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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

Drug-induced loci-specific and global demethylation. (A) MSP was applied to determine the methylation level of Trip10 and GSTp1 in MCF7 cells treated with each of the following derivatives at 7.5 or 30 μM: procainamide, 5-Aza, IM25, IM9, CI-4-1, CI-5-1, 4e, and 4o, after 5 days of treatment. Procainamide, 5-Aza, IM25, CI-4-1 and 4e significantly reduced the methylation level of GSTp1, while they appear to have weaker effect in demethylating Trip10 promoter (B) DMH analysis of the effect of 7.5 μM IM25 on genome-wide methylation. The higher Log2[Cy5/Cy3] values indicate stronger demethylation effects. (C) Genome-wide comparison of the demetylation after IM25 and 5-Aza treatment. DMH microarrays identified 5,506 more loci that are demethylated after IM25 treatment (open circles) than the result obtained after 5-Aza treatment (close gray circles). The regressed lines depicted the distribution of demethylation (dashed line for 5-Aza; solid line for IM25) and all the listed 5,506 loci were confirmed significant by ANOVA.
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Figure 4: Drug-induced loci-specific and global demethylation. (A) MSP was applied to determine the methylation level of Trip10 and GSTp1 in MCF7 cells treated with each of the following derivatives at 7.5 or 30 μM: procainamide, 5-Aza, IM25, IM9, CI-4-1, CI-5-1, 4e, and 4o, after 5 days of treatment. Procainamide, 5-Aza, IM25, CI-4-1 and 4e significantly reduced the methylation level of GSTp1, while they appear to have weaker effect in demethylating Trip10 promoter (B) DMH analysis of the effect of 7.5 μM IM25 on genome-wide methylation. The higher Log2[Cy5/Cy3] values indicate stronger demethylation effects. (C) Genome-wide comparison of the demetylation after IM25 and 5-Aza treatment. DMH microarrays identified 5,506 more loci that are demethylated after IM25 treatment (open circles) than the result obtained after 5-Aza treatment (close gray circles). The regressed lines depicted the distribution of demethylation (dashed line for 5-Aza; solid line for IM25) and all the listed 5,506 loci were confirmed significant by ANOVA.

Mentions: Both 5-Aza and procainamide have been shown to cause demethylation of GSTp1 (GSTπ1) in colon, prostate, and breast cancer cells [11-14]. Thus, we conducted MSP to determine the DNA methylation levels of Trip10 and GSTp1 in MCF7 cells treated with IM25, CI-4-1, and 4e vis-à-vis 5-Aza and procainamide. Among these testing compounds, IM25 exhibits the greatest potency in facilitating the demethylation of both Trip10 and GSTp1, CI-4-1 also decreases the methylation level of both genes, but to a lesser extent (Figure 4A). Compound 4e showed a slight demethylation of GSTp1 only at 30 μM. IM25 was chosen to further elucidate its genome-wide demethylation effects. Cells treated with either IM-25 or DMSO were harvested and subjected to DMH. As demonstrated in Figure 4B, IM25 was able to cause global demethylation in MCF7 cells. When comparing the global demethylation capacity between IM25 and 5-Aza, 5,506 more demethylated loci out of 244K target loci in Agilent array were identified (Figure 4C, significant loci confirmed by ANOVA). Therefore, the identified IM25 possesses equal or more demethylation capacity than the known drugs such as 5-Aza and procainamide.


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)

Drug-induced loci-specific and global demethylation. (A) MSP was applied to determine the methylation level of Trip10 and GSTp1 in MCF7 cells treated with each of the following derivatives at 7.5 or 30 μM: procainamide, 5-Aza, IM25, IM9, CI-4-1, CI-5-1, 4e, and 4o, after 5 days of treatment. Procainamide, 5-Aza, IM25, CI-4-1 and 4e significantly reduced the methylation level of GSTp1, while they appear to have weaker effect in demethylating Trip10 promoter (B) DMH analysis of the effect of 7.5 μM IM25 on genome-wide methylation. The higher Log2[Cy5/Cy3] values indicate stronger demethylation effects. (C) Genome-wide comparison of the demetylation after IM25 and 5-Aza treatment. DMH microarrays identified 5,506 more loci that are demethylated after IM25 treatment (open circles) than the result obtained after 5-Aza treatment (close gray circles). The regressed lines depicted the distribution of demethylation (dashed line for 5-Aza; solid line for IM25) and all the listed 5,506 loci were confirmed significant by ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 4: Drug-induced loci-specific and global demethylation. (A) MSP was applied to determine the methylation level of Trip10 and GSTp1 in MCF7 cells treated with each of the following derivatives at 7.5 or 30 μM: procainamide, 5-Aza, IM25, IM9, CI-4-1, CI-5-1, 4e, and 4o, after 5 days of treatment. Procainamide, 5-Aza, IM25, CI-4-1 and 4e significantly reduced the methylation level of GSTp1, while they appear to have weaker effect in demethylating Trip10 promoter (B) DMH analysis of the effect of 7.5 μM IM25 on genome-wide methylation. The higher Log2[Cy5/Cy3] values indicate stronger demethylation effects. (C) Genome-wide comparison of the demetylation after IM25 and 5-Aza treatment. DMH microarrays identified 5,506 more loci that are demethylated after IM25 treatment (open circles) than the result obtained after 5-Aza treatment (close gray circles). The regressed lines depicted the distribution of demethylation (dashed line for 5-Aza; solid line for IM25) and all the listed 5,506 loci were confirmed significant by ANOVA.
Mentions: Both 5-Aza and procainamide have been shown to cause demethylation of GSTp1 (GSTπ1) in colon, prostate, and breast cancer cells [11-14]. Thus, we conducted MSP to determine the DNA methylation levels of Trip10 and GSTp1 in MCF7 cells treated with IM25, CI-4-1, and 4e vis-à-vis 5-Aza and procainamide. Among these testing compounds, IM25 exhibits the greatest potency in facilitating the demethylation of both Trip10 and GSTp1, CI-4-1 also decreases the methylation level of both genes, but to a lesser extent (Figure 4A). Compound 4e showed a slight demethylation of GSTp1 only at 30 μM. IM25 was chosen to further elucidate its genome-wide demethylation effects. Cells treated with either IM-25 or DMSO were harvested and subjected to DMH. As demonstrated in Figure 4B, IM25 was able to cause global demethylation in MCF7 cells. When comparing the global demethylation capacity between IM25 and 5-Aza, 5,506 more demethylated loci out of 244K target loci in Agilent array were identified (Figure 4C, significant loci confirmed by ANOVA). Therefore, the identified IM25 possesses equal or more demethylation capacity than the known drugs such as 5-Aza and procainamide.

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