Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, induces potent anti-proliferative effects in prostate cancer cells. One mechanism that may contribute to the anti-proliferative effects of SFN is the modulation of epigenetic marks, such as inhibition of histone deacetylase (HDAC) enzymes. However, the effects of SFN on other common epigenetic marks such as DNA methylation are understudied. Promoter hyper-methylation of cyclin D2, a major regulator of cell cycle, is correlated with prostate cancer progression, and restoration of cyclin D2 expression exerts anti-proliferative effects on LnCap prostate cancer cells. Our study aimed to investigate the effects of SFN on DNA methylation status of cyclin D2 promoter, and how alteration in promoter methylation impacts cyclin D2 gene expression in LnCap cells. We found that SFN significantly decreased the expression of DNA methyltransferases (DNMTs), especially DNMT1 and DNMT3b. Furthermore, SFN significantly decreased methylation in cyclin D2 promoter regions containing c-Myc and multiple Sp1 binding sites. Reduced methlyation of cyclin D2 promoter corresponded to an increase in cyclin D2 transcript levels, suggesting that SFN may de-repress methylation-silenced cyclin D2 by impacting epigenetic pathways. Our results demonstrated the ability of SFN to epigenetically modulate cyclin D2 expression, and provide novel insights into the mechanisms by which SFN may regulate gene expression as a prostate cancer chemopreventive agent.

Isothiocyanates, such as sulforaphane and iberin, derived from glucosinolates (GLS) in cruciferous vegetables, are known to prevent and suppress cancer development. GLS can also be converted by bacteria to biologically inert nitriles, such as sulforaphane-nitrile (SFN-NIT) and iberin-nitrile (IBN-NIT), but the role of the gut microbiome in this process is relatively undescribed and SFN-NIT excretion in humans is unknown. An ex vivo fecal incubation model with in vitro digested broccoli sprouts and 16S sequencing was utilized to explore the role of the gut microbiome in SFN- and IBN-NIT production. SFN-NIT excretion was measured among human subjects following broccoli sprout consumption. The fecal culture model showed high inter-individual variability in nitrile production and identified two sub-populations of microbial communities among the fecal cultures, which coincided with a differing abundance of nitriles. The Clostridiaceae family was associated with high levels, while individuals with a low abundance of nitriles were more enriched with taxa from the Enterobacteriaceae family. High levels of inter-individual variation in urine SFN-NIT levels were also observed, with peak excretion of SFN-NIT at 24 h post broccoli sprout consumption. These results suggest that nitrile production from broccoli, as opposed to isothiocyanates, could be influenced by gut microbiome composition, potentially lowering efficacy of cruciferous vegetable interventions.

Xanthohumol (XN), a polyphenol found in the hop plant (Humulus lupulus), has antioxidant, anti-inflammatory, prebiotic, and anti-hyperlipidemic activity. Preclinical evidence suggests the gut microbiome is essential in mediating these bioactivities; however, relatively little is known about XN's impact on human gut microbiota in vivo. We conducted a randomized, triple-blinded, placebo-controlled clinical trial (ClinicalTrials.gov NCT03735420) to determine safety and tolerability of XN in healthy adults. Thirty healthy participants were randomized to 24 mg/day XN or placebo for 8 weeks. As secondary outcomes, quantification of bacterial metabolites and 16S rRNA gene sequencing were utilized to explore the relationships between XN supplementation, gut microbiota, and biomarkers of gut health. Although XN did not significantly change gut microbiota composition, it did re-shape individual taxa in an enterotype-dependent manner. High levels of inter-individual variation in metabolic profiles and bioavailability of XN metabolites were observed. Moreover, reductions in microbiota-derived bile acid metabolism were observed, which were specific to Prevotella and Ruminococcus enterotypes. These results suggest interactions between XN and gut microbiota in healthy adults are highly inter-individualized and potentially indicate that XN elicits effects on gut health in an enterotype-dependent manner.

Brassica vegetables contain a multitude of bioactive compounds that prevent and suppress cancer and promote health. Evidence suggests that the gut microbiome may be essential in the production of these compounds; however, the relationship between specific microbes and the abundance of metabolites produced during cruciferous vegetable digestion are still unclear. We utilized an ex vivo human fecal incubation model with in vitro digested broccoli sprouts (Broc), Brussels sprouts (Brus), a combination of the two vegetables (Combo), or a negative control (NC) to investigate microbial metabolites of cruciferous vegetables. We conducted untargeted metabolomics on the fecal cultures by LC-MS/MS and completed 16S rRNA gene sequencing. We identified 72 microbial genera in our samples, 29 of which were significantly differentially abundant between treatment groups. A total of 4499 metabolomic features were found to be significantly different between treatment groups (q ≤ 0.05, fold change > 2). Chemical enrichment analysis revealed 45 classes of compounds to be significantly enriched by brassicas, including long-chain fatty acids, coumaric acids, and peptides. Multi-block PLS-DA and a filtering method were used to identify microbe–metabolite interactions. We identified 373 metabolites from brassica, which had strong relationships with microbes, such as members of the family Clostridiaceae and genus Intestinibacter, that may be microbially derived.