Glucosinolates (GSLs) are plant secondary metabolites consisting of sulfur and nitrogen, commonly found in Brassicaceae crops, such as Arabidopsis thaliana. These compounds are known for their roles in plant defense mechanisms against pests and pathogens. 'Guilt-by-association' (GBA) approach predicts genes encoding proteins with similar function tend to share gene expression pattern generated from high throughput sequencing data. Recent studies have successfully identified GSL genes using GBA approach, followed by targeted verification of gene expression and metabolite data. Therefore, a GSL co-expression network was constructed using known GSL genes obtained from our in-house database, SuCComBase. DPClusO was used to identify subnetworks of the GSL co-expression network followed by Fisher's exact test leading to the discovery of a potential gene that encodes the ARIA-interacting double AP2-domain protein (ADAP) transcription factor (TF). Further functional analysis was performed using an effective gene silencing system known as CRES-T. By applying CRES-T, ADAP TF gene was fused to a plant-specific EAR-motif repressor domain (SRDX), which suppresses the expression of ADAP target genes. In this study, ADAP was proposed as a negative regulator in aliphatic GSL biosynthesis due to the over-expression of downstream aliphatic GSL genes (UGT74C1 and IPMI1) in ADAP-SRDX line. The significant over-expression of ADAP gene in the ADAP-SRDX line also suggests the behavior of the TF that negatively affects the expression of UGT74C1 and IPMI1 via a feedback mechanism in A. thaliana.
Glucosinolates (GSLs) are plant secondary metabolites comprising sulfur and nitrogen mainly found in plants from the order of Brassicales, such as broccoli, cabbage, and Arabidopsis thaliana. The activated forms of GSL play important roles in fighting against pathogens and have health benefits to humans. The increasing amount of data on A. thaliana generated from various omics technologies can be investigated more deeply in search of new genes or compounds involved in GSL biosynthesis and metabolism. This review describes a comprehensive inventory of A. thaliana GSLs identified from published literature and databases such as KNApSAcK, KEGG, and AraCyc. A total of 113 GSL genes encoding for 23 transcription components, 85 enzymes, and five protein transporters were experimentally characterized in the past two decades. Continuous efforts are still on going to identify all molecules related to the production of GSLs. A manually curated database known as SuCCombase (http://plant-scc.org) was developed to serve as a comprehensive GSL inventory. Realizing lack of information on the regulation of GSL biosynthesis and degradation mechanisms, this review also includes relevant information and their connections with crosstalk among various factors, such as light, sulfur metabolism, and nitrogen metabolism, not only in A. thaliana but also in other crucifers.
Cruciferous vegetables are a rich source of glucosinolates that have established anti-carcinogenic activity. Naturally-occurring glucosinolates and their derivative isothiocyanates (ITCs), generated as a result of their enzymatic degradation catalysed by myrosinase, have been linked to low cancer incidence in epidemiological studies, and in animal models isothiocyanates suppressed chemically-induced tumorigenesis. The prospective effect of isothiocyanates as anti-carcinogenic agent has been much explored as cytotoxic against wide array of cancer cell lines and being explored for the development of new anticancer drugs. However, the mechanisms of isothiocyanates in inducing apoptosis against tumor cell lines are still largely disregarded. A number of mechanisms are believed to be involved in the glucosinolate-induced suppression of carcinogenesis, including the induction of apoptosis, biotransformation of xenobiotic metabolism, oxidative stress, alteration of caspase activity, angiogenesis, histone deacytylation and cell cycle arrest. The molecular mechanisms through which isothiocyanates stimulate apoptosis in cancer cell lines have not so far been clearly defined. This review summarizes the underlying mechanisms through which isothiocyanates modify the apoptotic pathway leading to cell death.
