Scientific Research

Kensler, T., Wakabayashi, N. & Biswal, S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annual Review of Pharmacology and Toxicology 47, 89–116 (2007).

Link to article in: Annual Review of Pharmacology and Toxicology
PMID: 16968214

Key Points:

This excellent review article highlights the Nrf2 pathway and its role in the adaptive response and protection against acute and chronic cell injury provoked by environmental stresses. Keap1-Nrf2-ARE signaling plays a significant role in protecting cells from both internal and environmental stresses. Mice that lack the ability to produce Nrf2 have been shown to be excellent experimental model organisms used to investigate the importance of this pathway in relation to surviving through these stress conditions. Mice lacking Nrf2 have been shown to be more sensitive to the liver, lung, ovarian, and neurotoxic damage after being exposed to various environmental chemicals and drugs; as well as inflammatory stresses, and chronic exposure to cigarette smoke and other carcinogens. This review highlights the discovery and the molecular mechanisms behind Nrf2 function, followed by the potential roles that Nrf2 can play in combating chemically induced cancers, emphysema, asthma, pulmonary fibrosis, bacterial infection, neurodegenerative diseases, and cellular aging.

Yang, C., Zhang, X., Fan, H. & Liu, Y. Curcumin upregulates transcription factor Nrf2, HO-1 expression and protects rat brains against focal ischemia. Brain Research 1282, 133–41 (2009).

Link to article in: Brain Research
PMID: 19445907

Key Points:

Oxidative and cytotoxic damage play important roles in brain diseases relating to poor blood flow in the brain. Therefore blocking this damage may represent a viable option for treatment of these brain diseases. Curcumin is proved to produce a variety of biological effects through its antioxidant and anti-inflammatory properties and was tested in this study to determine whether treatment by curcumin could block brain damage caused by poor blood flow. Male rats subjected to reduced brain blood flow were used as model organisms in this trial. Here it was found that rats with restricted blood flow to the brain had elevated levels of Nrf2 in brain tissue. Rats with reduced blood flow to the brain that were treated with curcumin showed reduced brain damage and had higher levels of Nrf2 expression than rats with reduced blow-flow alone. The authors speculate that there is a correlation between the protective effects of the curcumin treatment and the upregulation of the Nrf2 protein levels in combating brain damage related to reduced blood flow.

Calabrese, V. et al. Cellular stress response: a novel target for chemoprevention and nutritional neuroprotection in aging, neurodegenerative disorders and longevity. Neurochemical Research 33, 2444–71 (2008).

Link to article in: Neurochemical Research
PMID: 18629638

Key Points:

The predominant molecular symptom of aging is the accumulation of altered gene products. This happens to cells in many ways including a disruption of the proper reduction-oxidation balance and the accumulation of misfolded and unfolded proteins inside cells. Within the aging brain, the neurological diseases of Alzheimer’s and Parkinson’s have several molecular symptoms in common including the production of abnormal proteins, mitochondrial dysfunction, and oxidative stress. While the cells in our bodies have mechanisms in place to cope with these issues, our overall heath, diet, and age can influence how well this protective system functions. Recently it has been shown that phytochemcials (curcumin is a phytochemical) can activate Nrf2 to promote the production of survival proteins to help combat symptoms of aging within the brain. This paper serves as a review of the importance of Nrf2 activated genes in cellular stress.

Kang, E. et al. Nrf2 regulates curcumin-induced aldose reductasee expression indirectly via nuclear factor-kappaB. Pharmacological Research: the official journal of the Italian Pharmacological Society 58, 15-21 (2008).

Link to article in: Pharmacological Research
PMID: 18588981

Key Points:

The osmotic response element (ORE) differs from the nuclear factor-kappaB (NF-kappaB) binding sequence by a single base pair; therefore, these researchers investigated the involvement of NF-kappaB in the induction of aldose reductase (AR) by curcumin. AR is a detoxification enzyme that is transcriptionally regulated by a variety of stimuli including reactive oxygen species. Curcumin, elicited an increase in the expression and promoter activity of the AR gene in a (Nrf2)-dependent manner. The interaction between Nrf2 and NF-kappaB in curcumin induced upregulation of AR gene expression is investigated in this study. Small interfering RNA (siRNA) against p65 (a member of the NF-Kappa B family) or BAY11-7082, an inhibitor of NF-kappaB, significantly suppressed the curcumin and/or Nrf2-induced increase in expression levels and promoter activity of the AR gene. BAY11-7082 or siRNA against p65 also attenuated the curcumin-induced increase in the promoter activity of the wild type AR-ORE(wt) gene, but not that of the mutated AR-ORE(mt), indicating that the ORE is essential for the response to NF-kappaB. The expression of p65, the promoter activity and DNA binding activity of NF-kappaB were enhanced in the presence of curcumin in cells that were transfected with Nrf2 compared to those treated with curcumin alone indicating that Nrf2 regulates curcumin-induced AR transcription indirectly via augmentation of NF-kappaB protein expression.

Mol Nutr Food Res. 2008 Jun;52 Suppl 1:S128-38.

Dinkova-Kostova AT, Talalay P.

Division of Clinical Pharmacology, Department of Medicine, The Johns Hopkins University School ofMedicine, Baltimore, MD, USA. a.dinkovakostova@dundee.ac.uk

Abstract

Cellular protection against oxidative and electrophile toxicities is provided by two types of small-molecule antioxidants: (i) direct antioxidants, which are redox active, short-lived, are sacrificed in the process of their antioxidant actions and need to be replenished or regenerated, and may evoke pro-oxidant effects; and (ii) indirect antioxidants, that may or may not be redox active. Indirect antioxidants activate the Keap1/Nrf2/ARE pathway resulting in transcriptional induction of a battery of cytoprotective proteins (also known as phase 2 enzymes) that act catalytically, are not consumed, have long half-lives, and are unlikely to evoke pro-oxidant effects. These protective systems are involved in a complex functional interplay, such that many cytoprotective proteins participate in the synthesis and/or regeneration of direct antioxidants, whereas some direct antioxidants are required for the catalytic functions of cytoprotective proteins. Importantly, many inducers of cytoprotective proteins have been isolated from edible plants, e. g., sulforaphane from broccoli and curcumin from turmeric. Both are pleiotropic agents with multiple biological activities that could collectively contribute to their protective effects in various animal studies, including models of carcinogenesis, hypertension, neuronal and retinal damage. In addition to inducing cytoprotective proteins, molecules like curcumin which contain Michael acceptor functionalities (olefins or acetylenes conjugated to electron withdrawing groups) and phenolic hydroxyl groups can scavenge directly and potently oxygen- and nitrogen-centered reactive intermediates. Such bifunctional antioxidants can play a dual protective role by: (i) scavenging hazardous oxidants directly and instantaneously, and (ii) inducing cytoprotective enzymes that in turn function to resolve the consequences of hazardous processes that are already in progress, and to ensure long-term protection against subsequent challenges.

Cell Mol Life Sci. 2008 Jun;65(11):1631-52.

