Free Sulforaphane Research Articles

Broccoli seedlings prevent glucose‐induced inflammation of peripheral blood mononuclear cells in humans

We studied the effect of anti‐inflammatory components in vegetables on postprandial inflammation using broccoli seedlings (Broccocress®, Koppert Cress BV, Monster, The Netherlands), which are rich in the isothiocyanate sulforaphane (SFN). In an NF‐κB reporter cell line, increasing concentrations of 2–15 μM SFN lead to inhibition of TNFα‐stimulated NF‐κB activation up to 98% (p<0.05). Five healthy subjects ingested a load of 75 gram glucose with or without a smoothie of broccoli seedlings (45 min. prior to glucose). NF‐κB activation in the nuclear fraction of peripheral blood mononuclear cells was determined by ELISA. 75 gram glucose leads to a 36.5% ± 20.3% increase in NF‐κB activation. In combination with the smoothie, NF‐κB was decreased by 64.8% (p<0.005) and 29.3% ± 9.3% (p<0.005) compared to high‐glucose‐induced and basal NF‐κB levels, respectively. The level of NF‐κB activation correlated to blood glucose levels after the oral glucose load, but not when the smoothie was ingested as well. SFN could be detected in plasma after 1.5 hrs. Total SFN (free SFN plus conjugated SFN) correlated to inhibition of NF‐κB. This suggests that broccoli seedlings are effective in reducing metabolic inflammation in humans.Grant Funding Source: Food Nutrition Delta project # FND09002

Abstract B64: A novel chemoprevention strategy using nanotechnology-based delivery of combinations of piroxicam, curcumin, and sulforaphane (PCS) on human pancreatic cancer cell lines

Abstract According to the American Cancer Society (ACS), an estimated 37,660 deaths will occur in 2011 due to complications from pancreatic cancer. The onset of pancreatic cancer is often devoid of symptoms and is mostly diagnosed at a stage where survival rate is less than 5%. Thus, chemoprevention could play an important role in the fight against this silent yet deadly disease. The objective of our study was to use combinations of chemopreventive agents namely piroxicam (a non-steroidal anti-inflammatory drug), curcumin (a natural spice with significant cancer chemopreventive properties) and sulforaphane (a potent broccoli extract) in very low doses to inhibit cell proliferation and promote apoptosis on two human pancreatic cancer cell lines, MIA PaCA-2 and Panc-1. More importantly, we compared the effects of the chemopreventive combinations in both, unmodified (free drug) and modified forms, encapsulated within solid-lipid nanoparticles (SLNs). SLNs were prepared using a process of hot-melt oil in water (o/w) emulsion technique using stearic acid as the lipid backbone to which the chemopreventive agents were added and then recovered as nanoparticles (∼200 nm size) after a process of freeze-drying. MTS cell proliferation, apoptosis and cell colony forming assays were conducted on cultured cells. For the MTS assay, upon 75 % confluence, 2.5 × 103 cells for MIA PaCa-2 and 4 × 103 cells for Panc-1 were transferred into each well of 96-well plates. The PCS chemopreventive agents alone or in combination were added to the cells and incubated for 72 h. Absorbance was recorded at 490 nm using an ELISA plate reader. For the apoptosis assay, 3 × 105 cells were cultured in 6-well plates for both MIA PaCa-2 and Panc-1 cell-lines. PCS, individual or combinations, were added and the plates were incubated for 72 h. Cells were analyzed using flow cytometry, measuring the fluorescence emission at 530 nm (FL1) and >575 nm (FL3). For the cell colony formation assays, MIA PaCa-2 and Panc-1 cells were seeded at a density of 1 ml containing 3 × 104 cells, incubated for a period of 24 h. After treatment with PCS alone and in combinations, cells were trypsinized then incubated for a period of 10 days. The number of colonies with >50 cells were counted under a dissecting microscope. IC50 values obtained for the unmodified and modified forms of piroxicam were (663.1μM; 52.85μM), curcumin (16.05μM; 4.93μM) respectively and free sulforaphane at 11.82 μM/L for MIA PaCa-2 cells. For Panc-1 cells, the IC50 values for unmodified and modified piroxicam were (389.3μM; 66.96μM), curcumin (24.01μM; 7.5μM) respectively and free sulforaphane at 17.81μM/L. For MIA PaCa-2 cell line, unmodified combinations of piroxicam (250μM) with curcumin (10μM) and sulforaphane (5μM) showed significant reduction in cell viability of > 60% using MTS assay. In comparison, lower concentrations of piroxicam SLN (25μM) and curcumin SLN (2.5μM) with free sulforaphane (5μM) mixtures showed decrease in cell viability by >47.4%. Flow cytometry analysis demonstrated apoptosis of 71% (unmodified) and 38.4% (modified) for the combinations, at the same concentration ranges as above. Panc-1 cell line treated with similar concentrations of unmodified and modified SLN combinations showed a >82% and >45.79% decrease in cell viability and 91% and 54.6% apoptotic cells, respectively. Colony formation assays in both cell lines showed a significant decrease (p value <0.0001) in cell counts. From this study, we believe the action of nanotechnology-based chemopreventive regimens on human pancreatic cell lines provides strong evidence of its potential as a viable tool to prevent the onset of pancreatic cancer. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B64.

