Experimental carcinogenesis with 7, 12-dimethylbenz(a)anthrazene (DMBA) and its inhibition with isothi-ocyanates / Carcinogénesis experimental con 7, 12-dimetilbenzantraceno (DMBA) y su inhibición con isitiocianatos

Introduction: DMBA is a chemical carcinogen that induces carcinomas within a few weeks of its application. We developed an experimental model of carcinogenesis induced by DMBA dissolved in 0,5% paraffin oil (DMBA-PO), verifying the inhibitory effect of the carcinogenicity of phenyl isothiocyanate (PhITC), phenethyl (PhnITC) and benzyl isothiocyanate (BITC). Material and Methods: For this, 88 hamsters were distributed into three groups: one exposed to DMBA-PO (Group 1, n=12), three subgroups (n=12) exposed to PhITC, PhnITC, BITC and DMBA-PO (Group 2, n=36) and four control subgroups (n=10) that were not exposed to the carcinogen in which PO (paraffin oil) and isothiocyanates were applied (Group 3, n=40). Results: The experiment had a duration of 20 weeks, at the end of which the inhibitory effect was established by comparing the lesions developed in the groups that received isothiocyanates with the group that was only treated with DMBA-PO. The carcinogenic effect of DMBA-PO is 100% (35 carcinomas) and the inhibitory effect was 0, whereas in the presence of isothiocyanates the carcinogenic effect decreases, with an inhibitory effect of 86% for BITC (5 carcinomas) and 74% for PhITC (9 carcinomas). Conclusion: The inhibitory effect for PhnITC is 80% in relation to invasive OSCC (1 carcinoma).

Introducción: El DMBA es un carcinógeno químico que induce carcinomas a las pocas semanas de su aplicación. Desarrollamos un modelo experimental de carcinogénesis inducida por DMBA disuelto en aceite de parafina al 0,5% (DMBA-Ap) comprobando el efecto inhibidor de la carcinogénesis de los isotiocianatos fenil (PhITC), fenetil (PhnITC) y bencil isotiocianato (BITC). Material y Métodos: Para ello, se distribuyeron 88 hámsteres en 3 grupos: uno expuesto al DMBA-Ap (Grupo 1, n=12), tres subgrupos (n=12) expuestos a PhITC, PhnITC, BITC y DMBA-Ap (Grupo 2, n=36) y cuatro subgrupos controles (n=10), no expuestos al carcinógeno en el que se aplicaron Ap e isotiocianatos (Grupo 3, n=40). Resultados:El experimento tuvo una duración de 20 semanas, al final de la cual se establece de forma comparativa el efecto inhibidor comparando las lesiones desarrolladas en los grupos que recibieron isotiocianatos con respecto al grupo tratado sólo con DMBA-Ap. El efecto carcinógeno del DMBA-Ap es del 100% (35 carcinomas) y el efecto inhibidor 0, mientras que en presencia de isotiocianatos el efecto carcinógeno disminuye, con un efecto inhibidor del 86% para BITC (5 carcinomas) y del 74% para el PhITC (9 carcinomas). Conclusión:El efecto inhibidor del PhnITC es del 80% en relación con el COCE invasivo (1 carcinoma).

Biofumigation with species of the Brassicaceae family: a review / Biofumigação com espécies da família Brassicaceae: uma revisão

ABSTRACT: Biofumigation involves the release of volatile biocidal compounds in the soil through the incorporation of certain plants and their residues. Species of the Brassicaceae family are the most widely used plants for biofumigation. These plants contain glucosinolates, which produce compounds, such as isothiocyanates, following enzymatic hydrolysis, with scientifically proven fungicidal effects. The most commonly used brassica species belong to the genera Brassica, Raphanus, Sinapis, and Eruca. In addition to the release of compounds in the soil, complementary mechanisms, such as the supply of organic matter and nutrients, and improvement of the soil structure, also play a role in biofumigation. In the past two decades, several studies on the use of brassica residues in biofumigation have been published, showing promising results in the management of soil pathogens (fungi and oomycetes, nematodes, bacteria, and protozoa), weed seeds, and insects. Usage of new biofumigation compounds has also been validated in recent years, including the development of patented technological products such as liquid formulations and pellets. The objective of this article was to review these new developments, beginning with concepts related to biofumigation, and to discuss the mechanisms of action of compounds involving brassica species and the recommendations on usage. Promising examples of the use of this technique are also presented, further detailing the advances in basic and applied knowledge on the subject.