Hatcher H, Planalp R, Cho J, Torti FM, Torti SV.

Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

Abstract

Curcumin is the active ingredient in the traditional herbal remedy and dietary spice turmeric (Curcuma longa). Curcumin has a surprisingly wide range of beneficial properties, including anti-inflammatory, antioxidant, chemopreventive and chemotherapeutic activity. The pleiotropic activities of curcumin derive from its complex chemistry as well as its ability to influence multiple signaling pathways, including survival pathways such as those regulated by NF-kappaB, Akt, and growth factors; cytoprotective pathways dependent on Nrf2; and metastatic and angiogenic pathways. Curcumin is a free radical scavenger and hydrogen donor, and exhibits both pro- and antioxidant activity. It also binds metals, particularly iron and copper, and can function as an iron chelator. Curcumin is remarkably non-toxic and exhibits limited bioavailability. Curcumin exhibits great promise as a therapeutic agent, and is currently in human clinical trials for a variety of conditions, including multiple myeloma, pancreatic cancer, myelodysplastic syndromes, colon cancer, psoriasis and Alzheimer’s disease.

Carcinogenesis. 2008 May;29(5):1022-32. Epub 2008 Mar 4.

Garg R, Gupta S, Maru GB.

Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai 410 208, India.

Abstract

Curcumin has been shown to possess anti-initiating and anti-promoting activity in experimental systems. However, the mechanisms of its actions are not fully elucidated in vivo. In the present study, mechanisms of curcumin-mediated anti-initiation were investigated in mice employing benzo[a]pyrene (B[a]P) as a model carcinogen. Dietary pretreatment of mice with chemopreventive doses of curcumin showed significant inhibition of B[a]P-induced enzyme activity, protein and messenger RNA (mRNA) levels of cytochrome P450 1A1/1A2 in liver and lungs. Although curcumin alone did not alter the basal levels of aryl hydrocarbon receptor (AhR), it significantly decreased the B[a]P-induced AhR protein levels, its phosphorylation, nuclear translocation and subsequent binding to DNA, thereby decreasing the transactivation of CYP1A. Dietary curcumin led to increase in NF-E2-related factor-2 (Nrf2) protein levels and enhanced its nuclear translocation in liver and lungs of mice as compared with controls. Additionally, increased binding of Nrf2 to antioxidant response element occurred in nuclear extracts from liver and lungs of mice pretreated with dietary curcumin. Induction of activity, protein and mRNA levels of glutathione S-transferase, its isoforms and NAD(P)H:quinone oxidoreductase-1 by dietary curcumin in mice paralleled the curcumin-mediated activation of Nrf2, leading to increased detoxification of B[a]P. In agreement with the observed curcumin-mediated decrease in B[a]P-induced phase I enzyme and concomitant induction of phase II enzymes, pretreatment with dietary curcumin resulted in significant reduction of B[a]P-induced DNA adduct, oxidative damage and inflammation. To conclude, curcumin exhibits anti-initiating effects via modulating the transcriptional regulators of phase I and phase II enzymes in mice.

PMID: 18321868 [PubMed – indexed for MEDLINE]Free Article

Food Chem Toxicol. 2008 Apr;46(4):1279-87. Epub 2007 Sep 26.

Farombi EO, Shrotriya S, Na HK, Kim SH, Surh YJ.

Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.

Abstract

Curcumin (diferuloymethane), a yellow colouring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), has been reported to possess anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic activities. Curcumin exerts its chemoprotective and chemopreventive effects via multiple mechanisms. It has been reported to induce expression of the antioxidant enzymes in various cell lines. Heme oxygenase-1 (HO-1) is an important antioxidant enzyme that plays a pivotal role in cytoprotection against noxious stimuli of both endogenous and exogenous origin. In the present study, we found that oral administration of curcumin at 200mg/kg dose for four consecutive days not only protected against dimethylnitrosamine (DMN)-induced hepatic injury, but also resulted in more than three-fold induction of HO-1 protein expression as well as activity in rat liver. Inhibition of HO-1 activity by zinc protoporphyrin-IX abrogated the hepatoprotective effect of curcumin against DMN toxicity. NF-E2-related factor 2 (Nrf2) plays a role in the cellular protection against oxidative stress through antioxidant response element (ARE)-directed induction of several phase-2 detoxifying and antioxidant enzymes including HO-1. Curcumin administration resulted in enhanced nuclear translocation and ARE-binding of Nrf2. Taken together, these findings suggest that curcumin protects against DMN-induced hepatotoxicity, at least in part, through ARE-driven induction of HO-1 expression.

Free Radic Biol Med. 2007 Aug 15;43(4):535-45. Epub 2007 May 16.

Kang ES, Woo IS, Kim HJ, Eun SY, Paek KS, Kim HJ, Chang KC, Lee JH, Lee HT, Kim JH, Nishinaka T, Yabe-Nishimura C, Seo HG.

Department of Pharmacology, Gyeongsang Institute of Health Science, College of Medicine, Gyeongsang National University, 92 Chilam-Dong, Jinju 660-751, Korea.

Abstract

Up-regulation of aldose reductase (AR) by reactive oxygen species (ROS) and aldehyde derivatives has been observed in vascular smooth muscle cells. However, the pathophysiological consequences of the induction of AR in vascular tissues are not fully elucidated. Herein we report that an herb-derived polyphenolic compound, curcumin, elicited a dose- and time-dependent increase in AR expression. Inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK) significantly suppressed the curcumin-augmented mRNA levels and promoter activity of the AR gene. Luciferase reporter assays indicated that an osmotic response element in the promoter was essential for the responsiveness to curcumin. Curcumin accelerated the nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2), and overexpression of Nrf2, but not the dominant negative Nrf2, enhanced the promoter activity of the AR gene. Cells preincubated with curcumin demonstrated resistance to ROS-induced apoptotic death. These effects were significantly attenuated in the presence of AR inhibitors or small interfering RNAs, indicating a protective role for AR against ROS-induced cell damage. Taken together, the activation of PI3K and p38 MAPK by curcumin augmented the expression of the AR gene via Nrf2, and increased AR activity may be an important cellular response against oxidative stress.

Neurochem Res. 2010 Aug;35(8):1231-8. Epub 2010 May 1.

Stridh MH, Correa F, Nodin C, Weber SG, Blomstrand F, Nilsson M, Sandberg M.

Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden. malin.stridh@physiol.gu.se

Abstract

Efflux of glutathione (GSH) from astrocytes has been suggested as a key factor for neuroprotection by astrocytes. Here we evaluated if the Nrf2 activator curcumin affects basal and stimulated (Ca(2+) omission) GSH efflux from cultures of astroglial cells. Stimulated efflux of GSH was observed at medium concentration of 0, 0.1 mM Ca(2+), but not at 0.2 or 0.3 mM Ca(2+). Astroglia treated with 30 microM curcumin increased the cellular content of GSH in parallel with elevated basal and stimulated efflux. Conversely treatment with buthionine sulfoximine lowered efflux of GSH. The efflux stimulated by Ca(2+)- omission was not affected by the P2X7-receptor antagonist Blue Brilliant G (100 nM) or the pannexin mimetic/blocking peptide (10)Panx1 but inhibited by the gap junction blocker carbenoxolone (100 microM) and a hemichannel blocker Gap26 (300 microM). RNAi directed against Nrf2 partly inhibited the effect of curcumin. The results show that elevated cellular GSH by curcumin treatment enhance efflux from astroglial cells, a process which appear to be a prerequisite for astroglial mediated neuroprotection.

