The histone demethylase JMJD2C constitutes a novel NFE2 target gene that is required for the survival of JAK2V617F mutated cells.

The transcription factor NFE2 is overexpressed in most patients with myeloproliferative neoplasms (MPN). Moreover, mutations in NFE2, found in a subset of MPN patients, strongly predispose for transformation to acute leukemia. Transgenic mice overexpressing NFE2 as well as mice harboring NFE2 mutations display an MPN phenotype and spontaneously develop leukemia. However, the molecular mechanisms effecting NFE2-driven leukemic transformation remain incompletely understood. Here we show that the pro-leukemic histone demethylase JMJD2C constitutes a novel NFE2 target gene. JMJD2C expression is elevated in MPN patients as well as in NFE2 transgenic mice. Moreover, we show that loss of JMJD2C selectively impairs proliferation of JAK2V617F mutated cells. Our data suggest that JMJD2C represents a promising drug target in MPN and provide a rationale for further investigation in preclinical and clinical settings.

Aucubin slows the development of osteoporosis by inhibiting osteoclast differentiation via the nuclear factor erythroid 2-related factor 2-mediated antioxidation pathway.

CONTEXT: Osteoporosis (OP) is a metabolic disease. We have previously demonstrated that aucubin (AU) has anti-OP effects that are due to its promotion of the formation of osteoblasts. OBJECTIVES: To investigate the mechanisms of anti-OP effects of AU. MATERIALS AND METHODS: C57BL/6 mice were randomly divided into control group, 30 mg/kg Dex-induced OP group (OP model group, 15 µg/kg oestradiol-treated positive control group, 5 or 45 mg/kg AU-treated group), and 45 mg/kg AU-alone-treated group. The administration lasted for 7 weeks. Subsequently, 1, 2.5 and 5 µM AU were incubated with 50 ng/mL RANKL-induced RAW264.7 cells for 7 days to observe osteoclast differentiation. The effect of AU was evaluated by analysing tissue lesions, biochemical factor and protein expression. RESULTS: The LD50 of AU was greater than 45 mg/kg. AU increased the number of trabeculae and reduced the loss of chondrocytes in OP mice. Compared to OP mice, AU-treated mice exhibited decreased serum concentrations of TRAP5b (19.6% to 28.4%), IL-1 (12.2% to 12.6%), IL-6 (12.1%) and ROS (5.9% to 10.7%) and increased serum concentrations of SOD (14.6% to 19.4%) and CAT (17.2% to 27.4%). AU treatment of RANKL-exposed RAW264.7 cells decreased the numbers of multi-nuclear TRAP-positive cells, reversed the over-expression of TRAP5, NFATc1 and CTSK. Furthermore, AU increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins in RANKL-exposed RAW264.7 cells. CONCLUSIONS: AU slows the development of OP via Nrf2-mediated antioxidant pathways, indicating the potential use of AU in OP therapy and other types of OP research.

ERK and p38 MAPK inhibition controls NF-E2 degradation and profibrotic signaling in renal proximal tubule cells.

AIMS: Transforming growth factor-ß (TGF-ß) mediates fibrotic manifestations of diabetic nephropathy. We demonstrated proteasomal degradation of anti-fibrotic protein, nuclear factor-erythroid derived 2 (NF-E2), in TGF-ß treated human renal proximal tubule (HK-11) cells and in diabetic mouse kidneys. The current study examined the role of mitogen-activated protein kinase (MAPK) pathways in mediating NF-E2 proteasomal degradation and stimulating profibrotic signaling in HK-11 cells. MAIN METHODS: HK-11 cells were pretreated with vehicle or appropriate proteasome and MAPK inhibitors, MG132 (0.5 µM), SB203580 (1 µM), PD98059 (25 µM) and SP600125 (10 µM), respectively, followed by treatment with/without TGF-ß (10 ng/ml, 24 h). Cell lysates and kidney homogenates from FVB and OVE26 mice treated with/without MG132 were immunoblotted with appropriate antibodies. pUse vector and pUse-NF-E2 cDNA were transfected in HK-11 cells and effects of TGF-ß on JNK MAPK phosphorylation (pJNK) was examined. KEY FINDINGS: We demonstrated activation of p38, ERK, and JNK MAPK pathways in TGF-ß treated HK-11 cells. Dual p38 and ERK MAPK blockade prevented TGF-ß-induced pSer82Hsp27, fibronectin and connective tissue growth factor (CTGF) expression while preserving NF-E2 expression. Blockade of JNK MAPK inhibited TGF-ß-induced CTGF expression without preserving NF-E2 expression. MG132 treatment prevented TGF-ß-induced pJNK in HK-11 cells and in type 1 diabetic OVE26 mouse kidneys, demonstrating that TGF-ß- and diabetes-induced pJNK occurs downstream of proteasome activation. A direct role for NF-E2 in modulating pJNK activation was demonstrated by NF-E2 over-expression. SIGNIFICANCE: ERK and p38 MAPK promotes NF-E2 proteasomal degradation while proteasome activation promotes pJNK and profibrotic signaling in renal proximal tubule cells.