A Novel pH-Sensitive Microsphere Composed of CM-Chitosan and Alginate for Sulforaphane Delivery

Sulforaphane (SF) has been proved to be an effective anticancer agent according to its experiments bothin vitroandin vivo. To date, there is few reported method to deliver SF for increasing its bioactivity and stability. In this study, a novel pH-sensitive microsphere composed of water-soluble carboxymethylated chitosan (CMCS) and alginate mixed with sodium sulfate was developed for SF delivery. Swelling studies and release characteristics under different pH values of microspheres were investigated. Then, the release of SF from test microspheres was studied in simulated gastric and segmented intestinal media. It has been found that the SF cumulated release in 5h was increased from 55.89% to 76.73% when the microspheres mixed with sodium sulfate. In addition, the stability of SF embedded in CMCS/alginate microspheres was also significantly improved. Under pH 7.4, free SF had a severe degradation of approximate 100% within 210 min, whereas the change of the SF in microspheres was only a decrease of about 10%. The results suggested that the microspheres of CMCS and alginate could be a suitable pH-sensitive carrier to increase the stability of SF in the segmented intestine.

Abstract 4591: Comparison of the inhibitory effects of unmodified and modified combinations of chemopreventive agents on MIA PaCa-2 and Panc-1 human pancreatic cancer cell lines

Abstract Pancreatic cancer is the fourth leading cause of death in the United States with approximately 40,000 estimated deaths in 2010. The objective of our study was to compare the effectiveness of chemopreventive combination regimens of aspirin, curcumin and sulforaphane (ACS) administered in unmodified (free drug) and in modified forms, encapsulated within solid lipid nanoparticles (SLNs), on the MIA PaCa-2 and Panc-1 human pancreatic cancer cell lines. MTS cell proliferation and apoptosis assay studies were conducted on cultured cells. For the MTS assay, upon 75 % confluence, 2.5 × 103 cells for MIA PaCa-2 and 4 × 103 cells for Panc-1 were transferred into each well of 96-well plates. The ACS chemopreventive agents alone or in combination were added to the cells and incubated for 72 h. Absorbance was recorded at 490 nm using an ELISA plate reader. For the apoptosis assay, 3 × 105 cells were cultured in 6-well plates for both Panc-1 and MIA PaCa-2 cell-lines. ACS, individual or combinations, were added and the plates were incubated for 72 h. Cells were analyzed using flow cytometry, measuring the fluorescence emission at 530 nm (FL1) and >575 nm (FL3). IC50 values obtained for the respective unmodified and modified forms of aspirin were (2.05mM/L; 66.08µM/L), curcumin (16.05µM/L; 4.93µM/L) and free sulforaphane at 11.82 µM/L for MIA PaCa-2 cells. Similarly, for Panc-1 cells, the IC50 values for unmodified and modified aspirin were (2.42mM/L; 90.23µM/L), curcumin (24.01 µM/L; 7.5µM/L) respectively and free sulforaphane at 17.81µM/L. For MIA PaCa-2 cell line, unmodified combinations of aspirin (1mM/L) with curcumin (10µM/L) and sulforaphane (5µM/L) showed significant reduction in cell viability of >60% using MTS assay. In comparison, aspirin SLN (25µM/L), curcumin SLN (2.5µM/L) and free sulforaphane (5µM/L) mixtures showed cell viability of >55%. Flow cytometry analysis demonstrated apoptosis of 63% (unmodified) and 50% (modified) for the combinations, at the same concentration ranges as above. For the Panc-1 cell line, treatment with similar concentrations of unmodified and modified SLN combinations showed a >75% and >60% decrease in cell viability (MTS assay) and 63% and 54% induction of apoptosis cells, respectively. Our results demonstrated that the drugs when encapsulated in SLNs at lower concentrations exhibited decreasing cell proliferation and increased induction of apoptosis within the pancreatic cancer cell lines. This data provides compelling evidence of the potential of the SLN carrier system in designing in-vivo studies for the pancreatic cancer chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4591. doi:10.1158/1538-7445.AM2011-4591

