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ABSTRACT: 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.
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).
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.
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.
Journal of Breast Cancer 03/2013; 16(1):23-31. · 0.32 Impact Factor
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ABSTRACT: Maspin is a serine protease inhibitor with tumor-suppressor activity. Maspin can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. Previous studies indicate that the loss of Maspin expression is closely linked to aberrant methylation of the Maspin promoter. We examined the promoter methylation status of Maspin in tumor and corresponding serum of breast cancer patients. In addition, protein expression of this gene was also assessed to determine possible correlation between promoter hypermethylation and gene silencing. Further, we investigated the correlation of Maspin expression with vascular endothelial growth factor (VEGF-A) and MTA1 expression. Maspin methylation was analyzed by methylation-specific PCR in 100 invasive ductal breast carcinoma patients' tumors and circulating DNA in a prospective study. Promoter hypermethylation was correlated with expression of the encoded protein in tumors by immunohistochemistry. Significant correlation was observed between promoter hypermethylation of Maspin (r = +0.88; p ≤ 0.0001) in tumors and paired sera. Significant association was found between Maspin promoter hypermethylation and loss of its protein expression (p = 0.01, OR = 3.1, 95% CI = 1.3-7.4). The expression of VEGF-A and MTA1 was lower in tumors with high Maspin expression compared to tumors with loss of Maspin expression. Our results indicate that aberrant promoter methylation is associated with loss of Maspin immunoreactivity in breast cancer tissues. Further, loss of Maspin expression is significantly correlated with increased expression of VEGF-A and MTA1.
Tumor Biology 02/2011; 32(1):23-32. · 1.94 Impact Factor
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ABSTRACT: The clinical relevance of frequent methylation of CpG islands of key cancer genes in breast cancer is being increasingly recognized. Our study aimed to evaluate the promoter methylation status of DNA repair genes-BRCA1MGMT and GSTP1 in tumor and circulating DNA of invasive ductal breast carcinoma patients.
Methylation-specific PCR was carried out to investigate the promoter methylation status of genes in tumor and circulating DNA of 100 breast cancer patients in a prospective study. The effect of promoter methylation on protein expression was evaluated by immunohistochemistry.
The frequency of tumor hypermethylation was 27% in BRCA1, 32% in MGMT and 25% in GSTP1 and correlated with methylation of these genes in paired serum DNA. Immunohistochemical analysis showed no detectable expression of BRCA1 and MGMT in 51/89 (57%) and 35/89 (39%) tumors, respectively. MGMT promoter methylation mediated gene silencing was associated with loss of its protein expression (p=0.002, O.R.=4.5, 95% C.I.=1.7-12.0). BRCA1 promoter methylation was not associated with loss of its protein expression, indicating that methylation is not the sole mechanism accounting for the loss/reduced BRCA1 protein expression. Importantly, GSTP1 and BRCA1 hypermethylation were found to be independent of other prognostic factors in predicting disease recurrence (p=0.02, HR=7.6, 95% C.I.=1.4-44.1; p=0.04, HR=6.2, 95% C.I.=1.1-35.7).
Our study underscores the potential utility of DNA methylation of these genes in serum as a promising biomarker and can serve as a surrogate for tumor DNA methylation for diagnosis and prognosis of breast cancer.
Life sciences 07/2010; 87(3-4):83-91. · 2.56 Impact Factor
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ABSTRACT: DNA methylation plays an important role in regulation of gene expression and is increasingly being recognized as a determinant of chemosensitivity of human cancers. With the aim of improving the chemotherapeutic efficacy of breast carcinoma, the effect of DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5-aza-CdR), on the chemosensitivity of anticancer drugs was investigated. The cytotoxicity of paclitaxel (PTX), adriamycin (ADR), and 5-fluorouracil (5-FU) was analyzed against human breast cancer cell lines, MDA MB 231 and MCF 7 cell lines using the MTT assay, and the synergy of 5-aza-CdR and these agents was determined by Drewinko's fraction method. The effects of each single agent or the combined treatment on cell cycle arrest were analyzed by flow cytometric analysis. We also investigated the effect of each single agent or the combined treatment of anticancer drugs with 5-aza-CdR on the methylation status of the selected genes by methylation specific PCR. In MDA MB 231 cells, a synergistic antiproliferative effect was observed with a combination of 10 microM 5-aza-CdR and these three anticancer drugs, while in MCF 7 cells, a semiadditive effect was observed. Treatment with 5-aza-CdR and anticancer drug resulted in partial demethylation of a panel of genes including RARbeta2, Slit2, GSTP1, and MGMT. Based on these findings, we propose that 5-aza-CdR enhances the chemosensitivity of anticancer drugs in breast cancer cells and may be a promising approach for increasing the chemotherapeutic potential of these anticancer agents for more effective management of breast carcinomas.
Molecular and Cellular Biochemistry 05/2010; 342(1-2):101-9. · 2.06 Impact Factor
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ABSTRACT: Multidrug resistance 1 (MDR1) gene encodes P-glycoprotein (P-gp), a transmembrane calcium-dependent efflux pump, implicated in drug resistance. In this prospective study, methylation status of MDR1 promoter and its correlation with clinicopathological parameters were evaluated in tumor and serum of breast cancer patients.
Methylation-specific PCR was carried out to investigate the promoter methylation status of MDR1 in tumor and serum of 100 patients with invasive ductal carcinomas of breast (IDCs). The effect of promoter methylation on protein expression was evaluated by immunohistochemistry.
MDR1 was hypomethylated in 47% tumors and 44% paired sera of IDC patients and correlated significantly with increased tumor size and advanced tumor stage. Promoter hypomethylation of MDR1 in serum DNA showed 98% specificity and 50% sensitivity.
Hypomethylation of MDR1 promoter in IDCs accounted for P-gp overexpression and aggressive biologic behavior in a subset of patients. Detection of these epigenetic changes in circulating DNA may not only enhance insight into the biological behavior of the primary tumor of an individual but may also provide valuable information regarding prognosis that can be readily monitored throughout the disease course.
Clinical biochemistry 10/2009; 43(4-5):373-9. · 2.02 Impact Factor
