Modifying Effects of Genetic Variations on the Association Between Dietary Isothiocyanate Exposure and Non-muscle Invasive Bladder Cancer Prognosis in the Be-Well Study.

SCOPE: Dietary isothiocyanate (ITC) exposure from cruciferous vegetable (CV) intake may improve non-muscle invasive bladder cancer (NMIBC) prognosis. This study aims to investigate whether genetic variations in key ITC-metabolizing/functioning genes modify the associations between dietary ITC exposure and NMIBC prognosis outcomes. METHODS AND RESULTS: In the Bladder Cancer Epidemiology, Wellness, and Lifestyle Study (Be-Well Study), a prospective cohort of 1472 incident NMIBC patients, dietary ITC exposure is assessed by self-reported CV intake and measured in plasma ITC-albumin adducts. Using Cox proportional hazards regression models, stratified by single nucleotide polymorphisms (SNPs) in nine key ITC-metabolizing/functioning genes, it is calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for recurrence and progression. The rs15561 in N-acetyltransferase 1 (NAT1) is alter the association between CV intake and progression risk. Multiple SNPs in nuclear factor E2-related factor 2 (NRF2) and nuclear factor kappa B (NFκB) are modify the associations between plasma ITC-albumin adduct level and progression risk (pint < 0.05). No significant association is observed with recurrence risk. Overall, >80% study participants are present with at least one protective genotype per gene, showing an average 65% reduction in progression risk with high dietary ITC exposure. CONCLUSION: Despite that genetic variations in ITC-metabolizing/functioning genes may modify the effect of dietary ITCs on NMIBC prognosis, dietary recommendation of CV consumption may help improve NMIBC survivorship.

The synthesis of broccoli sprout extract-loaded silk fibroin nanoparticles as efficient drug delivery vehicles: development and characterization.

Targeted drug delivery of biological molecules using the development of biocompatible, non-toxic and biodegradable nanocarriers can be a promising method for cancer therapy. In this study, silk fibroin protein nanoparticles (SFPNPs) were synthesized as a targeted delivery system for sulforaphane-rich broccoli sprout extract (BSE). The BSE-loaded SFPNPs were conjugated with polyethylene glycol and folic acid, and then their physicochemical properties were characterized via UV-Vis, XRD, FTIR, DLS, FE-SEM and EDX analyses. In vitro, the release profile, antioxidant and anticancer activities of NPs were also studied. The FE-SEM and DLS analyses indicated stable NPs with an average size of 88.5 nm and high zeta potential (-32 mV). The sulforaphane release profile from NPs was pH-dependent, with the maximum release value (70%) observed in simulated intestinal fluid (pH = 7.4). Encapsulation of BSE also decreased the release rate of sulforaphane from the capsules compared to free BSE. In vitro cytotoxicity of BSE and NPs on breast cancer cell lines (MCF-7) was concentration-dependent, and the IC50 for BSE and NPs were 54 and 210 µg ml-1, respectively. Moreover, the NPs demonstrated no appreciable cytotoxicity in normal mouse fibroblast (L929) cell lines. These results indicated that biocompatible NPs synthesized as controlled and long-term targeted drug delivery systems can be a potential candidate for breast cancer therapy.

Biphasic effect of sulforaphane on angiogenesis in hypoxia via modulation of both Nrf2 and mitochondrial dynamics.

