Phenethyl Isothiocyanate Research Articles

Phenethyl isothiocyanate protects against H2O2-induced insulin resistance in 3T3-L1 adipocytes.

Obesity is associated with systemic oxidative stress and leads to insulin resistance. Phenethyl isothiocyanate (PEITC), a natural dietary isothiocyanate, has been shown to have beneficial effects in improving cellular defense activities against oxidative stress through activation of nuclear factor erythroid-2 related factor 2 (Nrf2) pathway. However, little evidence exists if the antioxidative activity has beneficial effects on glucose metabolism. Here, we tested the preventive potential of PEITC for impaired insulin-induced glucose uptake by oxidative stress in 3T3-L1 adipocytes. Treatment with PEITC increased the expression of antioxidative enzymes regulated by Nrf2 such as γ-glutamylcysteine-synthetase, heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1 and glutathione S-transferase, and reduced oxidative stress induced by H2O2. Furthermore, PEITC restored impaired insulin-stimulated glucose uptake, translocation of glucose transporter 4 and insulin signaling by H2O2. These results indicate that PEITC protected insulin-regulated glucose metabolism impaired by oxidative stress through the antioxidative activity in 3T3-L1 adipocytes.

Effect of isothiocyanates, BITC and PEITC, on stress protein accumulation, protein aggregation and aggresome-like structure formation in Xenopus A6 kidney epithelial cells

Numerous studies have elucidated the health benefits of organosulfur compounds, known as isothiocyanates (ITCs), derived from cruciferous vegetables. As electrophiles, ITCs have the ability to directly bind and modify thiol-containing compounds such as glutathione and cellular protein, including tubulin. While the biochemical effects of ITCs have been well characterized, less information is available regarding their effects on the accumulation of stress-inducible heme oxygenase-1 (HO-1), heat shock proteins (HSPs) and the possible formation of aggregated protein due to thiol modification. The present study has examined the effect of the ITCs, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), on the accumulation of HO-1, HSP70 and HSP30 in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that both BITC and PEITC induced the accumulation of HO-1 and HSP70 whereas HSP30 levels were enhanced only in cells treated with BITC. Immunocytochemistry determined that ITC treatment induced F-actin disorganization and membrane ruffling and enhanced accumulation of HO-1 in the cytoplasm. Additionally, BITC induced enhanced levels of ubiquitinated protein, aggregated protein, and the collapse and fragmentation of microtubules. In comparison, treatment of cells with the proteasomal inhibitor, MG132, induced the accumulation of all three stress proteins, aggregated protein and aggresome-like structures. Finally, cells pretreated with BITC inhibited the formation of MG132-induced aggresome-like structures in the perinuclear region. This latter finding suggests that BITC-induced microtubule fragmentation may impede the movement of aggregated protein via microtubules and their subsequent coalescence into aggresome-like structures in the perinuclear region.

Phenethyl isothiocyanate decreases thymic stromal lymphopoietin-induced inflammatory reactions in mast cells

Phenethyl isothiocyanate (PEITC) has been reported to have anti-inflammatory and anti-carcinogenic properties. However, the anti-inflammatory effects of PEITC on thymic stromal lymphopoietin (TSLP)-induced inflammatory responses are uncertain. This study evaluates pharmacological activities of PEITC on inflammatory reactions in TSLP-stimulated mast cells. Human mast cell line HMC-1 was treated with PEITC (0.04, 0.4, and 4 µM) and subjected to inflammation by TSLP. Our results showed that PEITC significantly attenuated IL-13 and TNF-α levels increased by TSLP in HMC-1. PEITC significantly decreased TSLP-promoted HMC-1 proliferation and Ki67 mRNA expression. Protein levels of MDM2 and pSTAT6 increased by TSLP were significantly suppressed by PEITC in HMC-1. In addition, PEITC significantly enhanced protein levels of cleaved poly ADP-ribose polymerase and p53 decreased by TSLP. Based on the effects of PEITC on inflammation and proliferation in this study, it is possible that PEITC is a potential candidate to treat mast cells-mediated inflammatory disorders. Practical applications This report provides strong evidence that Phenethyl isothiocyanate (PEITC) which is a dietary constituent derived from cruciferous vegetables, may be considered an alternative agent for treatment of mast cells-mediated inflammatory disorders.