Glucoraphanin does not reduce plasma homocysteine in rats with sufficient Se supply via the induction of liver ARE-regulated glutathione biosynthesis enzymes

Data from human and animal trials have revealed contradictory results regarding the influence of selenium (Se) status on homocysteine (HCys) metabolism. It was hypothesised that sufficient Se reduces the flux of HCys through the transsulphuration pathway by decreasing the expression of glutathione (GSH) synthesising enzymes. Glucoraphanin (GRA) is a potent inducer of genes regulated via an antioxidant response element (ARE), including those of GSH biosynthesis. We tested the hypothesis that GRA supplementation to rat diets lowers plasma HCys levels by increasing GSH synthesis. Therefore 96 weaned albino rats were assigned to 8 groups of 12 and fed diets containing four different Se levels (15, 50, 150 and 450 μg kg(diet)(-1)), either without GRA (groups: C15, C50, C150 and C450) or in combination with 700 μmol GRA kg(diet)(-1) (groups G15, G50, G150 and G450). Rats fed the low Se diets C15 and G15 showed an impressive decrease of plasma HCys. Se supplementation increased plasma HCys and lowered GSH significantly by reducing the expression of GSH biosynthesis enzymes. As new molecular targets explaining these results, we found a significant down-regulation of the hepatic GSH exporter MRP4 and an up-regulation of the HCys exporter Slco1a4. In contrast to our hypothesis, GRA feeding did not reduce plasma HCys levels in Se supplemented rats (G50, G150 and 450) through inducing GSH biosynthesis enzymes and MRP4, but reduced their mRNA in some cases to a higher extent than Se alone. We conclude: 1. That the long-term supplementation of moderate GRA doses reduces ARE-driven gene expression in the liver by increasing the intestinal barrier against oxidative stress. 2. That the up-regulation of ARE-regulated genes in the liver largely depends on GRA cleavage to free sulforaphane and glucose by plant-derived myrosinase or bacterial β-glucosidases. As a consequence, higher dietary GRA concentrations should be used in future experiments to test if GRA or sulforaphane can be established as HCys lowering compounds.

Abstract 1897: Isothiocyanates and their metabolites in the plasma of mice fed broccoli sprouts isothiocyanate extract, broccoli sprouts, sulforaphane or erucin: A potential role in bladder cancer prevention

Abstract Background: Epidemiologic evidence suggests that cruciferous vegetables, particularly broccoli, may reduce bladder cancer risk. Our objectives were to characterize glucosinolates (GLUs) and their metabolites, isothiocyanates (ITCs), found in broccoli and broccoli sprouts, define bioactivity in bladder cancer cell lines in vitro, and to determine absorption and bioavailability in mice. Methods: Broccoli and broccoli sprouts GLUs and ITCs were characterized by HPLC-MS-MS and cyclocondensation assay. In vitro effects of non-hydrolyzed (GLUs) and hydrolyzed broccoli and broccoli sprouts (ITCs), and pure ITCs sulforaphane (SFN), erucin (ECN), iberin and allyl ITC, were studied on normal bladder urothelial cells (NBC) and a panel of human bladder cancer cell lines (BCC), representing the spectrum of bladder cancer biology (RT4: non-invasive; J82 and UMUC3: invasive). Cell viability (MTS and SRB assays), apoptosis (caspase-3/7 activity and PARP cleavage) and cell cycle analysis (flow cytometry) were performed. We defined absorption in control female mice and those fed diet containing 4% freeze-dried broccoli sprouts, or 2% freeze-dried broccoli sprouts extract; or gavaged daily with 295 µmol/kg sulforaphane or 295 µmol/kg erucin; for two weeks (n=16/group). Plasma mercapturic acid pathway metabolites of sulforaphane and erucin were extracted and then quantified by UPLC-MS/MS. Results: We observed significant dose-dependent decreases in cell viability of bladder cancer cell lines by broccoli or broccoli sprouts extracts as well as by pure ITCs. Broccoli sprout ITCs and SFN (IC50 = 2.52 µM) followed by ECN (IC50 = 2.24 µM) were the most potent inhibitors. Normal bladder urothelial cells were less sensitive than cancer cells. SFN and ECN resulted in a dose-dependent induction of apoptosis and modulation of the cell cycle in BCC. Metabolites of sulforaphane and erucin were absent in control mice and present in plasma of all treated groups, with dietary and pure phytochemical treatments, resulting in similar plasma metabolite concentrations (micromolar range). N-acetyl cysteine conjugates were found at highest concentrations, followed by cysteinyl and glutathione conjugates and small amounts of detectable cysteinyl glycine conjugates and free SFN. Evidence of inter-conversion of sulforaphane and erucin was seen. Conclusion: Broccoli extracts and pure ITCs inhibit cell viability, induce apoptosis and cell cycle arrest in non-invasive and invasive human bladder cancer cell lines. In vivo, dietary and gavaged ITCs are readily absorbed and metabolized to various conjugates. The distribution to bladder cancer xenografts will also be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1897.

