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Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein.

Koo J, Cabarcas-Petroski S, Petrie JL, Diette N, White RJ, Schramm L - BMC Cancer (2015)

Bottom Line: In addition, expression was compared between mice fed diets enriched or deprived of isoflavones.Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and selectively decreases methylation of the BRF2 promoter.In vivo relevance is suggested by the significantly elevated levels of BRF2 mRNA detected in female mice fed a high-isoflavone commercial diet.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, St. John's University, Queens, New York, 11439, USA.

ABSTRACT

Background: BRF2 is a transcription factor required for synthesis of a small group of non-coding RNAs by RNA polymerase III. Overexpression of BRF2 can transform human mammary epithelial cells. In both breast and lung cancers, the BRF2 gene is amplified and overexpressed and may serve as an oncogenic driver. Furthermore, elevated BRF2 can be independently prognostic of unfavorable survival. Dietary soy isoflavones increase metastasis to lungs in a model of breast cancer and a recent study reported significantly increased cell proliferation in breast cancer patients who used soy supplementation. The soy isoflavone daidzein is a major food-derived phytoestrogen that is structurally similar to estrogen. The putative estrogenic effect of soy raises concern that high consumption of soy foods by breast cancer patients may increase tumor growth.

Methods: Expression of BRF2 RNA and protein was assayed in ER-positive or -negative human breast cancer cells after exposure to daidzein. We also measured mRNA stability, promoter methylation and response to the demethylating agent 5-azacytidine. In addition, expression was compared between mice fed diets enriched or deprived of isoflavones.

Results: We demonstrate that the soy isoflavone daidzein specifically stimulates expression of BRF2 in ER-positive breast cancer cells, as well as the related factor BRF1. Induction is accompanied by increased levels of non-coding RNAs that are regulated by BRF2 and BRF1. Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and selectively decreases methylation of the BRF2 promoter. Functional significance of demethylation is supported by induction of BRF2 by the methyltransferase inhibitor 5-azacytidine. None of these effects are observed in an ER-negative breast cancer line, when tested in parallel with ER-positive breast cancer cells. In vivo relevance is suggested by the significantly elevated levels of BRF2 mRNA detected in female mice fed a high-isoflavone commercial diet. In striking contrast, BRF2 and BRF1 mRNA levels are suppressed in matched male mice fed the same isoflavone-enriched diet.

Conclusions: The BRF2 gene that is implicated in cancer can be induced in human breast cancer cells by the isoflavone daidzein, through promoter demethylation and/or mRNA stabilization. Dietary isoflavones may also induce BRF2 in female mice, whereas the converse occurs in males.

No MeSH data available.


Related in: MedlinePlus

Daidzein induces U6 snRNA and tRNAiMet specifically in MCF-7 cells without inducing proliferation. MCF-7 (a) and MDA-MB-231 (b) cells were treated with 0, 3, 10 μM daidzein for 48 h. U6 snRNA and tRNAiMet expression was then analysed by qRT-PCRusing the ΔΔCt method with RPS13 expression levels as a reference for normalization. Meta-analysis of three independent experiments performed in triplicate was completed using one-way ANOVA with a Tukey’s post-test with a 95 % confidence interval (Graphpad Prism 3.03); * = p <0.05; ** = p < 0.01; *** = p < 0.001. CellTiter-Glo® (Promega) was used to count MCF- 7 (c) and MDA-MB-231 cells (d) after 24, 48 and 72 h treatment with 0, 3 or 10 μM daidzein, as indicated. Each dose and time point was performed in triplicate. MDA-MD-231 cell proliferation significantly decreased with 10 μM daidzein after 48 h (p < 0.05) and 72 h (p < 0.01) treatment. MCF-7 cell proliferation significantly decreased at 48 h (p < 0.01) with 3 μM daidzein treatment. At 10 μM daidzein treatment cell proliferation was significantly inhibited in MCF-7 cells at 24 h (p < 0.05), 48 h (p < 0.01) and 72 h (p < 0.01)
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Fig3: Daidzein induces U6 snRNA and tRNAiMet specifically in MCF-7 cells without inducing proliferation. MCF-7 (a) and MDA-MB-231 (b) cells were treated with 0, 3, 10 μM daidzein for 48 h. U6 snRNA and tRNAiMet expression was then analysed by qRT-PCRusing the ΔΔCt method with RPS13 expression levels as a reference for normalization. Meta-analysis of three independent experiments performed in triplicate was completed using one-way ANOVA with a Tukey’s post-test with a 95 % confidence interval (Graphpad Prism 3.03); * = p <0.05; ** = p < 0.01; *** = p < 0.001. CellTiter-Glo® (Promega) was used to count MCF- 7 (c) and MDA-MB-231 cells (d) after 24, 48 and 72 h treatment with 0, 3 or 10 μM daidzein, as indicated. Each dose and time point was performed in triplicate. MDA-MD-231 cell proliferation significantly decreased with 10 μM daidzein after 48 h (p < 0.05) and 72 h (p < 0.01) treatment. MCF-7 cell proliferation significantly decreased at 48 h (p < 0.01) with 3 μM daidzein treatment. At 10 μM daidzein treatment cell proliferation was significantly inhibited in MCF-7 cells at 24 h (p < 0.05), 48 h (p < 0.01) and 72 h (p < 0.01)