Phenethyl Isothiocyanate (PEITC) and Benzyl Isothiocyanate (BITC) Inhibit Human Melanoma A375.S2 Cell Migration and Invasion by Affecting MAPK Signaling Pathway In Vitro.

Numerous evidence has shown that PEITC and BITC inhibit cancer cell migration and invasion. In this study, we investigated the anti-metastatic mechanisms of PEITC and BITC in human melanoma cancer A375.S2 cells in vitro. We used a cell viability assay, an in-vitro scratch wound healing assay, a transwell assay for cell migration and invasion, a gelatin zymography assay, western blotting and EMSA to examine the anti-metastatic mechanisms of PEITC and BITC in A375.S2 cells. Sublethal concentrations of PEITC (0, 1, 2 and 2.5 μM) and BITC (0, 0.5, 1 and 2 μM) inhibited mobility, migration and invasion of A375.S2 cells that were assayed by wound healing and Transwell filter. PEITC and BITC inhibited MMP-2 activity in A375.S2 cells, as assessed by gelatin zymography assay. Results from western blotting indicated that PEITC (2.5 μM) and BITC (2 μM) decreased the levels of p-p38 following 24 and 48 h treatment. PEITC (1-2.5 μM) reduced the levels of p-JNK1/2 proteins following 48-h treatment but BITC increased p-JNK1/2 levels following 24-h treatment. PEITC (2.5 μM) reduced the levels of p-ERK1/2 proteins following 48-h treatment but BITC (0.5-2 μM) increased p-ERK1/2 levels following 24- and 48-h treatment. PEITC and BITC affect cell migration and invasion of A375.S2 cells via MAPK pathway. PEITC and BITC inhibited MMP-2 activity. PEITC increased NF-κB expression but BITC decreased NF-κB expression in the nucleus. Furthermore, NF-κB p65 binding to DNA was decreased following 2.5 μM PEITC treatment, but increased following treatment with 1-2 μM. However, 0.5-2 μM BITC treatment decreased the binding of NF-κB to DNA in A375.S2 cells, as assessed by electrophoretic mobility shift (EMSA) assay. Based on these observations, we suggest that PEITC and BITC can be used as anti-metastastic agents of human melanoma cells in the future.

Emodin and rhein decrease levels of hypoxia-inducible factor-1α in human pancreatic cancer cells and attenuate cancer cachexia in athymic mice carrying these cells

The transcription factor hypoxia-inducible factor-1 (HIF-1) consists of oxygen-sensitive HIF-1α and constitutive HIF-1β. HIF-1α is undetectable in normal cells, but cancer cells frequently express HIF-1α to support their growth, angiogenesis, and high glycolysis (also known as the Warburg effect). The Warburg effect in cancer cells increases energy expenditure and thus participates in cancer-induced metabolic disorder, cancer cachexia. In the present study, we investigated whether two components of Rheum palmatum, emodin and rhein, inhibited HIF-1α expression in human pancreatic cancer cells and whether the inhibiting effect, if any, attenuated cancer cachexia. Using Western blotting, we demonstrated that emodin and rhein decreased HIF-1α expression in MiaPaCa2 and four other human pancreatic cancer cell lines. We also examined HIF-1α expression when MiaPaCa2 cells were exposed to PX-478, noscapine, and phenethyl isothiocyanate, as these compounds were known to inhibit HIF-1α expression in different cancer cells. PX-478 and noscapine inhibited HIF-1α expression to a less extent than emodin and rhein, and phenethyl isothiocyanate did not inhibit HIF-1α expression in tested concentrations. We obtained evidence that emodin and rhein decreased HIF-1α by decreasing its biosynthesis but not gene transcription or protein stability. When MiaPaCa2 cells were implanted in athymic mice, emodin and rhein inhibited cancer-cell growth and HIF-1α expression. In these athymic mice, emodin and rhein also attenuated two pathological constituents of cancer cachexia, namely high hepatic gluconeogenesis and skeletal-muscle proteolysis. In conclusion, emodin and rhein decrease pancreatic cancer cell's growth and HIF-1α expression and attenuate cancer cachexia in the athymic mice carrying the cancer cells.

Dietary Phenethyl Isothiocyanate Protects Mice from Colitis Associated Colon Cancer.

