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1.  The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced cell death 
Autophagy  2013;9(12):2087-2102.
Pharmacological inhibition of autophagic-lysosomal function has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Repurposing approved and abandoned non-oncological drugs is an alternative approach to the identification and development of anticancer therapeutics, and antimalarials that target autophagic-lysosomal functions have recently attracted considerable attention as candidates for oncological repurposing. Since cumulative research suggests that dependence on autophagy represents a specific vulnerability of malignant melanoma cells, we screened a focused compound library of antimalarials for antimelanoma activity. Here we report for the first time that amodiaquine (AQ), a clinical 4-aminoquinoline antimalarial with unexplored cancer-directed chemotherapeutic potential, causes autophagic-lysosomal and proliferative blockade in melanoma cells that surpasses that of its parent compound chloroquine. Monitoring an established set of protein markers (LAMP1, LC3-II, SQSTM1) and cell ultrastructural changes detected by electron microscopy, we observed that AQ treatment caused autophagic-lysosomal blockade in malignant A375 melanoma cells, a finding substantiated by detection of rapid inactivation of lysosomal cathepsins (CTSB, CTSL, CTSD). AQ-treatment was associated with early induction of energy crisis (ATP depletion) and sensitized melanoma cells to either starvation- or chemotherapeutic agent-induced cell death. AQ displayed potent antiproliferative effects, and gene expression array analysis revealed changes at the mRNA (CDKN1A, E2F1) and protein level (TP53, CDKN1A, CCND1, phospho-RB1 [Ser 780]/[Ser 807/811], E2F1) consistent with the observed proliferative blockade in S-phase. Taken together, our data suggest that the clinical antimalarial AQ is a promising candidate for repurposing efforts that aim at targeting autophagic-lysosomal function and proliferative control in malignant melanoma cells.
doi:10.4161/auto.26506
PMCID: PMC3981748  PMID: 24113242
malignant melanoma; amodiaquine; chloroquine; autophagy; lysosome; cathepsin; E2F1; CDKN1A
2.  Tanshinone I Activates the Nrf2-Dependent Antioxidant Response and Protects Against As(III)-Induced Lung Inflammation In Vitro and In Vivo 
Antioxidants & Redox Signaling  2013;19(14):1647-1661.
Abstract
Aims: The NF-E2 p45-related factor 2 (Nrf2) signaling pathway regulates the cellular antioxidant response and activation of Nrf2 has recently been shown to limit tissue damage from exposure to environmental toxicants, including As(III). In an attempt to identify improved molecular agents for systemic protection against environmental insults, we have focused on the identification of novel medicinal plant-derived Nrf2 activators. Results: Tanshinones [tanshinone I (T-I), tanshinone IIA, dihydrotanshinone, cryptotanshinone], phenanthrenequinone-based redox therapeutics derived from the medicinal herb Salvia miltiorrhiza, have been tested as experimental therapeutics for Nrf2-dependent cytoprotection. Using a dual luciferase reporter assay overexpressing wild-type or mutant Kelch-like ECH-associated protein-1 (Keap1), we demonstrate that T-I is a potent Keap1-C151-dependent Nrf2 activator that stabilizes Nrf2 by hindering its ubiquitination. In human bronchial epithelial cells exposed to As(III), T-I displays pronounced cytoprotective activity with upregulation of Nrf2-orchestrated gene expression. In Nrf2 wild-type mice, systemic administration of T-I attenuates As(III) induced inflammatory lung damage, a protective effect not observed in Nrf2 knockout mice. Innovation: Tanshinones have been identified as a novel class of Nrf2-inducers for antioxidant tissue protection in an in vivo As(III) inhalation model, that is relevant to low doses of environmental exposure. Conclusion: T-I represents a prototype Nrf2-activator that displays cytoprotective activity upon systemic administration targeting lung damage originating from environmental insults. T-I based Nrf2-directed systemic intervention may provide therapeutic benefit in protecting other organs against environmental insults. Antioxid. Redox Signal. 19, 1647–1661.
doi:10.1089/ars.2012.5117
PMCID: PMC3809600  PMID: 23394605
3.  Probing Mechanoregulation of Neuronal Differentiation by Plasma Lithography Patterned Elastomeric Substrates 
Scientific Reports  2014;4:6965.
Cells sense and interpret mechanical cues, including cell-cell and cell-substrate interactions, in the microenvironment to collectively regulate various physiological functions. Understanding the influences of these mechanical factors on cell behavior is critical for fundamental cell biology and for the development of novel strategies in regenerative medicine. Here, we demonstrate plasma lithography patterning on elastomeric substrates for elucidating the influences of mechanical cues on neuronal differentiation and neuritogenesis. The neuroblastoma cells form neuronal spheres on plasma-treated regions, which geometrically confine the cells over two weeks. The elastic modulus of the elastomer is controlled simultaneously by the crosslinker concentration. The cell-substrate mechanical interactions are also investigated by controlling the size of neuronal spheres with different cell seeding densities. These physical cues are shown to modulate with the formation of focal adhesions, neurite outgrowth, and the morphology of neuroblastoma. By systematic adjustment of these cues, along with computational biomechanical analysis, we demonstrate the interrelated mechanoregulatory effects of substrate elasticity and cell size. Taken together, our results reveal that the neuronal differentiation and neuritogenesis of neuroblastoma cells are collectively regulated via the cell-substrate mechanical interactions.
doi:10.1038/srep06965
PMCID: PMC4223667  PMID: 25376886
4.  Induction of autophagy contributes to cisplatin resistance in human ovarian cancer cells 
Molecular Medicine Reports  2014;11(1):91-98.