RESUMO: A biofumigação consiste na liberação de compostos biocidas voláteis no solo a partir da incorporação de determinadas plantas e de seus resíduos. As espécies da família Brassicaceae são as plantas mais utilizadas na biofumigação. Em sua constituição, possuem os glucosinolatos que, após hidrólise enzimática, produzem compostos como os isotiocianatos com efeito biofungicida comprovado cientificamente. As espécies de brássicas mais utilizadas pertencem aos gêneros Brassica, Raphanus, Sinapis e Eruca. Além da liberação de compostos no solo, mecanismos complementares como o fornecimento de matéria orgânica, nutrientes e melhoria da estrutura do solo, também desempenham papel complementar na biofumigação. Diversos estudos foram publicados nas últimas duas décadas com a utilização de resíduos de brássicas na biofumigação e apresentaram resultados promissores no manejo de patógenos de solo (fungos e oomicetos, nematóides, bactérias e protozoários), sementes de plantas daninhas e insetos. Novas formas de utilização também foram validadas nos últimos anos, inclusive com o desenvolvimento de produtos tecnológicos patenteados como formulações líquidas e pellets. Nesta revisão, objetivamos apresentar estes novos desdobramentos iniciando com os conceitos relacionados à biofumigação. Em seguida, apresentamos os mecanismos de ação e compostos envolvidos; as espécies de brássicas, produtos e recomendações para sua utilização; e exemplos promissores de adoção da técnica a nível mundial. Pretende-se, dessa forma, detalhar os avanços no conhecimento básico e aplicado do assunto.

Antitumor activities of new iso(thio)cyanates and their nitrogen and sulphur heterocyclic phosphorus derivatives

The cytotoxic activity of Iso(thio)cyanate derivatives and some new organophosphorus compounds were determined,using MTT assay against human hepatocellular carcinoma (HepG2) and adenocarcinoma breast (MCF-7) incomparison with the reference drug 5-flurouracil. All the selected products have been tested and showed concentrationdependent increase in the growth inhibition percentage against HepG2 and MCF-7. Where, the thietane derivativesrevealed anticancer activity with IC50 (20, 8.9 µg/ml) against HepG2 and (20, 10.3 µg/ml) against MCF-7; while,thiazinane compounds showed IC50 (12.7, 32.5 µg/ml) against HepG2 and (20 and 20 µg/ml) against MCF7. The newly synthesized azetidines showed anticancer activity with IC50 (13.5 and 32.5 µg/ml) against HepG2and IC50 (10 and 25.9 µg/ml) against MCF-7 cancer. Moreover, azetidinedione compound exhibited more potentactivity than the azetidinone with both types of cancer cell lines. In addition, the cytotoxic activity of the iso(thio)cyanates, and malonamic acid methyl ester compound were also investigated. 4-Methoxyphenyl isothiocyanate andmethylisothiocyanate, with IC50 (25.9, 12.3 and 40, 20 µg/ml), respectively, against HepG2 and MCF-7 cancer cells.1, 2-Dichloro-4-isocyanato-benzene was similar in potency to the known anticancer drug 5-flurouracil with IC50 (5.3µg/ml) versus 5 µg/ml for 5-flurouracil against MCF-7. While, malonamic acid methyl ester compound had no effecton both types of cancer cell lines.

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.

