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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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Genistein synergizes with vorinostat to induce apoptosis in prostate cancer cells. (A) Genistein treatment induces apoptosis in prostate cancer cells. Survival of androgen-independent PC-3 and DU145, androgen-dependent LNCaP, and androgen-repressed ARCaP-E and ARCaP-M cells in response to genistein, vorinostat, and combination treatment was measured using Annexin V/PI staining. Cells were treated with 20 μM genistein or DMSO for 6 days, 1 μM vorinostat for 2 days, and combination. Apoptosis was quantitated using Annexin V/PI staining. The data were quantitated showing total cell death. Data are presented as mean ± SE of triplicate experiments. (B) Proliferation of DU145, PC3, LNCAP, ARCaP-E, and ARCaP-M cells treated with genistein, vorinostat, and combination. Cells were treated with single agents vorinostat (1 μM), genistein (20 μM), or combination. Mean % proliferation ± SE of triplicate experiments are shown. Growth inhibition was measured using a hemocytometer following trypan blue staining. (C) Cell death of ARCaP-E and ARCaP-M cell treated with genistein, 5-deoxyazacytidine (5-aza), and vorinostat. Cells were treated with single agents, 1 μM vorinostat for 48 hrs, or in combination with genistein or 5-aza for 6 days. Total mean cell death ± SE of triplicate experiments are shown. (D) Cells were treated as in (C) and cell proliferation was assessed. Mean % proliferation ± SE of triplicate experiments are shown.
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Figure 2: Genistein synergizes with vorinostat to induce apoptosis in prostate cancer cells. (A) Genistein treatment induces apoptosis in prostate cancer cells. Survival of androgen-independent PC-3 and DU145, androgen-dependent LNCaP, and androgen-repressed ARCaP-E and ARCaP-M cells in response to genistein, vorinostat, and combination treatment was measured using Annexin V/PI staining. Cells were treated with 20 μM genistein or DMSO for 6 days, 1 μM vorinostat for 2 days, and combination. Apoptosis was quantitated using Annexin V/PI staining. The data were quantitated showing total cell death. Data are presented as mean ± SE of triplicate experiments. (B) Proliferation of DU145, PC3, LNCAP, ARCaP-E, and ARCaP-M cells treated with genistein, vorinostat, and combination. Cells were treated with single agents vorinostat (1 μM), genistein (20 μM), or combination. Mean % proliferation ± SE of triplicate experiments are shown. Growth inhibition was measured using a hemocytometer following trypan blue staining. (C) Cell death of ARCaP-E and ARCaP-M cell treated with genistein, 5-deoxyazacytidine (5-aza), and vorinostat. Cells were treated with single agents, 1 μM vorinostat for 48 hrs, or in combination with genistein or 5-aza for 6 days. Total mean cell death ± SE of triplicate experiments are shown. (D) Cells were treated as in (C) and cell proliferation was assessed. Mean % proliferation ± SE of triplicate experiments are shown.

Mentions: To determine genistein’s potential as a therapeutic agent in the treatment of prostate cancer, prostate cancer cell lines PC3, DU145, ARCaP-E, ARCaP-M and LNCaP were treated with 20 μM genistein for a total of six days, 1 μM vorinostat for 2 days, and a combination of genistein and vorinostat (Figure2A). Since genistein treatment increased histone acetylation, we hypothesized that it might cooperate with histone deacetylase (HDAC) inhibitors to induce apoptosis. Genistein exhibited only a minor (5–10 %) effect of increased cell death on these cells based on Annexin V/PI staining (Figure2A). There was an approximate increase of 8 % cell death in DU145 cells, 5 % cell death in ARCaP-E cells, 10 % cell death in LNCaP cells, and 8 % cell death in PC3 cells when compared to untreated DMSO cohorts. Nevertheless, we confirmed previous studies[37,38] indicating that genistein was quite effective in inhibiting cell proliferation (Figure2B). In addition, there was an increase in cell death of all prostate models when treated with vorinostat and combination genistein and vorinostat with the largest affect being in the ARCaP-E cell line model.