Cisplatin resistance is a major challenge in the clinical treatment of ovarian cancer, of which the underlying mechanisms remain unknown. The aim of the present study was to explore the role of autophagy in cisplatin resistance in ovarian cancer cells. A2780cp cisplatin-resistant ovarian carcinoma cells and the A2780 parental cell line, were used as a model throughout the present study. The cell viability was determined using a water soluble tetrazolium salt-8 assay, and western blot analysis was performed to determine the protein expression levels of microtubule-associated protein 1 light chain 3 (LC3 I and LC3 II), and Beclin 1. Beclin 1 small interfering (si)RNA and 3-methyladenine (3-MA) were used to determine whether inhibition of autophagy may re-sensitize cisplatin-resistant cells to cisplatin. The ultrastructural analysis of autophagosomes was performed using transmission electron microscopy, and apoptosis was measured by flow cytometry. In both A2780cp and A2780 cells, cisplatin induced the formation of autophagosomes and upregulated the expression levels of autophagy protein markers, LC3 II and Beclin 1. However, the levels of autophagy were significantly higher in A2780cp cells, as compared with the A2780 cells. The combined treatment of cisplatin with 3-MA, the autophagy pharmacological inhibitor, increased the cell death rate, but had no effects on apoptosis, as compared with cisplatin treatment alone in A2780cp cells. However, inhibition of autophagy by siRNA knockdown of Beclin 1 expression enhanced cisplatin-induced cell death and apoptosis. The findings of the present study suggest that autophagy has a protective role in human ovarian cancer cells, and that targeting autophagy may promote chemotherapeutic sensitivity.
doi:10.3892/mmr.2014.2671
PMCID: PMC4237096  PMID: 25322694
ovarian carcinoma; cisplatin; autophagy; resistance
5.  Nrf2 Modulates Contractile and Metabolic Properties of Skeletal Muscle in Streptozotocin-Induced Diabetic Atrophy 
Experimental cell research  2013;319(17):10.1016/j.yexcr.2013.07.015.
The role of Nrf2 in disease prevention and treatment is well documented, however the specific role of Nrf2 in skeletal muscle is not well described. The current study investigated whether Nrf2 plays a protective role in an STZ-induced model of skeletal muscle atrophy.
Modulation of Nrf2 through siRNA resulted in a more robust differentiation of C2C12s, whereas increasing Nrf2 with sulforaphane treatment inhibited differentiation. Diabetic muscle atrophy was not dramatically influenced by Nrf2 genotype, since no differences were observed in total atrophy (all fiber types combined) between WT+STZ and KO+STZ animals. Nrf2-KO animals however, illustrated alterations in muscle size of Fast, Type II myosin expressing fibers. KO+STZ animals show significant alterations in myosin isoform expression in the GAST. Similarly, KO controls mimic both WT+STZ and KO+STZ muscle alterations in mitochondrial subunit expression. PGC-1α, a well-established player in mitochondrial biogenesis and myosin isoform expression, was decreased in KO control, WT+STZ and KO+STZ SOL muscle. Similarly, PGC-1α protein levels are correlated with Nrf2 levels in C2C12s after modulation by Nrf2 siRNA or sulforaphane treatment.
We provide experimental evidence indicating Nrf2 plays a role in myocyte differentiation and governs molecular alterations in contractile and metabolic properties in an STZ-induced model of muscle atrophy.
doi:10.1016/j.yexcr.2013.07.015
PMCID: PMC3809009  PMID: 23896025
Nrf2; skeletal muscle; atrophy; myosin; metabolism
6.  Detection of mRNA in living cells by double-stranded locked nucleic acid probes 
The Analyst  2013;138(17):4777-4785.
Double-stranded probes are homogeneous biosensors for rapid detection of specific nucleotide sequences. These double-stranded probes have been applied in various molecular sensing applications, such as real-time polymerase chain reaction and detection of bacterial 16S rRNA. In this study, we present the design and optimization of double-stranded probes for single-cell gene expression analysis in living cells. With alternating DNA/LNA monomers for optimizing the stability and specificity, we show that the probe is stable in living cells for over 72 hours post-transfection and is capable of detecting changes in gene expression induced by external stimuli. The probes can be delivered to a large number of cells simultaneously by cationic liposomal transfection or to individual cells selectively by photothermal delivery. We also demonstrate that the probe quantifies intracellular mRNA in living cells through the use of an equilibrium analysis. With its effectiveness and performance, the double-stranded probe represents a broadly applicable approach for large-scale single-cell gene expression analysis toward numerous biomedical applications, such as systems biology, cancer, and drug screening.
doi:10.1039/c3an00722g
PMCID: PMC3736730  PMID: 23772441
7.  Nrf2 suppresses lupus nephritis through inhibition of oxidative injury and the NF-κB-mediated inflammatory response 
Kidney international  2013;85(2):333-343.
