Roderick H. Dashwood

Absorption and chemopreventive targets of sulforaphane in humans following consumption of broccoli sprouts or a myrosinase-treated broccoli sprout extract

SCOPEnSulforaphane (SFN), an isothiocyanate derived from crucifers, has numerous health benefits. SFN bioavailability from dietary sources is a critical determinant of its efficacy in humans. A key factor in SFN absorption is the release of SFN from its glucosinolate precursor, glucoraphanin, by myrosinase. Dietary supplements are used in clinical trials to deliver consistent SFN doses, but myrosinase is often inactivated in available supplements. We evaluated SFN absorption from a myrosinase-treated broccoli sprout extract (BSE) and are the first to report effects of twice daily, oral dosing on SFN exposure in healthy adults.nnnMETHODS AND RESULTSnSubjects consumed fresh broccoli sprouts or the BSE, each providing 200 μmol SFN daily, as a single dose and as two 100-μmol doses taken 12 h apart. Using HPLC-MS/MS, we detected ∼3 x higher SFN metabolite levels in plasma and urine of sprout consumers, indicating enhanced SFN absorption from sprouts. Twelve-hour dosing retained higher plasma SFN metabolite levels at later time points than 24-hour dosing. No dose responses were observed for molecular targets of SFN (i.e. heme oxygenase-1, histone deacetylase activity, p21).nnnCONCLUSIONnWe conclude that the dietary form and dosing schedule of SFN may impact SFN absorption and efficacy in human trials.

Nrf2 status affects tumor growth, HDAC3 gene promoter associations, and the response to sulforaphane in the colon

BackgroundThe dietary agent sulforaphane (SFN) has been reported to induce nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2)-dependent pathways as well as inhibiting histone deacetylase (HDAC) activity. The current investigation sought to examine the relationships between Nrf2 status and HDAC expression in preclinical and translational studies.ResultsWild type (WT) and Nrf2-deficient (Nrf2−/+) mice were treated with the colon carcinogen 1,2-dimethylhydrazinexa0(DMH) followed by 400xa0ppm SFN in the diet (nu2009=u200935 mice/group). WT mice were more susceptible than Nrf2−/+ mice to tumor induction in the colon. Tumors from WT mice had higher HDAC levels globally and locally on genes such as cyclin-dependant kinase inhibitor 2a (Cdkn2a/p16) that were dysregulated during tumor development. The average tumor burden was reduced by SFN from 62.7 to 26.0xa0mm3 in WT mice and from 14.6 to 11.7xa0mm3 in Nrf2−/+ mice. The decreased antitumor activity of SFN in Nrf2−/+ mice coincided with attenuated Cdkn2a promoter interactions involving HDAC3. HDAC3 knockdown in human colon cancer cells recapitulated the effects of SFN on p16 induction. Human subjects given a broccoli sprout extract supplement (200xa0μmol SFN equivalents), or reporting more than five cruciferous vegetable servings per week, had increased p16 expression that was inversely associated with HDAC3 in circulating peripheral blood mononuclear cells (PBMCs) and in biopsies obtained during screening colonoscopy.ConclusionsNrf2 expression varies widely in both normal human colon and human colon cancers and likely contributes to the overall rate of tumor growth in the large intestine. It remains to be determined whether this influences global HDAC protein expression levels, as well as local HDAC interactions on genes dysregulated during human colon tumor development. If corroborated in future studies, Nrf2 status might serve as a biomarker of HDAC inhibitor efficacy in clinical trials using single agent or combination modalities to slow, halt, or regress the progression to later stages of solid tumors and hematological malignancies.

Comparison of anti-inflammatory mechanisms of mango (Mangifera Indica L.) and pomegranate (Punica Granatum L.) in a preclinical model of colitis

SCOPEnTannin-rich fruits have been evaluated as alternative prevention strategies for colorectal cancer based on their anti-inflammatory properties. This study compared tannin-rich preparations from mango (rich in gallotannins) and pomegranate (rich in ellagitannins) in the dextran sodium sulfate-induced colitis model.nnnMETHODS AND RESULTSnIn rats, mango and pomegranate beverages decreased intestinal inflammation and the levels of pro-inflammatory cytokines in mucosa and serum. The mango beverage suppressed the ratio of phosphorylated/total protein expression of the IGF-1R-AKT/mTOR axis and downregulated mRNA expression of Igf1, Insr, and pik3cv. Pomegranate decreased p70S6K and RPS6, as well as Rps6ka2, Map2k2, and Mapk1 mRNA. In silico modeling indicated a high binding of docked of gallic acid to the catalytic domain of IGF-1R, which may suppress the activity of the enzyme. Ellagic acid docked effectively into the catalytic domains of both IGF-1R and EGFR. In vitro assays with lipopolysaccharide-treated CCD-18Co cells using polyphenolic extracts from each beverage, as well as pure compounds, corroborated the predictions made in silico.nnnCONCLUSIONnMango polyphenols inhibited the IGF-1R- AKT/mTOR axis, and pomegranate polyphenols downregulate the mTOR downstream pathway through reductions in ERK1/2. These results suggest that extracts rich in gallo- and ellagitannins act on different molecular targets in the protection against ulcerative colitis.

