Abstract
Dietary isothiocyanates derived from the consumption of broccoli and other cruciferous vegetables have been shown to prevent cancer in various animal and clinical studies. Sulforaphane has been extensively used in mouse liver and lung studies to assess both anticancer effects and oxidant‐induced gene transcriptional profiles. Sulforaphane imparts its antioxidant effects by activating the nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) pathway. NRF2 is a key transcriptional activator of antioxidant and phase II drug metabolism enzymes that regulate transcription by binding cis‐acting antioxidant response elements (AREs) found in gene promoter regions. Since genetic factors, such as loss of function deletions in drug metabolism genes like glutathione‐S‐transferases, can influence the effectiveness of chemopreventive or chemotherapeutic agents, we hypothesized that single nucleotide polymorphisms (SNPs) within ARE sequences located in NRF2 target genes may alter sulforaphane‐induced transcriptional regulation of these enzymes. To assess this, we exposed 60 unrelated, HapMap CEU cell lines to sulforaphane and measured global gene expression with Illumina gene expression arrays. We observed 2946 expression probes were either significantly induced (1436) or suppressed (1510) in at least 6 of the 60 cell lines by a fold change of 1.3 or greater (t‐test with FDR correction, p<0.09). Pathway analysis of induced genes indicated these genes are involved in oxidative stress response, apoptosis signaling and peroxide metabolism. SNP genotypes (identified by the HapMap project and our custom genotyping arrays) within a gene region were regressed against log2‐transformed intensity values for each gene. Of the 11,566 SNPs that were significantly associated with gene expression after sulforaphane treatment (linear regression, p<0.05), 972 SNPs were located within putative AREs identified by bioinformatics analysis and 55 SNPs were within 10kB of the transcriptional start site of a known ARE gene. To verify binding of NRF2 in these regions, NRF2‐bound DNA isolated from a sulforaphane treated lymphoblastoid cell line was immunoprecipitated and analyzed by Agilent ChIP‐on‐chip. We observed that 23 of the 1027 selected SNPs were located near NRF2 bound regions. Three of these SNPs were located directly within ChIP‐on‐chip probe regions. This study provides promising SNP candidates for follow‐up in molecular validation and clinical disease association studies. Functional SNPs that alter the binding potential and gene transactivation by NRF2 may help explain individual variation in the response to agents that modulate antioxidant and phase II enzyme regulation and subsequent clinical outcome.
Citation Information: Cancer Prev Res 2010;3(1 Suppl):B53.
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