We have previously reported alleviation of dextran sodium sulfate (DSS)-induced ulcerative colitis signs in phenethyl isothiocyanate (PEITC)-treated mice. Here we investigated chemoprotective activities of PEITC in mice with Azoxymethane-DSS induced colitis associated colon carcinogenesis. We also examined the molecular mediators associated with the PEITC effects using relevant cell lines. A 0.12% PEITC-enriched mouse-diet reduced mucosal and submucosal inflammation as well as glandular atypia by 12% and the frequency of adenocarcinoma by 17% with a concomitant improvement in overall disease activity indices compared to the diseased control group. Lipopolysaccharide-induced in vitro up-regulation of key mediators of inflammation, immune response, apoptosis, and cell proliferation were attenuated by 10 μM PEITC. Three of these mediators showed concentration-dependent reduction in respective mRNAs. Furthermore, PEITC inhibited Nuclear factor kappa B1 (NFκB1) proteins in a concentration-dependent manner. The NFκB1 mRNA expression inversely correlated (r = −0.940, p = 0.013) with tri-methylation of lysine 27 on histone 3 near its promoter region in a time-dependent manner. These results indicate that PEITC may slow down the development of colon carcinogenesis in an inflammatory intestinal setting which is potentially associated with epigenetic modulation of NFκB1 signaling.

Stability of Bioactive Compounds and Antioxidant Activity of Thai Cruciferous Vegetables during In Vitro Digestion

Raphanus sativus (L.) var. caudatus Alef (Thai rat-tailed radish), Brassica juncea (L.) Czern. (leaf mustard) and Brassica juncea (L.) Coss. var. sareptana Sinskaja (mustard green) are cruciferous vegetable commonly consumed in Thailand and Asian countries. The vegetables were extracted with different solvents namely methanol, hexane and water prior to total phenolic content (TPC), phenethyl isothiocyanate (PEITC) content and antioxidant activity by ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay were determined. Effects of gastrointestinal digestion on stability of these characteristics were investigated. It was found that the order of extraction efficiency for high bioactive compounds and antioxidant activity was water> methanol> hexane. Among vegetables, mustard green showed the highest TPC, PEITC, FRAP and DPPH values being 19.78±0.01 g GAE, 9.65 ± 1.08 µmol, 8.18±0.01 µM FeSO4 and 7.75±0.31 µM TE per 100g, respectively. Decreases in DPPH (30.6-53.5%), FRAP (49.0-88.0%) and PEITC (27.2-56.7%) values were slightly higher than TPC (27.9-41.6%) after in vitro digestion. It can be said that Thai cruciferous vegetables contain substantial chemical property and this may promote their health protection but the stability through digestive system should be warranted.

Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants.

Exploring the vicinity around a locus of a protein in sequence space may identify homologs with enhanced properties, which could become valuable in biotechnical and other applications. A rational approach to this pursuit is the use of ‘infologs’, i.e. synthetic sequences with specific substitutions capturing maximal sequence information derived from the evolutionary history of the protein family. Ninety-five such infolog genes of poplar glutathione transferase were synthesized and expressed in Escherichia coli, and the catalytic activities of the proteins determined with alternative substrates. Sequence–activity relationships derived from the infologs were used to design a second set of 47 infologs in which 90% of the members exceeded wild-type properties. Two mutants, C2 (V55I/E95D/D108E/A160V) and G5 (F13L/C70A/G122E), were further functionally characterized. The activities of the infologs with the alternative substrates 1-chloro-2,4-dinitrobenzene and phenethyl isothiocyanate, subject to different chemistries, were positively correlated, indicating that the examined mutations were affecting the overall catalytic competence without major shift in substrate discrimination. By contrast, the enhanced protein expressivity observed in many of the mutants were not similarly correlated with the activities. In conclusion, small libraries of well-defined infologs can be used to systematically explore sequence space to optimize proteins in multidimensional functional space.

Benzyl isothiocyanate and phenethyl isothiocyanate inhibit murine melanoma B16F10 cell migration and invasion in vitro.

Benzyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC) have been demonstrated to induce anticancer function in many human cancer cells and also inhibit cancer cell migration and invasion. However, there are no studies that show BITC and PEITC to inhibit cell migration and invasion in mouse melanoma B16F10 cells. In this study, we investigated anti-metastasis effects of BITC and PEITC in melanoma cancer cells invitro. Under sub-lethal concentrations (from 1, 2.5 up to 5µM), BITC and PEITC significantly inhibited cell mobility, migration and invasion nature of B16F10 cells. Gelatin zymography assay also showed that BITC and PEITC inhibited matrix metalloproteinase-2 (MMP-2) activity in B16F10 cells. PEITC reduced MAPK signaling associated proteins such as p-ERK1/2, p-p38 and p-JNK1/2 but BITC increased those MAPK signaling associated proteins. BITC and PEITC both suppressed the expression of RhoA, Ras, and SOS-1, however, PEITC increased FAK and GRB2 but BITC increased FAK at 48h. Furthermore, PEITC decreased the expression of MMP-2 and tissue inhibitors of matrix metalloproteinases (TIMP) but BITC increased them. PEITC inhibited NF-κB protein levels and DNA binding which was confirmed by electrophoretic mobility shift (EMSA) assay. Based on these observations, we suggest that BITC and PEITC can be used in anti-metastasis of melanoma cells in the future.

Development of validated high-temperature reverse-phase UHPLC-PDA analytical method for simultaneous analysis of five natural isothiocyanates in cruciferous vegetables

In the present study reverse-phase UHPLC-PDA technique was developed at 60°C for simultaneous quantification of allyl, 3-butenyl, 4-(methylthio)butyl, benzyl and phenethyl isothiocyanates. The validation parameter showed a very good linearity, with a correlation coefficient of 1.00 for all detected standard analytes. Also, high precision and accuracy were observed with lowest obtained values of 1.39% and 99.1%, respectively. Different varieties of three plants, viz. Brassica rapa var. rapa L., Raphanus sativus L. var. oleiformis Pers. and Eruca sativa Mill., were analyzed with this method. After analysis, 4-(methylthio)butyl isothiocyanate was observed to be the major component in the varieties of arugula. Allyl, benzyl and phenethyl isothiocyanates were detected in turnip varieties and, in addition, 3-butenyl isothiocyanate was detected in radish varieties.

Isothiocyanates suppress the invasion and metastasis of tumors by targeting FAK/MMP-9 activity.

Isothiocyanates, which are present as glucosinolate precursors in cruciferous vegetables, have strong activity against various cancers. Here, we compared the anti-metastatic effects of isothiocyanates (benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN)) by examining how they regulate MMP-9 expression. Isothiocyanates, particularly PEITC, suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 activity and invasion in various cancer cell lines. By contrast, N-methyl phenethylamine, a PEITC analog without an isothiocyanate functional group, had no effect. A reporter gene assay demonstrated that BITC, PEITC, and SFN suppressed TAP-induced MMP-9 expression by inhibiting AP-1 and NF-κB in U20S osteosarcoma cells. All three compounds reduced phosphorylation of FAK, ERK1/2, and Akt. In addition, MMP-9 expression was downregulated by inhibiting FAK, ERK1/2, and Akt. Isothiocyanates-mediated inhibition of FAK phosphorylation suppressed phosphorylation of ERK1/2 and Akt in U2OS and A549 cells, along with the translocation of p65 and c-Fos, suggesting that isothiocyanates inhibit MMP-9 expression and cell invasion by blocking phosphorylation of FAK. Furthermore, isothiocyanates, abolished MMP-9 expression and tumor metastasis in vivo with the following efficacy: PEITC>BITC>SFN. Thus, isothiocyanates act as anti-metastatic compounds that suppress MMP-9 activity/expression by inhibiting NF-κB and AP-1 via suppression of the FAK/ERK and FAK/Akt signaling pathways.

Abstract 5271: Prostate cancer chemoprevention by dietary isothiocyanates is associated with suppression of lipogenesis