Current Progress on The Neuroprotective Effects of Isothiocyanates in The Treatment for Experimental Parkinson's Disease / 中国药学杂志

Parkinson's disease (PD) is recognized as the second most common neurodegenerative disorder and is characterized by a slow and progressive degeneration of dopaminergic neurons in the substantia nigra. Despite intensive research, the mechanisms involved in neuronal loss are not completely understood yet; however, misfolded proteins, oxidative stress, excitotoxicity and inflammation play a pivotal role in the progression of the pathology. To date, no efficient therapy that arrest or slow down PD is available. Isothiocyanates (ITCs) have already shown interesting properties in detoxification, inflammation, apoptosis and cell cycle regulation through the induction of phase and phase Ⅱ enzyme. Moreover, ITCs may be able to modulate several key points in oxidative and inflammatory evolution. In this review, we describe ITCs as pleiotropic compounds capable of preventing and modulating the evolution of PD. In particular, we summarized the neuroprotective effects of sulforaphane (SFN), erucin (ER), phenethyl isothiocyanate (PEITC) and 6-(methylsulfinyl) hexyl isothiocyanate (6-MSITC) in the treatment for experimental PD.

A nested case-control study of cruciferous vegetables intake, urinary isothiocyanates level and lung cancer risk among men in urban Shanghai / 中华预防医学杂志

Objective@#To investigate the association between consumption of cruciferous vegetables (CV), level of urinary isothiocyanates (ITC) and the risk of lung cancer among man in urban Shanghai.@*Methods@#A nested case-control study was conducted within the Shanghai Men's Health Study. Using incidence density sampling with a 2∶1 control to case selection ratio, 885 controls were selected to match 443 lung cancer cases diagnosed prior December 31, 2010. A food-frequency questionnaire was administered to estimate CV consumption. The high performance liquid chromatography method was applied to measure urinary ITC level. The CV intake and urinary ITC level were divided into quartiles according to distribution of control group. The lowest quartile was as a reference group. Conditional logistic regression model was used to analyze the relationship between CV intake, urinary ITC level and the risk of lung cancer.@*Results@#The cruciferous vegetables intake median (P25, P75) in cases and controls were 80.05 (46.89, 129.04) and 97.68 (55.25, 151.72) g/d (Z=-3.93, P<0.001). The urinary ITC level were 1.256 (0.474, 3.836) and 1.244 (0.484, 3.004) μmol/g Cr (Z=-0.39, P=0.697). After adjusting for potential confounding factors such as age, education level, smoking and alcohol consumption, for urinary ITC level, the OR(95%CI) for the highest quartile(≥3.004 μmol/g Cr) was 1.25 (0.87-1.80) compared with the lowest quartile(<0.484 μmol/g). For CV intake, the OR(95%CI) for the highest quartile(≥151.71 g/d) was 0.66 (0.43-1.02) compared with the lowest quartile(<55.25 g/d).@*Conclusion@#No association was found between the CV intake, urinary ITC level and lung cancer risk in men.

Sulforaphene in Raphanus sativus L.var.caudatus Alef increased in late-bolting stage as well as anticancer activity

Objectives:To evaluate the concentration differences of sulforaphene and sulforaphane at various ages and in different pairs of Raphanus sativus L.var.caudatus with respect to their potential cancer preventive effect on HCT116 colon cancer cells.Methods:FTIR-ATR and GC-MS were used to characterize the isothiocyanates in the plant extracts followed by HPLC for quantification.Antiproliferation and apoptosis induction were determined by using MTT assay and flow cytometry,respectively.Results:The respective rank of anticancer activity ofRaphanus sativus were as follows:vegetative (3 week) ＜ older rosette (4 week) ＜ early-bolting (5 week) ＜ senescence (7 week) ＜ late-bolting (6 week).The low to high concentration of sulforaphene and sulforaphane occurred in the same stage order.Conclusions:The reproductive parts (flower,pod,and dry seed) of Raphanus sativus have the greatest isothiocyanate concentration,evidenced by a sulforaphene concentration higher than the sulforaphane.This result should inform the selection of the most appropriate harvesting stage and plant part for use as a potential chemopreventive agent.