Sulforaphane (SFN) is an isothiocyanate (ITC) derived from a glucosinolate, glucoraphinin found in cruciferous vegetables. There are few studies that focus on the role of SFN in angiogenesis under hypoxic conditions. The effect of SFN on angiogenesis and the underlying mechanisms including the roles of Nrf2 and mitochondrial dynamics were investigated using cultured human umbilical vein endothelial cells (HUVECs) in hypoxia. SFN at low doses (1.25-5 µM) increased hypoxia-induced HUVEC migration and tube formation, and alleviated hypoxia-induced retarded proliferation, but high doses (≥10 µM) exhibited an opposite effect. Under hypoxia, the expression of Nrf2 and heme oxygenase-1 was up-regulated by SFN treatment. Nrf2 knockdown abrogated SFN (2.5 µM)-induced tube formation and further potentiated the inhibitory effect of SFN (10 µM) on angiogenesis. Meanwhile, the mitochondrial function, morphology and expression of dynamic-related proteins suggested that low-dose SFN protected against hypoxia-induced mitochondrial injury and alleviated hypoxia-induced fission Nrf2-dependently without affecting the expression of key effector proteins (Drp1, Fis1, Mfn1/2 and Opa1), while high concentrations (≥10 µM SFN) aggravated hypoxia-induced mitochondrial injury, fission and Drp1 expression, and inhibited Mfn1/2 expression. These findings suggest that SFN biphasically affected the angiogenic capacity of hypoxia challenged HUVECs potentially via mechanisms involving an integrated modulation of Nrf2 and mitochondrial dynamics.

Effect of a sulforaphane supplement on muscle soreness and damage induced by eccentric exercise in young adults: A pilot study.

OBJECTIVE: Excessive exercise increases the production of reactive oxygen species in skeletal muscles. Sulforaphane activates nuclear factor erythroid 2-related factor 2 (Nrf2) and induces a protective effect against oxidative stress. In a recent report, sulforaphane intake suppressed exercise-induced oxidative stress and muscle damage in mice. However, the effect of sulforaphane intake on delayed onset muscle soreness after eccentric exercise in humans is unknown. We evaluated the effect of sulforaphane supplement intake in humans regarding the delayed onset muscle soreness (DOMS) after eccentric exercise. RESEARCH METHODS & PROCEDURES: To determine the duration of sulforaphane supplementation, continuous blood sampling was performed and NQO1 mRNA expression levels were analyzed. Sixteen young men were randomly divided into sulforaphane and control groups. The sulforaphane group received sulforaphane supplements. Each group performed six set of five eccentric exercise with the nondominant arm in elbow flexion with 70% maximum voluntary contraction. We assessed muscle soreness in the biceps using the visual analog scale, range of motion (ROM), muscle damage markers, and oxidative stress marker (malondialdehyde; MDA). RESULTS: Sulforaphane supplement intake for 2 weeks increased NQO1 mRNA expression in peripheral blood mononuclear cells (PBMCs). Muscle soreness on palpation and ROM were significantly lower 2 days after exercise in the sulforaphane group compared with the control group. Serum MDA showed significantly lower levels 2 days after exercise in the sulforaphane group compared with the control group. CONCLUSION: Our findings suggest that sulforaphane intake from 2 weeks before to 4 days after the exercise increased NQO1, a target gene of Nrf2, and suppressed DOMS after 2 days of eccentric exercise.

Persistence Enhancement of a Promising Tick Repellent, Benzyl Isothiocyanate, by Yeast Microcarriers.

Phenethyl isothiocyanate isolated from Armoracia rusticana root oil and its derivatives were tested at different doses in a bioassay designed to evaluate repellency against individual Haemaphysalis longicornis nymphs. Among the tested compounds, benzyl isothiocyanate exhibited repellency against H. longicornis nymphs at the lowest dose of 0.00625 mg/cm2, followed by phenethyl isothiocyanate (0.0125 mg/cm2) and phenyl isothiocyanate (0.025 mg/cm2). The behavioral responses of H. longicornis nymphs exposed to benzyl isothiocyanate and phenethyl isothiocyanate indicated that the mode of action of these compounds can be mainly attributed to the vapor phase. Encapsulated benzyl isothiocyanate showed repellency up to 120 min post-application at 0.1 mg/cm2, whereas pure benzyl isothiocyanate showed repellency up to 60 min post-application at 0.1 mg/cm2. The present study suggests that benzyl isothiocyanate is a potential repellent for protection against H. longicornis nymphs, and encapsulation in yeast cells may enhance the repellency effect.