Abstract Isothiocyanates (ITCs), including sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), have been studied extensively for chemoprevention of prostate cancer using both in vitro and in vivo models, but the underlying mechanism is not fully understood. Dependence on fatty acid metabolism is progressively recognized as a unique trait of prostate cancer. Using in vitro and in vivo prostate cancer models, we demonstrate for the first time, that prostate cancer chemoprevention by ITCs is associated with suppression of fatty acid metabolism. Exposure of human prostate cancer cells to plasma achievable concentrations of SFN resulted in a marked decrease in levels of key fatty acid metabolism proteins, including acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and carnitine palmitoyl transferase 1A (CPT1A), that was accompanied by suppression of their transcription. Consistent with the in vitro results, oral administration of prostate cancer chemopreventive concentrations of SFN (6 µmol per mouse, three times per week) resulted in downregulation of ACC1, FASN, and CPT1A proteins in prostate adenocarcinoma of TRAMP transgenic mice when compared with control. Levels of fatty acid metabolites (free fatty acid, triglycerides and phospholipids), lactate, and acetyl-CoA were also lower in the plasma and/or prostate adenocarcinoma of SFN-treated TRAMP mice in comparison with controls. Because fatty acid oxidation is an important step to supply energy to the prostate cancer cells, we explored the possibility of whether SFN could affect the fatty acid oxidation in prostate cancer cells. Indeed, SFN exposure significantly decreased the level of proteins involved in fatty acid oxidation in prostate cancer cells. Many of these changes were also observed with PEITC. In conclusion, it is reasonable to propose that suppression of fatty acid metabolism is an important mechanism in prostate cancer chemoprevention by ITCs. This study was supported by the grants RO1 CA115498-10 and RO1 CA101753-12 awarded by the National Cancer Institute. Citation Format: Krishna B. Singh, Shivendra V. Singh. Prostate cancer chemoprevention by dietary isothiocyanates is associated with suppression of lipogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5271. doi:10.1158/1538-7445.AM2017-5271

Do glucosinolate hydrolysis products reduce nitrous oxide emissions from urine affected soil?

New Zealand agriculture is predominantly comprised of pastoral grazing systems and deposition of animal excreta during grazing has been identified as a major source of nitrous oxide (N2O) emissions. Nitrification inhibitors have been shown to significantly reduce nitrous oxide emissions from grazing pastoral systems, and some plants have been identified as having nitrification inhibiting properties. Brassica crops are one such example as they contain the secondary metabolite glucosinolate (GLS) whose hydrolysis products are thought to slow soil nitrogen cycling. Forage brassicas have been increasingly used to supplement the diet of grazing animals. The aim of this study was to determine if GLS hydrolysis products (phenylethyl isothiocyanate, 4-pent-1-yl isothiocyanate, 2-propenyl nitrile, 2 propenyl isothiocyanate, 4-pentene nitrile) produced in brassica crops reduced N2O emissions from soil amended with urea or animal urine. In the laboratory, some GLS hydrolysis products added with urea to soil were found to decrease N2O emissions and the most effective product (phenylethyl isothiocyante) reduced N2O emissions by 51% during the study. There was some evidence that the reduction in N2O emissions found in the lab could be attributed to inhibition of nitrification. Results suggest that the inhibition by GLS hydrolysis products was short-lived and, if considered for use, multiple applications may be necessary to achieve effective inhibition of N2O emissions. This reduction, however, was not observed under field conditions. Further investigation is required to test more GLS hydrolysis products to fully understand their impact on N2O emissions from urine affected soil.

P 100 - Isothiocyanates trigger early disruption of mitochondrial function in cells overexpressing Bcl-2

Epidemiological evidence indicates that increased consumption of isothiocyanates is associated with reduced incidence of cancer. One mechanism to explain this is the ability of isothiocyanates to trigger apoptosis, including in cells that overexpress the anti-apoptotic protein Bcl-2, which is overexpressed in a number of cancers. This study aimed to examine the mechanisms by which phenethyl isothiocyanate (PEITC) induces apoptosis in Bcl-2 overexpressing (Bcl-2 OE) cells. Bcl-2 OE cells were more sensitive than wild-type cells to PEITC (LC50=8 μM vs. 12.5 μM). Bax-/-Bak-/-and Bim-/-Bid-/- cells were resistant to PEITC, indicating a classical apoptotic cell death. Cell death occurred more rapidly in the Bcl-2 OE cells, and increased levels of caspase activity and earlier activation were observed. PEITC had a greater effect on mitochondrial metabolic activity in the Bcl-2 OE cells; they had higher basal mitochondrial respiration, however, this was susceptible to inhibition by PEITC. There was also a significant decline in mitochondrial spare respiratory capacity. These effects occurred prior to caspase activation and cell death. Together, these results suggest that isothiocyanates bypass the action of Bcl-2 by acting directly on mitochondria to promote Bax/Bak-dependent apoptosis. This information may be valuable for designing novel isothiocyanates to target Bcl-2 OE cancer cells.

New Mild and Simple Approach to Isothiocyanates: A Class of Potent Anticancer Agents.

