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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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Related in: MedlinePlus

Genistein treatment induces hypermethylated Wnt-inhibitory genes via H3K9 acetylation, not CpG demethylation. (A) Wnt Inhibitory genes are methylated in prostate cancer patient samples. MSP of Wnt inhibitory genes was performed on genomic DNA derived from paraffin embedded prostate tissues in 8 prostate cancer samples. SOX7 was strongly methylated while WIF1, SFRP1, DKK3, and APC were partially methylated in multiple patient samples. U = unmethylated, M = methylated. (B) MSP analysis indicates no change in methylation status of APC, WIF1, SFRP1, SOX7, and DKK3 in DU145, ARCAPE, and PC3 cell lines after treatment with 20 μM genistein for 6 days. (C) Whole genome methylation profiling using the 27 K CpG Methylation Arrays was performed on ARCAPE and ARCAPM cells treated with DMSO and 20 μM genistein for 6 days. In addition, 5-aza-deoxy-cytidine was used as a positive control and PREC cells were used as a negative control. No significant changes in methylation were detected with genistein treatment. Unsupervised hierarchical clustering of 4,190 CpG loci with β-value > 0.5 is shown. (D) Bisulfite sequencing of 175 bp of the WIF1 CpG island across 13 CpG sites indicates high methylation in ARCAP-E and ARCAP-M cells with or without genistein treatment, and low methylation in PrEC cells. (E) Anti-acetyl histone H3-Lysine9 chromatin immunoprecipitation (acetyl-H3K9 ChIP) of ARCAP-E cells treated with DMSO control or genistein shows marked increases in acetyl-H3K9 following genistein treatment. (F) Immunoblot of HAT1 protein shows increased HAT1 protein in ARCaP-E and ARCaP-M after genistein treatment. (G) Gene expression with genistein treatment in ARCAPE, and ARCAPM cells. Gene expression of indicated Wnt inhibitory genes after treatment with genistein was determined using QPCR. The data are presented as fold change relative to DMSO control (mean ± SD, triplicate samples from five independent experiments). Significant p-values (p < 0.05) were computed using the student’s t-test with a two-tailed distribution and are indicated with an asterisk (*).
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Figure 1: Genistein treatment induces hypermethylated Wnt-inhibitory genes via H3K9 acetylation, not CpG demethylation. (A) Wnt Inhibitory genes are methylated in prostate cancer patient samples. MSP of Wnt inhibitory genes was performed on genomic DNA derived from paraffin embedded prostate tissues in 8 prostate cancer samples. SOX7 was strongly methylated while WIF1, SFRP1, DKK3, and APC were partially methylated in multiple patient samples. U = unmethylated, M = methylated. (B) MSP analysis indicates no change in methylation status of APC, WIF1, SFRP1, SOX7, and DKK3 in DU145, ARCAPE, and PC3 cell lines after treatment with 20 μM genistein for 6 days. (C) Whole genome methylation profiling using the 27 K CpG Methylation Arrays was performed on ARCAPE and ARCAPM cells treated with DMSO and 20 μM genistein for 6 days. In addition, 5-aza-deoxy-cytidine was used as a positive control and PREC cells were used as a negative control. No significant changes in methylation were detected with genistein treatment. Unsupervised hierarchical clustering of 4,190 CpG loci with β-value > 0.5 is shown. (D) Bisulfite sequencing of 175 bp of the WIF1 CpG island across 13 CpG sites indicates high methylation in ARCAP-E and ARCAP-M cells with or without genistein treatment, and low methylation in PrEC cells. (E) Anti-acetyl histone H3-Lysine9 chromatin immunoprecipitation (acetyl-H3K9 ChIP) of ARCAP-E cells treated with DMSO control or genistein shows marked increases in acetyl-H3K9 following genistein treatment. (F) Immunoblot of HAT1 protein shows increased HAT1 protein in ARCaP-E and ARCaP-M after genistein treatment. (G) Gene expression with genistein treatment in ARCAPE, and ARCAPM cells. Gene expression of indicated Wnt inhibitory genes after treatment with genistein was determined using QPCR. The data are presented as fold change relative to DMSO control (mean ± SD, triplicate samples from five independent experiments). Significant p-values (p < 0.05) were computed using the student’s t-test with a two-tailed distribution and are indicated with an asterisk (*).

Mentions: Tumor suppressor genes are often hypermethylated in prostate cancer patient tissue samples compared to normal tissues, and this methylation can correlate with prognosis[34,35]. To determine if Wnt Inhibitory genes are methylated in prostate cancer patient samples, we performed methylation specific PCR (MSP) on eight prostate cancer patient samples. We observed that SOX7 was highly methylated, whereas WIF1, SFRP1, DKK3, and APC were partially methylated in each of these samples (Figure1A).


