Isothiocyanate Research Articles

Application of polybrene to increase transfection efficiency of lentiviral vector in bone marrow derived dendritic cells

Objective To investigate the toxicity and optimal concentration of polybrene on lentiviral vector transduction of bone marrow derived dendritic cells (BMDCs). Methods The BMDCs suspensions in mice were prepared and randomly divided into experimental and control groups. The BMDCs in experimental groups were treated with different concentrations of polybrene, and those in control groups were added with phosphate buffer (PBS). Cell counting kit-8 (CCK-8) assay and annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) staining were performed to detect cells viability and apoptosis. Add to BMDCs suspensions were added with lentiviral vector [multiplicity of infection (MOI)=10] and the mixture was divided into experimental and control groups. The mixture in experimental groups was treated with different concentrations of polybrene, and that in control groups was given PBS. The expression of green fluorescent protein (GFP) was observed by fluorescence microscope every day. After the BMDCs were stimulated to mature, the GFP expression was detected using flow cytometry. The t test was used for comparison between two groups. Results The number of living cells in experimental group was significantly less than in control group when polybrene ≥11 mg/L [The absorbance (A) values were 0.881±0.007 vs. 1.031±0.017, t=7.832, P<0.05]. The GFP expression in experimental groups was significantly higher than in control groups. The highest GFP expression was observed when polybrene at 8 mg/L. Conclusion Polybrane shows dose-dependent toxicity on BMDCs, and in the safe range of concentration, polybrane can significantly increase the transfection efficacy of lentiviral vector to BMDCs and the optimal concentration is 8 mg/L. Key words: Polybrene; Lentiviral vector; Bone marrow derived dendritic cells

Efecto inhibitorio in vitro de cinco isotiocianatos sobre Rhizoctonia solani Kühn AG-3

&lt;p&gt;Evaluamos el efecto de cinco isotiocianatos (ITC) para controlar a Rhizoctonia solani Kühn AG-3, un hongo causante de enfermedades en plantas de importancia agrícola, como estudio preliminar para la implementación de la técnica de biofumigación en Colombia. Realizamos pruebas in vitro con diluciones de cinco ITC, con metanol como solvente, en cajas de Petri con medio PDA y calculamos las áreas de crecimiento micelial, el porcentaje de inhibición y la concentración efectiva media (CE50). La respuesta del patógeno fue dependiente de la concentración y del tipo de ITC aplicado. Todos los ITC en concentraciones al 10% presentaron efecto inhibitorio total. PEITC y BITC tuvieron un efecto fungistático hasta los seis y 27 días después de aplicados los tratamientos, respectivamente. PITC, AITC y MITC mantuvieron el efecto inhibitorio por más de un año después de aplicados los tratamientos. PITC fue más eficaz en controlar R. solani Kühn AG-3 que los otros ITC, mostrando el valor más bajo de la CE50 para la inhibición del crecimiento micelial.&lt;/p&gt;

Abstract A110: Isothiocyanate-containing hybrid androgen receptor (AR) antagonist downregulates AR and induces ferroptosis in GSH–deficient prostate cancer cells