Annu Rev Nutr. 2010 Apr 26. [Epub ahead of print]

Aggarwal BB.

Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; email aggarwal@mdanderson.org.

Abstract

Extensive research within the past two decades has revealed that obesity, a major risk factor for type 2 diabetes, atherosclerosis, cancer, and other chronic diseases, is a proinflammatory disease. Several spices have been shown to exhibit activity against obesity through antioxidant and anti-inflammatory mechanisms. Among them, curcumin, a yellow pigment derived from the spice turmeric (an essential component of curry powder), has been investigated most extensively as a treatment for obesity and obesity-related metabolic diseases. Curcumin directly interacts with adipocytes, pancreatic cells, hepatic stellate cells, macrophages, and muscle cells. There, it suppresses the proinflammatory transcription factor nuclear factor-kappa B, signal transducer and activators of transcription-3, and Wnt/beta-catenin, and it activates peroxisome proliferator-activated receptor-gamma and Nrf2 cell-signaling pathways, thus leading to the downregulation of adipokines, including tumor necrosis factor, interleukin-6, resistin, leptin, and monocyte chemotactic protein-1, and the upregulation of adiponectin and other gene products. These curcumin-induced alterations reverse insulin resistance, hyperglycemia, hyperlipidemia, and other symptoms linked to obesity. Other structurally homologous nutraceuticals, derived from red chili, cinnamon, cloves, black pepper, and ginger, also exhibit effects against obesity and insulin resistance. Expected final online publication date for the Annual Review of Nutrition Volume 30 is July 17, 2010. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates

Romeo, L. et al. The major green tea polyphenol, (-)-epigallocatechin-3-gallate, induces heme oxygenase in rat neurons and acts as an effective neuroprotective agent against oxidative stress. Journal of the American College of Nutrition 28 Suppl, 492S–499S (2009).

Link to article in: Journal of the American College of Nutrition
PMID: 20234037

Key Points:

Oxidative stress induced by hyperglycemia (high blood sugar levels) is a key factor in diabetic related diseases. Chemicals found in green tea called catechins have been found to trigger antioxidant response pathways to help improve glycemic control. In this study the authors research the catechin (-)-epigallocatechin-3-gallate (EGCG) for its ability to protect rat brain cells from oxidative stress damage. What they found was that within 6 hours of treating rat brain cells with EGCG, activation of Nrf2 was evident. Further, if they treated rat brain cells with EGCG for 12 hours followed by challenging the cells with glucose to mimic oxidative stress conditions caused by hyperglycemia, they saw enhanced cellular resistance to glucose mediated oxidative damage. Overall, the authors speculate that EGCG is likely activating Nrf2 which results in rat brain cells being in a state of protection against oxidative stress-induced cell death.

Kensler, T., et al. Keap1-Nrf2 signaling: a target for cancer prevention by sulforaphane. Topics in Current Chemistry 329, 163-77 (2013).

Link to article in: Topics in Current Chemistry
PMID: 22752583

Key Points:

Sulforaphane is a promising, naturally occurring chemical, currently being evaluated for several types of disease prevention. Sulforaphane interacts with a wide variety of cellular molecules. One of the strongest interactions is with a protein called Keap1. The function of Keap1 within the cell is to repress Nrf2 activity until times of intracellular stress. By directly interacting with Keap1, sulforaphane blocks the Keap1 mediated repression of Nrf2 allowing Nrf2 to become active. Activation of Nrf2 allows for the production of cytoprotective genes within the cell which results in enhanced resistance to carcinogenesis. Clinical evaluation of sulforaphane has been through testing various preparations of broccoli and broccoli sprouts (both naturally rich in sulforaphane). Results from clinical trials stated in this article show that use of broccoli sprouts within beverages may enhance the body’s ability to detoxify itself which may in turn decrease certain health risks including the developing cancer.

World J Gastroenterol. 2010 Jun 28;16(24):3002-10.

Zhao HD, Zhang F, Shen G, Li YB, Li YH, Jing HR, Ma LF, Yao JH, Tian XF.

Department of General Surgery, Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China.

Abstract

AIM: To investigate the effect of sulforaphane (SFN) on regulation of NF-E2-related factor-2 (Nrf2)-antioxidant response element (ARE) pathway in liver injury induced by intestinal ischemia/reperfusion (I/R). METHODS: Rats were divided randomly into four experimental groups: control, SFN control, intestinal I/R and SFN pretreatment groups (n = 8 in each group). The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h and 2 h reperfusion. In the SFN pretreatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg SFN 1 h before the operation. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and glutathione peroxidase (GSH-Px) activity were assayed. The liver transcription factor Nrf2 and heme oxygenase-1 (HO-1) were determined by immunohistochemical analysis and Western blotting analysis. RESULTS: Intestinal I/R induced intestinal and liver injury, characterized by histological changes as well as a significant increase in serum AST and ALT levels (AST: 260.13 +/- 40.17 U/L vs 186.00 +/- 24.21 U/L, P < 0.01; ALT: 139.63 +/- 11.35 U/L vs 48.38 +/- 10.73 U/L, P < 0.01), all of which were reduced by pretreatment with SFN, respectively (AST: 260.13 +/- 40.17 U/L vs 216.63 +/- 22.65 U/L, P < 0.05; ALT: 139.63 +/- 11.35 U/L vs 97.63 +/- 15.56 U/L, P < 0.01). The activity of SOD in the liver tissue decreased after intestinal I/R (P < 0.01), which was enhanced by SFN pretreatment (P < 0.05). In addition, compared with the control group, SFN markedly reduced liver tissue MPO activity (P < 0.05) and elevated liver tissue GSH and GSH-Px activity (P < 0.05, P < 0.05), which was in parallel with the increased level of liver Nrf2 and HO-1 expression. CONCLUSION: SFN pretreatment attenuates liver injury induced by intestinal I/R in rats, attributable to the antioxidant effect through Nrf2-ARE pathway.

J Neurosci Res. 2010 May 1;88(6):1355-63.

Soane L, Li Dai W, Fiskum G, Bambrick LL.