Mentions: If the observed induction of BRF1 and BRF2 is functionally significant, we would expect to see changes in expression of pol III products that depend upon these subunits for their transcription. Indeed, both U6 snRNA, dependent on BRF2, and tRNAiMet, requiring BRF1, show significant induction in MCF-7 cells by 10 μM daidzein (Fig. 3a). As with BRF1 and BRF2, neither of these pol III transcripts is induced when MDA-MB-231 cells are treated in the same way (Fig. 3b). As pol III activity is generally coupled to cell proliferation, we tested if daidzein has a mitogenic effect under our assay conditions. However, cell viability assays provided no evidence of enhanced proliferation when either cell line was exposed to 10 μM daidzein (Figs. 3c and d); indeed, significant suppression was observed after 72 h, in agreement with previous studies noting daidzein treatments greater than 1 μM inhibit breast cancer cell proliferation [43]. The selective induction of U6 snRNA and tRNAiMet therefore appears not to reflect a mitogenic response, but correlates with increases in BRF1 and BRF2.Fig. 3


Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein.

Koo J, Cabarcas-Petroski S, Petrie JL, Diette N, White RJ, Schramm L - BMC Cancer (2015)

Daidzein induces U6 snRNA and tRNAiMet specifically in MCF-7 cells without inducing proliferation. MCF-7 (a) and MDA-MB-231 (b) cells were treated with 0, 3, 10 μM daidzein for 48 h. U6 snRNA and tRNAiMet expression was then analysed by qRT-PCRusing the ΔΔCt method with RPS13 expression levels as a reference for normalization. Meta-analysis of three independent experiments performed in triplicate was completed using one-way ANOVA with a Tukey’s post-test with a 95 % confidence interval (Graphpad Prism 3.03); * = p <0.05; ** = p < 0.01; *** = p < 0.001. CellTiter-Glo® (Promega) was used to count MCF- 7 (c) and MDA-MB-231 cells (d) after 24, 48 and 72 h treatment with 0, 3 or 10 μM daidzein, as indicated. Each dose and time point was performed in triplicate. MDA-MD-231 cell proliferation significantly decreased with 10 μM daidzein after 48 h (p < 0.05) and 72 h (p < 0.01) treatment. MCF-7 cell proliferation significantly decreased at 48 h (p < 0.01) with 3 μM daidzein treatment. At 10 μM daidzein treatment cell proliferation was significantly inhibited in MCF-7 cells at 24 h (p < 0.05), 48 h (p < 0.01) and 72 h (p < 0.01)
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Related In: Results  -  Collection