Fasudil ameliorates cognitive deficits, oxidative stress and neuronal apoptosis via inhibiting ROCK/MAPK and activating Nrf2 signalling pathways in APP/PS1 mice.

Alzheimer's disease (AD) is a severe neurodegenerative disorder of the central nervous system (CNS) characterized by neuron loss and dementia. Previous abundant evidence demonstrates that the first critical step in the course of AD is the state of oxidative stress and the neuronal loss is closely related to the interaction of several signalling pathways. The neuroprotective efficacy of Rho-associated protein kinase (ROCK) inhibitor in the treatment of AD has been reported, but its exact mechanism has not been well elucidated. The purpose of this study is to investigate the therapeutic effects of Fasudil on amyloid precursor protein/presenilin-1 (APP/PS1) mice and to discover the potential underlying mechanism. Sixteen 8-month-old APP/PS1 mice were divided into model and Fasudil treatment groups and 8 wild-type mice were used as a normal control group. After the behavioural test, all mice were sacrificed for immunofluorescence and other biochemical tests. The results showed that the administration of Fasudil improved learning and memory ability, elevated the concentration of antioxidative substances and decreased lipid peroxides, as well as inhibited neuronal apoptosis by increasing the expression of B-cell lymphoma-2 (Bcl-2) (p < 0.05), reducing Bcl-2 Associated X (Bax) (p < 0.05) and cleaved caspase-3 (p < 0.05) of APP/PS1 mice. Moreover, Fasudil treatment also ameliorated the phosphorylation of p38 (p < 0.01), c-Jun N-terminal kinase (JNK) (p < 0.001) and extracellular regulated protein kinases (ERK) (p < 0.001), and accelerated the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) (p < 0.01) expression and its antioxidative downstream molecules (p < 0.05, p < 0.05, and p < 0.05, respectively). Data from the present study demonstrate that Fasudil significantly restored cognitive function, restrained oxidative stress and reduced neuronal apoptosis in the hippocampus, probably by inhibiting ROCK/MAPK and activating Nrf2 signalling pathways in APP/PS1 mice.

Harnessing the Benefits of Neuroinflammation: Generation of Macrophages/Microglia with Prominent Remyelinating Properties.

Excessive inflammation within the CNS is injurious, but an immune response is also required for regeneration. Macrophages and microglia adopt different properties depending on their microenvironment, and exposure to IL4 and IL13 has been used to elicit repair. Unexpectedly, while LPS-exposed macrophages and microglia killed neural cells in culture, the addition of LPS to IL4/IL13-treated macrophages and microglia profoundly elevated IL10, repair metabolites, heparin binding epidermal growth factor trophic factor, antioxidants, and matrix-remodeling proteases. In C57BL/6 female mice, the generation of M(LPS/IL4/IL13) macrophages required TLR4 and MyD88 signaling, downstream activation of phosphatidylinositol-3 kinase/mTOR and MAP kinases, and convergence on phospho-CREB, STAT6, and NFE2. Following mouse spinal cord demyelination, local LPS/IL4/IL13 deposition markedly increased lesional phagocytic macrophages/microglia, lactate and heparin binding epidermal growth factor, matrix remodeling, oligodendrogenesis, and remyelination. Our data show that a prominent reparative state of macrophages/microglia is generated by the unexpected integration of pro- and anti-inflammatory activation cues. The results have translational potential, as the LPS/IL4/IL13 mixture could be locally applied to a focal CNS injury to enhance neural regeneration and recovery.SIGNIFICANCE STATEMENT The combination of LPS and regulatory IL4 and IL13 signaling in macrophages and microglia produces a previously unknown and particularly reparative phenotype devoid of pro-inflammatory neurotoxic features. The local administration of LPS/IL4/IL13 into spinal cord lesion elicits profound oligodendrogenesis and remyelination. The careful use of LPS and IL4/IL13 mixture could harness the known benefits of neuroinflammation to enable repair in neurologic insults.

CEACAM3 decreases asthma exacerbations and modulates respiratory syncytial virus latent infection in children.