Sulforaphane Causes Cell Cycle Arrest and Apoptosis in Human Glioblastoma U87MG and U373MG Cell Lines under Hypoxic Conditions.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary brain tumor. The median survival rate from diagnosis ranges from 15 to 17 months because the tumor is resistant to most therapeutic strategies. GBM exhibits microvascular hyperplasia and pronounced necrosis triggered by hypoxia. Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, has already demonstrated the ability to inhibit cell proliferation, by provoking cell cycle arrest, and leading to apoptosis in many cell lines. In this study, we investigated the antineoplastic effects of SFN [20-80 µM for 48 h] in GBM cells under normoxic and hypoxic conditions. Cell viability assays, flow cytometry, and Western blot results revealed that SFN could induce apoptosis of GBM cells in a dose-dependent manner, under both conditions. In particular, SFN significantly induced caspase 3/7 activation and DNA fragmentation. Moreover, our results demonstrated that SFN suppressed GBM cells proliferation by arresting the cell cycle at the S-phase, also under hypoxic condition, and that these effects may be due in part to its ability to induce oxidative stress by reducing glutathione levels and to increase the phosphorylation of extracellular signal-regulated kinases (ERKs). Overall, we hypothesized that SFN treatment might serve as a potential therapeutic strategy, alone or in combination, against GBM.

Nanoencapsulation of sulforaphane in broccoli membrane vesicles and their in vitro antiproliferative activity.

CONTEXT: The development of nanocarriers of plant origin, such as plant cell membranes, has recently been investigated. Also, plant bioactive compounds as sulforaphane (SFN) from broccoli have recognized antioxidant or anticancer properties. OBJECTIVE: To investigate the capacity of membrane vesicles from broccoli (BM-vesicles) to encapsulate SFN and their application in the cancer cell line. MATERIALS AND METHODS: Physicochemical analysis was carried out to characterize BM-vesicles through different approaches: dynamic light scattering, transmission electron microscopy, stopped-flow analysis, and proteomic analysis. They were applied at different concentrations (BM-vesicles at 0.04-0.00315% of protein and SFN at 5, 25, and 100 µM) in SK-MEL-28 cells during 24 h for studying cytotoxicity and gene expression. RESULTS: The entrapment efficiency was 41%. The anticancer activity tested in cells showed a decrease in proliferation when SFN in BM-vesicles was utilized. Expression patterns when SFN was applied in an encapsulated form showed a reduction of cancer markers and an increase of AQP3. Also, the metabolism of SFN occurred inside of cells, and higher SFN penetrated when it was encapsulated. DISCUSSION: The results showed that encapsulated SFN was better absorbed by melanoma cells providing metabolism products and a reduction of cancer molecular markers. Also aquaporin, AQP3 was pointed to as an important marker since it appeared to play a key role in homeostasis due to the importance of water transport in biological processes. CONCLUSION: These results indicate that SFN and SFN encapsulated in BM-vesicles have a high activity for the inhibition of melanocyte development. Therefore, BM-vesicles could serve as nanocarriers for drugs.

Allyl isothiocyanate attenuates LED light-induced retinal damage in rats: exploration for the potential molecular mechanisms.

PURPOSE: Environmental light pollution due to artificial light may increase the rate and severity of retinal diseases, and plant-based nutritional interventions with antioxidant properties have the potential to reverse this phenomenon. We aimed to investigate the potential effects of allyl isothiocyanate (AITC) against white light-emitting diode (LED)-induced retinal degeneration (RD) in the rats. METHODS: Twenty-eight male rats were allocated as: (i) Control, (ii) LED, (iii) LED + AITC (10 mg/kg BW), (iv) LED + AITC (20 mg/kg BW). Rats were administered with AITC for 28 days, followed by two days of intense environmental LED light (750 Lux) exposure to the eyes. Animals were sacrificed immediately at the end of the study, then the blood and eyeballs were taken for the biochemical, western blotting, and histopathology examinations. RESULTS: AITC lowered the serum and retina malondialdehyde (MDA) levels while significantly (p < 0.05) improving the retinal antioxidant enzyme activities in a dose-dependent manner. AITC improved retinal and outer nuclear layer (ONL) thickness as compared to the LED group (p < 0.05). AITC increased the levels of Bax, caspase-3, HO-1, GAP43, and VEGF, while decreasing IL-1ß, IL-6, NF-κB, Bcl-2, GFAP, Grp78, activating ATF4 and ATF6 as compared to the LED group (p < 0.05). CONCLUSION: In conclusion, four weeks of AITC administration to the rats showed specific protective effects against two days of intense LED light-induced retinal damage; through antiinflammatory, antioxidant, anti-apoptotic, and modulating mitochondrial metabolic pathways.