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Fig3: Daidzein induces U6 snRNA and tRNAiMet specifically in MCF-7 cells without inducing proliferation. MCF-7 (a) and MDA-MB-231 (b) cells were treated with 0, 3, 10 μM daidzein for 48 h. U6 snRNA and tRNAiMet expression was then analysed by qRT-PCRusing the ΔΔCt method with RPS13 expression levels as a reference for normalization. Meta-analysis of three independent experiments performed in triplicate was completed using one-way ANOVA with a Tukey’s post-test with a 95 % confidence interval (Graphpad Prism 3.03); * = p <0.05; ** = p < 0.01; *** = p < 0.001. CellTiter-Glo® (Promega) was used to count MCF- 7 (c) and MDA-MB-231 cells (d) after 24, 48 and 72 h treatment with 0, 3 or 10 μM daidzein, as indicated. Each dose and time point was performed in triplicate. MDA-MD-231 cell proliferation significantly decreased with 10 μM daidzein after 48 h (p < 0.05) and 72 h (p < 0.01) treatment. MCF-7 cell proliferation significantly decreased at 48 h (p < 0.01) with 3 μM daidzein treatment. At 10 μM daidzein treatment cell proliferation was significantly inhibited in MCF-7 cells at 24 h (p < 0.05), 48 h (p < 0.01) and 72 h (p < 0.01)
Mentions: If the observed induction of BRF1 and BRF2 is functionally significant, we would expect to see changes in expression of pol III products that depend upon these subunits for their transcription. Indeed, both U6 snRNA, dependent on BRF2, and tRNAiMet, requiring BRF1, show significant induction in MCF-7 cells by 10 μM daidzein (Fig. 3a). As with BRF1 and BRF2, neither of these pol III transcripts is induced when MDA-MB-231 cells are treated in the same way (Fig. 3b). As pol III activity is generally coupled to cell proliferation, we tested if daidzein has a mitogenic effect under our assay conditions. However, cell viability assays provided no evidence of enhanced proliferation when either cell line was exposed to 10 μM daidzein (Figs. 3c and d); indeed, significant suppression was observed after 72 h, in agreement with previous studies noting daidzein treatments greater than 1 μM inhibit breast cancer cell proliferation [43]. The selective induction of U6 snRNA and tRNAiMet therefore appears not to reflect a mitogenic response, but correlates with increases in BRF1 and BRF2.Fig. 3

Bottom Line: In addition, expression was compared between mice fed diets enriched or deprived of isoflavones.Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and selectively decreases methylation of the BRF2 promoter.In vivo relevance is suggested by the significantly elevated levels of BRF2 mRNA detected in female mice fed a high-isoflavone commercial diet.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, St. John's University, Queens, New York, 11439, USA.

ABSTRACT

Background: BRF2 is a transcription factor required for synthesis of a small group of non-coding RNAs by RNA polymerase III. Overexpression of BRF2 can transform human mammary epithelial cells. In both breast and lung cancers, the BRF2 gene is amplified and overexpressed and may serve as an oncogenic driver. Furthermore, elevated BRF2 can be independently prognostic of unfavorable survival. Dietary soy isoflavones increase metastasis to lungs in a model of breast cancer and a recent study reported significantly increased cell proliferation in breast cancer patients who used soy supplementation. The soy isoflavone daidzein is a major food-derived phytoestrogen that is structurally similar to estrogen. The putative estrogenic effect of soy raises concern that high consumption of soy foods by breast cancer patients may increase tumor growth.

Methods: Expression of BRF2 RNA and protein was assayed in ER-positive or -negative human breast cancer cells after exposure to daidzein. We also measured mRNA stability, promoter methylation and response to the demethylating agent 5-azacytidine. In addition, expression was compared between mice fed diets enriched or deprived of isoflavones.

Results: We demonstrate that the soy isoflavone daidzein specifically stimulates expression of BRF2 in ER-positive breast cancer cells, as well as the related factor BRF1. Induction is accompanied by increased levels of non-coding RNAs that are regulated by BRF2 and BRF1. Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and selectively decreases methylation of the BRF2 promoter. Functional significance of demethylation is supported by induction of BRF2 by the methyltransferase inhibitor 5-azacytidine. None of these effects are observed in an ER-negative breast cancer line, when tested in parallel with ER-positive breast cancer cells. In vivo relevance is suggested by the significantly elevated levels of BRF2 mRNA detected in female mice fed a high-isoflavone commercial diet. In striking contrast, BRF2 and BRF1 mRNA levels are suppressed in matched male mice fed the same isoflavone-enriched diet.

Conclusions: The BRF2 gene that is implicated in cancer can be induced in human breast cancer cells by the isoflavone daidzein, through promoter demethylation and/or mRNA stabilization. Dietary isoflavones may also induce BRF2 in female mice, whereas the converse occurs in males.

No MeSH data available.


Related in: MedlinePlus