Sulforaphane (SFN), an aliphatic isothiocyanate, is a known cancer chemopreventive agent. Aiming to investigate anti-inflammatory mechanisms of SFN, we here report a potent decrease in lipopolysaccharide (LPS)-induced secretion of pro-inflammatory and pro-carcinogenic signaling factors in cultured Raw 264.7 macrophages after SFN treatment, i.e. NO, prostaglandin E₂, and tumor necrosis factor α. SFN did not directly interact with NO, nor did it inhibit inducible nitric-oxide synthase enzymatic activity. Western blot analyses revealed time- and dose-dependent reduction of LPS-induced inducible nitric-oxide synthase as well as Cox-2 protein expression, which was suppressed at the transcriptional level. To reveal the target of SFN beyond its anti-inflammatory action, we performed electrophoretic mobility shift assay analyses of transcription factor-DNA binding. Consequently, nuclear factor κB (NF-κB), a pivotal transcription factor in LPS-stimulated pro-inflammatory response, was identified as the key mediator. SFN selectively reduced DNA binding of NF-κB without interfering with LPS-induced degradation of the inhibitor of NF-κB nor with nuclear translocation of NF-κB. Because SFN can interact with thiol groups by dithiocarbamate formation, it may impair the redox-sensitive DNA binding and transactivation of NF-κB. Sulforaphane could either directly inactivate NF-κB subunits by binding to essential Cys residues or interact with glutathione or other redox regulators like thioredoxin and Ref-1 relevant for NF-κB function. Our data provide novel evidence that anti-inflammatory mechanisms contribute to sulforaphane-mediated cancer chemoprevention.

DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.