Abstract Androgen receptor (AR) over-expression, mutation and the emergence of AR splice variants (AR-Vs) promote disease-driving AR signaling in castration-resistant prostate cancer (CRPC). In addition to the sustained AR activation, apoptosis evasion adds extra hurdle for treating hormone refractory prostate cancer (PCa). To overcome these challenges, we firstly designed isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and then rationally combined ITC-ARi with glutathione (GSH) biosynthesis inhibitor buthionine sulphoximine (BSO) to efficiently downregulate AR/AR-V and induce ferroptosis in enzalutamide (Enz)-resistant PCa cells. Naturally occurring ITCs (e.g., sulforaphane and phenylethyl isothiocyanate) display pleotropic anti-PCa activities. A representative ITC-ARi 2-63 is designed by incorporating ITC into an AR ligand scaffold. The sulfhydryl reactivity of ITC is transiently masked as N-acetyl cysteine (NAC) conjugate that gradually releases parental free ITC in aqueous solution. 2-63 antagonizes AR transactivation, downregulates AR/AR-V7, and upregulates cellular stress markers Hsp70 and heme oxygenase-1 (HO-1), indicating the disruption of heat shock proteins (Hsps) and the activation of Nrf2 pathway. Because the anticancer effects of ITC/ITC-NAC conjugate are attenuated by GSH-participated conjugation or thiol exchange, we rationally combined 2-63 with BSO for enhanced anti-PCa activities. 2-63 and BSO synergistically reduce viability of multiple PCa cell lines (VCaP, C4-2 and 22Rv-1), and the noncancerous prostatic RWPE-1 cell is much less affected. Because 2-63 plus BSO does not upregulate typical apoptosis markers, we investigated alternative cell death mechanism in combinatorial treatment. Drug combination-caused cell viability loss is effectively rescued by iron chelator deferoxamine (DFO), antioxidants α-tocopherol (α-Toc) and ferrostatin-1 (Fer-1), or the inhibitor of HO-1 Zinc (II) protoporphyrin IX (ZnPP). Both Fer-1 and α-Toc are potent radical scavengers blocking lipid hydroperoxide generation, and ZnPP suppresses HO-1-catalyzed upregulation of intracellular Fe2+. The reverse of growth suppression by DFO, antioxidants and ZnPP supports the induction of ferroptosis, a regulated non-apoptotic cell death caused by reactive oxygen species (ROS)- and iron-dependent lipid peroxidation. 2-63 and BSO also more efficiently downregulate AR/AR-V than 2-63 alone in Enz-resistant PCa cells. Interestingly, ferroptosis-preventing DFO and Fer-1 do not rescue AR/AR-V7 and are unable to suppress the upregulation of Hsp70 and HO-1. Our results suggest that the synergism of 2-63 and BSO occurs through increasing drug accessibility to cellular targets, expanding availability of iron and potentially affecting glutathione peroxidase 4 (GPX4) activity, the most important cellular defense to eliminate lipid hydroperoxide. Combining ITC-ARi and GSH-depleting agents could be a new concept leading to effective CRPC treatment. Citation Format: Zhihui Qin, Liping Xu, Siyu Ou. Isothiocyanate-containing hybrid androgen receptor (AR) antagonist downregulates AR and induces ferroptosis in GSH–deficient prostate cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A110. doi:10.1158/1535-7163.TARG-19-A110

Organic Isothiocyanates as Hydrogen Sulfide Donors.

Significance: Hydrogen sulfide (H2S), the "new entry" in the series of endogenous gasotransmitters, plays a fundamental role in regulating the biological functions of various organs and systems. Consequently, the lack of adequate levels of H2S may represent the etiopathogenetic factor of multiple pathological alterations. In these diseases, the use of H2S donors represents a precious and innovative opportunity. Recent Advances: Natural isothiocyanates (ITCs), sulfur compounds typical of some botanical species, have long been investigated because of their intriguing pharmacological profile. Recently, the ITC moiety has been proposed as a new H2S-donor chemotype (with a l-cysteine-mediated reaction). Based on this recent discovery, we can clearly observe that almost all the effects of natural ITCs can be explained by the H2S release. Consistently, the ITC function was also used as an original H2S-releasing moiety for the design of synthetic H2S donors and original "pharmacological hybrids." Very recently, the chemical mechanism of H2S release, resulting from the reaction between l-cysteine and some ITCs, has been elucidated. Critical Issues: Available literature gives convincing demonstration that H2S is the real player in ITC pharmacology. Further, countless studies have been carried out on natural ITCs, but this versatile moiety has been used only rarely for the design of synthetic H2S donors with optimal drug-like properties. Future Directions: The development of more ITC-based synthetic H2S donors with optimal drug-like properties and selectivity toward specific tissues/pathologies seem to represent a stimulating and indispensable prospect of future experimental activities.