HDAC8 and STAT3 repress BMF gene activity in colon cancer cells

Histone deacetylase (HDAC) inhibitors are undergoing clinical trials as anticancer agents, but some exhibit resistance mechanisms linked to anti-apoptotic Bcl-2 functions, such as BH3-only protein silencing. HDAC inhibitors that reactivate BH3-only family members might offer an improved therapeutic approach. We show here that a novel seleno-α-keto acid triggers global histone acetylation in human colon cancer cells and activates apoptosis in a p21-independent manner. Profiling of multiple survival factors identified a critical role for the BH3-only member Bcl-2-modifying factor (Bmf). On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300. Treatment with a p300 inhibitor or transient overexpression of exogenous HDAC8 interfered with BMF induction, whereas RNAi-mediated silencing of STAT3 activated the target gene. This is the first report to identify a direct target gene of HDAC8 repression, namely, BMF. Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis. These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy.

Streptococcus gallolyticus subsp. gallolyticus promotes colorectal tumor development

Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of β-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of β-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and β-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications.

Long noncoding RNAs and sulforaphane: a target for chemoprevention and suppression of prostate cancer

Long noncoding RNAs (lncRNAs) have emerged as important in cancer development and progression. The impact of diet on lncRNA expression is largely unknown. Sulforaphane (SFN), obtained from vegetables like broccoli, can prevent and suppress cancer formation. Here we tested the hypothesis that SFN attenuates the expression of cancer-associated lncRNAs. We analyzed whole-genome RNA-sequencing data of normal human prostate epithelial cells and prostate cancer cells treated with 15 μM SFN or dimethylsulfoxide. SFN significantly altered expression of ~100 lncRNAs in each cell type and normalized the expression of some lncRNAs that were differentially expressed in cancer cells. SFN-mediated alterations in lncRNA expression correlated with genes that regulate cell cycle, signal transduction and metabolism. LINC01116 was functionally investigated because it was overexpressed in several cancers, and was transcriptionally repressed after SFN treatment. Knockdown of LINC01116 with siRNA decreased proliferation of prostate cancer cells and significantly up-regulated several genes including GAPDH (regulates glycolysis), MAP1LC3B2 (autophagy) and H2AFY (chromatin structure). A four-fold decrease in the ability of the cancer cells to form colonies was found when the LINC01116 gene was disrupted through a CRISPR/CAS9 method, further supporting an oncogenic function for LINC01116 in PC-3 cells. We identified a novel isoform of LINC01116 and bioinformatically investigated the possibility that LINC01116 could interact with target genes via ssRNA:dsDNA triplexes. Our data reveal that chemicals from the diet can influence the expression of functionally important lncRNAs, and suggest a novel mechanism by which SFN may prevent and suppress prostate cancer.

Oncogenic targets Mmp7, S100a9, Nppb and Aldh1a3 from transcriptome profiling of FAP and Pirc adenomas are downregulated in response to tumor suppression by Clotam

Intervention strategies in familial adenomatous polyposis (FAP) patients and other high‐risk colorectal cancer (CRC) populations have highlighted a critical need for endoscopy combined with safe and effective preventive agents. We performed transcriptome profiling of colorectal adenomas from FAP patients and the polyposis in rat colon (Pirc) preclinical model, and prioritized molecular targets for prevention studies in vivo. At clinically relevant doses in the Pirc model, the drug Clotam (tolfenamic acid, TA) was highly effective at suppressing tumorigenesis both in the colon and in the small intestine, when administered alone or in combination with Sulindac. Cell proliferation in the colonic crypts was reduced significantly by TA, coincident with increased cleaved caspase‐3 and decreased Survivin, β‐catenin, cyclin D1 and matrix metalloproteinase 7. From the list of differentially expressed genes prioritized by transcriptome profiling, Mmp7, S100a9, Nppb and Aldh1a3 were defined as key oncogene candidates downregulated in colon tumors after TA treatment. Monthly colonoscopies revealed the rapid onset of tumor suppression by TA in the Pirc model, and the temporal changes in Mmp7, S100a9, Nppb and Aldh1a3, highlighting their value as potential early biomarkers for prevention in the clinical setting. We conclude that TA, an “old drug” repurposed from migraine, offers an exciting new therapeutic avenue in FAP and other high‐risk CRC patient populations.