The generation of reactive oxygen species plays a pivotal role in both acute and chronic glomerular injuries in patients with lupus nephritis. Since the transcription factor Nrf2 is a major regulator of the antioxidant response and is a primary cellular defense mechanism we sought to determine a role of Nrf2 in the progression of lupus nephritis. Pathological analyses of renal biopsies from patients with different types of lupus nephritis showed oxidative damage in the glomeruli, accompanied by an active Nrf2 antioxidant response. A murine lupus nephritis model using Nrf2+/+ and Nrf2−/− mice was established using pristine injection. In this model, Nrf2−/− mice suffered from greater renal damage and had more severe pathological alterations in the kidney. In addition, Nrf2+/+ mice showed ameliorative renal function when treated with sulforaphane, an Nrf2 inducer. Nrf2−/− mice had higher expression of TGFβ1, fibronectin and iNOS. In primary mouse mesangial cells, the nephritogenic monoclonal antibody R4A activated the NF-κB pathway and increased the level of reactive oxygen species, iNOS, TGFβ1 and fibronectin. Knockdown of Nrf2 expression aggravated all aforementioned responses induced by R4A. Thus, these results suggest that Nrf2 improves lupus nephritis by neutralizing reactive oxygen species and by negatively regulating the NF-κB and TGFβ1 signaling pathways.
doi:10.1038/ki.2013.343
PMCID: PMC3992978  PMID: 24025640
lupus nephritis; Nrf2; ROS; NF-κB; TGFβ1; iNOS
8.  Single cell antimicrobial susceptibility testing by confined microchannels and electrokinetic loading 
Analytical chemistry  2013;85(8):3971-3976.
Multidrug-resistant pathogens are an emerging global health problem. In addition to the need of developing new antibiotics in the pipeline, the ability to rapidly determine the antibiotic resistance profiles of bacteria represents one of the most crucial steps toward the management of infectious diseases and the prevention of multidrug-resistant pathogens. Here, we report a single cell antimicrobial susceptibility testing (AST) approach for rapid determination of the antibiotic resistance of bacterial pathogens. By confining individual bacteria in gas permeable microchannels with dimensions comparable to a single bacterium, the antibiotic resistance of the bacteria can be monitored in real-time at the single cell level. To facilitate the dynamic loading of the bacteria into the confined microchannels for observation, AC electrokinetics is demonstrated for capturing bacteria to defined locations in high-conductivity AST buffer. The electrokinetic technique achieves a loading efficiency of about 75% with a negligible effect on the bacterial growth rate. To optimize the protocol for single cell AST, the bacterial growth rate of individual bacteria under different antibiotic conditions has been determined systematically. The applicability of single cell AST is demonstrated by the rapid determination of the antimicrobial resistant profiles of uropathogenic clinical isolates in Mueller-Hinton media and in urine. The antibiotic resistance profiles of bacteria can be determined in less than one hour compared to days in standard culture-based AST techniques.
doi:10.1021/ac4004248
PMCID: PMC4102406  PMID: 23445209
9.  Tumor protein translationally controlled 1 is a p53 target gene that promotes cell survival 
Cell Cycle  2013;12(14):2321-2328.
Tumor suppressor p53 maintains genome stability by differentially activating target genes that control diverse cellular responses, such as the antioxidant response, cell cycle arrest and apoptosis. Despite the fact that many p53 downstream genes have been well characterized, novel p53 target genes are continuously being identified. Here, we report that Tpt1 is a direct target gene of p53. We found that p53 upregulates the transcription of Tpt1 and identified a p53-responsive element in the promoter of the mouse Tpt1 gene. Furthermore, p53-dependent induction of Tpt1 was able to reduce oxidative stress, minimize apoptosis, and promote cell survival in response to H2O2 challenge. In addition, a positive correlation between the expression of p53 and Tpt1 only existed in normal lung tissues, not in lung tumors. Such positive correlation was also found in lung cell lines that contain wild-type p53, but not mutated p53. Based on the important role of Tpt1 in cancer development, chemoresistance, and cancer reversion, identification of Tpt1 as a direct target gene of p53 not only adds to the complexity of the p53 network, but may also open up a new avenue for cancer prevention and intervention.
doi:10.4161/cc.25404
PMCID: PMC3755082  PMID: 24067374
p53; Tpt1; TCTP; cancer
10.  USP15 negatively regulates Nrf2 through deubiquitination of Keap1 
Molecular cell  2013;51(1):68-79.
Summary
Nrf2 is a master regulator of the antioxidant response. Under basal conditions Nrf2 is polyubiquitinated by the Keap1-Cul3-E3 ligase and degraded by the 26S-proteasome. In response to Nrf2 inducers there is a switch in polyubiquitination from Nrf2 to Keap1. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or Keap1 remains unknown. Here we report that the deubiquitinating enzyme, USP15, specifically deubiquitinates Keap1, which suppresses the Nrf2 pathway. We demonstrated that deubiquitinated-Keap1 incorporates into the Keap1-Cul3-E3 ligase complex more efficiently, enhancing the complex stability and enzymatic activity. Consequently, there is an increase in Nrf2 protein degradation and a reduction in Nrf2 target gene expression. Furthermore, USP15-siRNA enhances chemoresistance of cells through upregulation of Nrf2. These findings further our understanding of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy.
