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Genistein cooperates with the histone deacetylase inhibitor vorinostat to induce cell death in prostate cancer cells.

Phillip CJ, Giardina CK, Bilir B, Cutler DJ, Lai YH, Kucuk O, Moreno CS - BMC Cancer (2012)

Bottom Line: Contrary to earlier reports, genistein did not have an effect on CpG methylation at 20 μM, but it did affect histone H3K9 acetylation and induced increased expression of histone acetyltransferase 1 (HAT1).In addition, genistein also had differential effects on survival and cooperated with the histone deacteylase inhibitor vorinostat to induce cell death and inhibit proliferation.Our results suggest that there are a number of pathways that are affected with genistein and vorinostat treatment such as Wnt, TNF, G2/M DNA damage checkpoint, and androgen signaling pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA.

ABSTRACT

Background: Among American men, prostate cancer is the most common, non-cutaneous malignancy that accounted for an estimated 241,000 new cases and 34,000 deaths in 2011. Previous studies have suggested that Wnt pathway inhibitory genes are silenced by CpG hypermethylation, and other studies have suggested that genistein can demethylate hypermethylated DNA. Genistein is a soy isoflavone with diverse effects on cellular proliferation, survival, and gene expression that suggest it could be a potential therapeutic agent for prostate cancer. We undertook the present study to investigate the effects of genistein on the epigenome of prostate cancer cells and to discover novel combination approaches of other compounds with genistein that might be of translational utility. Here, we have investigated the effects of genistein on several prostate cancer cell lines, including the ARCaP-E/ARCaP-M model of the epithelial to mesenchymal transition (EMT), to analyze effects on their epigenetic state. In addition, we investigated the effects of combined treatment of genistein with the histone deacetylase inhibitor vorinostat on survival in prostate cancer cells.

Methods: Using whole genome expression profiling and whole genome methylation profiling, we have determined the genome-wide differences in genetic and epigenetic responses to genistein in prostate cancer cells before and after undergoing the EMT. Also, cells were treated with genistein, vorinostat, and combination treatment, where cell death and cell proliferation was determined.

Results: Contrary to earlier reports, genistein did not have an effect on CpG methylation at 20 μM, but it did affect histone H3K9 acetylation and induced increased expression of histone acetyltransferase 1 (HAT1). In addition, genistein also had differential effects on survival and cooperated with the histone deacteylase inhibitor vorinostat to induce cell death and inhibit proliferation.

Conclusion: Our results suggest that there are a number of pathways that are affected with genistein and vorinostat treatment such as Wnt, TNF, G2/M DNA damage checkpoint, and androgen signaling pathways. In addition, genistein cooperates with vorinostat to induce cell death in prostate cancer cell lines with a greater effect on early stage prostate cancer.

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Whole genome expression profiling of prostate cancer cells treated with genistein, vorinostat, or the combination. (A) Hierarchical clustering of 1077 probes strongly affected by treatment with genistein and vorinostat. (B) Ingenuity Pathway Analysis (IPA) Network of genes annotated for function in Apoptosis. Red indicates increased expression after treatment, and green indicates reduced expression. Solid lines represent direct interactions and dashed lines represent indirect interactions. (C) IPA Network of genes annotated for function in DNA Repair and Cell Cycle. (D) QPCR data from a subset of genes with functions in apoptosis pathways normalized to ARCAP-E control cells. (E) QPCR confirmation of down regulation of TGFB1I1/ARA55, SLUG, BIRC7/Livin, and HES1 following genistein treatment. Data are presented as mean ± SE of triplicate experiments.
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Figure 3: Whole genome expression profiling of prostate cancer cells treated with genistein, vorinostat, or the combination. (A) Hierarchical clustering of 1077 probes strongly affected by treatment with genistein and vorinostat. (B) Ingenuity Pathway Analysis (IPA) Network of genes annotated for function in Apoptosis. Red indicates increased expression after treatment, and green indicates reduced expression. Solid lines represent direct interactions and dashed lines represent indirect interactions. (C) IPA Network of genes annotated for function in DNA Repair and Cell Cycle. (D) QPCR data from a subset of genes with functions in apoptosis pathways normalized to ARCAP-E control cells. (E) QPCR confirmation of down regulation of TGFB1I1/ARA55, SLUG, BIRC7/Livin, and HES1 following genistein treatment. Data are presented as mean ± SE of triplicate experiments.