A novel isothiocyanate derivative inhibits HIV-1 gene expression and replication by modulating the nuclear matrix associated protein SMAR1

The essential role of SMAR1 in HIV-1 transcription and LTR driven gene expression suggests SMAR1 as an HIV dependency factor (HDF) and a potential anti-HIV therapeutic target. Here, we report for the first time, anti-HIV activity of 8 novel isothiocyanate (ITC) derivatives that differentially stabilise SMAR1. Out of 8 novel ITC derivatives, SCS-OCL-381 was observed to inhibit HIV-1 replication most significantly at the noncytotoxic concentration in reporter T-cell line, CEM-GFP. Further, the highly conserved anti-HIV activity of SCS-OCL-381 is a cell type, virus isolate and viral load independent phenomena and is approximately 3 fold more effective than the representative ITC, Sulforaphane (SFN). Further, SCS-OCL-381 does not hamper the activity of viral enzymes reverse transcriptase, integrase and protease. Mechanistically, SCS-OCL-381 stabilises SMAR1 which, otherwise undergoes proteasomal degradation upon HIV-1 infection in T-cells. This stabilisation results in the recruitment of repressor complex on HIV-1 LTR resulting in repression of LTR mediated transcription and gene expression. These inhibitory consequences were further confirmed by reporter based LTR activity assays in different cell lines. Taken together, these findings highlight the anti-HIV potential of novel ITC derivatives by the stabilisation of SMAR1 and strongly support further in vivo characterisation and potential translational applications of SCS-OCL-381.

NRF2-Independent Regulation of Intestinal Constitutive Androstane Receptor by the Pro-Oxidants Cadmium and Isothiocyanate in hUGT1 Mice.

Environmental toxicants such as heavy metals from contaminated water or soil and isothiocyanates (ITC) from dietary sources act as pro-oxidants by directly generating reactive oxygen species (ROS) or through depleting cellular antioxidants such as glutathione. Toxicants can alter drug metabolism, and it was reported that CYP2B10 and UGT1A1 are induced by phenethyl isothiocyanate (PEITC) through the constitutive androstane receptor (CAR). The possibility that nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of the antioxidant response, could coactivate CAR was investigated in neonatal hUGT1/Nrf2 -/- mice. Neonatal mice were treated with PEITC or cadmium (Cd2+) by oral gavage for 2 days. Both PEITC and Cd2+ induced UGT1A1 RNA and protein in intestinal tissues in both hUGT1/Nrf2 +/- and hUGT1/Nrf2 -/- neonates, indicating NRF2-independent regulation of UGT1A1. Increases in CYP2B10 RNA in intestinal tissues were observed following PEITC or Cd2+ exposure. Activation of intestinal CAR by Cd2+ exposure was directly assessed by nuclear fractionation and Western blot analyses at 0.5, 1, 2, and 4 hours after treatment in hUGT1 neonates and after 48 hours in hUGT1/Nrf2 +/- and hUGT1/Nrf2 -/- neonates. CAR localized to the nucleus independently of NRF2 48 hours after exposure. Substantial CAR localization to the nucleus occurred at the 2- and 4-hour time points, coinciding with a decrease in phosphorylation of cytoplasmic extracellular signal-regulated kinases 1 and 2 and a nuclear increase in P38/p-P38 content. This suggests that a novel oxidative stress-MAPK-CAR axis exists with phenotypic consequences. SIGNIFICANCE STATEMENT: Pro-oxidant toxicants can alter drug metabolism through activation of CAR, independent of the NRF2-KEAP1 signaling pathway. Changes in proteins associated with drug metabolism and linked to increases in intestinal maturation are mediated through an oxidative stress-MAPK-CAR axis.

Quantification of Total Glucosinolates and Isothiocyanates for Common Brassicaceous Vegetables Consumed in the US Market Using Cyclocondensation and Thiocyanate Ion Measurement Methods

Glucosinolates are a group of important sulfur and nitrogen containing plant secondary metabolites found in Brassicaceous vegetables that are associated with many health benefits. There is a growing interest in assessing dietary glucosinolates and their degradation products’ isothiocyanates exposure to humans. Determination of glucosinolates in plants is a very challenging task. In this study, the total glucosinolates from 22 commonly consumed Brassicaceous vegetables in the US market were determined by employing 1,2-benzenedithiol cyclocondensation for the determination of isothiocyanates (ITCs) from non-indole glucosinolates and a colorimetric method for the determination of thiocyanate ion (SCN−) from indole glucosinolates. The total glucosinolates were determined from the sum of these two methods. In the 22 investigated vegetables, the highest level of total ITCs was found in horse radish (5189.0 ± 544.8 nmol/g dry weight) and the highest level of total SCN− level was found in brussel sprouts (1294.0 ± 141.6 nmol/g dry weight). The total glucosinolates ranged from 18.6 ± 3.2 to 2271.2 ± 215.3 nmol/g dry weight. The total glucosinolate and ITC data are necessary for the USDA glucosinolates database and important for establishing the relationship of glucosinolates to human nutrition.