doi:10.1016/j.molcel.2013.04.022
PMCID: PMC3732832  PMID: 23727018
Nrf2; Keap1; USP15; Cul3; ubiquitination; deubiquitination; antioxidant response; chemoresistance
11.  Nrf2 induces cisplatin resistance through activation of autophagy in ovarian carcinoma 
Cisplatin resistance is a major problem affecting ovarian carcinoma treatment. NF-E2-related factor 2 (Nrf2), a nuclear transcription factor, plays an important role in chemotherapy resistance. However, the underlying mechanism by which Nrf2 mediates cisplatin chemoresistance is unclear. Methods: The human ovarian carcinoma cell line, A2780, and its cisplatin-resistant variant, A2780cp were cultivated. Cell viability was determined with WST-8 assay. Western blot was applied to detect the expression of Nrf2, Nrf2 target genes, and autophagy-related proteins. RNA interference was used to knock down target genes. Annexin V and propidium iodide (PI) staining was utilized to quantify apoptosis. The ultrastructural analysis of autophagosomes was performed by transmission electron microscopy (TEM). Results: Nrf2 and its targeting genes, NQO1 and HO-1, are overexpressed in A2780cp cells compared with A2780 cells. Knocking down Nrf2 sensitized A2780cp cells to cisplatin treatment and decreased autophagy-related genes, Atg3, Atg6, Atg12 and p62 in both mRNA and protein levels. Furthermore, we demonstrated that in both cell lines cisplatin could induce the formation of autophagosomes and upregulate the expression of autophagy-related genes Atg3, Atg6 and Atg12. Treatment with an autophagy inhibitor, 3-Methyladenine (3-MA), or beclin 1 siRNA enhanced cisplatin-induced cell death in A2780cp cells, suggesting that inhibition of autophagy renders resistant cells to be more sensitive to cisplatin. Taken together, Nrf2 signaling may regulate cisplatin resistance by activating autophagy. Conclusions: Nrf2-activated autophagy may function as a novel mechanism causing cisplatin-resistance.
PMCID: PMC4014230  PMID: 24817946
Ovarian carcinoma; Nrf2; autophagy; cisplatin; chemoresistance
12.  Arsenic Inhibits Autophagic Flux, Activating the Nrf2-Keap1 Pathway in a p62-Dependent Manner 
Molecular and Cellular Biology  2013;33(12):2436-2446.
The Nrf2-Keap1 signaling pathway is a protective mechanism promoting cell survival. Activation of the Nrf2 pathway by natural compounds has been proven to be an effective strategy for chemoprevention. Interestingly, a cancer-promoting function of Nrf2 has recently been observed in many types of tumors due to deregulation of the Nrf2-Keap1 axis, which leads to constitutive activation of Nrf2. Here, we report a novel mechanism of Nrf2 activation by arsenic that is distinct from that of chemopreventive compounds. Arsenic deregulates the autophagic pathway through blockage of autophagic flux, resulting in accumulation of autophagosomes and sequestration of p62, Keap1, and LC3. Thus, arsenic activates Nrf2 through a noncanonical mechanism (p62 dependent), leading to a chronic, sustained activation of Nrf2. In contrast, activation of Nrf2 by sulforaphane (SF) and tert-butylhydroquinone (tBHQ) depends upon Keap1-C151 and not p62 (the canonical mechanism). More importantly, SF and tBHQ do not have any effect on autophagy. In fact, SF and tBHQ alleviate arsenic-mediated deregulation of autophagy. Collectively, these findings provide evidence that arsenic causes prolonged activation of Nrf2 through autophagy dysfunction, possibly providing a scenario similar to that of constitutive activation of Nrf2 found in certain human cancers. This may represent a previously unrecognized mechanism underlying arsenic toxicity and carcinogenicity in humans.
doi:10.1128/MCB.01748-12
PMCID: PMC3700105  PMID: 23589329
13.  The Nrf2-inducers tanshinone I and dihydrotanshinone protect human skin cells and reconstructed human skin against solar simulated UV☆ 
Redox Biology  2013;1(1):532-541.
Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photocarcinogenesis and photoaging, and an urgent need exists for improved strategies for skin photoprotection. The redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defense against environmental electrophilic insult, has recently emerged as an important determinant of cutaneous damage from solar UV, and the concept of pharmacological activation of Nrf2 has attracted considerable attention as a novel approach to skin photoprotection. In this study, we examined feasibility of using tanshinones, a novel class of phenanthrenequinone-based cytoprotective Nrf2 inducers derived from the medicinal plant Salvia miltiorrhiza, for protection of cultured human skin cells and reconstructed human skin against solar simulated UV. Using a dual luciferase reporter assay in human Hs27 dermal fibroblasts pronounced transcriptional activation of Nrf2 by four major tanshinones [tanshinone I (T-I), dihydrotanshinone (DHT), tanshinone IIA (T-II-A) and cryptotanshinone (CT)] was detected. In fibroblasts, the more potent tanshinones T-I and DHT caused a significant increase in Nrf2 protein half-life via blockage of ubiquitination, ultimately resulting in upregulated expression of cytoprotective Nrf2 target genes (GCLC, NQO1) with the elevation of cellular glutathione levels. Similar tanshinone-induced changes were also observed in HaCaT keratinocytes. T-I and DHT pretreatment caused significant suppression of skin cell death induced by solar simulated UV and riboflavin-sensitized UVA. Moreover, feasibility of tanshinone-based cutaneous photoprotection was tested employing a human skin reconstruct exposed to solar simulated UV (80 mJ/cm2 UVB; 1.53 J/cm2 UVA). The occurrence of markers of epidermal solar insult (cleaved procaspase 3, pycnotic nuclei, eosinophilic cytoplasm, acellular cavities) was significantly attenuated in DHT-treated reconstructs that displayed increased immunohistochemical staining for Nrf2 and γ-GCS together with the elevation of total glutathione levels. Taken together, our data suggest the feasibility of achieving tanshinone-based cutaneous Nrf2-activation and photoprotection.