In our current work, acetyl chloride-mediated synthesis of phenethyl isothiocyanate (PEITC) derivatives proves to be convenient and provides the expected products at good to excellent yields. Biological evaluation and structure-activity relationship analysis found that the novel compound 7 showed the best anticancer activity against human cancer cell line Panc1 and HGC27 compared with PEITC. Compounds 6 and 7 induced more apoptosis in pancreatic cancer cells but less toxicity in non-cancer cells. Further biological study demonstrated that 7 substantially increased intracellular reactive oxygen species (ROS) and depleted glutathione (GSH), leading to an oxidative stress to kill cancer cell.

Inhibition of cancer growth in vitro and in vivo by a novel ROS-modulating agent with ability to eliminate stem-like cancer cells

Reactive oxygen species (ROS) have a crucial role in cell signaling and cellular functions. Mounting evidences suggest that abnormal increase of ROS is often observed in cancer cells and that this biochemical feature can be exploited for selective killing of the malignant cells. A naturally occurring compound phenethyl isothiocyanate (PEITC) has been shown to have promising anticancer activity by modulating intracellular ROS. Here we report a novel synthetic analog of PEITC with superior in vitro and in vivo antitumor effects. Mechanistic study showed that LBL21 induced a rapid depletion of intracellular glutathione (GSH), leading to abnormal ROS accumulation and mitochondrial dysfunction, evident by a decrease in mitochondrial respiration and transmembrane potential. Importantly, LBL21 exhibited the ability to abrogate stem cell-like cancer side population (SP) cells in non-small cell lung cancer A549 cells associated with a downregulation of stem cell markers including OCT4, ABCG2, SOX2 and CD133. Functionally, LBL21 inhibited the ability of cancer cells to form colonies in vitro and develop tumor in vivo. The therapeutic efficacy of LBL21 was further demonstrated in mice bearing A549 lung cancer xenografts. Our study suggests that the novel ROS-modulating agent LBL21 has promising anticancer activity with an advantage of elimination of stem-like cancer cells. This compound merits further study to evaluate its potential for use in cancer treatment.

Fatty Acid Synthesis Intermediates Represent Novel Noninvasive Biomarkers of Prostate Cancer Chemoprevention by Phenethyl Isothiocyanate.

Increased de novo synthesis of fatty acids is a distinctive feature of prostate cancer, which continues to be a leading cause of cancer-related deaths among American men. Therefore, inhibition of de novo fatty acid synthesis represents an attractive strategy for chemoprevention of prostate cancer. We have shown previously that dietary feeding of phenethyl isothiocyanate (PEITC), a phytochemical derived from edible cruciferous vegetables such as watercress, inhibits incidence and burden of poorly differentiated prostate cancer in transgenic adenocarcinoma of mouse prostate (TRAMP) model. The current study was designed to test the hypothesis of whether fatty acid intermediate(s) can serve as noninvasive biomarker(s) of prostate cancer chemoprevention by PEITC using archived plasma and tumor specimens from the TRAMP study as well as cellular models of prostate cancer. Exposure of prostate cancer cells (LNCaP and 22Rv1) to pharmacologic concentrations of PEITC resulted in downregulation of key fatty acid metabolism proteins, including acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and carnitine palmitoyltransferase 1A (CPT1A). The mRNA expression of FASN and CPT1A as well as acetyl-CoA levels were decreased by PEITC treatment in both cell lines. PEITC administration to TRAMP mice also resulted in a significant decrease in tumor expression of FASN protein. Consistent with these findings, the levels of total free fatty acids, total phospholipids, triglyceride, and ATP were significantly lower in the plasma and/or prostate tumors of PEITC-treated TRAMP mice compared with controls. The current study is the first to implicate inhibition of fatty acid synthesis in prostate cancer chemoprevention by PEITC. Cancer Prev Res; 10(5); 279-89. ©2017 AACR.

Identification of deubiquitinase targets of isothiocyanates using SILAC-assisted quantitative mass spectrometry.

