Abstract A47: Establishing candidate biomarkers for the pharmacodynamic action of sulforaphane in the breast

Abstract

Abstract The goal of this study is the discovery of biomarkers reflecting the pharmacodynamic action of sulforaphane (SFN), initially using normal human mammary epithelial MCF10A cells and then in tissue obtained from healthy patients undergoing reduction mammoplasty. The isothiocyanate SFN is formed by the hydrolysis of glucoraphanin, a water soluble glucosinolate found in cruciferous vegetables with especially high levels measured in 3 day old broccoli sprouts. Chemoprevention by SFN is achieved in part through the upregulation of cytoprotective enzymes via the Keap1/Nrf2 pathway. Preliminary dose response and time course studies in MCF10A cells established that SFN upregulated cytoprotective enzymes as indicated by increased levels of NQO1 transcripts, protein and activity. NQO1 induction was dependent on the Keap1/Nrf2 pathway since NQO1 transcripts, protein and activity were enhanced by KEAP1 siRNA knockdown in MCF10As. For the biomarker discovery phase SFN treated and KEAP1 knockdown MCF10A cells were analyzed by microarray to determine transcriptomic changes in response to pharmacological and genetic stimulation of the Keap1/Nrf2 pathway. The two experimental approaches were analyzed independently and then compared. The predominant class of upregulated genes common to both SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidant genes including NQO1, heme oxygenase (decycling) 1, sequestosome 1, thioredoxin reductase 1 and peroxiredoxin 1. The most highly upregulated genes in this class were the aldo-keto reductase family members including AKR1B1, AKR1B10, AKR1C1 and AKR1C3. Other classes of genes with overlapping regulation were glutathione metabolism genes such as GCLC and GCLM, molecular transport, cell growth and cell death regulation genes. Some members of the xenobiotic metabolism, molecular transport, cell growth and cell death regulation classes of genes were also regulated exclusively by SFN. Additionally histone cluster and chromatin remodeling genes were downregulated by SFN. Potential biomarker genes from the microarray study include NQO1, GCLC, SQSTM1 and AKR1C1. An accompanying proteomic study for biomarker discovery using Stable Isotopic Labeling with Amino Acids in Cell Culture (SILAC) is underway. The transcriptomic and proteomic changes in response to SFN treatment and KEAP1 knockdown will be compared to determine if the changes seen on the transcriptomic level are translated to the proteomic level. Candidate biomarkers identified from microarray and SILAC studies reflecting the pharmacodynamic action of SFN in human cells will be analyzed by Western blot, qRT-PCR and enzyme assays in tissue from reduction mammoplasty patients. These women have been randomized in a clinical trial to receive either a broccoli sprout preparation containing 100 micromole SFN or a placebo beverage daily for 10 days prior to surgery. Supported by DOD grant BC073262 and NIH grants P50 CA088843 and U54 RR020839. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A47.