Graphical abstract
Highlights
•Tanshinones are phenanthrenequinone-based Nrf2 inducers active in human skin cells.•Tanshinones upregulate Nrf2 target gene expression with the elevation of glutathione.•Dihydrotanshinone protects cultured human skin cells against solar simulated UV.•Dihydrotanshinone protects reconstructed human skin against acute photodamage.
doi:10.1016/j.redox.2013.10.004
PMCID: PMC3836278  PMID: 24273736
CHX, cycloheximide; CT, cryptotanshinone; DHT, dihydrotanshinone; DMEM, Dulbecco's modified Eagle's medium; γ-GCS, gamma-glutamate-cysteine ligase; H&E, hematoxylin and eosin; HMOX1, heme oxygenase-1; IHC, immunohistochemistry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor-E2-related factor 2; ROS, reactive oxygen species; SF, sulforaphane; SLL, solar simulated UV light; T-I, tanshinone I; T-II-A, tanshinone IIA; UVA, ultraviolet; UVB, ultraviolet B; Tanshinone I; Dihydrotanshinone; Nrf2; Solar simulated ultraviolet light; Skin photoprotection
14.  Sulforaphane prevents pulmonary damage in response to inhaled arsenic by activating the Nrf2-defense response 
Toxicology and applied pharmacology  2012;265(3):292-299.
Exposure to arsenic is associated with an increased risk of lung disease. Novel strategies are needed to reduce the adverse health effects associated with arsenic exposure in the lung. Nrf2, a transcription factor that mediates an adaptive cellular defense response, is effective in detoxifying environmental insults and prevents a broad spectrum of diseases induced by environmental exposure to harmful substances. In this report, we tested whether Nrf2 activation protects mice from arsenic-induced toxicity. We used an in vivo arsenic inhalation model that is highly relevant to low environmental human exposure to arsenic-containing dusts. Two-week exposure to arsenic-containing dust resulted in pathological alterations, oxidative DNA damage, and mild apoptotic cell death in the lung; all of which were blocked by sulforaphane (SF) in an Nrf2-dependent manner. Mechanistically, SF-mediated activation of Nrf2 alleviated inflammatory responses by modulating cytokine production. This study provides strong evidence that dietary intervention targeting Nrf2 activation is a feasible approach to reduce adverse health effects associated with arsenic exposure.
doi:10.1016/j.taap.2012.08.028
PMCID: PMC3725323  PMID: 22975029
Nrf2; Keap1; Arsenic; Antioxidant response
15.  Arsenic-Mediated Activation of the Nrf2-Keap1 Antioxidant Pathway 
Arsenic is present in the environment and has become a worldwide health concern due to its toxicity and carcinogenicity. However, the specific mechanism(s) by which arsenic elicits its toxic effects has yet to be fully elucidated. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been recognized as the master regulator of a cellular defense mechanism against toxic insults. This review highlights studies demonstrating that arsenic activates the Nrf2-Keap1 antioxidant pathway by a distinct mechanism from that of natural compounds such as sulforaphane (SF) found in broccoli sprouts or tert-butylhyrdoquinone (tBHQ), a natural antioxidant commonly used as a food preservative. Evidence also suggests that arsenic prolongs Nrf2 activation and may mimic constitutive activation of Nrf2, which has been found in several human cancers due to disruption of the Nrf2-Keap1 axis. The current literature strongly suggests that activation of Nrf2 by arsenic potentially contributes to, rather than protects against, arsenic toxicity and carcinogenicity. The mechanism(s) by which known Nrf2 activators, such as the natural chemopreventive compounds SF and lipoic acid, protect against the deleterious effects caused by arsenic will also be discussed. These findings will provide insight to further understand how arsenic promotes a prolonged Nrf2 response, which will lead to the identification of novel molecular markers and development of rational therapies for the prevention or intervention of arsenic-induced diseases. The National Institute of Environmental Health Science (NIEHS) Outstanding New Environmental Scientist (ONES) award has provided the opportunity to review the progress both in the fields of arsenic toxicology and Nrf2 biology. Much of the funding has led to (1) the novel discovery that arsenic activates the Nrf2 pathway by a mechanism different to that of other Nrf2 activators, such as sulforaphane and tert-butylhydroquinone, (2) activation of Nrf2 by chemopreventive compounds protects against arsenic toxicity and carcinogenicity both in vitro and in vivo, (3) constitutive activation of Nrf2 by disrupting Keap1-mediated negative regulation contributes to cancer and chemoresistance, (4) p62-mediated sequestration of Keap1 activates the Nrf2 pathway, and (5) arsenic-mediated Nrf2 activation may be through a p62-dependent mechanism. All of these findings have been published and are discussed in this review. This award has laid the foundation for my laboratory to further investigate the molecular mechanism(s) that regulate the Nrf2 pathway and how it may play an integral role in arsenic toxicity. Moreover, understanding the biology behind arsenic toxicity and carcinogenicity will help in the discovery of potential strategies to prevent or control arsenic-mediated adverse effects.
doi:10.1002/jbt.21463
PMCID: PMC3725327  PMID: 23188707
Nrf2; Arsenic; Keap1; Oxidative stress; p62; Autophagy; Chemoprevention
16.  Cinnamoyl-based Nrf2-Activators Targeting Human Skin Cell Photo-oxidative Stress 
Free radical biology & medicine  2008;45(4):385-395.
