J Breast Cancer.  2013 Mar;16(1):23-31.

Expression of DNA Methyltransferases in Breast Cancer Patients and to Analyze the Effect of Natural Compounds on DNA Methyltransferases and Associated Proteins

• Mirza S
• Sharma G
• Parshad R
• Gupta SD
• Pandya P
• Ralhan R

Abstract

PURPOSE
The DNA methylation mediated by specific DNA methyltransferases (DNMTs), results in the epigenetic silencing of multiple genes which are implicated in human breast cancer. We hypothesized that the natural compounds modulate the expression of DNMTs and their associated proteins in the breast cancer cell lines and affect the methylation mediated gene silencing.
METHODS
The DNMTs transcript expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in the tumors and the adjacent normal breast tissues of the patients with invasive ductal breast carcinoma. We tested the hypothesis that the natural compounds, viz., epigallocatechin gallate (EGCG), genistein, withaferin A, curcumin, resveratrol, and guggulsterone, have demethylation potential. To investigate this hypothesis, we analyzed the DNMTs expression at the transcript levels, followed by the analysis of DNMT1 and its associated proteins (HDAC1, MeCP2, and MBD2).
RESULTS
The increased DNMTs transcripts expression, viz., DNMT1, DNMT3a, and DNMT3b, in the breast cancer tissues suggest involvement of the DNMTs in the breast carcinogenesis. Quantitative RT-PCR analysis revealed that the treatment with natural compounds, viz., EGCG, genistein, withaferin A, curcumin, resveratrol, and guggulsterone, resulted in a significant decrease in the transcript levels of all the DNMTs investigated. Importantly, these natural compounds decreased the protein levels of DNMT1, HDAC1, and MeCP2.
CONCLUSION
Our results demonstrate that the natural compounds, EGCG, genistein, withaferin A, curcumin, resveratrol, and guggulsterone, have the potential to reverse the epigenetic changes. Moreover, their lack of toxicity makes these natural compounds promising candidates for the chemoprevention of the breast cancer. In-depth future mechanistic studies aimed to elucidate how these compounds affect the gene transcription are warranted.

Keyword

Breast neoplasms; Chemoprevention; DNA methylation; Epigenomics

MeSH Terms

Breast
Breast Neoplasms
Catechin
Cell Line
Chemoprevention
Curcumin
DNA
DNA Methylation
Epigenomics
Genistein
Humans
Methylation
Methyltransferases
Pregnenediones
Proteins
Stilbenes
Withanolides
Catechin
Curcumin
DNA
Genistein
Methyltransferases
Pregnenediones
Proteins
Stilbenes
Withanolides

Figure

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  Figure 1 Expression levels of DNMT1, DNMT 3a, and DNMT3b in breast cancer tissues. The levels of DNMT1, DNMT3a, and DNMT3b mRNA were observed to be 1.2- to 4.4-folds, 1.1- to 3.77-folds, and 1.06- to 4.01-folds elevated in most of the breast cancer tissues as compared to the adjacent normal breast tissues.

• 

  Figure 2 Effect of natural compounds on cell viability of breast cancer cells. The effect of natural compounds, viz., curcumin, EGCG, resveratrol, genistein, and guggulsterone on the viability of breast cancer cells was determined by MTT assay. (A) MCF 7. (B) MDA MB 231. (C) Effect of withaferin A on MCF 7 and MDA MB 231 cells. Mean values from the three experiments±standard error of mean (SEM) are shown.

• 

  Figure 3 Effect of natural compounds on the methylation status of ERα, TMS1, PRB, Maspin, RARβ2, SLIT2, Cyclin D2, and MGMT. MCF 7 and MDA MB 231 cells were treated with EGCG, genistein, withaferin A, curcumin, resveratrol, guggulsterone (conc. of each natural compound used equals their IC50 value) and decitabine (at conc. of 10 µM for MDA MB 231 cells, and 12 µM for MCF 7 cells) for 96 hours. MSP was performed for ERα, TMS1, PRB, Maspin, Cyclin D2, and MGMT in MDA MB 231 cells and RARβ2 and SLIT2 in MCF 7 cells.

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  Figure 4 Effect of natural compounds on the DNMT transcript levels, viz., DNMT1, DNMT3a, and DNMT3b in human breast cancer cell lines. MCF 7 and MDA MB 231 cells were treated with EGCG, genistein, withaferin A, curcumin, resveratrol, guggulsterone (conc. of each compound used equals their IC50 value) and decitabine (at conc. of 10 µM for MDA MB 231 cells and 12 µM for MCF 7 cells) for 96 hours and then real time polymerase chain reaction analysis was performed for the transcript levels of DNMT1 (A), DNMT3a (B), and DNMT3b (C).

• 

  Figure 5 Effect of natural compounds on the expression of p21WAF1, DNMT1, HDAC1, and methyl-CpG binding proteins (MeCP2 and MBD2) in human breast cancer cell lines. MCF 7 and MDA MB 231 cells were treated with EGCG, genistein, withaferin A, curcumin, resveratrol, guggulsterone (conc. of each compound equals their IC50 value) and decitabine (at conc. of 10 µM for MDA MB 231 cells and 12 µM for MCF 7 cells) for 96 hours and then Western blot analysis was performed for DNMT1, HDAC1, MeCP2, MBD2, and p21WAF1 in MCF 7 cells (A) and MDA MB 231 cells (B). The fold change values were calculated as a relative change in comparison to the control cells treated with DMSO (expression equals 1).

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