Sinigrin, a precursor of allyl isothiocyanate, present in the Raphanus sativus exhibits diverse biological activities, and has an immense role against cancer proliferation. Therefore, the objective of this study was to quantify the sinigrin in the R. sativus roots using developed and validated RP-HPLC method and further evaluated its' anticancer activity. To achieve the objective, the roots of R. sativus were lyophilized to obtain a stable powder, which were extracted and passed through an ion-exchange column to obtain sinigrin-rich fraction. The RP-HPLC method using C18 analytical column was used for chromatographic separation and quantification of sinigrin in the prepared fraction, which was attained using the mobile phase consisting of 20 mM tetrabutylammonium: acetonitrile (80:20%, v/v at pH 7.0) at a flow rate of 0.5 mL/min. The chromatographic peak for sinigrin was showed at 3.592 min for pure sinigrin, where a good linearity was achieved within the concentration range of 50 to 800 µg/mL (R2 > 0.99), with an excellent accuracy (-1.37% and -1.29%) and precision (1.43% and 0.94%), for intra and inter-day, respectively. Finally, the MTT assay was performed for the sinigrin-rich fraction using three different human cancer cell lines, viz. prostate cancer (DU-145), colon adenocarcinoma (HCT-15), and melanoma (A-375). The cell-based assays were extended to conduct apoptotic and caspase-3 activities, to determine the mechanism of action of sinigrin in the treatment of cancer. MTT assay showed IC50 values of 15.88, 21.42, and 24.58 µg/mL for DU-145, HCT-15, and A-375 cell lines, respectively. Increased cellular apoptosis and caspase-3 expression were observed with sinigrin-rich fraction, indicating significant increase in overexpression of caspase-3 in DU-145 cells. In conclusion, a simple, sensitive, fast, and accurate RP-HPLC method was developed for the estimation of sinigrin in the prepared fraction. The data observed here indicate that sinigrin can be beneficial in treating prostate cancer possibly by inducing apoptosis.
Gluconasturtiin, a glucosinolate present in watercress, is hydrolysed by myrosinase to form gluconasturtiin-isothiocyanate (GNST-ITC), which has potential chemopreventive effects; however, the underlying mechanisms of action have not been explored, mainly in human cell lines. The purpose of the study is to evaluate the cytotoxicity of GNST-ITC and to further assess its potential to induce apoptosis. GNST-ITC inhibited cell proliferation in both human hepatocarcinoma (HepG2) and human breast adenocarcinoma (MCF-7) cells with IC50 values of 7.83 µM and 5.02 µM, respectively. Morphological changes as a result of GNST-ITC-induced apoptosis showed chromatin condensation, nuclear fragmentation, and membrane blebbing. Additionally, Annexin V assay showed proportion of cells in early and late apoptosis upon exposure to GNST-ITC in a time-dependent manner. To delineate the mechanism of apoptosis, cell cycle arrest and expression of caspases were studied. GNST-ITC induced a time-dependent G2/M phase arrest, with reduction of 82% and 93% in HepG2 and MCF-7 cell lines, respectively. The same treatment also led to the subsequent expression of caspase-3/7 and -9 in both cells demonstrating mitochondrial-associated cell death. Collectively, these results reveal that GNST-ITC can inhibit cell proliferation and can induce cell death in HepG2 and MCF-7 cancer cells via apoptosis, highlighting its potential development as an anticancer agent.