Abstract B33: Regulation and function of Nrf2-associated long noncoding RNA

We hypothesized that the long non coding RNA (lncRNA), Loc344887, is induced by Nrf2 activation and acts as a transcriptional co-activator of the Nrf2 target gene NQO1. Nrf2 is a transcription factor that is important in oxidative stress responses. Upon oxidative stress, Nrf2 is released from its binding partner Keap1. Nrf2 translocates to the nucleus, partners with small Maf proteins, binds to antioxidant response elements (ARE) and induces detoxifying and metabolizing genes such as NQO1, HMOX1, and GSTs. We propose that Loc344887 is acting as a co-activator of Nrf2, either directly in the Nrf2-Maf complex, or indirectly by some other downstream mechanism, in order to regulate NQO1 expression. Loc344887 has significantly reduced expression in human colon tumors when compared to normal colon tissue (TCGA database). Human HCT116 colon cancer cells and CCD841 non-cancer colonic epithelial cells replicate this expression difference between tumor and non-transformed. We used these cell lines to study the regulation and function of Loc344887. HCT116 and CCD841 cells were treated with 15 μM sulforaphane (SFN), a dietary isothiocyanate known to have chemopreventative properties due to Nrf2 activation. Loc344887 increased 40-fold in HCT116 cells, but was not induced in CCD841 cells. Loc344887 was also induced by oltipraz and tert-butylhydroquinone, known Nrf2 activators. Keap1 knockdown upregulated Loc344887, and inducibility of Loc344887 by SFN was lost when Nrf2 was knocked down using RNAi. The gene promoter of Loc344887 contains putative AREs, and chromatin immunoprecipitation assays identified putative Nrf2 and MafK binding regions. Because Loc344887 is regulated by Nrf2, we hypothesized that Loc344887 may be involved in NRF2 gene activation. Knockdown of Loc344887 caused a loss of NQO1 induction by both SFN and Keap1 knockdown; however, this effect was not seen for HMOX1. Time-course assays showed that Loc344887 is induced as early as 1 h post-SFN treatment and peaks at ~8 h, similar to HMOX1. NQO1, however, was induced much slower, peaking after 24 h. This suggests that other factor(s), such as Loc344887, needs to be present for full induction to occur. This study shows that Loc344887 is an Nrf2 target gene and is required for complete NQO1 induction, but not for HMOX1 activation. Thus, there is a differential response for Nrf2 target genes that depends, in part, on lincRNAs as co-regulators. Further studies are required to access the mechanism of NQO1 regulation. We hypothesize that once Loc344887 is induced, it acts as a scaffold for MafK, Nrf2 and/or other transcription factors on the NQO1 promoter. Loc344887 localizes primarily to the nucleus, and preliminary RNA immunoprecipitation experiments show that Loc344887 binds directly to MafK. Further studies will also test whether Loc344887 is required for regulation of other Nrf2 target genes, or if other lncRNAs are involved in Nrf2 target gene induction. Studies supported in part by Chancellor9s Research Initiative funding from Texas AM 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B33.

Untargeted Metabolomic Screen Reveals Changes in Human Plasma Metabolite Profiles Following Consumption of Fresh Broccoli Sprouts

SCOPEnSeveral lines of evidence suggest that the consumption of cruciferous vegetables is beneficial to human health. Yet, underlying mechanisms and key molecular targets that are involved with achieving these benefits in humans are still not fully understood. To accelerate this research, we conduct a human study to identify potential molecular targets of crucifers for further study. This study aims to characterize plasma metabolite profiles in humans before and after consuming fresh broccoli sprouts (a rich dietary source of bioactive sulforaphane).nnnMETHODS AND RESULTSnTen healthy adults consume fresh broccoli sprouts (containing 200 μmol sulforaphane equivalents) at time 0 and provide blood samples at 0, 3, 6, 12, 24, and 48 h. An untargeted metabolomics screen reveals that levels of several plasma metabolites are significantly different before and after sprout intake, including fatty acids (14:0, 14:1, 16:0, 16:1, 18:0, and 18:1), glutathione, glutamine, cysteine, dehydroepiandrosterone, and deoxyuridine monophosphate. Evaluation of all time points is conducted using paired t-test (R software) and repeated measures analysis of variance for a within-subject design (Progenesis QI).nnnCONCLUSIONnThis investigation identifies several potential molecular targets of crucifers that may aid in studying established and emerging health benefits of consuming cruciferous vegetables and related bioactive compounds.

Measuring Histone Deacetylase Inhibition in the Brain

Histone deacetylases (HDACs) represent a family of enzymes that are targets for epigenetic modulation of genomic activity and may be beneficial in the treatment of many diseases, including cancer and central nervous system disorders. In animal models, HDAC inhibitors have neuroprotective, antiepileptogenic, and antidepressant effects. Assaying HDAC activity provides a robust method for identifying HDAC inhibitors and for assessing their effects under various physiological conditions or after pathological insults. In this unit, a simple and sensitive assay for measuring HDAC activity is described. HDAC activity in tissue lysates can be assessed fluorometrically using a Boc‐Lys(Ac) HDAC activity kit. HDACs catalyze the deacetylation of the substrate Boc‐Lys(Ac)‐AMC. Addition of a trypsin‐containing developer converts the deacetylated product to a quantifiable fluorophore that can be used both as a screening method to identify putative HDAC inhibitors and to assess the effects of these inhibitors on tissue and animal epigenetic‐modulated phenotypes.