Pterostilbene-isothiocyanate inhibits breast cancer metastasis by selectively blocking IKK-ß/NEMO interaction in cancer cells.

Metastasis, the main cause of breast cancer-associated fatalities, relies on many regular pathways involved in normal cell physiology and metabolism, thus, making it challenging to identify disease-specific therapeutic target(s). Chemically synthesized anti-metastatic agents are preferred for their fast and robust actions. However, these agents have adverse side effects, thus, increasingly favouring the identification of phytocompounds as suitable alternatives. Resveratrol and pterostilbene have long been established as potent anti-cancer agents. Earlier studies from our laboratory documented the anti-cancer activities associated with pterostilbene-isothiocyanate (PTER-ITC), a derivative of pterostilbene. The current study focuses on evaluating the anti-metastatic property of PTER-ITC and the underlying mechanism, by employing in silico, in vitro, and in vivo approaches. The significant anti-metastatic activity of PTER-ITC was observed in vitro against breast cancer metastatic cell line (MDA-MB-231) and in vivo in the 4T1 cell-induced metastatic mice model. Epithelial-mesenchymal transition (EMT), a hallmark of metastasis regulated by the transcription factors, Snail1 and Twist, was found to be reverted in vitro by PTER-ITC treatment. PTER-ITC blocked the activation of NF-κB/p65 and its concomitant nuclear translocation, resulting in the transcriptional repression of its target genes, Snail1 and Twist. PTER-ITC prevented the formation of IKK complex, central to NF-κB activation, by binding to the NEMO-binding domain (NBD) of IKK-ß and inhibiting its interaction with NEMO (NF-κB essential modulator). According to our observations, PTER-ITC attenuated NF-κB activation selectively in cancerous cells. In conclusion, this study demonstrated that PTER-ITC is a potent anti-metastatic agent capable of targeting physiologically important pathways in a cancer-specific manner.

Phenethyl isothiocyanate attenuates diabetic nephropathy via modulation of glycative/oxidative/inflammatory signaling in diabetic rats.

Diabetic nephropathy (DN) is a diabetic complication characterized by disruption of renal microvasculature, reactive oxygen species accumulation and increased inflammation, all of which contribute to renal injury. Phenethyl isothiocyanate (PEITC) is a naturally occurring isothiocyanate well known for its antioxidant and anti-inflammatory effects, yet its reno-preventive effects against DN has not been investigated. The current study looked into the in vivo reno-protective effects of PEITC in STZ-induced DN in rats. PEITC (3, 10 and 30 mg/kg) was administered orally for 8 weeks post DM establishment. PEITC treatment significantly improved kidney and liver functions, renal histopathological features, tissue fibrosis, macrophage infiltration and blood glucose levels compared to DN control. Mechanistically, PEITC treatment alleviated DN-induced renal damage via modulating glycation and oxidative stresses and inflammatory response. As such, PEITC activated glyoxalase 1 (GLO1) that induced a retraction in renal tissue expression of advanced glycation end products (AGEs) and its receptor (RAGE). PEITC activated nuclear erythroid 2-related factor 2 (Nrf2) and increased expression of its downstream targets, hemeoxygenase-1 (HO-1) and gamma glutamate-cysteine (γ-GCS). Additionally, PEITC treatment decreased the expression of Nrf2 repressor protein, keap1. The anti-inflammatory effect of PEITC was driven, at least in part, via reducing the NLRP3 inflammasome activation as indicated by down regulation of NLRP3, TXNIP, capsase-1 and IL-1ß, TNF-alpha and IL-6. In conclusion; PEITC attenuated DN progression in a dose dependent manner mainly via interruption of AGE/RAGE and NLPR3/TXNIP/NrF2 crosstalk.