Sulforaphene in Raphanus sativus L. var. caudatus Alef increased in late-bolting stage as well as anticancer activity

Objectives To evaluate the concentration differences of sulforaphene and sulforaphane at various ages and in different parts of Raphanus sativus L. var. caudatus with respect to their potential cancer preventive effect on HCT116 colon cancer cells. Methods FTIR–ATR and GC–MS were used to characterize the isothiocyanates in the plant extracts followed by HPLC for quantification. Antiproliferation and apoptosis induction were determined by using MTT assay and flow cytometry, respectively. Results The respective rank of anticancer activity of Raphanus sativus were as follows: vegetative (3 week) < older rosette (4 week) < early-bolting (5 week) < senescence (7 week) < late-bolting (6 week). The low to high concentration of sulforaphene and sulforaphane occurred in the same stage order. Conclusions The reproductive parts (flower, pod, and dry seed) of Raphanus sativus have the greatest isothiocyanate concentration, evidenced by a sulforaphene concentration higher than the sulforaphane. This result should inform the selection of the most appropriate harvesting stage and plant part for use as a potential chemopreventive agent.

Inhibition effects of sulforaphane-induced autophagy flux to cell proliferation on ex vivo human capsular bags / 中华实验眼科杂志

Background Sulforaphane (SFN) is an effective chemopreventive agent and can regulate the biological molecular mechanisms to inhibit the overgrowth of cells.Autophagy is a biological process of maintaining cellular internal environment.Understanding the affection of SFN to biological behavior of human lens epithelial cells (LECs) and the association of SFN with autophagy is helpful for the prevention and target treatment of posterior capsule opacification (PCO).Objective This study was to investigate the eradication effeccts of SFN on residual lens cell population in vitro posterior capsule opacification (PCO) model and evaluate the mechanism of SFN-induced cell death.Methods In vitro human capsular bag models were generated from fresh donor eyes by phacoemulsification and were cultured in EMEM containing 2％ fetal bovine serum (FBS).Different concentrations of SFN (0,1,10 and 100 μ mol) were added in the medium for 30 days respectively according to grouping,and the growth of LECs was observed by optical microscope and immunofluorescence technique.FHL124,a human LEC line,was cultured with EMEM containing 5％ FBS and divided into 0,1,10,30 and 100 μmol SFN groups.Lactate dehydrogenase (LDH) release rate in the medium was detected to evaluate cell damage/death.The migration of the cells on capsular bags was assessed by scratch test.The ultrastructure and number of autophagosomes were examined under the transmission electron microscope.The expression of LC3 in the cells were detected using Western blot in the presence or absence of autophagy inhibitors.Results The cell coverage rates on the capsular bags were significantly lower in the 10 and 100 μ mol/L SFN groups than those in the 0 and 1 μmol/L SFN groups,with a statistically significant difference among the groups (F =48.57,P ＜ 0.01).Immunofluorescence showed that the density of F-actin-and Vimentin-positive cells was evidently decreased in the 10 and 100 μmol/L SFN groups compared with 0 and 1 μ mol/L SFN groups.The releasing levels of LDH (absorbancy) were 0.19± 0.03,0.39±0.06,0.56±0.07,0.68±0.08 and 0.89±0.09 in the 0,1,10,30 and 100 μ mol/L SFN groups,respectively,and the releasing level of LDH was gradually increased in the 10 and 100 μ mol/L SFN groups in comparison with the 1 μmol/LSFN group (all at P＜0.01).With the increase of SFN concentration,the reduction rate of scratched area decreased with the increase of SFN concentration,and the decrease of scratch area was significantly lower than that of adjacent low mass concentration group and the differences were statistically significant (P＜0.05).The relative expressions of LC3-Ⅱ protein were 0.423±0.003,14.543±0.024,0.668±0.024 and 0.576±0.056 in the blank control group,SFN group,SFN + 3-MA group and 3-MA group,respectively,and the relative expressions of LC3-Ⅱ protein were significantly lower in the SFN+3-MA group and 3-MA group than those in the SFN group (all at P＜0.01).The number of autophagosomes was 4.07±0.32,4.13±0.34,9.21 ±0.53 and 21.02± 1.34 in the blank control group,and 1,10,100 μmol/L SFN groups,and the number of autophagosomes in the 10 and 100 μ mol/L SFN groups was significantly higher than that in the blank control group and 1 μmol/L SFN group (all at P＜0.01).Conclusions SFN mediates LECs death by promoting autophagy in ex vivo capsular bags,and SFN may be a novel agent of potential chemopreventive and target treatment for PCO.