Department of Anesthesiology, Center for Shock, Trauma, and Anesthesiology Research (STAR), University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

Abstract

Oxidative stress is a mediator of cell death following cerebral ischemia/reperfusion and heme toxicity, which can be an important pathogenic factor in acute brain injury. Induced expression of phase II detoxification enzymes through activation of the antioxidant response element (ARE)/Nrf2 pathway has emerged as a promising approach for neuroprotection. Little is known, however, about the neuroprotective potential of this strategy against injury in immature brain cells. In this study, we tested the hypothesis that sulforaphane (SFP), a naturally occurring isothiocyanate that is also a known activator of the ARE/Nrf2 antioxidant pathway, can protect immature neurons from oxidative stress-induced death. The hypothesis was tested with primary mouse hippocampal neurons exposed to either O(2) and glucose deprivation (OGD) or hemin. Treatment of immature neurons with SFP immediately after the OGD during reoxygenation was effective in protecting immature neurons from delayed cell death. Exposure of immature hippocampal neurons to hemin induced significant cell death, and both pre- and cotreatment with SFP were remarkably effective in blocking cytotoxicity. RT-PCR analysis indicated that several Nrf2-dependent cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamate-cysteine ligase modifier subunit (GCLM), which is involved in glutathione biosynthesis, were up-regulated following SFP treatment both in control neurons and following exposure to OGD and hemin. These results indicate that SFP activates the ARE/Nrf2 pathway of antioxidant defense and protects immature neurons from death caused by stress paradigms relevant to those associated with ischemic and traumatic injury to the immature brain. (c) 2009 Wiley-Liss, Inc.

Parkinsonism Relat Disord. 2009 Dec;15 Suppl 3:S189-94.

Department of Neurology & Neuroscience, Weill Cornell Medical College, New York, NY 10065-4870, USA. fbeal@mail.med.cornell.edu

Abstract

A large body of evidence from postmortem brain tissue and genetic analysis in humans, as well as biochemical and pathological studies in animal models of neurodegeneration suggest that mitochondrial dysfunction is a key pathological mechanism in Parkinson’s Disease (PD). Mitochondrial dysfunction leads to oxidative stress, damage to mitochondrial DNA, mitochondrial DNA deletions, altered mitochondrial morphology, alterations in mitochondrial fission and fusion and ultimately neuronal demise. Therapeutic approaches targeting mitochondrial dysfunction and oxidative damage, therefore, hold great promise in PD. A number of agents, which target energy metabolism, are presently in therapeutic trials in PD. Both creatine and Coenzyme Q10 (CoQ10) are being tested in phase III clinical trials. In addition, preclinical studies in animal models have shown efficacy of mitochondrial-targeted antioxidants and the SS peptides. A promising approach for increasing antioxidant defenses is to transcriptionally increase the activity of the Nrf2/ARE pathway, which activates transcription of anti-inflammatory and antioxidant genes. A number of agents including sulforaphane, curcumin and triterpenoids have been shown to activate this pathway and to produce neuroprotective effects. Lastly, newly identified therapeutic targets include peroxisomal proliferator activated receptor gamma-coactivator (PGC-1alpha) and sirtuins. These pathways provide promise for future therapeutic developments in the treatment of PD.

Ping Z, Liu W, Kang Z, Cai J, Wang Q, Cheng N, Wang S, Wang S, Zhang JH, Sun X.

Department of Clinical Laboratory, Changzhou No. 2 People’s Hospital, Jiangsu, 213003, PR China.

Abstract

Neonatal hypoxia-ischemia (HI) brain injury involves reactive oxygen species (ROS) and inflammatory responses. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, has cytoprotective effects against oxidative stress and its effect was mediated by NF-E2-related factor-2 (Nrf2), a transcription factor, and heme oxygenase 1 (HO-1) which is one of Nrf2 downstream target genes. This study was undertaken to investigate the neuroprotective mechanisms of SFN in a neonatal HI rat model. Seven-day-old rat pups were subjected to left common carotid artery ligation and hypoxia (8% oxygen at 37 degrees C) for 90min. SFN (5mg/kg) was systemically administered 30min before HI insult. Brain injury was assessed by 2,3,5-triphenyltetrazoliumchloride (TTC), Nissl, TUNEL staining, malondialdehyde (MDA), 8OH-dG level, and caspase-3 activity in the cortex and hippocampus. SFN pretreatment increased the expression of Nrf2 and HO-1 in the brain and reduced infarct ratio at 24h after HI. The number of TUNEL-positive neurons as well as activated macroglia and the amount of 8OH-dG, were markedly reduced after SFN treatment, accompanied by suppressed caspase-3 activity and reduced lipid peroxidation (MDA) level. These results demonstrated that SFN could exert neuroprotective effects through increasing Nrf2 and HO-1 expression. Copyright © 2010 Elsevier B.V. All rights reserved.

Glia. 2009 Apr 15;57(6):645-56.

Danilov CA, Chandrasekaran K, Racz J, Soane L, Zielke C, Fiskum G.

Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Abstract

Oxidative stress is an important molecular mechanism of astrocyte injury and death following ischemia/reperfusion and may be an effective target of intervention. One therapeutic strategy for detoxifying the many different reactive oxygen and nitrogen species that are produced under these conditions is induction of the Phase II gene response by the use of chemicals or conditions that promote the translocation of the transcriptional activating factor NRF2 from the cytosol to the nucleus, where it binds to genomic antioxidant response elements. This study tested the hypothesis that pre- or post-treatment of cultured cortical astrocytes with sulforaphane, an alkylating agent known to activate the NRF2 pathway of gene expression protects against death of astrocytes caused by transient exposure to O(2) and glucose deprivation (OGD). Rat cortical astrocytes were exposed to 5 muM sulforaphane either 48 h prior to, or for 48 h after a 4-h period of OGD. Both pre- and post-treatments significantly reduced cell death at 48 h after OGD. Immunostaining for 8-hydroxy-2-deoxyguanosine, a marker of DNA/RNA oxidation, was reduced at 4 h reoxygenation with sulforaphane pretreatment. Sulforaphane exposure was followed by an increase in cellular and nuclear NRF2 immunoreactivity. Moreover, sulforaphane also increased the mRNA, protein level, and enzyme activity of NAD(P)H/Quinone Oxidoreductase1, a known target of NRF2 transcriptional activation. We conclude that sulforaphane stimulates the NRF2 pathway of antioxidant gene expression in astrocytes and protects them from cell death in an in vitro model of ischemia/reperfusion. (c) 2008 Wiley-Liss, Inc.

Proc Natl Acad Sci U S A. 2010 May 25;107(21):9590-5. Epub 2010 May 3.

Ahn YH, Hwang Y, Liu H, Wang XJ, Zhang Y, Stephenson KK, Boronina TN, Cole RN, Dinkova-Kostova AT, Talalay P, Cole PA.