Strong experimental evidence suggests the involvement of photo-oxidative stress mediated by reactive oxygen species as a crucial mechanism of solar damage relevant to human skin photoaging and photocarcinogenesis. Based on the established role of antioxidant response element (ARE)-mediated gene expression in cancer chemoprevention, we tested the hypothesis that small molecule Nrf2-activators may serve a photo-chemopreventive role by targeting skin cell photo-oxidative stress. A luciferase-based reporter gene assay was used as a primary screen for the identification of novel agents that modulate the Nrf2-Keap1 signaling pathway. A series of cinnamoyl-based electrophilic Michael acceptors including cinnamic aldehyde and methyl-1-cinnamoyl-5-oxo-2-pyrrolidine-carboxylate was identified as potent Nrf2-activators. Hit confirmation was performed in a secondary screen, based on immunodetection of Nrf2 protein upregulation in human Hs27 skin fibroblasts, HaCaT keratinocytes, and primary skin keratinocytes. Bioefficacy profiling of positive test compounds in skin cells demonstrated compound-induced upregulation of hemeoxygenase I and NAD(P)H-quinone oxidoreductase, two Nrf2 target genes involved in the cellular antioxidant response. Pretreatment with cinnamoyl-based Nrf2-activators suppressed intracellular oxidative stress and protected against photo-oxidative induction of apoptosis in skin cells exposed to high doses of singlet oxygen. Our pilot studies suggest feasibility of developing cinnamoyl-based Nrf2-activators as novel photo-chemopreventive agents targeting skin cell photo-oxidative stress.
doi:10.1016/j.freeradbiomed.2008.04.023
PMCID: PMC3710742  PMID: 18482591
Nrf2; skin cancer; photo-oxidative stress; photo-chemoprevention; Michael acceptor; cinnamic aldehyde; singlet oxygen
17.  Nrf2 Pathway Regulates Multidrug-Resistance-Associated Protein 1 in Small Cell Lung Cancer 
PLoS ONE  2013;8(5):e63404.
Although multidrug-resistance-associated protein-1 (MRP1) is a major contributor to multi-drug resistance (MDR), the regulatory mechanism of Mrp1 still remains unclear. Nrf2 is a transcription factor that regulates cellular defense response through antioxidant response elements (AREs) in normal tissues. Recently, Nrf2 has emerged as an important contributor to chemo-resistance in tumor tissues. In the present study, the role of Nrf2-ARE pathway on regulation of Mrp1 was investigated. Compared with H69 lung cancer cells, H69AR cells with MDR showed significantly higher Nrf2-ARE pathway activity and expression of Mrp1 as well. When Nrf2 was knocked down in H69AR cells, MRP1's expression decreased accordingly. Moreover, those H69AR cells with reduced Nrf2 level restored sensitivity to chemo-drugs. To explore how Nrf2-ARE pathway regulates Mrp1, the promoter of Mrp1 gene was searched, and two putative AREs—ARE1 and ARE2—were found. Using reporter gene and ChIP assay, both ARE1 and ARE2 showed response to and interaction with Nrf2. In 40 cases of cancer tissues, the expression of Nrf2 and MRP1 was measured by immunohistochemistry (IHC). As the quantitive data of IHC indicated, both Nrf2 and MRP1 showed significantly higher expression in tumor tissue than adjacent non-tumor tissue. And more important, the correlation analysis of the two genes proved that their expression was correlative. Taken together, theses data suggested that Nrf2-ARE pathway is required for the regulatory expression of Mrp1 and implicated Nrf2 as a new therapeutic target for MDR.
doi:10.1371/journal.pone.0063404
PMCID: PMC3646742  PMID: 23667609
18.  Nrf2 Is Crucial to Graft Survival in a Rodent Model of Heart Transplantation 
Currently, the sole treatment option for patients with heart failure is transplantation. The battle of prolonging graft survival and modulating innate and adaptive immune responses is still being waged in the clinic and in research labs. The transcription factor Nrf2 controls major cell survival pathways and is central to moderating inflammation and immune responses. In this study the effect of Nrf2 levels in host recipient C57BL/6 mice on Balb/c allogeneic graft survival was examined. Importantly, Nrf2−/− recipient mice could not support the graft for longer than 7.5 days on average, whereas activation of Nrf2 by sulforaphane in Nrf2+/+ hosts prolonged graft survival to 13 days. Several immune cells in the spleen of recipient mice were unchanged; however, CD11b+ macrophages were significantly increased in Nrf2−/− mice. In addition, IL-17 mRNA levels were elevated in grafts transplanted into Nrf2−/− mice. Although Nrf2 appears to play a crucial role in graft survival, the exact mechanism is yet to be fully understood.
doi:10.1155/2013/919313
PMCID: PMC3603380  PMID: 23533698
19.  Therapeutic Potential of Nrf2 Activators in Streptozotocin-Induced Diabetic Nephropathy 
Diabetes  2011;60(11):3055-3066.
OBJECTIVE
To determine whether dietary compounds targeting NFE2-related factor 2 (Nrf2) activation can be used to attenuate renal damage and preserve renal function during the course of streptozotocin (STZ)-induced diabetic nephropathy.