Various modes of action of dietary phytochemicals, sulforaphane and phenethyl isothiocyanate, on pathogenic bacteria

Isothiocyanates (ITCs) derived from cruciferous plants reveal antibacterial activity, although detailed mechanism is not fully elucidated. Recently it has been reported that ITCs induce the stringent response in Escherichia coli strains. The aim of this work was to determine whether two isothiocyanates, sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), similarly as in E. coli induce stringent response in Bacillus subtilis, model Gram(+) bacterium, and test their potency against a panel of clinical isolates belonging to Gram(+) or Gram(−) groups. Minimal inhibitory concentrations were determined as well as effect of ITCs on membranes integrity, synthesis of DNA, RNA and stringent response alarmones was assessed. SFN and PEITC are effective against B. subtilis and bacterial isolates, namely E. coli, K. pneumonia, S. aureus, S. epidermidis and E. faecalis. Interestingly, in B. subtilis and E. faecalis the inhibition of growth and nucleic acids synthesis is independent of ppGpp accumulation. In bacteria, which do not induce the stringent response in the presence of ITCs, membrane integrity disruption is observed. Thus, ITCs are effective against different pathogenic bacteria and act by at least two mechanisms depending on bacteria species.

Chemical Aspects of Biological Activity of Isothiocyanates and Indoles, the Products of Glucosinolate Decomposition.

There is growing evidence that cancer chemoprevention employing natural, bioactive compounds may halt or at least slow down the different stages of carcinogenesis. A particularly advantageous effect is attributed to derivatives of sulfur-organic phytochemicals, such as glucosinolates (GLs) synthesized mainly in Brassicaceae plant family. GLs are hydrolysed enzymatically to bioactive isothiocyanates (ITC) and indoles, which exhibit strong anti-inflammatory and anti-carcinogenic activity. Highly bioavailable electrophilic ITC are of particular interest, as they can react with nucleophilic groups of important biomolecules to form dithiocarbamates, thiocarbamates and thioureas. These modifications seem responsible for the chemopreventive activity, but also for genotoxicity and mutagenicity. It was documented that ITC can permanently bind to important biomolecules such as glutathione, cytoskeleton proteins, transcription factors NF-κB and Nrf2, thiol-disulfide oxidoreductases, proteasome proteins or heat shock proteins. Furthermore, ITC may also affect epigenetic regulation of gene expression, e.g. by inhibition of histone deacetylases. Some other derivatives of glucosinolates, especially indoles, are able to form covalent bonds with nucleobases in DNA, which may result in genotoxicity and mutagenicity. This article summarizes the current state of knowledge about glucosinolates and their degradation products in terms of possible interactions with reactive groups of cellular molecules.

Alyssin and Iberin in Cruciferous Vegetables Exert Anticancer Activity in HepG2 by Increasing Intracellular Reactive Oxygen Species and Tubulin Depolymerization

To determine the chemopreventive potential of alyssin and iberin, the in vitro anticancer activities and molecular targets of isothiocyanates (ITCs) were measured and compared to sulforaphane in hepatocellular carcinoma cell HepG2. The SR-FTIR spectra observed a similar pattern vis-à-vis the biomolecular alteration amongst the ITCs-treated cells suggesting a similar mode of action. All of the ITCs in this study cause cancer cell death through both apoptosis and necrosis in concentration dependent manner (20–80 μM). We found no interactions of any of the ITCs studied with DNA. Notwithstanding, all of the ITCs studied increased intracellular reactive oxygen species (ROS) and suppressed tubulin polymerization, which led to cell-cycle arrest in the S and G2/M phase. Alyssin possessed the most potent anticancer ability; possibly due to its ability to increase intracellular ROS rather than tubulin depolymerization. Nevertheless, the structural influence of alkyl chain length on anticancer capabilities of ITCs remains inconclusive. The results of this study indicate an optional, potent ITC (viz., alyssin) because of its underlying mechanisms against hepatic cancer. As a consequence, further selection and development of effective chemotherapeutic ITCs is recommended.