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Abstract

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane], a naturally occurring isothiocyanate derived from cruciferous vegetables, is a highly potent inducer of phase 2 cytoprotective enzymes and can protect against electrophiles including carcinogens, oxidative stress, and inflammation. The mechanism of action of sulforaphane is believed to involve modifications of critical cysteine residues of Keap1, which lead to stabilization of Nrf2 to activate the antioxidant response element of phase 2 enzymes. However, the dithiocarbamate functional group formed by a reversible reaction between isothiocyanate of sulforaphane and sulfhydryl nucleophiles of Keap1 is kinetically labile, and such modification in intact cells has not yet been demonstrated. Here we designed sulforaphane analogs with replacement of the reactive isothiocyanate by the more gentle electrophilic sulfoxythiocarbamate group that also selectively targets cysteine residues in proteins but forms stable thiocarbamate adducts. Twenty-four sulfoxythiocarbamate analogs were synthesized that retain the structural features important for high potency in sulforaphane analogs: the sulfoxide or keto group and its appropriate distance to electrophilic functional group. Evaluation in various cell lines including hepatoma cells, retinal pigment epithelial cells, and keratinocytes as well as in mouse skin shows that these analogs maintain high potency and efficacy for phase 2 enzyme induction as well as the inhibitory effect on lipopolysaccharide-induced nitric oxide formation like sulforaphane. We further show in living cells that a sulfoxythiocarbamate analog can label Keap1 on several key cysteine residues as well as other cellular proteins offering new insights into the mechanism of chemoprotection.

J Investig Med. 2010 Jun;58(5):730-6.

Qi L, Xiangdong Z, Hongmei Y, Xiaohong N, Xiaoyan X.

Second Affiliated Hospital of Chongqing Medical University, China.

Abstract

Mucin 5AC (MUC5AC) is one of the main airway mucins implicated in pulmonary diseases with mucous hypersecretion. Neutrophil elastase (NE), a serine protease released by neutrophils, is known to induce MUC5AC synthesis by increasing reactive oxygen species (ROS) generation. Nuclear factor erythroid-2 related factor 2 (Nrf2), a basic-region leucine-zipper transcription factor, is believed to protect against ROS damage by activating a series of antioxidant enzymes; Nrf2 is also reported to reduce NE activity. The aim of our study was to examine the relationship between Nrf2 expression and NE-induced MUC5AC production. We used a small interfering RNA to inhibit Nrf2 expression. The Nrf2 gene and protein expressions were assessed by quantitative real-time polymerase chain reaction, and Western blotting, respectively. The ROS generation was examined using a kit. The expression of MUC5AC was assessed using enzyme-linked immunosorbent assay. The results showed that NCI-H292 epithelial cells, in which the Nrf2 gene expression repressed, were highly predisposed to NE stimulation, with marked exacerbation of ROS generation and reduced secretory leukocyte protease inhibitor production, resulting in high MUC5AC expression. Pretreatment with the potent Nrf2 activator sulforaphane had the reverse effect. These results demonstrate that Nrf2 is a novel nuclear factor involved in down-regulating MUC5AC synthesis by inhibiting ROS generation and augmenting proteinase inhibitor production.

Pharmacol Res. 2010 Apr;61(4):342-8. Epub 2009 Dec 3.

Piao CS, Gao S, Lee GH, Kim do S, Park BH, Chae SW, Chae HJ, Kim SH.

Department of Pharmacology, Diabetes Research Center, Chonbuk National University Medical School, Jeonju, Republic of Korea.

Abstract

Reactive oxygen species are important mediators that exert a toxic effect during ischemia-reperfusion (I/R) injury of various organs. Sulforaphane is known to be an indirect antioxidant that acts by inducing Nrf2-dependent phase 2 enzymes. In this study, we investigated whether sulforaphane protects heart against I/R injury. Sprague-Dawley rats received sulforaphane (500microg/kg/day) or vehicle intraperitoneally for 3 days and global ischemia was performed using isolated perfused Langendorff hearts. Hearts were perfused with Krebs-bicarbonate buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Treatment with sulforaphane inhibited an increase in the post-ischemic left ventricular end-diastolic pressure (LVEDP) and improved the post-ischemic left ventricular developed pressure (LVDP), +/-dP/dt, and coronary flow as compared with the untreated control hearts. Pretreatment with 5-hydroxydecanoic acid (5-HD), a mitochondrial K(ATP) channel blocker, for 10min before ischemia attenuated the improvement of LVEDP, LVDP, +/-dP/dt, and coronary flow induced by sulforaphane. Sulforaphane markedly decreased the infarcted size and attenuated the increased lactate dehydrogenase level in effluent during reperfusion. Pretreatment with 5-HD also blocked these protective effects of sulforaphane. Post-ischemia increased the concentration of atrial natriuretic peptide in coronary effluent, which attenuated by sulforaphane treatment. Decreases on Mn-superoxide dismutase (SOD), catalase, and heme oxygenase-1 levels by I/R were increased by sulforaphane treatment and pretreatment of 5-HD blocked the sulforaphane effects. Increases in Bax and caspase-3 levels, and decrease in Bcl-2 level by I/R were attenuated by sulforaphane treatment. These results suggest that the protective effects of sulforaphane against I/R injury may be partly mediated through mitochondrial K(ATP) channels and antioxidant pathway.

Ungvari Z, Bagi Z, Feher A, Recchia FA, Sonntag WE, Pearson K, de Cabo R, Csiszar A.

Dept. of Geriatric Medicine, Reynolds Oklahoma Center on Aging, University of Oklahoma Health Sciences Center, 975 NE 10th St., Oklahoma City, OK 73104, USA. zoltan-ungvari@ouhsc.edu

Comment in:

• Am J Physiol Heart Circ Physiol. 2010 Jul;299(1):H10-2.

Abstract

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. Resveratrol was also shown to confer vasoprotection in animal models of type 2 diabetes and aging. However, the mechanisms by which resveratrol exerts its antioxidative vasculoprotective effects are not completely understood. Using a nuclear factor-E(2)-related factor-2 (Nrf2)/antioxidant response element-driven luciferase reporter gene assay, we found that in cultured coronary arterial endothelial cells, resveratrol, in a dose-dependent manner, significantly increases transcriptional activity of Nrf2. Accordingly, resveratrol significantly upregulates the expression of the Nrf2 target genes NAD(P)H:quinone oxidoreductase 1, gamma-glutamylcysteine synthetase, and heme oxygenase-1. Resveratrol treatment also significantly attenuated high glucose (30 mM)-induced mitochondrial and cellular oxidative stress (assessed by flow cytometry using MitoSox and dihydroethidine staining). The aforementioned effects of resveratrol were significantly attenuated by the small interfering RNA downregulation of Nrf2 or the overexpression of Kelch-like erythroid cell-derived protein 1, which inactivates Nrf2. To test the effects of resveratrol in vivo, we used mice fed a high-fat diet (HFD), which exhibit increased vascular oxidative stress associated with an impaired endothelial function. In HFD-fed Nrf2(+/+) mice, resveratrol treatment attenuates oxidative stress (assessed by the Amplex red assay), improves acetylcholine-induced vasodilation, and inhibits apoptosis (assessed by measuring caspase-3 activity and DNA fragmentation) in branches of the femoral artery. In contrast, the aforementioned endothelial protective effects of resveratrol were diminished in HFD-fed Nrf2(-/-) mice. Taken together, our results indicate that resveratrol both in vitro and in vivo confers endothelial protective effects which are mediated by the activation of Nrf2.

Trends Neurosci. 2006 Nov;29(11):632-9. Epub 2006 Sep 26.