RESEARCH DESIGN AND METHODS
Diabetes was induced in Nrf2+/+ and Nrf2−/− mice by STZ injection. Sulforaphane (SF) or cinnamic aldehyde (CA) was administered 2 weeks after STZ injection and metabolic indices and renal structure and function were assessed (18 weeks). Markers of diabetes including blood glucose, insulin, polydipsia, polyuria, and weight loss were measured. Pathological alterations and oxidative damage in glomeruli were also determined. Changes in protein expression of the Nrf2 pathway, as well as transforming growth factor-β1 (TGF-β1), fibronectin (FN), collagen IV, and p21/WAF1Cip1 (p21) were analyzed. The molecular mechanisms of Nrf2-mediated protection were investigated in an in vitro model using human renal mesangial cells (HRMCs).
RESULTS
SF or CA significantly attenuated common metabolic disorder symptoms associated with diabetes in Nrf2+/+ but not in Nrf2−/− mice, indicating SF and CA function through specific activation of the Nrf2 pathway. Furthermore, SF or CA improved renal performance and minimized pathological alterations in the glomerulus of STZ-Nrf2+/+ mice. Nrf2 activation reduced oxidative damage and suppressed the expression of TGF-β1, extracellular matrix proteins and p21 both in vivo and in HRMCs. In addition, Nrf2 activation reverted p21-mediated growth inhibition and hypertrophy of HRMCs under hyperglycemic conditions.
CONCLUSIONS
We provide experimental evidence indicating that dietary compounds targeting Nrf2 activation can be used therapeutically to improve metabolic disorder and relieve renal damage induced by diabetes.
doi:10.2337/db11-0807
PMCID: PMC3198067  PMID: 22025779
20.  PALB2 Interacts with KEAP1 To Promote NRF2 Nuclear Accumulation and Function 
Molecular and Cellular Biology  2012;32(8):1506-1517.
PALB2/FANCN is mutated in breast and pancreatic cancers and Fanconi anemia (FA). It controls the intranuclear localization, stability, and DNA repair function of BRCA2 and links BRCA1 and BRCA2 in DNA homologous recombination repair and breast cancer suppression. Here, we show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the master antioxidant transcription factor NRF2. PALB2 shares with NRF2 a highly conserved ETGE-type KEAP1 binding motif and can effectively compete with NRF2 for KEAP1 binding. PALB2 promotes NRF2 accumulation and function in the nucleus and lowers the cellular reactive oxygen species (ROS) level. In addition, PALB2 also regulates the rate of NRF2 export from the nucleus following induction. Our findings identify PALB2 as a regulator of cellular redox homeostasis and provide a new link between oxidative stress and the development of cancer and FA.
doi:10.1128/MCB.06271-11
PMCID: PMC3318596  PMID: 22331464
21.  Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition 
PLoS ONE  2012;7(7):e41831.
mTOR inhibitors are used clinically to treat renal cancer but are not curative. Here we show that autophagy is a resistance mechanism of human renal cell carcinoma (RCC) cell lines to mTOR inhibitors. RCC cell lines have high basal autophagy that is required for survival to mTOR inhibition. In RCC4 cells, inhibition of mTOR with CCI-779 stimulates autophagy and eliminates RIP kinases (RIPKs) and this is blocked by autophagy inhibition, which induces RIPK- and ROS-dependent necroptosis in vitro and suppresses xenograft growth. Autophagy of mitochondria is required for cell survival since mTOR inhibition turns off Nrf2 antioxidant defense. Thus, coordinate mTOR and autophagy inhibition leads to an imbalance between ROS production and defense, causing necroptosis that may enhance cancer treatment efficacy.
doi:10.1371/journal.pone.0041831
PMCID: PMC3406086  PMID: 22848625
22.  KPNA6 (Importin α7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿  
Molecular and Cellular Biology  2011;31(9):1800-1811.
The transcription factor Nrf2 has emerged as a master regulator of cellular redox homeostasis. As an adaptive response to oxidative stress, Nrf2 activates the transcription of a battery of genes encoding antioxidants, detoxification enzymes, and xenobiotic transporters by binding the cis-antioxidant response element in the promoter regions of genes. The magnitude and duration of inducible Nrf2 signaling is delicately controlled at multiple levels by Keap1, which targets Nrf2 for redox-sensitive ubiquitin-mediated degradation in the cytoplasm and exports Nrf2 from the nucleus. However, it is not clear how Keap1 gains access to the nucleus. In this study, we show that Keap1 is constantly shuttling between the nucleus and the cytoplasm under physiological conditions. The nuclear import of Keap1 requires its C-terminal Kelch domain and is independent of Nrf1 and Nrf2. We have determined that importin α7, also known as karyopherin α6 (KPNA6), directly interacts with the Kelch domain of Keap1. Overexpression of KPNA6 facilitates Keap1 nuclear import and attenuates Nrf2 signaling, whereas knockdown of KPNA6 slows down Keap1 nuclear import and enhances the Nrf2-mediated adaptive response induced by oxidative stress. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus during the postinduction phase, therefore promoting restoration of the Nrf2 protein to basal levels. These findings demonstrate that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.
doi:10.1128/MCB.05036-11
PMCID: PMC3133232  PMID: 21383067
23.  High levels of Nrf2 determine chemoresistance in type II endometrial cancer 
Cancer research  2010;70(13):5486-5496.
