Anna Hsu

Bioavailability and inter-conversion of sulforaphane and erucin in human subjects consuming broccoli sprouts or broccoli supplement in a cross-over study design

Broccoli consumption may reduce the risk of various cancers and many broccoli supplements are now available. The bioavailability and excretion of the mercapturic acid pathway metabolites isothiocyanates after human consumption of broccoli supplements has not been tested. Two important isothiocyanates from broccoli are sulforaphane and erucin. We employed a cross-over study design in which 12 subjects consumed 40 g of fresh broccoli sprouts followed by a 1 month washout period and then the same 12 subjects consumed 6 pills of a broccoli supplement. As negative controls for isothiocyanate consumption four additional subjects consumed alfalfa sprouts during the first phase and placebo pills during the second. Blood and urine samples were collected for 48h during each phase and analyzed for sulforaphane and erucin metabolites using LC-MS/MS. The bioavailability of sulforaphane and erucin is dramatically lower when subjects consume broccoli supplements compared to fresh broccoli sprouts. The peaks in plasma concentrations and urinary excretion were also delayed when subjects consumed the broccoli supplement. GSTP1 polymorphisms did not affect the metabolism or excretion of sulforaphane or erucin. Sulforaphane and erucin are able to interconvert in vivo and this interconversion is consistent within each subject but variable between subjects. This study confirms that consumption of broccoli supplements devoid of myrosinase activity does not produce equivalent plasma concentrations of the bioactive isothiocyanate metabolites compared to broccoli sprouts. This has implications for people who consume the recommended serving size (1 pill) of a broccoli supplement and believe they are getting equivalent doses of isothiocyanates.

Differential effects of sulforaphane on histone deacetylases, cell cycle arrest and apoptosis in normal prostate cells versus hyperplastic and cancerous prostate cells

SCOPE Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables such as broccoli. The ability of SFN to inhibit histone deacetylase (HDAC) enzymes may be one mechanism by which it acts as a chemoprevention agent. The ability of a chemopreventive agent to specifically cause cytotoxicity in cancer and not normal cells is an important factor in determining its safety and clinical relevance. METHODS AND RESULTS We characterized the effects of SFN in normal (PrEC), benign hyperplasia (BPH1) and cancerous (LnCap and PC3) prostate epithelial cells. We observed that 15 μM SFN selectively induced cell cycle arrest and apoptosis in BPH1, LnCap and PC3 cells but not PrEC cells. SFN treatment also selectively decreased HDAC activity, and Class I and II HDAC proteins, increased acetylated histone H3 at the promoter for P21, induced p21 expression and increased tubulin acetylation in prostate cancer cells. HDAC6 over-expression was able to reverse SFN-induced cyotoxicity. In PrEC cells, SFN caused only a transient reduction in HDAC activity with no change in any other endpoints tested. The differences in sensitivity to SFN in PrEC and PC3 are likely not due to differences in SFN metabolism or differences in phase 2 enzyme induction. CONCLUSION SFN exerts differential effects on cell proliferation, HDAC activity and downstream targets in normal and cancer cells.

Promoter de-methylation of cyclin D2 by sulforaphane in prostate cancer cells

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.

Differential effects of whole soy extract and soy isoflavones on apoptosis in prostate cancer cells.

Previous studies have suggested that soy isoflavones exert anticarcinogenic effects against prostate cancer. We propose that soy extracts, containing a mixture of soy isoflavones and other bioactive components, would be a more potent chemo-preventive agent than individual soy isoflavones. We compared the apoptotic effects of whole soy extracts and individual soy isoflavones, genistein and daidzein, on prostate cancer cells. The soy extract contained 50% w/w of total isoflavones with approximately 1:5.5:3.5 ratios of genistin, daidzin and glycitin, respectively. Benign prostate hyperplasia (BPH-1), LnCap and PC3 cells were treated with varying concentrations of soy extract, genistein or daidzein and analyzed for cell cycle alterations and induction of apoptosis. At equal concentrations (25 μmol/L), soy extract induced a significantly higher percentage of cells undergoing apoptosis than genistein or daidzein (P < 0.001). No significant changes in cell cycle arrest or apoptosis were observed in non-cancerous BPH-1 cells treated with soy extract, suggesting that the effects of soy extract may be tumor cell specific. On the contrary, both genistein and daidzein induced apoptosis in BPH-1 cells, suggesting that individual isoflavones may have cytotoxicity in non-cancerous cells. Soy extracts also increased Bax expression in PC3 cells, but no significant changes in nuclear factor κB (NFκB) activation were detected, suggesting that the induction of apoptosis was independent of the NFκB pathway. Food products that bear a combination of active compounds may be more efficacious and safer as chemo-preventive agents than individual compounds. This ‘whole-food’-based approach is significant for the development of public health recommendations for prostate cancer prevention.

Effects of sulforaphane and 3,3'-diindolylmethane on genome-wide promoter methylation in normal prostate epithelial cells and prostate cancer cells.

