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 (*).