Mattson MP, Cheng A.

Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD 21224, USA. mattsonm@grc.nia.nih.gov

Abstract

Diets rich in vegetables and fruits are associated with reduced risk of several major diseases, including neurodegenerative disorders. Although some beneficial phytochemicals might function solely as antioxidants, it is becoming clear that many of the beneficial chemicals in vegetables and fruits evolved as toxins (to dissuade insects and other predators) that, at subtoxic doses, activate adaptive cellular stress-response pathways in a variety of cells including neurons. Examples of such ‘preconditioning’ or ‘neurohormesis’ pathways include those involving cell-survival signaling kinases, the transcription factors NRF2 and CREB, and histone deacetylases of the sirtuin family. In these ways, neurohormetic phytochemicals such as resveratrol, sulforaphanes and curcumin might protect neurons against injury and disease by stimulating the production of antioxidant enzymes, neurotrophic factors, protein chaperones and other proteins that help cells to withstand stress. Thus, as we discuss in this review, highly conserved longevity and survival pathways in neurons are the targets of many phytochemicals.

Int J Biochem Cell Biol. 2010 Jan 12. [Epub ahead of print]

Zhang H, Shih A, Rinna A, Forman HJ.

University of California at Merced, Merced, CA 95343, United States.

Abstract

Resveratrol, a polyphenolic compound rich in grapes and red wine, has been reported to protect cells against oxidative damage and cell death by increasing cellular antioxidant/detoxification capacity. Cigarette smoking is a major risk factor for respiratory diseases and oxidative damage is implicated in its pathogenesis. Here we investigated the enhancement of antioxidant capacity by resveratrol and its potential protection against cell death caused by cigarette smoke in human bronchial epithelial cells (HBE1). At concentrations that did not affect cell growth, resveratrol activated Nrf2 signaling and increased the expression of NAD(P)H:quinone reductase-1, heme oxygenase-1, and the catalytic subunit of glutamate cysteine ligase. Surprisingly, instead of protecting against cell death, resveratrol significantly enhanced cigarette smoke extract-induced apoptosis. To define the underlying mechanism, the effect of resveratrol on caspase activity was examined and it was found that resveratrol significantly enhanced cigarette smoke-stimulated caspase activity. In conclusion, results from this study suggest that although resveratrol increased antioxidant and detoxification capacity, it increased rather than protected against cigarette smoke-induced apoptosis. Copyright © 2010 Elsevier Ltd. All rights reserved.

Ther Adv Respir Dis. 2008 Dec;2(6):351-74.

Rahman I.

Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, NY, USA. irfan_rahman@urmc.rochester.edu

Abstract

Chronic obstructive pulmonary disease (COPD) is associated with a high incidence of morbidity and mortality. Cigarette smoke-induced oxidative stress is intimately associated with the progression and exacerbation of COPD and therefore targeting oxidative stress with antioxidants or boosting the endogenous levels of antioxidants is likely to have beneficial outcome in the treatment of COPD. Among the various antioxidants tried so far, thiol antioxidants and mucolytic agents, such as glutathione, N-acetyl-L-cysteine, N-acystelyn, erdosteine, fudosteine and carbocysteine; Nrf2 activators; and dietary polyphenols (curcumin, resveratrol, and green tea catechins/quercetin) have been reported to increase intracellular thiol status along with induction of GSH biosynthesis. Such an elevation in the thiol status in turn leads to detoxification of free radicals and oxidants as well as inhibition of ongoing inflammatory responses. In addition, specific spin traps, such as alpha-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (ECSOD mimetic), porphyrins (AEOL 10150 and AEOL 10113), and a SOD mimetic M40419 have also been reported to inhibit cigarette smoke-induced inflammatory responses in vivo in the lung. Since a variety of oxidants, free radicals and aldehydes are implicated in the pathogenesis of COPD, it is possible that therapeutic administration of multiple antioxidants and mucolytics will be effective in management of COPD. However, a successful outcome will critically depend upon the choice of antioxidant therapy for a particular clinical phenotype of COPD, whose pathophysiology should be first properly understood. This article will review the various approaches adopted to enhance lung antioxidant levels, antioxidant therapeutic advances and recent past clinical trials of antioxidant compounds in COPD.

Antioxid Redox Signal. 2009 Mar;11(3):425-38.

Vincent AM, Kato K, McLean LL, Soules ME, Feldman EL.

Department of Neurology, University of Michigan, Ann Arbor, Michigan 48109, USA. andreav@umich.edu

Abstract

Elevated blood glucose is a key initiator of mechanisms leading to diabetic neuropathy. Increases in glucose induce acute mitochondrial oxidative stress in dorsal root ganglion (DRG) neurons, the sensory neurons normally affected in diabetic neuropathy, whereas Schwann cells are largely unaffected. We propose that activation of an antioxidant response in DRG neurons would prevent glucose-induced injury. In this study, mild oxidative stress (1 microM H2O2) leads to the activation of the transcription factor Nrf2 and expression of antioxidant (phase II) enzymes. DRG neurons are thus protected from subsequent hyperglycemia-induced injury, as determined by activation of caspase 3 and the TUNEL assay. Schwann cells display high basal antioxidant enzyme expression and respond to hyperglycemia and mild oxidative stress via further increases in these enzymes. The botanical compounds resveratrol and sulforaphane activate the antioxidant response in DRG neurons. Other drugs that protect DRG neurons and block mitochondrial superoxide, identified in a compound screen, have differential ability to activate the antioxidant response. Multiple cellular targets exist for the prevention of hyperglycemic oxidative stress in DRG neurons, and these form the basis for new therapeutic strategies against diabetic neuropathy.

Eur J Pharmacol. 2008 Sep 4;591(1-3):66-72. Epub 2008 Jun 22.

Rubiolo JA, Mithieux G, Vega FV.

Departamento de Fisiología, Facultad de Veterinaria Universidad de Santiago de Compostela, 27002, Lugo, Spain.

Abstract

Oxidative stress is recognized as an important factor in the development of liver pathologies. The reactive oxygen species endogenously generated or as a consequence of xenobiotic metabolism are eliminated by enzymatic and nonenzymatic cellular systems. Besides endogen defences, the antioxidant consumption in the diet has an important role in the protection against the development of diseases product of oxidative damage. Resveratrol is a naturally occurring compound which is part of the human diet. This molecule has been shown to have many biological properties, including antioxidant activity. We decided to test if resveratrol could protect primary hepatocytes in culture from oxidative stress damage and if so, to determine if this compound affects the cellular detoxifying systems and their regulation through the Nrf2 transcription factor that regulates the expression of antioxidant and phase II detoxifying enzymes. Cell death by necrosis was detected by measuring the activity of lactate dehydrogenase liberated to the medium. The activities of antioxidant and phase II enzymes were measured using previously described methods. Activation of the Nrf2 transcription factor was studied by confocal microscopy and the Nrf2 and its coding mRNA levels were determined by western blot and quantitative PCR respectively. Resveratrol pre-treatment effectively protected hepatocytes in culture exposed to oxidative stress, increasing the activities of catalase, superoxide dismutase, glutathione peroxidase, NADPH quinone oxidoreductase and glutathione-S-transferase. Resveratrol increases the level of Nrf2 and induces its translocation to the nucleus. Also, it increases the concentration of the coding mRNA for Nrf2. In this work we show that resveratrol could be a useful drug for the protection of liver cells from oxidative stress induced damage.