Mentions: To determine the genome-wide effects of genistein, vorinostat, and combined treatment on gene expression, we conducted whole genome expression profiling of ARCaP-E and ARCaP-M cells using Illumina HT-12 v3 Expression BeadChips. Genistein treatment had a larger effect on ARCaP-E cells (291 genes induced and 144 genes repressed) than on ARCaP-M cells (31 genes induced and 33 repressed). Vorinostat impacted more genes than genistein in both ARCaP-E cells (820 genes induced and 1046 genes repressed) and ARCaP-M cells (1296 genes induced and 883 genes repressed). As expected, the largest changes in gene expression were observed by combined treatment with genistein and vorinostat for ARCaP-E cells (1978 genes induced and 1758 genes repressed) and ARCaP-M cells (1503 genes induced and 1161 genes repressed) (Figure3A). Gene ontology enrichment analysis using the DAVID knowledgebase[39] and Ingenuity Pathway Analysis (IPA)[40], demonstrated that the affected genes were highly enriched in genes involved in DNA damage, cell cycle arrest, and apoptosis (Tables1 and2). Interestingly, IPA analysis of genes affected by combined genistein and vorinostat treatment identified a gene network with the pro-apoptotic Tumor Necrosis Factor alpha (TNFα) as a major hub (Figure3B), including the pro-survival gene BIRC7 (or Livin) which was downregulated 4.8-fold. In addition, genes involved in the G2/M cell cycle and response to DNA damage was also identified (Figure3C-D). For example, four members of the minichromosome maintenance complex (MCM) essential for DNA replication are strongly upregulated, as are BRCA1, BARD1, RAD23B, and XRCC2. QPCR analysis following genistein treatment confirmed reduced levels of BIRC7, as well as SLUG, HES1, and TGFB1I1 (or ARA55), and several genes associated with apoptosis (Figure3E). These data indicate that genistein affects cell survival and proliferation via multiple mechanisms including the TNFα-NFκB and ATM-CHEK2-BRCA1 pathways. Additionally, genes involved in chromatin modifications and histone acetylations such as HAT1 were also impacted by genistein treatment (Table1 and Additional File3: Figure S2).


Genistein cooperates with the histone deacetylase inhibitor vorinostat to induce cell death in prostate cancer cells.

Phillip CJ, Giardina CK, Bilir B, Cutler DJ, Lai YH, Kucuk O, Moreno CS - BMC Cancer (2012)

Whole genome expression profiling of prostate cancer cells treated with genistein, vorinostat, or the combination. (A) Hierarchical clustering of 1077 probes strongly affected by treatment with genistein and vorinostat. (B) Ingenuity Pathway Analysis (IPA) Network of genes annotated for function in Apoptosis. Red indicates increased expression after treatment, and green indicates reduced expression. Solid lines represent direct interactions and dashed lines represent indirect interactions. (C) IPA Network of genes annotated for function in DNA Repair and Cell Cycle. (D) QPCR data from a subset of genes with functions in apoptosis pathways normalized to ARCAP-E control cells. (E) QPCR confirmation of down regulation of TGFB1I1/ARA55, SLUG, BIRC7/Livin, and HES1 following genistein treatment. Data are presented as mean ± SE of triplicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Whole genome expression profiling of prostate cancer cells treated with genistein, vorinostat, or the combination. (A) Hierarchical clustering of 1077 probes strongly affected by treatment with genistein and vorinostat. (B) Ingenuity Pathway Analysis (IPA) Network of genes annotated for function in Apoptosis. Red indicates increased expression after treatment, and green indicates reduced expression. Solid lines represent direct interactions and dashed lines represent indirect interactions. (C) IPA Network of genes annotated for function in DNA Repair and Cell Cycle. (D) QPCR data from a subset of genes with functions in apoptosis pathways normalized to ARCAP-E control cells. (E) QPCR confirmation of down regulation of TGFB1I1/ARA55, SLUG, BIRC7/Livin, and HES1 following genistein treatment. Data are presented as mean ± SE of triplicate experiments.
Mentions: To determine the genome-wide effects of genistein, vorinostat, and combined treatment on gene expression, we conducted whole genome expression profiling of ARCaP-E and ARCaP-M cells using Illumina HT-12 v3 Expression BeadChips. Genistein treatment had a larger effect on ARCaP-E cells (291 genes induced and 144 genes repressed) than on ARCaP-M cells (31 genes induced and 33 repressed). Vorinostat impacted more genes than genistein in both ARCaP-E cells (820 genes induced and 1046 genes repressed) and ARCaP-M cells (1296 genes induced and 883 genes repressed). As expected, the largest changes in gene expression were observed by combined treatment with genistein and vorinostat for ARCaP-E cells (1978 genes induced and 1758 genes repressed) and ARCaP-M cells (1503 genes induced and 1161 genes repressed) (Figure3A). Gene ontology enrichment analysis using the DAVID knowledgebase[39] and Ingenuity Pathway Analysis (IPA)[40], demonstrated that the affected genes were highly enriched in genes involved in DNA damage, cell cycle arrest, and apoptosis (Tables1 and2). Interestingly, IPA analysis of genes affected by combined genistein and vorinostat treatment identified a gene network with the pro-apoptotic Tumor Necrosis Factor alpha (TNFα) as a major hub (Figure3B), including the pro-survival gene BIRC7 (or Livin) which was downregulated 4.8-fold. In addition, genes involved in the G2/M cell cycle and response to DNA damage was also identified (Figure3C-D). For example, four members of the minichromosome maintenance complex (MCM) essential for DNA replication are strongly upregulated, as are BRCA1, BARD1, RAD23B, and XRCC2. QPCR analysis following genistein treatment confirmed reduced levels of BIRC7, as well as SLUG, HES1, and TGFB1I1 (or ARA55), and several genes associated with apoptosis (Figure3E). These data indicate that genistein affects cell survival and proliferation via multiple mechanisms including the TNFα-NFκB and ATM-CHEK2-BRCA1 pathways. Additionally, genes involved in chromatin modifications and histone acetylations such as HAT1 were also impacted by genistein treatment (Table1 and Additional File3: Figure S2).