BACKGROUND: Respiratory syncytial virus (RSV) is associated with childhood asthma. Nevertheless, not all children exposed to RSV develop asthma symptoms, possibly because genes modulate the effects of RSV on asthma exacerbations. OBJECTIVE: The purpose of this study was to identify genes that modulate the effect of RSV latent infection on asthma exacerbations. METHODS: We performed a meta-analysis to investigate differentially expressed genes (DEGs) of RSV infection from Gene Expression Omnibus datasets. Expression quantitative trait loci (eQTL) methods were applied to select single nucleotide polymorphisms (SNPs) that were associated with DEGs. Gene-based analysis was used to identify SNPs that were significantly associated with asthma exacerbations in the Taiwanese Consortium of Childhood Asthma Study (TCCAS), and validation was attempted in an independent cohort, the Childhood Asthma Management Program (CAMP). Gene-RSV interaction analyses were performed to investigate the association between the interaction of SNPs and RSV latent infection on asthma exacerbations. RESULTS: A total of 352 significant DEGs were found by meta-analysis of RSV-related genes. We used 38 123 SNPs related to DEGs to investigate the genetic main effects on asthma exacerbations. We found that eight RSV-related genes (GADD45A, GYPB, MS4A3, NFE2, RNASE3, EPB41L3, CEACAM6 and CEACAM3) were significantly associated with asthma exacerbations in TCCAS and also validated in CAMP. In TCCAS, rs7251960 (CEACAM3) significantly modulated the effect of RSV latent infection on asthma exacerbations (false-discovery rate <0.05). The rs7251960 variant was associated with CEACAM3 mRNA expression in lung tissue (p for trend=1.2×10-7). CEACAM3 mRNA was reduced in nasal mucosa from subjects with asthma exacerbations in two independent datasets. CONCLUSIONS: rs7251960 is an eQTL for CEACAM3, and CEACAM3 mRNA expression is reduced in subjects experiencing asthma exacerbations. CEACAM3 may be a modulator of RSV latent infection on asthma exacerbations.

Nuclear factor erythroid 2 (NF-E2) p45-related factor 2 interferes with homeodomain-interacting protein kinase 2/p53 activity to impair solid tumors chemosensitivity.

Resistance to chemotherapy represents a major hurdle to successful cancer treatment. A key role for efficient response to anticancer therapies is played by TP53 oncosuppressor gene that indeed is mutated in 50% of human cancers or inactivated at protein level in the remaining 50%. Homeodomain-interacting protein kinase 2 (HIPK2) is the wild-type p53 (wtp53) apoptotic activator, and its inhibition by hypoxia or hyperglycemia may contribute to tumor chemoresistance mainly by impairing p53 apoptotic activity. Another important molecule able to induce chemoresistance is nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) transcription factor, whose activation by oxidative and/or electrophilic stress regulates a transcriptional antioxidant program allowing cancer cells to adapt and survive to stresses. NRF2 may shift from cytoprotective to tumor-promoting function, according to tumor phases. NRF2 may crosstalk with both wtp53 and mutant p53 (mutp53), inhibiting the wtp53 apoptotic function and strengthening the mutp53 oncogenic function. NRF2 has also been shown to induce HIPK2 mRNA expression cooperating in inducing cytoprotection. Although HIPK2, p53, and NRF2 have been individually extensively studied, their interplay has not been clearly addressed yet. On the basis of the background and our results, we aim at hypothesizing the unexpected pro-survival activity played by the NRF2/HIPK2/p53 interplay that can be hijacked by cancer cells to bypass drugs cytotoxicity.

The transcription factor NFE2 enhances expression of the hematopoietic master regulators SCL/TAL1 and GATA2.

Activity of the transcription factor NFE2 is elevated in the majority of patients with myeloproliferative neoplasms (MPNs), either by overexpression of the wild-type alleles or by the presence of an activating mutation. In murine models, enhanced NFE2 activity causes an MPN phenotype with spontaneous transformation to acute leukemia. However, little is known about the downstream target genes activated by augmented NFE2 levels. Here, we describe that NFE2 regulates expression of the hematopoietic master regulators GATA2 and SCL/TAL1, which are in turn overexpressed in primary MPN cells, suggesting that concomitant aberrant activation of several transcription factors coordinately contributes to the cellular expansion characteristic of these disorders.

Loss of NF-E2 expression contributes to the induction of profibrotic signaling in diabetic kidneys.