Allyl Isothiocyanate: A TAS2R38 Receptor-Dependent Immune Modulator at the Interface Between Personalized Medicine and Nutrition.

Understanding individual responses to nutrition and medicine is of growing interest and importance. There is evidence that differences in bitter taste receptor (TAS2R) genes which give rise to two frequent haplotypes, TAS2R38-PAV (functional) and TAS2R38-AVI (non-functional), may impact inter-individual differences in health status. We here analyzed the relevance of the TAS2R38 receptor in the regulation of the human immune response using the TAS2R38 agonist allyl isothiocyanate (AITC) from Brassica plants. A differential response in calcium mobilization upon AITC treatment in leucocytes from healthy humans confirmed a relevance of TAS2R38 functionality, independent from cation channel TRPV1 or TRPA1 activation. We further identified a TAS2R38-dependence of MAPK and AKT signaling activity, bactericidal (toxicity against E. coli) and anti-inflammatory activity (TNF-alpha inhibition upon cell stimulation). These in vitro results were derived at relevant human plasma levels in the low micro molar range as shown here in a human intervention trial with AITC-containing food.

Anticancer activities of dietary benzyl isothiocyanate: A comprehensive review.

Benzyl isothiocyanate (BITC) is one of the common isothiocyanates found in cruciferous vegetables such as broccoli, cabbage or watercress. Preclinical studies report of its effectiveness in the prevention and treatment against several cancers. This review aims to report and discuss findings on anticancer activities of BITC and its modes of action against 14 types of cancer. A literature search was conducted using the keywords "BITC" and "anticancer" from PubMed, Google Scholar and CINAHL Plus to obtain relevant research articles. This review highlights the anticancer efficacy of BITC through modulation of various signaling pathways involved in apoptosis, cell proliferation, cell cycle arrest, metastasis, angiogenesis, autophagy and the effects of BITC in combination with other drugs. With the available pharmacology evidence, we conclude that further studies are needed to validate its effectiveness in humans for further development and translation into prophylaxis or therapy by promoting optimal therapeutic effects and minimizing toxicity in cancer treatment.

Translational and pharmacokinetic-pharmacodynamic application for the clinical development of GDC-0334, a novel TRPA1 inhibitor.

GDC-0334 is a novel small molecule inhibitor of transient receptor potential cation channel member A1 (TRPA1), a promising therapeutic target for many nervous system and respiratory diseases. The pharmacokinetic (PK) profile and pharmacodynamic (PD) effects of GDC-0334 were evaluated in this first-in-human (FIH) study. A starting single dose of 25 mg was selected based on integrated preclinical PK, PD, and toxicology data following oral administration of GDC-0334 in guinea pigs, rats, dogs, and monkeys. Human PK and PK-PD of GDC-0334 were characterized after single and multiple oral dosing using a population modeling approach. The ability of GDC-0334 to inhibit dermal blood flow (DBF) induced by topical administration of allyl isothiocyanate (AITC) was evaluated as a target-engagement biomarker. Quantitative models were developed iteratively to refine the parameter estimates of the dose-concentration-effect relationships through stepwise estimation and extrapolation. Human PK analyses revealed that bioavailability, absorption rate constant, and lag time increase when GDC-0334 was administered with food. The inhibitory effect of GDC-0334 on the AITC-induced DBF biomarker exhibited a clear sigmoid-Emax relationship with GDC-0334 plasma concentrations in humans. This study leveraged emerging preclinical and clinical data to enable iterative refinement of GDC-0334 mathematical models throughout the FIH study for dose selection in subsequent cohorts throughout the study. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? GDC-0334 is a novel, small molecule TRPA1 inhibitor and a pharmacokinetic-pharmacodynamic (PK-PD) modeling strategy could be implemented in a systematic and step-wise manner to build and learn from emerging data for early clinical development. WHAT QUESTION DID THIS STUDY ADDRESS? Can noncompartmental and population-based analyses be used to describe the PK and PD characteristics of GDC-0334 in preclinical and clinical studies? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? GDC-0334 exposure generally increased with dose in rats, dogs, and monkeys. The starting dose (25 mg) in the clinical study was determined based on the preclinical data. GDC-0334 exhibited linear PK in humans and the bioavailability was increased with food. The inhibitory effect of GDC-0334 on dermal blood flow induced by the TRPA1 agonist allyl isothiocyanate in humans indicates a clear PK-PD relationship. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? The models developed based on TRPA1 agonist-induced dermal blood flow inhibition data can be used to predict PK-PD relationships in future preclinical and clinical studies evaluating new drug entities that target TRPA1.