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)

Genistein treatment induces hypermethylated Wnt-inhibitory genes via H3K9 acetylation, not CpG demethylation. (A) Wnt Inhibitory genes are methylated in prostate cancer patient samples. MSP of Wnt inhibitory genes was performed on genomic DNA derived from paraffin embedded prostate tissues in 8 prostate cancer samples. SOX7 was strongly methylated while WIF1, SFRP1, DKK3, and APC were partially methylated in multiple patient samples. U = unmethylated, M = methylated. (B) MSP analysis indicates no change in methylation status of APC, WIF1, SFRP1, SOX7, and DKK3 in DU145, ARCAPE, and PC3 cell lines after treatment with 20 μM genistein for 6 days. (C) Whole genome methylation profiling using the 27 K CpG Methylation Arrays was performed on ARCAPE and ARCAPM cells treated with DMSO and 20 μM genistein for 6 days. In addition, 5-aza-deoxy-cytidine was used as a positive control and PREC cells were used as a negative control. No significant changes in methylation were detected with genistein treatment. Unsupervised hierarchical clustering of 4,190 CpG loci with β-value > 0.5 is shown. (D) Bisulfite sequencing of 175 bp of the WIF1 CpG island across 13 CpG sites indicates high methylation in ARCAP-E and ARCAP-M cells with or without genistein treatment, and low methylation in PrEC cells. (E) Anti-acetyl histone H3-Lysine9 chromatin immunoprecipitation (acetyl-H3K9 ChIP) of ARCAP-E cells treated with DMSO control or genistein shows marked increases in acetyl-H3K9 following genistein treatment. (F) Immunoblot of HAT1 protein shows increased HAT1 protein in ARCaP-E and ARCaP-M after genistein treatment. (G) Gene expression with genistein treatment in ARCAPE, and ARCAPM cells. Gene expression of indicated Wnt inhibitory genes after treatment with genistein was determined using QPCR. The data are presented as fold change relative to DMSO control (mean ± SD, triplicate samples from five independent experiments). Significant p-values (p < 0.05) were computed using the student’s t-test with a two-tailed distribution and are indicated with an asterisk (*).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Genistein treatment induces hypermethylated Wnt-inhibitory genes via H3K9 acetylation, not CpG demethylation. (A) Wnt Inhibitory genes are methylated in prostate cancer patient samples. MSP of Wnt inhibitory genes was performed on genomic DNA derived from paraffin embedded prostate tissues in 8 prostate cancer samples. SOX7 was strongly methylated while WIF1, SFRP1, DKK3, and APC were partially methylated in multiple patient samples. U = unmethylated, M = methylated. (B) MSP analysis indicates no change in methylation status of APC, WIF1, SFRP1, SOX7, and DKK3 in DU145, ARCAPE, and PC3 cell lines after treatment with 20 μM genistein for 6 days. (C) Whole genome methylation profiling using the 27 K CpG Methylation Arrays was performed on ARCAPE and ARCAPM cells treated with DMSO and 20 μM genistein for 6 days. In addition, 5-aza-deoxy-cytidine was used as a positive control and PREC cells were used as a negative control. No significant changes in methylation were detected with genistein treatment. Unsupervised hierarchical clustering of 4,190 CpG loci with β-value > 0.5 is shown. (D) Bisulfite sequencing of 175 bp of the WIF1 CpG island across 13 CpG sites indicates high methylation in ARCAP-E and ARCAP-M cells with or without genistein treatment, and low methylation in PrEC cells. (E) Anti-acetyl histone H3-Lysine9 chromatin immunoprecipitation (acetyl-H3K9 ChIP) of ARCAP-E cells treated with DMSO control or genistein shows marked increases in acetyl-H3K9 following genistein treatment. (F) Immunoblot of HAT1 protein shows increased HAT1 protein in ARCaP-E and ARCaP-M after genistein treatment. (G) Gene expression with genistein treatment in ARCAPE, and ARCAPM cells. Gene expression of indicated Wnt inhibitory genes after treatment with genistein was determined using QPCR. The data are presented as fold change relative to DMSO control (mean ± SD, triplicate samples from five independent experiments). Significant p-values (p < 0.05) were computed using the student’s t-test with a two-tailed distribution and are indicated with an asterisk (*).
Mentions: Tumor suppressor genes are often hypermethylated in prostate cancer patient tissue samples compared to normal tissues, and this methylation can correlate with prognosis[34,35]. To determine if Wnt Inhibitory genes are methylated in prostate cancer patient samples, we performed methylation specific PCR (MSP) on eight prostate cancer patient samples. We observed that SOX7 was highly methylated, whereas WIF1, SFRP1, DKK3, and APC were partially methylated in each of these samples (Figure1A).

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