Callus was induced from mangosteen (Garcinia mangostana L.) young purple-red leaves on Murashige and Skoog basal medium with various combinations of plant growth regulators. Murashige and Skoog medium with 4.44 µM 6-benzylaminopurine and 4.52 µM 2,4-dichlorophenoxyacetic acid was the best for friable callus induction. This friable callus was used for the initiation of cell suspension culture. The effects of different combinations of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid, carbon sources and inoculum sizes were tested. It was found that combination of 2.22 µM 6-benzylaminopurine + 2.26 µM 2,4-dichlorophenoxyacetic acid, glucose (30 g/l) and 1.5 g/50 ml inoculum size was the best for cell growth. Callus and cell suspension cultures were then treated either with 100 µM methyl jasmonate as an elicitor for 5 days, or 0.5 g/l casein hydrolysate as an organic supplement for 7 days. Metabolites were then extracted and profiled using liquid chromatography-time of flight mass spectrometry. Multivariate discriminant analyses revealed significant metabolite differences (P ≤ 0.05) for callus and suspension cells treated either with methyl jasmonate or casein hydrolysate. Based on MS/MS data, methyl jasmonate stimulated the production of an alkaloid (thalsimine) and fatty acid (phosphatidyl ethanolamine) in suspension cells while in callus, an alkaloid (thiacremonone) and glucosinolate (7-methylthioheptanaldoxime) was produced. Meanwhile casein hydrolysate stimulated the production of alkaloids such as 3ß,6ß-dihydroxynortropane and cis-hinokiresinol and triterpenoids such as schidigerasaponin and talinumoside in suspension cells. This study provides evidence on the potential of secondary metabolite production from in vitro culture of mangosteen.
As a cytosolic transcription factor, the aryl hydrocarbon (Ah) receptor is involved in several patho- physiological events leading to immunosuppression and cancer; hence antagonists of the Ah receptor may possess chemoprevention properties. It is known to modulate carcinogen-metabolising enzymes, for instance the CYP1 family of cytochromes P450 and quinone reductase, both important in the biotransformation of many chemical carcinogens via regulating phase I and phase II enzyme systems. Utilising chemically-activated luciferase expression (CALUX) assay it was revealed that intact glucosinolates, glucoraphanin and glucoerucin, isolated from Brassica oleracea L. var. acephala sabellica and Eruca sativa ripe seeds, respectively, are such antagonists. Both glucosinolates were poor ligands for the Ah receptor; however, they effectively antagonised activation of the receptor by the avid ligand benzo[a]pyrene. Indeed, intact glucosinolate glucoraphanin was a more potent antagonist to the receptor than glucoerucin. It can be concluded that both glucosinolates effectively act as antagonists for the Ah receptor, and this may contribute to their established chemoprevention potency.
Crucifer vegetables, Brassicaceae and other species of the order Brassicales, e.g., Moringaceae that are commonly consumed as spice and food, have been reported to have potential benefits for the treatment and prevention of several health disorders. Though epidemiologically inconclusive, investigations have shown that consumption of those vegetables may result in reducing and preventing the risks associated with neurodegenerative disease development and may also exert other biological protections in humans. The neuroprotective effects of these vegetables have been ascribed to their secondary metabolites, glucosinolates (GLs), and their related hydrolytic products, isothiocyanates (ITCs) that are largely investigated for their various medicinal effects. Extensive pre-clinical studies have revealed more than a few molecular mechanisms of action elucidating multiple biological effects of GLs hydrolytic products. This review summarizes the most significant and up-to-date in vitro and in vivo neuroprotective actions of sulforaphane (SFN), moringin (MG), phenethyl isothiocyanate (PEITC), 6-(methylsulfinyl) hexyl isothiocyanate (6-MSITC) and erucin (ER) in neurodegenerative diseases.
Moringa oleifera Lam, family Moringaceae, is a perennial plant which is called various names, but is locally known in Malaysia as "murungai" or "kelor". Glucomoringin, a glucosinolate with from M. oleifera is a major secondary metabolite compound. The seeds and leaves of the plant are reported to have the highest amount of glucosinolates. M. oleifera is well known for its many uses health and benefits. It is claimed to have nutritional, medicinal and chemopreventive potentials. Chemopreventive effects of M. oleifera are expected due to the existence of glucosinolate which it is reported to have the ability to induce apoptosis in anticancer studies. Furthermore, chemopreventive value of M. oleifera has been demonstrated in studies utilizing its leaf extract to inhibit the growth of human cancer cell lines. This review highlights the advantages of M. oleifera targeting chemoprevention where glucosinolates could help to slow the process of carcinogenesis through several molecular targets. It is also includes inhibition of carcinogen activation and induction of carcinogen detoxification, anti-inflammatory, anti-tumor cell proliferation, induction of apoptosis and inhibition of tumor angiogenesis. Finally, for synergistic effects of M. oleifera with other drugs and safety, essential for chemoprevention, it is important that it safe to be consumed by human body and works well. Although there is promising evidence about M. oleifera in chemoprevention, extensive research needs to be done due to the expected rise of cancer in coming years and to gain more information about the mechanisms involved in M. oleifera influence, which could be a good source to inhibit several major mechanisms involved in cancer development.