Effects of in vivo treatment of mice with sulforaphane on repair of DNA pyridyloxylbutylation.

The phytochemical sulforaphane (SF) has gained interest for its apparent association with reduced cancer risk and other cytoprotective properties, at least some of which are attributed to activation of the transcription factor Nrf2. Repair of bulky DNA adducts is important for mitigating carcinogenesis from exogenous DNA damaging agents, but it is unknown whether in vivo treatment with SF affects adduct repair. At 12 h following a single oral dose of 100 mg/kg SF, an almost doubling in activity for repair of pyridyloxobutylated DNA was observed in CD-1 mouse liver nuclear extracts, but not in lung extracts. This change at 12 h in repair activity was preceded by the induction of Nrf2-regulated genes but not accompanied by changes in levels of the specific nucleotide excision repair (NER) proteins XPC, XPA, XPB and p53 or in binding of hepatic XPC, XPA and XPB to damaged DNA. SF also did not significantly alter histone deacetylase activity as measured by acetylated histone H3 levels, or stimulate formation of γ-H2A.X, a marker of DNA damage. A significant reduction in oxidative DNA damage, as measured by 8-OHdG (a biomarker of oxidative DNA damage), was observed only in DNA from the lungs of SF-treated mice 3 h post-dosing. These results suggest that the ability of SF to increase bulky adduct repair activity is organ-selective and is consistent with activation of the Nrf2 signaling pathway.

A TRPA1 inhibitor suppresses neurogenic inflammation and airway contraction for asthma treatment.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

Sulforaphane-cisplatin combination inhibits the stemness and metastatic potential of TNBCs via down regulation of sirtuins-mediated EMT signaling axis.

BACKGROUND: Sulforaphane (SFN) is a naturally occurring organosulfur compound found in cruciferous vegetables such as broccoli, brussels sprouts and cabbage. SFN is known for its multiple therapeutic properties, such as HDAC inhibitory, chemo preventive and anti-cancer effects. Cisplatin (CIS) has limited effect against metastatic triple-negative breast cancer (TNBC). Additionally, CIS impose severe side effects to normal cells, and later TNBC cells develops resistance. Studies suggest that the overexpression of sirtuins (SIRTs) promotes CIS resistance and metastasis by activating epithelial-to-mesenchymal transition (EMT) pathway in TNBC. PURPOSE: In view of the above information, we investigated the therapeutic efficacy of SFN, in combination with CIS against TNBC metastasis and CIS resistance. METHODS: The anti-cancerous effect of SFN-CIS combination on human TNBC cell lines was demonstrated by utilizing MTT assay and, apoptosis and cell cycle assay followed by FACS analysis. The synergistic effect of SFN-CIS combination on the experimental metastasis was demonstrated by utilizing migration, invasion, chemotaxis, mammosphere and colony formation assay on human TNBC MDA-MB-231 and MDA-MB-468 cells. The role of SIRTs-mediated EMT signaling axis in the metastasis and chemoresistance was investigated by western blotting technique as well as sirtuin activity tests. This was further validated by using Chromatin immunoprecipitation (ChIP) analysis. RESULTS: We found that SFN-CIS combination synergistically inhibits cellular growth of MDA-MB-231 and MDA-MB-468 cells. More importantly, SFN was found to protect normal kidney cells from CIS-induced toxicity. Further, SFN-CIS combination was found to synergistically inhibit metastatic-events via significantly altering EMT markers which was further associated with the suppression of SIRTs functions in TNBC cells. ChIP analysis validated that SFN-CIS combination suppresses EMT mechanism through altered chromatin modifications at E-cadherin promoter resulting in its re-expression. CONCLUSION: The results of the current study suggests that CIS when supplemented with SFN, inhibits metastasis and stemness potential of TNBC cells by down regulating SIRTs-mediated EMT cascade. Overall this study affirms that, this novel combination could be a promising strategy against SIRT-mediated TNBC metastasis and CIS-resistance.