Type II endometrial cancer, which mainly presents as serous and clear cell types, has proved to be the most malignant and recurrent carcinoma among various female genital malignancies. The transcription factor, Nrf2, was first described as having chemopreventive activity. Activation of the Nrf2-mediated cellular defense response protects cells against the toxic and carcinogenic effects of environmental insults by upregulating an array of genes that detoxify reactive oxygen species (ROS) and restore cellular redox homeostasis. However, the cancer-promoting role of Nrf2 has recently been revealed. Nrf2 is constitutively upregulated in several types of human cancer tissues and cancer cell lines. Furthermore, inhibition of Nrf2 expression sensitizes cancer cells to chemotherapeutic drugs. In this study, the constitutive level of Nrf2 was compared in different types of human endometrial tumors. It was found that Nrf2 was highly expressed in endometrial serous carcinoma (ESC), whereas complex hyperplasia (CH) and endometrial endometrioid carcinoma (EEC) had no or marginal expression of Nrf2. Likewise, the ESC derived SPEC-2 cell line had a higher level of Nrf2 expression and was more resistant to the toxic effects of cisplatin and paclitaxel than that of the Ishikawa cell line, which was generated from EEC. Silencing of Nrf2 rendered SPEC-2 cells more susceptible to chemotherapeutic drugs while it had a limited effect on Ishikawa cells. Inhibition of Nrf2 expression by overexpressing Keap1 sensitized SPEC-2 cells or SPEC-2-derived xenografts to chemotherapeutic treatments using both cell culture and SCID mouse models. Collectively, we provide a molecular basis for the use of Nrf2 inhibitors to increase the efficacy of chemotherapeutic drugs and to combat chemoresistance, the biggest obstacle in chemotherapy.
doi:10.1158/0008-5472.CAN-10-0713
PMCID: PMC2896449  PMID: 20530669
Nrf2; chemoresistance; and endometrial cancer
24.  The Cinnamon-derived Dietary Factor Cinnamic Aldehyde Activates the Nrf2-dependent Antioxidant Response in Human Epithelial Colon Cells 
Molecules (Basel, Switzerland)  2010;15(5):3338-3355.
Colorectal cancer (CRC) is a major cause of tumor-related morbidity and mortality worldwide. Recent research suggests that pharmacological intervention using dietary factors that activate the redox sensitive Nrf2/Keap1-ARE signaling pathway may represent a promising strategy for chemoprevention of human cancer including CRC. In our search for dietary Nrf2 activators with potential chemopreventive activity targeting CRC, we have focused our studies on trans-cinnamic aldehyde (cinnamaldeyde, CA), the key flavor compound in cinnamon essential oil. Here we demonstrate that CA and an ethanolic extract (CE) prepared from Cinnamomum cassia bark, standardized for CA content by GC-MS analysis, display equipotent activity as inducers of Nrf2 transcriptional activity. In human colon cancer cells (HCT116, HT29) and non-immortalized primary fetal colon cells (FHC), CA- and CE-treatment upregulated cellular protein levels of Nrf2 and established Nrf2 targets involved in the antioxidant response including heme oxygenase 1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCS, catalytic subunit). CA- and CE-pretreatment strongly upregulated cellular glutathione levels and protected HCT116 cells against hydrogen peroxide-induced genotoxicity and arsenic-induced oxidative insult. Taken together our data demonstrate that the cinnamon-derived food factor CA is a potent activator of the Nrf2-orchestrated antioxidant response in cultured human epithelial colon cells. CA may therefore represent an underappreciated chemopreventive dietary factor targeting colorectal carcinogenesis.
doi:10.3390/molecules15053338
PMCID: PMC3101712  PMID: 20657484
colon cancer; Nrf2-activator; cinnamic aldehyde; antioxidant response
25.  The Protective Role of Nrf2 in Streptozotocin-Induced Diabetic Nephropathy 
Diabetes  2010;59(4):850-860.
OBJECTIVE
Diabetic nephropathy is one of the major causes of renal failure, which is accompanied by the production of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls the antioxidant response essential for maintaining cellular redox homeostasis. Here, we report our findings demonstrating a protective role of Nrf2 against diabetic nephropathy.
RESEARCH DESIGN AND METHODS
We explore the protective role of Nrf2 against diabetic nephropathy using human kidney biopsy tissues from diabetic nephropathy patients, a streptozotocin-induced diabetic nephropathy model in Nrf2−/− mice, and cultured human mesangial cells.
RESULTS
The glomeruli of human diabetic nephropathy patients were under oxidative stress and had elevated Nrf2 levels. In the animal study, Nrf2 was demonstrated to be crucial in ameliorating streptozotocin-induced renal damage. This is evident by Nrf2−/− mice having higher ROS production and suffering from greater oxidative DNA damage and renal injury compared with Nrf2+/+ mice. Mechanistic studies in both in vivo and in vitro systems showed that the Nrf2-mediated protection against diabetic nephropathy is, at least, partially through inhibition of transforming growth factor-β1 (TGF-β1) and reduction of extracellular matrix production. In human renal mesangial cells, high glucose induced ROS production and activated expression of Nrf2 and its downstream genes. Furthermore, activation or overexpression of Nrf2 inhibited the promoter activity of TGF-β1 in a dose-dependent manner, whereas knockdown of Nrf2 by siRNA enhanced TGF-β1 transcription and fibronectin production.
CONCLUSIONS
This work clearly indicates a protective role of Nrf2 in diabetic nephropathy, suggesting that dietary or therapeutic activation of Nrf2 could be used as a strategy to prevent or slow down the progression of diabetic nephropathy.
doi:10.2337/db09-1342
PMCID: PMC2844833  PMID: 20103708

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