AIMS: This study aimed to examine the anti-fibrotic role of Nuclear Factor-Erythroid derived 2 (NF-E2) in human renal tubule (HK-11) cells and in type 1 and type 2 diabetic (T1D, T2D) mouse kidneys. MAIN METHODS: Anti-fibrotic effects of NF-E2 were examined in transforming growth factor-ß (TGF-ß) treated HK-11 cells by over-expressing/silencing NF-E2 expression and determining its effects on profibrotic signaling. NF-E2 proteasomal degradation was confirmed by proteasome inhibition in HK-11 cells and diabetic mice. Clinical relevance of changes in NF-E2 expression to fibrotic changes in the kidney were assessed in T1D and T2D mouse kidneys. KEY FINDINGS: NF-E2 expression was significantly decreased in TGF-ß treated HK-11 cells and in kidneys of diabetic mice with concurrent increase in expression of fibrotic proteins. TGF-ß treatment of HK-11 cells did not inhibit NF-E2 mRNA expression, suggesting that the post-translational changes may contribute to NF-E2 protein degradation. The down-regulation of NF-E2 expression was attributed to its proteasomal degradation, as TGF-ß- and diabetes-induced NF-E2 down regulation was prevented by proteasome inhibitor treatment. In HK-11 cells TGF-ß treatment decreased E-cadherin expression and induced pSer82Hsp27/NF-E2 association, likely to promote NF-E2 degradation, as Hsp27 can target proteins to the proteasome. A critical role for NF-E2 in regulation of renal fibrosis was demonstrated as over-expression of NF-E2 or silencing NF-E2 expression, decreased or increased profibrotic proteins in TGF-ß-treated HK-11 cells, respectively. SIGNIFICANCE: NF-E2, a novel anti-fibrotic protein, is down-regulated in diabetic kidneys. Preserving/inducing NF-E2 expression in diabetic kidneys may provide a therapeutic potential to combat DN.

Reductive Stress Causes Pathological Cardiac Remodeling and Diastolic Dysfunction.

Aims: Redox homeostasis is tightly controlled and regulates key cellular signaling pathways. The cell's antioxidant response provides a natural defense against oxidative stress, but excessive antioxidant generation leads to reductive stress (RS). This study elucidated how chronic RS, caused by constitutive activation of nuclear erythroid related factor-2 (caNrf2)-dependent antioxidant system, drives pathological myocardial remodeling. Results: Upregulation of antioxidant transcripts and proteins in caNrf2-TG hearts (TGL and TGH; transgenic-low and -high) dose dependently increased glutathione (GSH) redox potential and resulted in RS, which over time caused pathological cardiac remodeling identified as hypertrophic cardiomyopathy (HCM) with abnormally increased ejection fraction and diastolic dysfunction in TGH mice at 6 months of age. While the TGH mice exhibited 60% mortality at 18 months of age, the rate of survival in TGL was comparable with nontransgenic (NTG) littermates. Moreover, TGH mice had severe cardiac remodeling at ∼6 months of age, while TGL mice did not develop comparable phenotypes until 15 months, suggesting that even moderate RS may lead to irreversible damages of the heart over time. Pharmacologically blocking GSH biosynthesis using BSO (l-buthionine-SR-sulfoximine) at an early age (∼1.5 months) prevented RS and rescued the TGH mice from pathological cardiac remodeling. Here we demonstrate that chronic RS causes pathological cardiomyopathy with diastolic dysfunction in mice due to sustained activation of antioxidant signaling. Innovation and Conclusion: Our findings demonstrate that chronic RS is intolerable and adequate to induce heart failure (HF). Antioxidant-based therapeutic approaches for human HF should consider a thorough evaluation of redox state before the treatment.

Comparison of the neurotoxicity associated with cobalt nanoparticles and cobalt chloride in Wistar rats.

Cobalt nanoparticles (CoNPs) have been widely used in industry given their physical, chemical and magnetic properties; however, CoNPs may cause neurological symptoms and diseases in human, yet their mechanisms of toxicity remain unknown. Here, we used male Wistar rats to investigate differences in the toxic effects associated with CoNPs and CoCl2. Upon exposure to CoCl2, and 96 nm or 123 nm CoNPs at the same concentration, the Co2+ content in CoCl2 group was significantly higher than that in either the CoNPs groups in brain tissues and blood, but lower in liver. Significant neural damage was observed in both hippocampus and cortex of the temporal lobe. Increase malondialdehyde (MDA) content and CASPASE 9 protein level were associated both with CoCl2 and CoNPs treatments, consistent with lipid perioxidation and apoptosis. Heme oxygenase-1 and (NF-E2) p45-related factor-2 protein levels were elevated in response to 96 nm CoNPs exposure. In PC12 cells, NRF2 downregulation led to reduced cell viability and increased apoptotic rate. In conclusion, both CoNPs and CoCl2 cause adverse neural effects, with nanoparticles showing greater neurotoxic potency. In addition, NRF2 protects neural cells from damage induced by CoCl2 and CoNPs by activating downstream antioxidant responses.