Effects of honokiol on activation of transient receptor potential channel in rat spinal dorsal root ganglion cells and pruritus in mouse models

Objective To evaluate the effect of honokiol on the activation of transient receptor potential (TRP) channel in rat spinal dorsal root ganglion cells and pruritus in mouse models. Methods Healthy male ICR mice aged 4-6 weeks were used to establish histamine-induced and acetone/ether/water (AEW) -induced itching models separately. Totally, mice were randomly divided into 7 groups (histamine-induced model experiment) or 6 groups (AEW-induced model experiment) : normal control group and model group both gavaged with sodium chloride physiological solution, solvent group gavaged with sodium carboxymethylcellulose solution, chlorphenamine group (only set up in the histamine-induced model experiment) gavaged with chlorphenamine, 50, 25 and 12.5 mg/kg honokiol groups gavaged with 50, 25 and 12.5 mg/kg honokiol respectively. In the histamine-induced model experiment, the mice were all injected with histamine except the normal control group injected with sodium chloride physiological solution 24 hours after the gavage treatment, while the mice in the AEW-induced model experiment were all topically treated with AEW except the normal control group topically treated with sodium chloride physiological solution for 4 days, followed by gavage with different drugs. The anti-itch effect of each treatment was evaluated by counting the scratching frequency within 30 minutes. Rat spinal dorsal root ganglion (DRG) cells were isolated and subjected to a primary culture. Then, the DRG cells were divided into 6 groups: capsaicin or allyl isothiocyanate (AITC) -induced model group pre-incubated with Hank′s balanced salt solution (HBSS) , 500 μmol/L capsazepine or 10 μmol/L HSC030031 group pre-incubated with capsazepine or HSC030031, solvent group pre-incubated with dimethyl sulfoxide (DMSO) , 3 honokiol groups pre-incubated with 7.81, 15.63 and 31.25 mg/L honokiol respectively, and Ca2+ fluorescence imaging system was used to observe changes of Ca2+ influx in these cells after capsaicin or AITC stimulation. Statistical analysis was carried out with SPSS 20.0 software by using one-way analysis of variance and Dunnett-t test. Results In the histamine-induced mouse models, the scratching frequency was significantly lower in the 50 and 25 mg/kg honokiol groups than in the model group (21.88 and 21.14 vs. 63.70, t=3.48, 3.49 respectively, both P=0.003) , while no significant difference in the scratching frequency was observed between the 12.5 mg/kg honokiol group and the model group (t=2.01, P=0.062) . After the treatment with 50 mg/kg honokiol in the AEW-induced mouse models, the scratching frequency significantly decreased compared with the model group (61.4 vs. 101.17, t=0.45, P=0.009) , while there were no significant differences among the 25, 12.5 mg/kg honokiol groups and the model group (all P > 0.05) . Compared with the capsaicin or AITC-induced model group, the increase of Ca2+ fluorescence signal in the DRG cells was significantly inhibited in the 31.25 mg/L honokiol group: at the 45th second, the rate of relative fluorescence intensity change (ΔF/F0) was 1.11 in the model group, but-0.11 in the 31.25 mg/L honokiol group in the capsaicin-induced model experiment, and 0.56 in the model group, but 0.00 in the 31.25 mg/L honokiol group in the AITC-induced model experiment. Conclusion Honokiol shows an inhibitory effect on mouse models of pruritus induced by histaminergic or non-histaminergic factors, likely by inhibiting Ca2+ influx through activated TRPV1 and TRPA1 channels in the DRG cells. Key words: Pruritus; Honokiol; Transient receptor potential channels; Disease models, animal

Influence of Cooking Methods on Glucosinolates and Isothiocyanates Content in Novel Cruciferous Foods.

Brassica vegetables are of great interest due to their antioxidant and anti-inflammatory activity, being responsible for the glucosinolates (GLS) and their hydroxylated derivatives, the isothiocyanates (ITC). Nevertheless, these compounds are quite unstable when these vegetables are cooked. In order to study this fact, the influence of several common domestic cooking practices on the degradation of GLS and ITC in two novel Brassica spp.: broccolini (Brassica oleracea var italica Group x alboglabra Group) and kale (Brassica oleracea var. sabellica L.) was determined. On one hand, results showed that both varieties were rich in health-promoter compounds, broccolini being a good source of glucoraphanin and sulforaphane (≈79 and 2.5 mg 100 g−1 fresh weight (F.W.), respectively), and kale rich in glucoiberin and iberin (≈12 and 0.8 mg 100 g−1 F.W., respectively). On the other hand, regarding cooking treatments, stir-frying and steaming were suitable techniques to preserve GLS and ITC (≥50% of the uncooked samples), while boiling was deleterious for the retention of these bioactive compounds (20–40% of the uncooked samples). Accordingly, the appropriate cooking method should be considered an important factor to preserve the health-promoting effects in these trending Brassica.