Sulforaphane Inhibits Osteoclastogenesis via Suppression of the Autophagic Pathway.

Previous studies have demonstrated that sulforaphane (SFN) is a promising agent against osteoclastic bone destruction. However, the mechanism underlying its anti-osteoclastogenic activity is still unclear. Herein, for the first time, we explored the potential role of autophagy in SFN-mediated anti-osteoclastogenesis in vitro and in vivo. We established an osteoclastogenesis model using receptor activator of nuclear factor kappa-ß ligand (RANKL)-induced RAW264.7 cells and bone marrow macrophages (BMMs). Tartrate-resistant acid phosphatase (TRAP) staining showed the formation of osteoclasts. We observed autophagosomes by transmission electron microscopy (TEM). In vitro, we found that SFN inhibited osteoclastogenesis (number of osteoclasts: 22.67 ± 0.88 in the SFN (0) group vs. 20.33 ± 1.45 in the SFN (1 µM) group vs. 13.00 ± 1.00 in the SFN (2.5 µM) group vs. 6.66 ± 1.20 in the SFN (2.5 µM) group), decreased the number of autophagosomes, and suppressed the accumulation of several autophagic proteins in osteoclast precursors. The activation of autophagy by rapamycin (RAP) almost reversed the SFN-elicited anti-osteoclastogenesis (number of osteoclasts: 22.67 ± 0.88 in the control group vs. 13.00 ± 1.00 in the SFN group vs. 17.33 ± 0.33 in the SFN+RAP group). Furthermore, Western blot (WB) analysis revealed that SFN inhibited the phosphorylation of c-Jun N-terminal kinase (JNK). The JNK activator anisomycin significantly promoted autophagy, whereas the inhibitor SP600125 markedly suppressed autophagic activation in pre-osteoclasts. Microcomputed tomography (CT), immunohistochemistry (IHC), and immunofluorescence (IF) were used to analyze the results in vivo. Consistent with the in vitro results, we found that the administration of SFN could decrease the number of osteoclasts and the expression of autophagic light chain 3 (LC3) and protect against lipopolysaccharide (LPS)-induced calvarial erosion. Our findings highlight autophagy as a crucial mechanism of SFN-mediated anti-osteoclastogenesis and show that the JNK signaling pathway participates in this process.

Sulforaphane Bioavailability and Effects on Blood Pressure in Women with Pregnancy Hypertension.

Sulforaphane, an isothiocyanate found in cruciferous vegetables such as broccoli, shows promise as an adjuvant therapy for preeclampsia. To inform future clinical trials, we set out to determine the bioavailability of sulforaphane in non-pregnant and preeclamptic women. In six healthy female volunteers, we performed a crossover trial to compare the bioavailability of sulforaphane and metabolites afforded by an activated and non-activated broccoli extract preparation. We then undertook a dose escalation study of the activated broccoli extract in 12 women with pregnancy hypertension. In non-pregnant women, an equivalent dose of activated broccoli extract gave higher levels of sulforaphane and metabolites than a non-activated extract (p < 0.0001) and greater area under the curve (AUC) (3559 nM vs. 2172 nM, p = 0.03). Compared to non-pregnant women, in women with preeclampsia, the same dose of activated extract gave lower levels of total metabolites (p < 0.000) and AUC (3559 nM vs. 1653 nM, p = 0.007). Doubling the dose of the activated extract in women with preeclampsia doubled levels of sulforaphane and metabolites (p = 0.02) and AUC (1653 nM vs. 3333 nM, p = 0.02). In women with preeclampsia, activated broccoli extract was associated with modest decreases in diastolic blood pressure (p = 0.05) and circulating levels of sFlt-1 (p = 0.0002). A myrosinase-activated sulforaphane formulation affords better sulforaphane bioavailability than a non-activated formulation. Higher doses of sulforaphane are required to achieve likely effective doses in pregnant women than in non-pregnant women. Sulforaphane may improve endothelial function and blood pressure in women with pregnancy hypertension.