Aliphatic glucosinolate is an important secondary metabolite responsible in plant defense mechanism and carcinogenic
activity. It plays a crucial role in plant adaptation towards changes in the environment such as salinity and drought.
However, in many plant genomes, there are thousands of genes encoding proteins still with putative functions and
incomplete annotations. Therefore, the genome of Arabidopsis thaliana was selected to be investigated further to identify
any putative genes that are potentially involved in the aliphatic glucosinolate biosynthesis pathway, most of its gene are
with incomplete annotation. Known genes for aliphatic glucosinolates were retrieved from KEGG and AraCyc databases.
Three co-expression databases i.e., ATTED-II, GeneMANIA and STRING were used to perform the co-expression network
analysis. The integrated co-expression network was then being clustered, annotated and visualized using Cytoscape plugin,
MCODE and ClueGO. Then, the regulatory network of A. thaliana from AtRegNet was mapped onto the co-expression
network to build the transcriptional regulatory network. This study showed that a total of 506 genes were co-expressed
with the 61 aliphatic glucosinolate biosynthesis genes. Five transcription factors have been predicted to be involved
in the biosynthetic pathway of aliphatic glucosinolate, namely SEPALLATA 3 (SEP3), PHYTOCHROME INTERACTING FACTOR
3-like 5 (AtbHLH15/PIL5), ELONGATED HYPOCOTYL 5 (HY5), AGAMOUS-like 15 (AGL15) and GLABRA 3 (GL3). Meanwhile,
three other genes with high potential to be involved in the aliphatic glucosinolates biosynthetic pathway were identified,
i.e., methylthioalkylmalate-like synthase 4 (MAML-4) and aspartate aminotransferase (ASP1 and ASP4). These findings
can be used to complete the aliphatic glucosinolate biosynthetic pathway in A. thaliana and to update the information
on the glucosinolate-related pathways in public metabolic databases.
Moringa is a genus of medicinal plants that has been used traditionally to cure wounds and various diseases such as colds and diabetes. In addition, the genus is also consumed as a source of nutrients and widely used for purifying water. The genus consists of 13 species that have been widely cultivated throughout Asia and Africa for their multiple uses. The purpose of this review is to provide updated and categorized information on the traditional uses, phytochemistry, biological activities, and toxicological research ofMoringaspecies in order to explore their therapeutic potential and evaluate future research opportunities. The literature reviewed for this paper was obtained from PubMed, ScienceDirect, and Google Scholar journal papers published from 1983 to March 2017.Moringaspecies are well-known for their antioxidant, anti-inflammatory, anticancer, and antihyperglycemic activities. Most of their biological activity is caused by their high content of flavonoids, glucosides, and glucosinolates. By documenting the traditional uses and biological activities ofMoringaspecies, we hope to support new research on these plants, especially on those species whose biological properties have not been studied to date.