Sulforaphene and sulforaphane in commonly consumed cruciferous plants contributed to antiproliferation in HCT116 colon cancer cells

Objective: To analyze two isothiocyanates (sulforaphene and sulforaphane) and their antiproliferative effect of 11 indigenous cruciferous vegetables. Methods: Phytoconstituents identification was conducted by high performance liquid chromatography and gas chromatography-mass spectrometer techniques. The antiproliferation was evaluated in colon cancer cell line HCT116 by MTT assay. Results: Isothiocyanate identification by high performance liquid chromatography showed that broccoli, cabbage, "Khi-Hood" (Raphanus sativus L. var. caudatus Alef) and Chinese radish contained isothiocyanates sulforaphane. Sulforaphene and sulforaphane in broccoli, cabbage and "Khi-Hood" were characterized by the gas chromatography-mass spectrometer analysis. Antiproliferation screening by MTT assay found that the potent plants which possessed IC

Protection of sulforaphane against hydrogen peroxide-induced bovine trabecular meshwork cell apoptosis / 中华实验眼科杂志

Background Evidences indicated that oxidative stress damage is an essential pathological process in primary open angle glaucoma.Sulforaphane (SFN) can play an antioxidative stress role to many tissues and cells by activating Nrf2/ARE single pathway.However,whether SFN has a protective role to oxidative stress induced damage of trabecular meshwork cells is still unclear.Objective This study was to investigate the antioxidant effect of SFN against H2O2-induced oxidative damage in bovine trabecular meshwork cells.Methods Trabecular cells were isolated from fresh black bovine eyeballs and primarily cultured and passaged.The third generation of cells were incubated to 96-well dish at a density of 1 ×103/well for 24 hours and divided into 4 groups.The cells were incubated using 100 μl serum-free medium in the blank control group.Oxidative damage models were established by adding 100 μmol/L H2O2(100 μl) in medium in the H2O2 group.The cells were cultured with the medium containing 10 μmol/L SFN (100 μl) in the SFN group,and 100 μl H2O2 at the final concentration of 100 μmol/L was added in the SFN-treated cell medium in the SFN +H2O2 group.The cell vitality in various groups was assayed by using cell counting kit-8 (CCK-8).The apoptosis rate of the cells was detected by Annexin V-FITC/PI double-staining with flow cytometry.Results Cultured cells showed a spindle shape with uniform size,abundant cytoplasm,numberous pigmented particles and big nucleolus.The relative cell vitality reduced to (67.00± 1.27)％ and (80.00±6.25)％ in the H2O2 group and SFN+H2O2 group in comparison with 100％ in the blank control group,and the cell vitality in the SFN+ H2O2 group was lower than that in the SFN group but higher than that in the H2 O2 group (both at P＜0.01).The mean apoptosis rate was (11.33 ±0.77) ％,(32.31 ± 1.03) ％,(10.44 ±0.68) ％ and (17.68 ±0.21) ％ in the blank control group,H2 O2 group,SFN group and SFN+H2O2 group,respectively,showing a significant difference among the groups (F=539.96,P＜0.01),and the apoptosis rate in the SFN+H2O2 group was significantly lower than that in the H2O2 group but higher than that in the blank control group and SFN group (all at P＜0.01).Conclusions SFN can improve the antioxidative stress ability of trabecular meshwork cells and alleviate the damage induced by oxidative stress.