Susceptibility to the acute toxicity of acrylonitrile in streptozotocin-induced diabetic rats: protective effect of phenethyl isothiocyanate, a phytochemical CYP2E1 inhibitor.

Diabetes mellitus is a significant global public health issue. The diabetic state not only precipitates chronic disease but also has the potential to change the toxicity of drugs and chemicals. Acrylonitrile (AN) is a potent neurotoxin widely used in industrial products. This study used a streptozotocin (STZ)-induced diabetic rat model to examine the role of cytochrome P450 2E1 (CYP2E1) in acute AN toxicity. The protective effect of phenethyl isothiocyanate (PEITC), a phytochemical inhibitor of CYP2E1, was also investigated. A higher incidence of convulsions and loss of the righting reflex, and decreased rates of survival, as well as elevated CYP2E1 activity, were observed in diabetic rats treated with AN when compared to those in non-diabetic rats, suggesting that diabetes confers susceptibility to the acute toxicity of AN. Pretreatment with PEITC (20-80 mg/kg) followed by AN injection alleviated the acute toxicity of AN in diabetic rats as evidenced by the decreased incidence of convulsions and loss of righting reflex, and increased rates of survival. PEITC pretreatment at 40 and 80 mg/kg decreased hepatic CYP2E1 activity in AN-exposed diabetic rats. PEITC pretreatment (20 mg/kg) increased the glutathione (GSH) content and glutathione S-transferase (GST) activity and further decreased ROS levels in AN-exposed diabetic rats. Collectively, STZ-induced diabetic rats were more sensitive to AN-induced acute toxicity mainly due to CYP2E1 induction, and PEITC pretreatment significantly alleviated the acute toxicity of AN in STZ-induced diabetic rats. PEITC might be considered as a potential effective chemo-preventive agent against AN-induced acute toxicity in individuals with an underlying diabetic condition.

Sulforaphane and Vitamin E Protect From Glucotoxic Neurodegeneration and Lifespan Reduction In C. Elegans.

Caenorhabditis elegans is an established model organism in neurodegeneration and aging research. Oxidative stress and formation of advanced glycation endproducts (AGEs), as they occur under hyperglycemic conditions in diabetes mellitus, contribute to neuronal damage and lifespan reduction. Sulforaphane (SFN) is an indirect antioxidant, alpha-tocopherol (vitamin E) is a direct antioxidant that acts as a free radical scavenger. Aim of this study is to investigate the protective effects of SFN and vitamin E against glucotoxic damages to the neuronal system and lifespan in C. elegans. Culture conditions that mimic clinical hyperglycemia increased the formation of reactive oxygen species (ROS) (p<0.001) and the accumulation of methylglyoxal-derived advanced glycation endproducts (MG-derived AGEs) (p<0.01) with subsequent neuronal damage and neuronal dysfunction, ultimately leading to a significant shortening of lifespan (p<0.01). Treatment with both, 20 µmol/l SFN and 200 µg/ml vitamin E, completely prevented the increase in ROS and MG-derived AGEs, abolished the glucotoxic effects on neuronal structure and function, and preserved lifespan, resulting in a life expectancy similar to untreated controls. These data emphasize the relevance of indirect and direct antioxidants as potential therapeutic options for the prevention of glucotoxic pathologies.