Am J Physiol Lung Cell Mol Physiol. 2008 Mar;294(3):L478-88. Epub 2007 Dec 27.

Kode A, Rajendrasozhan S, Caito S, Yang SR, Megson IL, Rahman I.

Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Box 850,601 Elmwood Ave., Rochester, NY 14642, USA.

Abstract

Nuclear erythroid-related factor 2 (Nrf2), a redox-sensitive transcription factor, is involved in transcriptional regulation of many antioxidant genes, including glutamate-cysteine ligase (GCL). Cigarette smoke (CS) is known to cause oxidative stress and deplete glutathione (GSH) levels in alveolar epithelial cells. We hypothesized that resveratrol, a polyphenolic phytoalexin, has antioxidant signaling properties by inducing GSH biosynthesis via the activation of Nrf2 and protects lung epithelial cells against CS-mediated oxidative stress. Treatment of human primary small airway epithelial and human alveolar epithelial (A549) cells with CS extract (CSE) dose dependently decreased GSH levels and GCL activity, effects that were associated with enhanced production of reactive oxygen species. Resveratrol restored CSE-depleted GSH levels by upregulation of GCL via activation of Nrf2 and also quenched CSE-induced release of reactive oxygen species. Interestingly, CSE failed to induce nuclear translocation of Nrf2 in A549 and small airway epithelial cells. On the contrary, Nrf2 was localized in the cytosol of alveolar and airway epithelial cells due to CSE-mediated posttranslational modifications such as aldehyde/carbonyl adduct formation and nitration. On the other hand, resveratrol attenuated CSE-mediated Nrf2 modifications, thereby inducing its nuclear translocation associated with GCL gene transcription, as demonstrated by GCL-promoter reporter and Nrf2 small interfering RNA approaches. Thus resveratrol attenuates CSE-mediated GSH depletion by inducing GSH synthesis and protects epithelial cells by reversing CSE-induced posttranslational modifications of Nrf2. These data may have implications in dietary modulation of antioxidants in treatment of chronic obstructive pulmonary disease.

Arch Biochem Biophys. 2009 Jan 1;481(1):110-5. Epub 2008 Oct 22.

Zhang H, Shih A, Rinna A, Forman HJ.

School of Natural Sciences, University of California-Merced, Merced, CA 95340, USA.

Abstract

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined

Mol Nutr Food Res. 2010 May 19. [Epub ahead of print]

Kim JW, Lim SC, Lee MY, Lee JW, Oh WK, Kim SK, Kang KW.

BK21 Project Team, College of Pharmacy, Chosun University, Gwangju, Republic of Korea.

Abstract

Neointima, defined as abnormal growth of the intimal layer of blood vessels, is believed to be a critical event in the development of vascular occlusive disease. Although resveratrol’s inhibitory effects on proliferation and migration of vascular smooth muscle cells has been reported, its activity on neointimal formation is still unclear. Oral administration of trans-resveratrol significantly suppressed intimal hyperplasia in a wire-injured femoral artery mouse model. In cultured vascular smooth muscle cells, trans-resveratrol inhibited platelet-derived growth factor-stimulated DNA synthesis and cell proliferation with down-regulation of cyclin D and pRB. Moreover, platelet-derived growth factor-induced production of reactive oxygen species was inhibited by trans-resveratrol and the compound induced heme oxygenase-1 (HO-1). The anti-proliferative activity of trans-resveratrol was reversed by an HO-1 inhibitor, ZnPPIX. Subcellular fractionation and reporter gene analyses revealed that trans-resveratrol increased the level of nuclear Nrf2 and antioxidant response element reporter activity, and that these were essential for the induction of HO-1. Trans-resveratrol also enhanced the activities of phosphatidyl inositol 3-kinase and extracellular signal regulated kinase, and phosphatidyl inositol 3-kinase was required for Nrf2/antioxidant response element-dependent HO-1 induction. These data have significant implications for the elucidation of the pharmacological mechanism by which trans-resveratrol prevents vascular occlusive diseases.

Vascul Pharmacol. 2010 Jul-Aug;53(1-2):61-7. Epub 2010 Apr 14.

Park ES, Lim Y, Hong JT, Yoo HS, Lee CK, Pyo MY, Yun YP.

College of Pharmacy, Research Center for Bioresource and Health, CBITRC, Chungbuk National University, Cheongju 361-763, Republic of Korea.

Abstract

Vascular smooth muscle cells (VSMCs) are the main cellular component in the arterial wall, and abnormal proliferation of VSMCs plays a central role in the pathogenesis of atherosclerosis and restenosis after angioplasty, and possibly in the development of hypertension. Pterostilbene, a natural dimethylated analog of resveratrol, is known to have diverse pharmacological activities including anti-cancer, anti-inflammation and anti-oxidant activities. The present study was designed to investigate the effects of pterostilbene on platelet-derived growth factor (PDGF)-BB-induced VSMCs proliferation as well as the molecular mechanisms of the antiproliferative effects. The cell growth of VSMCs was determined by cell counting and [(3)H]thymidine incorporation assays. Pterostilbene significantly inhibited the DNA synthesis and proliferation of PDGF-BB-stimulated VSMCs in a concentration-dependent manner. The inhibition percentages of pterostilbene at 1, 3 and 5microM to VSMCs proliferation were 68.5, 80.7 and 94.6%, respectively. The DNA synthesis of pterostilbene at 1, 3 and 5microM in VSMCs was inhibited by 47.4, 76.7 and 100%, respectively. Pterostilbene inhibited the PDGF-BB-stimulated phosphorylation of Akt kinase. However, pterostilbene did not change the expression of extracellular signal-related kinase (ERK) 1/2, PLCgamma1, phosphatidylinositol (PI)3 kinase and PDGF-Rbeta phosphorylation. In addition, pterostilbene down-regulated the cell cycle-related proteins including the expression of cyclin-dependent kinase (CDK) 2, cyclin E, CDK4, cyclin D1, retinoblastoma (Rb) proteins and proliferative cell nuclear antigen (PCNA). These findings suggest that the inhibition of pterostilbene to the cell proliferation and DNA synthesis of PDGF-BB-stimulated VSMCs may be mediated by the suppression of Akt kinase. Furthermore, pterostilbene may be a potential anti-proliferative agent for the treatment of atherosclerosis and angioplasty restenosis. Copyright 2010 Elsevier Inc. All rights reserved

Toxicol In Vitro. 2010 Jun;24(4):1215-28. Epub 2010 Feb 10.

Chakraborty A, Gupta N, Ghosh K, Roy P.

Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, Uttarakhand, India.