Analysis of a suppressive subtractive hybridization library of Alternaria alternata resistant to 2-propenyl isothiocyanate

Background Isothiocyanates (ITCs) are natural products obtained from plants of the Brassicas family. They represent an environmentally friendly alternative for the control of phytopathogenic fungi. However, as it has been observed with synthetic fungicides, the possibility of inducing ITC-resistant strains is a major concern. It is, therefore, essential to understanding the molecular mechanisms of fungal resistance to ITCs. We analyzed a subtractive library containing 180 clones of an Alternaria alternata strain resistant to 2-propenyl ITC (2-pITC). After their sequencing, 141 expressed sequence tags (ESTs) were identified using the BlastX algorithm. The sequence assembly was carried out using CAP3 software; the functional annotation and metabolic pathways identification were performed using the Blast2GO program. Results The bioinformatics analysis revealed 124 reads with similarities to proteins involved in transcriptional control, defense and stress pathways, cell wall integrity maintenance, detoxification, organization and cytoskeleton destabilization; exocytosis, transport, DNA damage control, ribosome maintenance, and RNA processing. In addition, transcripts corresponding to enzymes as oxidoreductases, transferases, hydrolases, lyases, and ligases, were detected. Degradation pathways for styrene, aminobenzoate, and toluene were induced, as well as the biosynthesis of phenylpropanoid and several types of N-glycan. Conclusions The fungal response showed that natural compounds could induce tolerance/resistance mechanisms in organisms in the same manner as synthetic chemical products. The response of A. alternata to the toxicity of 2-pITC is a sophisticated phenomenon including the induction of signaling cascades targeting a broad set of cellular processes. Whole-transcriptome approaches are needed to elucidate completely the fungal response to 2-pITC.

Effect of sulforaphane on human small cell lung cancer NCI-H446 cells proliferation, invasion and the activity of MMP-9 / 中国医师杂志

Objective To investigate the effects of sulforaphane (SFN) on proliferation and invasion of human small cell lung cancer NCI-H446 and the activity of matrix metalloproteinase (MMP) -9.Methods NCI-H446 cells were cultured with 0,25,50,100 μmol/L SFN for 24 ～ 72 h,then MTT assay was employed to detect cell proliferation.Chamber invasion assay was used to study the cell invasion,and gelatin zymography assay was implied in MMP-9 enzyme activity.Results After treatment of 25,50,100μmol/L SFN,the growth of NCI-H446 cells were inhibited.When cells were incubated with 25,50,100μmol/L of SFN for 72h,the inhibition ratio was ( 11.1 ± 2.26 ) ％,( 25.2 ± 3.24 ) ％ and ( 44.6 ±4.2) ％,respectively,the difference was statistically significant compared with the solvent control group ( t =10.685,8.417,5.264,P ＜0.05 ).Chamber invasion assay showed that NCI-H446 cell invasion could be reduced.25,50,100 μmol/L of SFN could decrease the trans-membrane cells to (48.6 ± 1.84)％,(35.4 ± 2.22) ％ and (27.8 ± 1.36) ％,and it was statistically significant compared with the solvent control group ( t =6.341,5.562,4.925,P ＜0.05 ),respectively.In addition,MMP-9 activity was significantly inhibited by SFN.25,50,100 μmol/L of SFN could decrease the gray value of MMP-9 to 764 ±18.4,685 ± 14.74 and 638 ± 21.54 ( control group 822 ± 12.53,t =4.971,7.582,11.235,respectively,P ＜0.05).Conclusions SFN can inhibit NCI-H446 cells growth,invasion and the activity ofMMP-9.