Bottom Line: Contrary to earlier reports, genistein did not have an effect on CpG methylation at 20 μM, but it did affect histone H3K9 acetylation and induced increased expression of histone acetyltransferase 1 (HAT1).In addition, genistein also had differential effects on survival and cooperated with the histone deacteylase inhibitor vorinostat to induce cell death and inhibit proliferation.Our results suggest that there are a number of pathways that are affected with genistein and vorinostat treatment such as Wnt, TNF, G2/M DNA damage checkpoint, and androgen signaling pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA.

ABSTRACT

Background: Among American men, prostate cancer is the most common, non-cutaneous malignancy that accounted for an estimated 241,000 new cases and 34,000 deaths in 2011. Previous studies have suggested that Wnt pathway inhibitory genes are silenced by CpG hypermethylation, and other studies have suggested that genistein can demethylate hypermethylated DNA. Genistein is a soy isoflavone with diverse effects on cellular proliferation, survival, and gene expression that suggest it could be a potential therapeutic agent for prostate cancer. We undertook the present study to investigate the effects of genistein on the epigenome of prostate cancer cells and to discover novel combination approaches of other compounds with genistein that might be of translational utility. Here, we have investigated the effects of genistein on several prostate cancer cell lines, including the ARCaP-E/ARCaP-M model of the epithelial to mesenchymal transition (EMT), to analyze effects on their epigenetic state. In addition, we investigated the effects of combined treatment of genistein with the histone deacetylase inhibitor vorinostat on survival in prostate cancer cells.

Methods: Using whole genome expression profiling and whole genome methylation profiling, we have determined the genome-wide differences in genetic and epigenetic responses to genistein in prostate cancer cells before and after undergoing the EMT. Also, cells were treated with genistein, vorinostat, and combination treatment, where cell death and cell proliferation was determined.

Results: Contrary to earlier reports, genistein did not have an effect on CpG methylation at 20 μM, but it did affect histone H3K9 acetylation and induced increased expression of histone acetyltransferase 1 (HAT1). In addition, genistein also had differential effects on survival and cooperated with the histone deacteylase inhibitor vorinostat to induce cell death and inhibit proliferation.

Conclusion: Our results suggest that there are a number of pathways that are affected with genistein and vorinostat treatment such as Wnt, TNF, G2/M DNA damage checkpoint, and androgen signaling pathways. In addition, genistein cooperates with vorinostat to induce cell death in prostate cancer cell lines with a greater effect on early stage prostate cancer.

Show MeSH
Related in: MedlinePlus