Natural and Synthetic Isothiocyanates Possess Anticancer Potential Against Liver and Prostate Cancer In Vitro.

Isothiocyanates (ITCs) are phytochemicals with potential cancer-preventative properties derived from the breakdown of glucosinolates that exist in cruciferous vegetables. Studies, to date, have demonstrated that various ITCs possess the ability to act as anticancer agents in different cancer types. This study investigated the anticancer properties of dietary ITCs (allyl-ITC, benzyl-ITC, phenylethyl-ITC) and synthetic (phenylbutyl-ITC and phenylhexyl-ITC) on liver and prostate carcinoma cells in vitro. The effects of ITCs on cellular viability, migration, invasion, clonogenicity, apoptosis induction and reactive oxygen species generation were assessed in HepG2, DU145 and 22Rv1 cells. All ITCs reduced metabolic activity in each cell line with the most significant being phenylethyl-ITC. Both dietary and synthetic ITCs suppressed the migratory and invasive potential of all cell lines, inhibited colony-forming capability and induced apoptosis. Phenylethyl-ITC exposure resulted in the significant generation of reactive oxygen species. These data highlight the potential advantages of utilizing ITCs to delay the carcinogenic process and the potential for dietary and synthetic ITCs to act as anticancer agents.

MFS transporter from Botrytis cinerea provides tolerance to glucosinolate-breakdown products and is required for pathogenicity

Glucosinolates accumulate mainly in cruciferous plants and their hydrolysis-derived products play important roles in plant resistance against pathogens. The pathogen Botrytis cinerea has variable sensitivity to glucosinolates, but the mechanisms by which it responds to them are mostly unknown. Exposure of B. cinerea to glucosinolate-breakdown products induces expression of the Major Facilitator Superfamily transporter, mfsG, which functions in fungitoxic compound efflux. Inoculation of B. cinerea on wild-type Arabidopsis thaliana plants induces mfsG expression to higher levels than on glucosinolate-deficient A. thaliana mutants. A B. cinerea strain lacking functional mfsG transporter is deficient in efflux ability. It accumulates more isothiocyanates (ITCs) and is therefore more sensitive to this compound in vitro; it is also less virulent to glucosinolates-containing plants. Moreover, mfsG mediates ITC efflux in Saccharomyces cerevisiae cells, thereby conferring tolerance to ITCs in the yeast. These findings suggest that mfsG transporter is a virulence factor that increases tolerance to glucosinolates.

Glucosinolates metabolism and redox state of rocket ( Eruca sativa Mill.) during germination

Isothiocyanates (ITCs) exhibit anticancer and antioxidant activity. ITCs are formed from the catalysis of glucosinolates (GLs) by myrosinase (MYR) in Brassicaceae vegetables. This study investigated the GL content, MYR activity, ITC formation, and redox state of rocket upon germination. Banye and Dongsheng were selected from four cultivars by determining growth state and ITC metabolism. In both cultivars, GL levels were the highest on the second day of germination and significantly declined on the eighth day. The 4-day-old sprouts had the highest ITC level. MYR exhibited maximal activity in Banye and Dongsheng on the fourth and the second day of germination, respectively. Regarding the antioxidant system, the sprouts had more chlorophyll, total phenol, and anthocyanin content, and they had higher DPPH scavenging effects compared with raw seeds. Moreover, Dongsheng showed greater scavenging capacity than Banye in general. Practical applications Rocket belongs to the Brassicaceae family, which has been widely considered a good source of bioactive components to prevent tumor formation and reduce the incidence of cardiovascular disease and cancer given the abundance of glucosinolate (GLs). GLs could be catalyzed by myrosinases (MYR) and converted into biologically active products including isothiocyanates (ITCs). After germination, MYR is activated and more ITCs are produced. Using rocket sprouts as a daily diet or raw material is beneficial for human health.