GATA-1: A potential novel biomarker for the differentiation of essential thrombocythemia and myelofibrosis.

Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. ABSTRACT: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.

Altered NFE2 activity predisposes to leukemic transformation and myelosarcoma with AML-specific aberrations.

In acute myeloid leukemia (AML), acquired genetic aberrations carry prognostic implications and guide therapeutic decisions. Clinical algorithms have been improved by the incorporation of novel aberrations. Here, we report the presence and functional characterization of mutations in the transcription factor NFE2 in patients with AML and in a patient with myelosarcoma. We previously described NFE2 mutations in patients with myeloproliferative neoplasms and demonstrated that expression of mutant NFE2 in mice causes a myeloproliferative phenotype. Now, we show that, during follow-up, 34% of these mice transform to leukemia presenting with or without concomitant myelosarcomas, or develop isolated myelosarcomas. These myelosarcomas and leukemias acquired AML-specific alterations, including the murine equivalent of trisomy 8, loss of the AML commonly deleted region on chromosome 5q, and mutations in the tumor suppressor Trp53 Our data show that mutations in NFE2 predispose to the acquisition of secondary changes promoting the development of myelosarcoma and/or AML.

CD226 is involved in megakaryocyte activation and early-stage differentiation.

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia.

Essentials Loss of fibrinogen in zebrafish has been previously shown to result in adult onset hemorrhage Hemostatic defects were discovered in early fga-/- embryos but well tolerated until adulthood Afibrinogenemia and thrombocytopenia results in synthetic lethality in zebrafish. Testing human FGA variants of uncertain significance in zebrafish identified causative mutations SUMMARY: Background Mutations in the alpha chain of fibrinogen (FGA), such as deficiencies in other fibrinogen subunits, lead to rare inherited autosomal recessive hemostatic disorders. These range from asymptomatic to catastrophic life-threatening bleeds and the molecular basis of inherited fibrinogen deficiencies is only partially understood. Zinc finger nucleases have been used to produce mutations in zebrafish fga, resulting in overt adult-onset hemorrhage and reduced survival. Objectives To determine the age of onset of hemostatic defects in afibrinogenemic zebrafish and model human fibrinogen deficiencies. Methods TALEN genome editing (transcription activator-like effector nucleases) was used to generate a zebrafish fga mutant. Hemostatic defects were assessed through survival, gross anatomical and histological observation and laser-induced endothelial injury. Human FGA variants with unknown pathologies were engineered into the orthologous positions in zebrafish fga. Results Loss of Fga decreased survival and resulted in synthetic lethality when combined with thrombocytopenia. Zebrafish fga mutants exhibit a severe hemostatic defect by 3 days of life, but without visible hemorrhage. Induced thrombus formation through venous endothelial injury was completely absent in mutant embryos and larvae. This hemostatic defect was restored by microinjection of wild-type fga cDNA plasmid or purified human fibrinogen. This system was used to determine whether unknown human variants were pathological by engineering them into fga. Conclusions These studies confirm that loss of fibrinogen in zebrafish results in the absence of hemostasis from the embryonic period through adulthood. When combined with thrombocytopenia, zebrafish exhibit synthetic lethality, demonstrating that thrombocytes are necessary for survival in response to hemorrhage.

Phenethyl Isothiocyanate, a Dual Activator of Transcription Factors NRF2 and HSF1.

Cruciferous vegetables are rich sources of glucosinolates which are the biogenic precursor molecules of isothiocyanates (ITCs). The relationship between the consumption of cruciferous vegetables and chemoprotection has been widely documented in epidemiological studies. Phenethyl isothiocyanate (PEITC) occurs as its glucosinolate precursor gluconasturtiin in the cruciferous vegetable watercress (Nasturtium officinale). PEITC has multiple biological effects, including activation of cytoprotective pathways, such as those mediated by the transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) and the transcription factor heat shock factor 1 (HSF1), and can cause changes in the epigenome. However, at high concentrations, PEITC leads to accumulation of reactive oxygen species and cytoskeletal changes, resulting in cytotoxicity. Underlying these activities is the sulfhydryl reactivity of PEITC with cysteine residues in its protein targets. This chemical reactivity highlights the critical importance of the dose of PEITC for achieving on-target selectivity, which should be carefully considered in the design of future clinical trials.