The potential of isothiocyanates to antagonize the carcinogenicity of structurally diverse chemicals has been established in animals. A feasible mechanism of action involves protecting DNA by reducing the availability of the genotoxic metabolites of chemical carcinogens by either inhibiting their generation and/or stimulating their detoxification. In vivo as well as in vitro studies conducted in rat/human primary hepatocytes and precision-cut tissue slices have revealed that isothiocyanates can impair cytochrome P450 activity, including the CYP1 family which is the most active in the bioactivation of carcinogens, by virtue of being mechanism-based inactivators. The aromatic phenethyl isothiocyanate is the most effective of those studied, whereas aliphatic isothiocyanates such as sulforaphane and erucin necessitate high doses in order to manifest such effects that may not always be achievable through the diet. In all systems studied, isothiocyanates are strong inducers of detoxification enzyme systems including quinone reductase, glutathione S-transferase, epoxide hydrolase, and UDP-glucuronosyl transferase. Indeed, in smokers phenethyl isothiocyanate intake increases the urinary excretion of inactive mercapturate metabolites of toxic chemicals present in tobacco. Glucosinolates, the precursors of isothiocyanates, have also the potential to upregulate detoxification enzyme systems, but their contribution to the cancer chemoprevention linked to cruciferous vegetable consumption remains to be evaluated.
Glucoraphanin is the main glucosinolate found in broccoli and other cruciferous vegetables (Brassicaceae). The objective of the study was to evaluate whether glucoraphanin and its breakdown product sulforaphane, are potent modulators of various phase I and phase II enzymes involved in carcinogen-metabolising enzyme systems in vitro. The glucosinolate glucoraphanin was isolated from cruciferous vegetables and exposed to human hepatoma cell line HepG2 at various concentrations (0-25 μM) for 24 hours. Glucoraphanin at higher concentration (25 μM) decreased dealkylation of methoxyresorufin, a marker for cytochrome P4501 activity; supplementation of the incubation medium with myrosinase (0.018 U), the enzyme that converts glucosinolate to its corresponding isothiocyanate, showed minimal induction in this enzyme activity at concentration 10 μM. Quinone reductase and glutathione S-transferase activities were unaffected by this glucosinolate; however, supplementation of the incubation medium with myrosinase elevated quinone reductase activity. It may be inferred that the breakdown product of glucoraphanin, in this case sulforaphane, is superior than its precursor in modulating carcinogen- metabolising enzyme systems in vitro and this is likely to impact on the chemopreventive activity linked to cruciferous vegetable consumption.
Cruciferous vegetable consumption correlates with reduced risk of cancer. This chemopreventative activity may involve glucosinolates and their hydrolysis products. Glucosinolate-derived isothiocyanates have been studied for their toxicity and chemopreventative properties, but other hydrolysis products (epithionitriles and nitriles) have not been thoroughly examined. We report that these hydrolysis products differ in their cytotoxicity to human cells, with toxicity most strongly associated with isothiocyanates rather than epithionitriles and nitriles. We explored mechanisms of this differential cytotoxicity by examining the role of oxidative metabolism, oxidative stress, mitochondrial permeability, reduced glutathione levels, cell cycle arrest and apoptosis. 2-Propenylisothiocyanate and 3-butenylisothiocyanate both inhibited cytochome P450 1A (CYP1A) enzyme activity in CYP expressing MCL-5 cells at high cytotoxic doses. Incubation of MCL-5 cells with non-cytotoxic doses of 2-propenylisothiocyanate for 24h resulted in a dose-dependent inhibition of ethoxyresorufin O-deethylase, yet failed to affect CYP1A1 mRNA expression indicating interference with enzyme activity rather than inhibition of transcription. Increased reactive oxygen species (ROS) production was observed only for 2-propenylisothiocyanate treatment. 2-Propenylisothiocyanate treatment lowered reduced glutathione levels whereas no changes were noted with 3,4-epithiobutylnitrile. Cell cycle analysis showed that 2-propenylisothiocyanate induced a G2/M block whereas other hydrolysis products showed only marginal effects. We found that 2-propenylisothiocyanate and 3-butenylisothiocyanate induced cell death predominantly via necrosis whereas, 3,4-epithiobutylnitrile promoted both necrosis and apoptosis. Thus the activity of glucosinolate hydrolysis products includes cytotoxicity that is compound-class specific and may contribute to their putative chemoprotection properties.
