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Identification of novel DNA methylation inhibitors via a two-component reporter gene system.

Lin YS, Shaw AY, Wang SG, Hsu CC, Teng IW, Tseng MJ, Huang TH, Chen CS, Leu YW, Hsiao SH - J. Biomed. Sci. (2011)

Bottom Line: Alternatively, procainamide has emerged as a potential DNA demethylating agent for clinical translation.While procainamide is much safer than 5-aza-2'-deoxycytidine, it requires high concentrations to be effective in DNA demethylation in suppressing cancer cell growth.A lead agent IM25, which exhibits substantially higher potency in GSTp1 DNA demethylation with lower cytotoxicity in MCF7 cells relative to procainamide and 5-aza-2'-deoxycytidine, was identified by the screening platform.

View Article: PubMed Central - HTML - PubMed

Affiliation: Human Epigenomics Center, Department of Life Science, Institute of Molecular Biology, National Chung Cheng University, Chia-Yi, 621, Taiwan.

ABSTRACT

Background: Targeting abnormal DNA methylation represents a therapeutically relevant strategy for cancer treatment as demonstrated by the US Food and Drug Administration approval of the DNA methyltransferase inhibitors azacytidine and 5-aza-2'-deoxycytidine for the treatment of myelodysplastic syndromes. But their use is associated with increased incidences of bone marrow suppression. Alternatively, procainamide has emerged as a potential DNA demethylating agent for clinical translation. While procainamide is much safer than 5-aza-2'-deoxycytidine, it requires high concentrations to be effective in DNA demethylation in suppressing cancer cell growth. Thus, our laboratories have embarked on the pharmacological exploitation of procainamide to develop potent DNA methylation inhibitors through lead optimization.

Methods: We report the use of a DNA methylation two-component enhanced green fluorescent protein reporter system as a screening platform to identify novel DNA methylation inhibitors from a compound library containing procainamide derivatives.

Results: A lead agent IM25, which exhibits substantially higher potency in GSTp1 DNA demethylation with lower cytotoxicity in MCF7 cells relative to procainamide and 5-aza-2'-deoxycytidine, was identified by the screening platform.

Conclusions: Our data provide a proof-of-concept that procainamide could be pharmacologically exploited to develop novel DNA methylation inhibitors, of which the translational potential in cancer therapy/prevention is currently under investigation.

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Related in: MedlinePlus

Potency and safety of DNA demethylating agents. (A) Comparison of the dose-dependent suppressive effects of 5-Aza, procainamide, IM25, and IM9 on the viability of MCF7 cells by MTT assays. Columns, mean (n = 3); error bars, S.D. (B) Parallel ELISA analysis of the suppressive effects on EGFP expression in the two-component reporter system. Columns, mean (n = 3); error bars, S.D. (C) Western blot analysis of the effects of procainamide, 5-Aza, IM9, and IM25, each at 7.5 μM, on EGFP expression.
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Figure 3: Potency and safety of DNA demethylating agents. (A) Comparison of the dose-dependent suppressive effects of 5-Aza, procainamide, IM25, and IM9 on the viability of MCF7 cells by MTT assays. Columns, mean (n = 3); error bars, S.D. (B) Parallel ELISA analysis of the suppressive effects on EGFP expression in the two-component reporter system. Columns, mean (n = 3); error bars, S.D. (C) Western blot analysis of the effects of procainamide, 5-Aza, IM9, and IM25, each at 7.5 μM, on EGFP expression.

Mentions: We further validated this two-component EGFP reporter gene system by exposing the transfected MCF7 cells to different concentrations of 5-Aza and procainamide. After 5-day treatment, both agents showed dose-dependent, repressive effects on EGFP expression levels relative to DMSO control, as visualized by fluorescence microscopy, with relative potency of 5-Aza greater than procainamide (Additional file 1: Figure S2 and Figure 2B). Equally important, as these two drugs did not cause morphological changes [light microscopy (Vis), Additional file 1: Figure S2] or suppression of cell viability (Figure 3A, Statistics in Additional file 1: Table S2), these findings indicate that this reduction in EGFP expression was not caused by drug-induced cell death, suggesting a direct consequence of DNA demethylation.


