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Fig. 7 | Clinical Epigenetics

Fig. 7

From: Combined inhibition of histone deacetylase and cytidine deaminase improves epigenetic potency of decitabine in colorectal adenocarcinomas

Fig. 7

Western immunoblotting of DNMT1, p53, JAK1 and acetylated lysine residues of H3 following decitabine and PBA treatment of colon cancer cell lines. A We queried the TCGA public repository and retrieved gene expression data of DNMT1 and the decitabine uptake transporter SLC15A1. We obtained data for 275 COAD patient samples and compared their expression to histologically proven adjacent normal tissue. This revealed DNMT1 to be significantly upregulated in tumor samples; however, SLC15A1 decreased the expression in tumor samples. B1 Gene expression of DNMT1 in colon cancer cells following decitabine treatment for 72 h. Shown are the results of RT-qPCR assays. Decitabine treatment caused a small but significant increase in DNMT1 gene expression. B2 Gene expression of the decitabine uptake transporter SLC15A1/ PEPT1 following decitabine, PBA and the combined decitabine/PBA treatment. Treatment of Caco-2 cells with PBA and/or the combined decitabine/PBA treatment caused induced expression of SLC15A. C1 Western blotting of p53 protein in HCT-116 wild type and p53 null cells. C2 Western blotting of DNMT1, p53 and JAK1 protein following decitabine treatment of colon cancer cells. Decitabine treatment of colon cancer cells inhibited DNMT1 protein and to a lesser extent the JAK1 protein. Conversely, decitabine treatment of colon cancer cells induced p53 protein expression. The quantification of the WB experiments is shown as histogram. Statistical significance *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C3 Western blotting of DNMT1 and p53 protein following PBA treatment of colon cancer cells. PBA treatment did not influence DNMT1 protein expression; however, induced p53 protein expression in Caco-2 and HCT-116-p53wt cells. The quantification of the WB experiments is shown as histogram. Statistical significance: ****p < 0.0001. D Western blotting of unacetylated and acetylated H3 protein following PBA treatment of colon cancer cells. PBA treatment of Caco-2, HCT-116-p53wt and HCT-116-p53null cells repressed unacetylated H3; however, it strongly induced protein expression of acetylated H3. Shown are immunoblots with antibodies specific for lysine residues 9, 14, 18, 27 and 56 of the H3 protein. The quantification of the WB experiments is shown as histogram. Statistical significance ****p < 0.0001. E Acetylation of the decitabine uptake transporter SLC15A1 by CBP/p300. We used the GPS-Pail lysine acetylation prediction tool to identify acetylation sites of the SLC15A1/PEPT1 protein sequence. This defined K706 as a candidate. Independent research confirmed the HDAC inhibitors SAHA and TSA to restore acyltransferase activity of histone lysine acetyltransferase p300 and CBP and to recover SLC15A1 expression in colorectal cancer cells [40]

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