The Metabolic Fate of Purified Glucoraphanin in F344 Rats

Dietary broccoli is commonly eaten cooked, exposing individuals to intact glucoraphanin rather than to its hydrolysis product, the anticarcinogenic isothiocyanate sulforaphane, since cooking destroys the hydrolyzing enzyme myrosinase. There is little information on the absorption and metabolism of glucoraphanin, due partly to the lack of purified compound. In this study, glucoraphanin was purified from broccoli seed and 150 mumol/kg was administered to male F344 rats. Glucoraphanin (5% of an oral dose) was recovered intact in urine, showing that it is absorbed intact, and no glucoraphanin or metabolites were found in feces. Total urinary products accounted for 20 and 45% of oral and intraperitonneal doses, respectively, including sulforaphane N-acetyl cysteine conjugate (12.5 and 2%), free sulforaphane (0.65 and 0.77%), sulforaphane nitrile (2 and 1.4%), and erucin (0.1 and 0.1%), respectively. Both glucoraphanin and its reduced form glucoerucin were identified in bile following intravenous glucoraphanin administration. We conclude that orally administered glucoraphanin is absorbed intact, undergoes enterohepatic circulation, and is hydrolyzed in the gut in F344 rats.

Induction of Quinone Reductase by Sulforaphane and Sulforaphane N-Acetylcysteine Conjugate in Murine Hepatoma Cells

Broccoli belongs to a group of vegetables termed cruciferous vegetables and characterized by their glucosinolate content. These glucosinolates are secondary metabolites that, upon hydrolysis, release bioactive isothiocyanates (ITCs). Bioactive ITCs are considered to protect the body from cancer by inducing detoxification enzymes such as quinone reductase (QR). This has the potential to make dietary choice a powerful strategy for achieving protection against carcinogenesis, mutagenesis, and other forms of toxicity from xenobiotic electrophiles and reactive forms of oxygen. The bioactive ITC sulforaphane (SF) is the hydrolysis product of glucoraphanin, the predominant aliphatic glucosinolate in broccoli. Because SF appears more potent than many other ITCs in induction of detoxification enzymes, it may have potential as a dietary cancer-preventative agent. One potential concern is that SF is highly reactive and has a very short half-life in the body, forming a glutathione conjugate that is further metabolized to the N-acetyl-L-cysteine conjugate (SF-NAC), the major excretory product found in the urine. However, the conjugate is a reversible complex, able to release free SF. The objective of this study was to compare QR-inducing activity by SF and its major metabolite SF-NAC, in murine hepatoma cells. Both SF and SF-NAC caused dose-related cell growth inhibition and QR induction. SF, 1 and 2 microM, resulted in a 3.0- and 3.5-fold induction of QR, respectively, and the same concentrations of SF-NAC caused a similar, although somewhat greater, induction of QR, 3.8- and 4.5-fold, respectively. These results strengthen the basis for considering that an effective therapeutic form of SF may be the ITC conjugate, formed in situ or given in place of purified ITC as prophylactic treatment to individuals at high risk for cancer.