Identification of novel DNA methylation inhibitors via a two-component reporter gene system.

Lin YS, Shaw AY, Wang SG, Hsu CC, Teng IW, Tseng MJ, Huang TH, Chen CS, Leu YW, Hsiao SH - J. Biomed. Sci. (2011)

Potency and safety of DNA demethylating agents. (A) Comparison of the dose-dependent suppressive effects of 5-Aza, procainamide, IM25, and IM9 on the viability of MCF7 cells by MTT assays. Columns, mean (n = 3); error bars, S.D. (B) Parallel ELISA analysis of the suppressive effects on EGFP expression in the two-component reporter system. Columns, mean (n = 3); error bars, S.D. (C) Western blot analysis of the effects of procainamide, 5-Aza, IM9, and IM25, each at 7.5 μM, on EGFP expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3025941&req=5

Figure 3: Potency and safety of DNA demethylating agents. (A) Comparison of the dose-dependent suppressive effects of 5-Aza, procainamide, IM25, and IM9 on the viability of MCF7 cells by MTT assays. Columns, mean (n = 3); error bars, S.D. (B) Parallel ELISA analysis of the suppressive effects on EGFP expression in the two-component reporter system. Columns, mean (n = 3); error bars, S.D. (C) Western blot analysis of the effects of procainamide, 5-Aza, IM9, and IM25, each at 7.5 μM, on EGFP expression.
Mentions: We further validated this two-component EGFP reporter gene system by exposing the transfected MCF7 cells to different concentrations of 5-Aza and procainamide. After 5-day treatment, both agents showed dose-dependent, repressive effects on EGFP expression levels relative to DMSO control, as visualized by fluorescence microscopy, with relative potency of 5-Aza greater than procainamide (Additional file 1: Figure S2 and Figure 2B). Equally important, as these two drugs did not cause morphological changes [light microscopy (Vis), Additional file 1: Figure S2] or suppression of cell viability (Figure 3A, Statistics in Additional file 1: Table S2), these findings indicate that this reduction in EGFP expression was not caused by drug-induced cell death, suggesting a direct consequence of DNA demethylation.

Bottom Line: Alternatively, procainamide has emerged as a potential DNA demethylating agent for clinical translation.While procainamide is much safer than 5-aza-2'-deoxycytidine, it requires high concentrations to be effective in DNA demethylation in suppressing cancer cell growth.A lead agent IM25, which exhibits substantially higher potency in GSTp1 DNA demethylation with lower cytotoxicity in MCF7 cells relative to procainamide and 5-aza-2'-deoxycytidine, was identified by the screening platform.

View Article: PubMed Central - HTML - PubMed

Affiliation: Human Epigenomics Center, Department of Life Science, Institute of Molecular Biology, National Chung Cheng University, Chia-Yi, 621, Taiwan.

ABSTRACT

Background: Targeting abnormal DNA methylation represents a therapeutically relevant strategy for cancer treatment as demonstrated by the US Food and Drug Administration approval of the DNA methyltransferase inhibitors azacytidine and 5-aza-2'-deoxycytidine for the treatment of myelodysplastic syndromes. But their use is associated with increased incidences of bone marrow suppression. Alternatively, procainamide has emerged as a potential DNA demethylating agent for clinical translation. While procainamide is much safer than 5-aza-2'-deoxycytidine, it requires high concentrations to be effective in DNA demethylation in suppressing cancer cell growth. Thus, our laboratories have embarked on the pharmacological exploitation of procainamide to develop potent DNA methylation inhibitors through lead optimization.

Methods: We report the use of a DNA methylation two-component enhanced green fluorescent protein reporter system as a screening platform to identify novel DNA methylation inhibitors from a compound library containing procainamide derivatives.

Results: A lead agent IM25, which exhibits substantially higher potency in GSTp1 DNA demethylation with lower cytotoxicity in MCF7 cells relative to procainamide and 5-aza-2'-deoxycytidine, was identified by the screening platform.

Conclusions: Our data provide a proof-of-concept that procainamide could be pharmacologically exploited to develop novel DNA methylation inhibitors, of which the translational potential in cancer therapy/prevention is currently under investigation.

Show MeSH
Related in: MedlinePlus