In this study, gum of Araucaria heterophylla was collected. The collected gum was subjected for extraction of polysaccharide using solvent extraction system. Thus, extracted polysaccharide was further purified using solvent method and was characterized using UV-Vis spectroscopy, Phenol sulfuric acid assay, FTIR, TGA, TLC and GC-MS. The gum derived polysaccharide was found to have the following sugars Rhamnose, Allose, Glucosinolate, Threose, Idosan, Galactose and Arabinose. The extracted polysaccharide was tested for various in-vitro bioactive studies such as antibacterial activity, antioxidant activity and anticancer activity. The polysaccharide was found to have antioxidant and anticancer activity. Further, the polysaccharide was subjected for carboxymethylation to favor the nanocarrier synthesis, where it was chelated using Sodium Tri Meta Phosphate (STMP) to form nanocarriers. The nanocarriers so formed were loaded with curcumin and were characterized using FTIR, SEM, EDX and AFM. Both the loaded and unloaded nanocarriers were studied for its in-vitro cytotoxic effect against the MCF7 human breast cancer cell lines. The nanocarriers were found to deliver the drug efficiently against the cancer cell line used in this study.
Glucoraphenin, a glucosinolate present in large quantities in radish is hydrolysed by myrosinase to form the isothiocyanate sulforaphene, which is believed to be responsible for its chemopreventive activity; however, the underlying mechanisms of action have not been investigated, particularly in human cell lines. The aim of the study is to assess the cytotoxicity of sulforaphene in HepG2 cells and evaluate its potential to enhance apoptosis. The cytotoxicity of sulforaphene in HepG2 cells was carried out ensuing an initial screening with two other cell lines, MFC-7 and HT-29, where sulforaphene displayed highest toxicity in HepG2 cells following incubation at 24, 48 and 72 h. In contrast, the intact glucosinolate showed no cytotoxicity. Morphological studies indicated that sulforaphene stimulated apoptosis as exemplified by cell shrinkage, blebbing, chromatin condensation, and nuclear fragmentation. The Annexin V assay revealed significant increases in apoptosis and the same treatment increased the activity of caspases -3/7 and -9, whereas a decline in caspase-8 was observed. Impairment of cell proliferation was indicated by cell cycle arrest at the Sub G₀/G₁ phase as compared to the other phases. It may be concluded that sulforaphene, but not its parent glucosinolate, glucoraphenin, causes cytotoxicity and stimulates apoptosis in HepG2 cells.
Moringa oleifera is a miracle plant with many nutritional and medicinal properties. Chemopreventive values of the combined mixture of moringa leaves and seed residue (MOLSr) at different ratios (M1S9, M1S1 and M9S1) were investigated. MOLSr extracts were subjected to phytochemical screening, antioxidant assays, metabolite profiling and cytotoxic activity on the primary mammary epithelial cells (PMECs), non-malignant Chang's liver cells and various human cancer cell lines (including breast, cervical, colon and liver cancer cell lines). The MOLSr ratio with the most potent cytotoxic activity was used in xenograft mice injected with MDA-MB-231 cells for in vivo tumorigenicity study as well as further protein and gene expression studies. M1S9, specifically composed of saponin and amino acid, retained the lowest antioxidant activity but the highest glucosinolate content as compared to other ratios. Cell viability decreased significantly in MCF-7 breast cancer cells and PMECs after treatment with M1S9. Solid tumor from MDA-MB-231 xenograft mice was inhibited by up to 64.5% at third week after treatment with high-dose M1S9. High-dose M1S9 significantly decreased the expression of calcineurin (CaN) and vascular endothelial cell growth factor (VEGF) proteins as well as the secreted frizzled-related protein 1 (SFRP1) and solute carrier family 39 member 6 (SLC39A6) genes. This study provides new scientific evidence for the chemoprevention potential of MOLSr extracts in a breast cancer model; however, the precise mechanism warrants further investigation.