Epigenetic changes, including aberrant DNA methylation, result in altered gene expression and play an important role in carcinogenesis. Phytochemicals such as sulforaphane (SFN) and 3,3′-diindolylmethane (DIM) are promising chemopreventive agents for the treatment of prostate cancer. Both have been shown to induce re-expression of genes, including tumor suppressor genes silenced in cancer cells, via modulation of epigenetic marks including DNA methylation. However, it remained unclear the effects SFN and DIM on DNA methylation at a genomic scale. The goal of this study was to determine the genome-wide effects of SFN and DIM on promoter methylation in normal prostate epithelial cells and prostate cancer cells. Both SFN and DIM treatment decreased DNA methyltransferase expression in normal prostate epithelial cells (PrEC), and androgen-dependent (LnCAP) and androgen-independent (PC3) prostate cancer cells. The effects of SFN and DIM on promoter methylation profiles in normal PrEC, LnCAP and PC3 prostate cancer cells were determined using methyl-DNA immunoprecipitation followed by genome-wide DNA methylation array. We showed widespread changes in promoter methylation patterns, including both increased and decreased methylation, in all three prostate cell lines in response to SFN or DIM treatments. In particular, SFN and DIM altered promoter methylation in distinct sets of genes in PrEC, LnCAP, and PC3 cells, but shared similar gene targets within a single cell line. We further showed that SFN and DIM reversed many of the cancer-associated methylation alterations, including aberrantly methylated genes that are dysregulated or are highly involved in cancer progression. Overall, our data suggested that both SFN and DIM are epigenetic modulators that have broad and complex effects on DNA methylation profiles in both normal and cancerous prostate epithelial cells. Results from our study may provide new insights into the epigenetic mechanisms by which SFN and DIM exert their cancer chemopreventive effects.

Dietary soy and tea mitigate chronic inflammation and prostate cancer via NFκB pathway in the Noble rat model

Chronic inflammation and nuclear factor-kappa B (NFκB) have been implicated in prostate cancer development; thus, dietary factors that inhibit NFκB may serve as effective chemo-preventative agents. Prostate cancer risk is significantly lower in Asian countries compared to the United States, which has prompted interest in the potential chemopreventative action of Asian dietary components such as soy and green tea. This study examined the effects of dietary soy and tea on NFκB activation and inflammation in vivo using a hormone-induced rat model for prostate cancer. Male Noble rats implanted with estradiol and testosterone were divided into 4 dietary groups: control, soy, tea, or soy+tea. NFκB activation and inflammatory cytokines were measured post implantation. The combination of soy and tea suppressed NFκB p50 binding activity and protein levels via induction of IκBα. Soy and tea also decreased prostate inflammatory infiltration, increased Bax/BcL2 ratio and decreased protein expression of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1β compared to control. Soy and tea attenuated prostate malignancy by decreasing prostate hyperplasia. These effects were not apparent in groups treated with soy or tea alone. The ongoing in vivo studies thus far suggest that combination of foods, such as soy and tea, may inhibit hormone-induced proinflammatory NFκB signals that contribute to prostate cancer development.

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

SCOPE Sulforaphane (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. METHODS AND RESULTS Subjects 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). CONCLUSION We conclude that the dietary form and dosing schedule of SFN may impact SFN absorption and efficacy in human trials.

Anti-inflammatory activity of soy and tea in prostate cancer prevention

Prostate cancer is the leading cancer-related cause of death for men in the USA. Prostate cancer risk is significantly lower in Asian countries compared with the USA, which has prompted interest in the potential chemo-preventive action of soy and green tea that are more predominant in Asian diets. It has been proposed that chronic inflammation is a major risk factor of prostate cancer, acting as both an initiator and promoter. Specifically, the nuclear factor-kappa B (NF-κB) pathway has been implicated as an important mediator between chronic inflammation, cell proliferation and prostate cancer. Dietary factors that inhibit inflammation and NF-κB may serve as effective chemo-preventive agents. Recent studies have demonstrated that soy and green tea have anti-inflammatory properties, and may have the potential to block the inflammatory response during cancer progression. This minireview discusses the relationship between chronic inflammation and prostate cancer, emphasizing on the significance of NF-κB, and further explores the anti-inflammatory effects of soy and green tea. Finally, we propose that dietary strategies that incorporate these bioactive food components as whole foods may be a more effective means to target pathways that contribute to prostate cancer development.

Sulforaphane modulates telomerase activity via epigenetic regulation in prostate cancer cell lines.

Epidemiologic studies have revealed that diets rich in sulforaphane (SFN), an isothiocyanate present in cruciferous vegetables, are associated with a marked decrease in prostate cancer incidence. The chemo-preventive role of SFN is associated with its histone de-acetylase inhibitor activity. However, the effect of SFN on chromatin composition and dynamic folding, especially in relation to HDAC inhibitor activity, remains poorly understood. In this study, we found that SFN can inhibit the expression and activity of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, in 2 prostate cancer cell lines. This decrease in gene expression is correlated with SFN-induced changes in chromatin structure and composition. The SFN-mediated changes in levels of histone post-translational modifications, more specifically acetylation of histone H3 lysine 18 and di-methylation of histone H3 lysine 4, 2 modifications linked with high risk of prostate cancer recurrence, were associated with regulatory elements within the hTERT promoter region. Chromatin condensation may also play a role in SFN-mediated hTERT repression, since expression and recruitment of MeCP2, a known chromatin compactor, were altered in SFN treated prostate cancer cells. Chromatin immuno-precipitation (ChIP) of MeCP2 showed enrichment over regions of the hTERT promoter with increased nucleosome density. These combined results strongly support a role for SFN in the mediation of epigenetic events leading to the repression of hTERT in prostate cancer cells. This ability of SFN to modify chromatin composition and structure associated with target gene expression provides a new model by which dietary phytochemicals may exert their chemoprevention activity.

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

SCOPE Several 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). METHODS AND RESULTS Ten 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). CONCLUSION This 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.