Cruciferous vegetables such as broccoli and kale have well documented chemopreventative and anticancer effects that are attributed to the presence of isothiocyanates (ITCs). ITCs modulate the levels of many oncogenic proteins, but the molecular mechanisms of ITC action are not understood. We previously reported that phenethyl isothiocyanate (PEITC) inhibits two deubiquitinases (DUBs), USP9x and UCH37. DUBs regulate many cellular processes and DUB dysregulation is linked to the pathogenesis of human diseases including cancer, neurodegeneration, and inflammation. Using SILAC assisted quantitative mass spectrometry, here we identify 9 new PEITC-DUB targets: USP1, USP3, USP10, USP11, USP16, USP22, USP40, USP48 and VCPIP1. Seven of these PEITC-sensitive DUBs have well-recognized roles in DNA repair or chromatin remodeling. PEITC both inhibits USP1 and increases its ubiquitination and degradation, thus decreasing USP1 activity by two mechanisms. The loss of USP1 activity increases the level of mono-ubiquitinated DNA clamp PCNA, impairing DNA repair. Both the inhibition/degradation of USP1 and the increase in mono-ubiquitinated PCNA are new activities for PEITC that can explain the previously recognized ability of ITCs to enhance cancer cell sensitivity to cisplatin treatment. Our work also demonstrates that PEITC reduces the mono-ubiquityl histones H2A and H2B. Understanding the mechanism of action of ITCs should facilitate their use as therapeutic agents.

Isothiocyanates induce UGT1A1 in humanized UGT1 mice in a CAR dependent fashion that is highly dependent upon oxidative stress

Isothiocyanates, such as phenethyl isothiocyanate (PEITC), are formed following the consumption of cruciferous vegetables and generate reactive oxygen species (ROS) that lead to the induction of cytoprotective genes such as the UDP-glucuronosyltransferases (UGTs). The induction of ROS activates the Nrf2-Keap 1 pathway leading to the induction of genes through antioxidant response elements (AREs). UGT1A1, the sole enzyme responsible for the metabolism of bilirubin, can be induced following activation of Nrf2. When neonatal humanized UGT1 (hUGT1) mice, which exhibit severe levels of total serum bilirubin (TSB) because of a developmental delay in expression of the UGT1A1 gene, were treated with PEITC, TSB levels were reduced. Liver and intestinal UGT1A1 were induced, along with murine CYP2B10, a consensus CAR target gene. In both neonatal and adult hUGT1/Car−/− mice, PEITC was unable to induce CYP2B10. A similar result was observed following analysis of UGT1A1 expression in liver. However, TSB levels were still reduced in hUGT1/Car−/− neonatal mice because of ROS induction of intestinal UGT1A1. When oxidative stress was blocked by exposing mice to N-acetylcysteine, induction of liver UGT1A1 and CYP2B10 by PEITC was prevented. Thus, new findings in this report link an important role in CAR activation that is dependent upon oxidative stress.

Targeting Colorectal Cancer Proliferation, Stemness and Metastatic Potential Using Brassicaceae Extracts Enriched in Isothiocyanates: A 3D Cell Model-Based Study.

Colorectal cancer (CRC) recurrence is often attributable to circulating tumor cells and/or cancer stem cells (CSCs) that resist to conventional therapies and foster tumor progression. Isothiocyanates (ITCs) derived from Brassicaceae vegetables have demonstrated anticancer effects in CRC, however little is known about their effect in CSCs and tumor initiation properties. Here we examined the effect of ITCs-enriched Brassicaceae extracts derived from watercress and broccoli in cell proliferation, CSC phenotype and metastasis using a previously developed three-dimensional HT29 cell model with CSC-like traits. Both extracts were phytochemically characterized and their antiproliferative effect in HT29 monolayers was explored. Next, we performed cell proliferation assays and flow cytometry analysis in HT29 spheroids treated with watercress and broccoli extracts and respective main ITCs, phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). Soft agar assays and relative quantitative expression analysis of stemness markers and Wnt/β-catenin signaling players were performed to evaluate the effect of these phytochemicals in stemness and metastasis. Our results showed that both Brassicaceae extracts and ITCs exert antiproliferative effects in HT29 spheroids, arresting cell cycle at G2/M, possibly due to ITC-induced DNA damage. Colony formation and expression of LGR5 and CD133 cancer stemness markers were significantly reduced. Only watercress extract and PEITC decreased ALDH1 activity in a dose-dependent manner, as well as β-catenin expression. Our research provides new insights on CRC therapy using ITC-enriched Brassicaceae extracts, specially watercress extract, to target CSCs and circulating tumor cells by impairing cell proliferation, ALDH1-mediated chemo-resistance, anoikis evasion, self-renewal and metastatic potential.