Dynamic variation of glucosinolates and isothiocyanates in broccoli sprouts during hydrolysis

In this study, the glucosinolates (GLs) content and isothiocyanates (ITCs) formation in broccoli sprouts and their dynamic variation during hydrolysis were investigated. The results showed that 7 kinds of GLs were detected and glucoraphanin (GRA) was the dominating component. After hydrolysis, three kinds of ITCs and 2 kinds of nitriles were detected in broccoli sprouts, however, the corresponding nitriles of 4-isothiocyanato-1-butene and 1-isothiocyanato-butane were not detectable. This inferred that the inter-transformation among ITCs existed during sprouts hydrolysis. According to the identification, 4-isothiocyanato-1-butene had another source in addition to gluconapin (GNA), 1-isothiocyanato-butane stemmed from glucoerucin (GER). Moreover, the affinity of MYR in broccoli sprouts was stronger to GER than to GRA and other GLs.

Impact of Indian mustard growth and incorporation on annual weed population dynamics and communities

SummaryBiofumigation from Brassica cover crops may be used to control soilborne pests and weeds. A study was conducted to understand the influence of biofumigation on key processes of annual weed population dynamics. Five combinations of Indian mustard (M) and oat (O) cover crop treatments were assessed in a 3 year field study at two locations in Québec, Canada. Treatments included four spring/fall cover crop combinations (M/M, M/O, O/M, O/O) and a weedy check control with no cover crop. Prior to mowing and incorporation of cover crops, weed identification, count and biomass measurements were recorded to evaluate the total weed density, to calculate the relative neighbour effect (RNE) and weed diversity metrics and to perform principal co‐ordinates analyses. Indian mustard cover crops had no impact on weed establishment in 2014 due to low biofumigant potential compared to the oat cover crop. In 2015 and 2016, Indian mustard isothiocyanate (ITC) production increased and weed establishment within the Indian mustard cover crop decreased. Moreover, post‐cover crop incorporation decreased the next year spring weed emergence. Allelopathic interference of Indian mustard was significant when plant tissues produced more than 600 μg of allyl‐ITC g−1. It is now possible to rationalise the use of Brassica cover crops and biofumigation for weed control with an enhanced understanding of the impact of biofumigation on key processes of weed population dynamics.

Effect of silencing troponin I3 gene expression on biological property of rat embryonic H9C2 cardiomyocytes

Objective To investigate the effect of silencing troponin I3 (Tnni3) gene expression on biological property of rat embryonic H9C2 cardiomyocytes. Methods The rat embryonic H9C2 cardiomyocytes were cultured and divided into 2 groups: control group transfected with negative control small interfering RNA (NC-siRNA group) and experimental group transfected with Tnni3 small interfering RNA (Tnni3-siRNA group). At 48 h, 72 h after transfection, the cells were collected, and real time quantitative polymerase chain reaction (qPCR)was used to detect the mRNA expressions of Tnni3 and Caspase-3, and Western blot was used to detect the protein expressions of Tnni3, Cyclin A1 and Cyclin B1.Annexin V-fluorescein isothiocyanate(FITC) apoptosis detection kit was used to analyze cell apoptosis.Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) solution and cell cycle was detected by flow cytometry. Results At 48 h post-transfection with Tnni3-siRNA, H9C2 cells exhibited a significant decrease in Tnni3 mRNA (0.27±0.05 vs. 1.00±0.00) and protein (0.18±0.03 vs. 1.00±0.00) compared with those transfected with NC-siRNA, and the differences were statistically significant (t=25.26, 47.40, all P<0.01). Apoptotic cells were observed in the NC-siRNA group and the Tnni3-siRNA group.At 72 h post-transfection, the percentage of apoptotic cells significantly increased in H9C2 cells transfected with Tnni3-siRNA [(11.30±1.85)% vs. (0.33±0.15)%] compared with those transfected with NC-siRNA, an increased expression of Caspase-3 mRNA was also observed in Tnni3-siRNA-transfected H9C2 cells (1.39±0.13 vs. 1.00±0.00), and the differences were statistically significant (t=10.24, 5.19, all P<0.01). Compared with NC-siRNA-transfected H9C2 cells, a time-dependent reduction in cell proliferation was observed in Tnni3-siRNA-transfected H9C2 cells (48 h: 0.32±0.06 vs. 0.46±0.03; 72 h: 0.31±0.01 vs. 0.63±0.04; 96 h: 0.36±0.01 vs 0.75±0.04), and the differences were statistically significant (t=3.62, 13.45, 16.39, all P<0.01). At 72 h post-transfection with Tnni3-siRNA, the percentage of G1 phase, S phase and G2 phase cells was (71.25±3.82)%, (18.28±2.78)% and (9.94±1.09)%, respectively.There was a significant increase in the proportion of G2 phase cells [(9.94±1.09)% vs. (4.54±0.99)%] in H9C2 cells transfected with Tnni3-siRNA compared with those transfected with NC-siRNA, an increased expression of Cyclin A1 protein (1.89±0.09 vs.1.00±0.00) and a decreased expression of Cyclin B1 protein (0.47±0.06 vs.1.00±0.00) were observed in Tnni3-siRNA-transfected H9C2 cells, respectively, and the differences were statistically significant (t=6.35, 17.12, 15.32, all P<0.01). Conclusions Silencing Tnni3 gene expression in rat embryonic H9C2 cardiomyocytes can induce cell apoptosis, suppress cell proliferation, and led to G2 cell cycle arrest. Key words: Troponin I3 gene; Cell apoptosis; Cell proliferation; Cell cycle