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 synergizes with vorinostat to induce apoptosis in prostate cancer cells. (A) Genistein treatment induces apoptosis in prostate cancer cells. Survival of androgen-independent PC-3 and DU145, androgen-dependent LNCaP, and androgen-repressed ARCaP-E and ARCaP-M cells in response to genistein, vorinostat, and combination treatment was measured using Annexin V/PI staining. Cells were treated with 20 μM genistein or DMSO for 6 days, 1 μM vorinostat for 2 days, and combination. Apoptosis was quantitated using Annexin V/PI staining. The data were quantitated showing total cell death. Data are presented as mean ± SE of triplicate experiments. (B) Proliferation of DU145, PC3, LNCAP, ARCaP-E, and ARCaP-M cells treated with genistein, vorinostat, and combination. Cells were treated with single agents vorinostat (1 μM), genistein (20 μM), or combination. Mean % proliferation ± SE of triplicate experiments are shown. Growth inhibition was measured using a hemocytometer following trypan blue staining. (C) Cell death of ARCaP-E and ARCaP-M cell treated with genistein, 5-deoxyazacytidine (5-aza), and vorinostat. Cells were treated with single agents, 1 μM vorinostat for 48 hrs, or in combination with genistein or 5-aza for 6 days. Total mean cell death ± SE of triplicate experiments are shown. (D) Cells were treated as in (C) and cell proliferation was assessed. Mean % proliferation ± SE of triplicate experiments are shown.
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Figure 2: Genistein synergizes with vorinostat to induce apoptosis in prostate cancer cells. (A) Genistein treatment induces apoptosis in prostate cancer cells. Survival of androgen-independent PC-3 and DU145, androgen-dependent LNCaP, and androgen-repressed ARCaP-E and ARCaP-M cells in response to genistein, vorinostat, and combination treatment was measured using Annexin V/PI staining. Cells were treated with 20 μM genistein or DMSO for 6 days, 1 μM vorinostat for 2 days, and combination. Apoptosis was quantitated using Annexin V/PI staining. The data were quantitated showing total cell death. Data are presented as mean ± SE of triplicate experiments. (B) Proliferation of DU145, PC3, LNCAP, ARCaP-E, and ARCaP-M cells treated with genistein, vorinostat, and combination. Cells were treated with single agents vorinostat (1 μM), genistein (20 μM), or combination. Mean % proliferation ± SE of triplicate experiments are shown. Growth inhibition was measured using a hemocytometer following trypan blue staining. (C) Cell death of ARCaP-E and ARCaP-M cell treated with genistein, 5-deoxyazacytidine (5-aza), and vorinostat. Cells were treated with single agents, 1 μM vorinostat for 48 hrs, or in combination with genistein or 5-aza for 6 days. Total mean cell death ± SE of triplicate experiments are shown. (D) Cells were treated as in (C) and cell proliferation was assessed. Mean % proliferation ± SE of triplicate experiments are shown.
Mentions: To determine genistein’s potential as a therapeutic agent in the treatment of prostate cancer, prostate cancer cell lines PC3, DU145, ARCaP-E, ARCaP-M and LNCaP were treated with 20 μM genistein for a total of six days, 1 μM vorinostat for 2 days, and a combination of genistein and vorinostat (Figure2A). Since genistein treatment increased histone acetylation, we hypothesized that it might cooperate with histone deacetylase (HDAC) inhibitors to induce apoptosis. Genistein exhibited only a minor (5–10 %) effect of increased cell death on these cells based on Annexin V/PI staining (Figure2A). There was an approximate increase of 8 % cell death in DU145 cells, 5 % cell death in ARCaP-E cells, 10 % cell death in LNCaP cells, and 8 % cell death in PC3 cells when compared to untreated DMSO cohorts. Nevertheless, we confirmed previous studies[37,38] indicating that genistein was quite effective in inhibiting cell proliferation (Figure2B). In addition, there was an increase in cell death of all prostate models when treated with vorinostat and combination genistein and vorinostat with the largest affect being in the ARCaP-E cell line model.

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