues.ResultsIncubation of colon and lung cancer cell lines with IL6, but not other cytokines, caused hMSH3, but no other mismatch repair proteins, to move from the nucleus to the cytosol after generation of oxidative stress; inhibition of IL6 signaling prevented this shift. Expression of constitutively active STAT3 also caused hMSH3 to translocate from the nucleus to the cytoplasm in cancer cell lines. Incubation of cells with IL6 led to tetranucleotide frameshifts, the signature for EMAST. EMAST-positive colorectal tumors had significantly higher levels of IL6 than EMAST-negative tumors.ConclusionsIL6 signaling disrupts the nuclear localization of hMSH3 and DNA repair, leading to EMAST in cancer cell lines. Inflammatory cytokines might therefore promote genetic alterations in human cancer cells.

Project description:Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influence the expression of sFRP4. Polycomb group (PcG) proteins are epigenetic transcriptional repressors that selectively repress gene expression by forming polycomb repressive complexes (PRCs). Enhancer of zeste homolog 2 (EZH2), the core component of PRC2, is a histone-lysine N-methyltransferase that interacts with DNA methyltransferases. In the present study, the promoter DNA methylation status of sFRP4 in CRC cell lines was analyzed and the underlying mechanisms of action governing this modification was investigated. Firstly, the DNA methylation status of the sFRP4 promoter in CRC cell lines was assessed using methylation-specific PCR. Subsequently, the mRNA and protein levels of sFRP4 were measured using real-time qPCR and western blot analysis, respectively, to determine whether the DNA methylation status of the sFRP4 promoter is correlated with its transcriptional levels. To screen for important epigenetic modifiers that may regulate the promoter DNA methylation status of sFRP4, the expression levels of PcG proteins were examined by gene array analysis. ChIP-qPCR was performed to test whether the selected PcG proteins directly bind the promoter region of sFRP4. Finally, the downregulated PcG proteins EZH2, chromobox 7 (CBX7) and jumonji and AT-rich interaction domain containing 2 (JARID2) were identified and their association with sFRP4 expression levels and Wnt/?-catenin signaling pathway activity were investigated. The present study revealed that sFRP4 was hypermethylated in the promoter region and downregulated during the progression of the CRC cell lines from Dukes A to Dukes C. Expression levels of PcG proteins EZH2, CBX7 and JARID2 were upregulated and positively associated with the aberrantly activated Wnt signaling pathway in the CRC cell lines. EZH2, CBX7 and JARID2 were all enriched in the sFRP4 promoter region in CRC cells. EZH2 downregulation did not affect the promoter DNA methylation status of sFRP4 but increased its expression levels and decreased CRC cell proliferation. DNA methylation controls the expression of sFRP4. EZH2 regulates sFRP4 expression without affecting the DNA hypermethylation of the sFRP4 promoter and influences CRC cell proliferation and Wnt/?-catenin signaling pathway activities.

Dataset Information

S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression.

Publications

S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression.

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