The structure of Trametes versicolor glutathione transferase Omega 3S bound to its conjugation product glutathionyl-phenethylthiocarbamate reveals plasticity of its active site.

Trametes versicolor glutathione transferase Omega 3S (TvGSTO3S) catalyzes the conjugation of isothiocyanates (ITC) with glutathione (GSH). Previously, this isoform was investigated in depth both biochemically and structurally. Structural analysis of complexes revealed the presence of a GSH binding site (G site) and a deep hydrophobic binding site (H site) able to bind plant polyphenols. In the present study, crystals of apo TvGSTO3S were soaked with glutathionyl-phenethylthiocarbamate, the product of the reaction between GSH and phenethyl isothiocyanate (PEITC). On the basis of this crystal structure, we show that the phenethyl moiety binds in a new site at loop β2 -α2 while the glutathionyl part exhibits a particular conformation that occupies both the G site and the entrance to the H site. This binding mode is allowed by a conformational change of the loop β2 -α2 at the enzyme active site. It forms a hydrophobic slit that stabilizes the phenethyl group at a distinct site from the previously described H site. Structural comparison of TvGSTO3S with drosophila DmGSTD2 suggests that this flexible loop could be the region that binds PEITC for both isoforms. These structural features are discussed in a catalytic context.

Moringin from Moringa Oleifera Seeds Inhibits Growth, Arrests Cell-Cycle, and Induces Apoptosis of SH-SY5Y Human Neuroblastoma Cells through the Modulation of NF-κB and Apoptotic Related Factors.

In the last decades, glucosinolates (GLs), precursors of isothiocyanates (ITCs), have been studied mostly for their chemopreventive and chemotherapeutic properties. The aim of our research was to study the antiproliferative effect of 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate (glucomoringin; GMG) bioactivated by myrosinase enzyme to form the corresponding isothiocyanate 4-(α-L-rhamnopyranosyloxy) benzyl C (moringin) in SH-SY5Y human neuroblastoma cells. We found that moringin significantly reduced SH-SY5Y cell growth in a time and concentration-dependent (p < 0.05, 0.01, and 0.001 vs. ctrl, after treatment with 16.4 µM moringin for 24, 48, and 72 h, respectively) manner through a mechanism involving the activation of apoptotic machinery. In addition, it altered the normal progression of cells through the cell cycle, increasing the cell population in both G2 and S phases, as well as decreasing that in the G1 phase. Studying the drug mechanism of action, we found that moringin was able to increase the expression of p53, p21, and Bax at both the protein and transcriptional level. Moreover, exposure of SH-SY5Y cells to moringin significantly increased the gene expression of both caspase 3 and 9 and enhanced their cleavage, thereby initiating an intrinsic apoptotic cascade. Finally, moringin inhibited nuclear translocation of NF-κB. Our study demonstrates the ability of moringin to reduce the growth of SH-SY5Y cells and reveals its mechanism of action, suggesting its promising role as an anticancer drug.