Abstract

Pterostilbene, a dimethyl ester derivative of resveratrol, may act as an cytotoxic and hence as an anti-cancer agent. The present study was conducted to test the anti-cancer activity of pterostilbene purified from Pterocarpus marsupium on breast (MCF-7) and prostate (PC3) cancer cell lines. The purified pterostilbene was found to cause apoptosis in both the cell lines, which was marked by DNA fragmentation, formation of apoptotic bodies and membrane distortions. Apoptosis probably was due to the production of reactive oxygen species in MCF-7 and nitric oxide over production in PC3 cells. Even the drug detoxifying anti-oxidant enzymes could not nullify the effect of pterostilbene as required by the cancer cells for survival. Pterostilbene was found to inhibit the cell proliferating factors like Akt, Bcl-2 and induced the mitochondrial apoptotic signals like Bax, and the series of caspases. It also inhibited Matrix metalloproteinase 9 (MMP9) and alpha-methylacyl-CoA recemase (AMACR), two very well known metastasis inducers. In conclusion, pterostilbene has multiple target sites to induce apoptosis. Hence, after proper validation it can be used as a potential agent for the cure of breast and prostate cancer. Copyright 2010 Elsevier Ltd. All rights reserved.

J Gastrointest Surg. 2010 May;14(5):873-9. Epub 2010 Feb 6.

Mannal PW, Alosi JA, Schneider JG, McDonald DE, McFadden DW.

University of Vermont/Fletcher Allen Health Care, Burlington, VT, USA. Patrick.Mannal@vtmednet.org

Abstract

INTRODUCTION: Stilbenes are phenolic compounds present in grapes and blueberries. Resveratrol, a naturally occurring compound present in grapes, has been shown to have potent antioxidant properties as well as an ability to induce apoptosis. Resveratrol has also been reported to have significant inhibitory effects against a variety of primary tumors including breast, colon, and prostate. Pterostilbene, a naturally occurring analogue of resveratrol found in blueberries, also has antioxidant and antiproliferative properties. It is also substantially more bioavailable orally than resveratrol. These effects have not been studied in pancreatic cancer. We hypothesized that pterostilbene would inhibit pancreatic cancer cell growth in vitro. MATERIALS AND METHODS: Two pancreatic cancer cell lines (MIA PaCa and PANC-1) were cultured using standard techniques. Cells were treated with graduated doses of pterostilbene ranging from 10 to 100 microM. Cell viability was measured by MTT at 24, 48, and 72 h. RESULTS: Pterostilbene decreases cell viability in both cancer cell lines in a concentration- and time-dependent manner. Higher doses (75-100 microM) caused a significant reduction in cell viability at 24 and 48 h. However, by 72 h, all tested concentrations of pterostilbene (10 to 100 microM) resulted in significantly reduced cell viability in both pancreatic cancer cell lines in a dose-dependent fashion. Pterostilbene caused a dose-dependent 10-63% inhibition in MIA PaCa-2 cells and 10-75% inhibition in PANC-1 cells. DISCUSSION: Treatment of pancreatic cancer cells in vitro with Pterostilbene leads to inhibition of cell proliferation and/or cell death, cell cycle arrrest, mitochondrial membrane depolarization, and activation of effector caspases. This naturally occurring agent may have a role in treating pancreatic cancer. CONCLUSIONS: Pterostilbene inhibits the growth of pancreatic cancer in vitro. Further, in vitro mechanistic studies and in vivo experiments are warranted to determine its potential for the treatment of pancreatic cancer.

Plant Foods Hum Nutr. 2010 Mar;65(1):57-63.

Mikstacka R, Rimando AM, Ignatowicz E.

Department of Chemical Technology of Drugs, Poznań University of Medical Sciences, Poznań, Poland. rmikstac@ump.edu.pl

Abstract

There is evidence that a diet rich in fruit and vegetables may reduce the risk of cancer and other degenerative diseases. However, potential health impact of bioactive phytochemicals is limited by their low amount and relatively poor bioavailability. It has been suggested that the health benefits associated with fruit and red wine consumption could be due to the whole antioxidant pool of the diet microcomponents. In this study, the antioxidant activities of trans-resveratrol, pterostilbene and quercetin, and the effect of their combination were investigated in human erythrocytes in vitro. H(2)O(2)-induced lipid peroxidation was assessed by measuring the amount of thiobarbituric acid reactive species. Quercetin and pterostilbene protected erythrocyte membranes against lipid peroxidation (IC(50) values = 64 +/- 8.7 microM and 44.5 +/- 7.8 microM, respectively). Resveratrol was significantly less effective. However, the three compounds protected the erythocytes against hemolysis and GSH (reduced glutathione) depletion to the same extent. Combinations consisting of two compounds (molar ratio 1:1) influenced lipid peroxidation in a concentration-dependent manner. At lower concentrations, resveratrol with quercetin or pterostilbene inhibited synergistically the oxidative injury of membrane lipids At higher concentrations, an additive effect was observed. These protective effects may partially explain the health benefit of these bioactive microcomponents when together in the diet.

Mol Nutr Food Res. 2010 Mar;54(3):335-44.

Wang TT, Schoene NW, Kim YS, Mizuno CS, Rimando AM.

Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705, USA. tom.wang@ars.usda.gov

Abstract

Stilbenes are phytoalexins that become activated when plants are stressed. These compounds exist in foods and are widely consumed. Resveratrol is a grape-derived stilbene, which possesses a wide range of health-promoting activities, including anticancer properties. Several other stilbenes structurally similar to resveratrol are also available in food, but their biological activities remain largely unknown. In this study, we compared the effects of resveratrol and its natural derivatives pterostilbene, trans-resveratrol trimethylether, trans-pinostilbene and trans-desoxyrhapontigenin on androgen-responsive human prostate cancer LNCaP cells. We found that these compounds exert differential effects on LNCaP cell growth, cell cycle and apoptosis. Trans-resveratrol trimethylether appeared to be the most potent compound among the stilbenes tested. Treatment of LNCaP cells with trans-resveratrol trimethylether resulted in G2/M blockage while other compounds, including resveratrol, induced G1/S arrest. Moreover, different from other compounds, trans-resveratrol trimethylether induced apoptosis. At the molecular level, the effects of these compounds on cell cycle correlated with induction of the cyclin-dependent kinase inhibitor 1A and B mRNA levels. Additionally, these compounds also inhibited both androgen- as well as estrogen-mediated pathways. These results provide mechanistic information on how resveratrol and its methylether analogs may act to contribute to potential antiprostate cancer activity.

Carcinogenesis. 2010 Jan 8. [Epub ahead of print]

Paul S, Decastro A, Lee HJ, Smolarek AK, So JY, Simi B, Wang CX, Zhou R, Rimando AM, Suh N.

Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, U.S.A.

Abstract

Stilbenes are phytochemicals present in grapes, berries, peanuts and red wine. A widely studied stilbene, resveratrol (trans-3,5,4′-trihydroxystilbene), has been shown to exert anti-oxidant, anti-inflammatory, chemopreventive, and anti-aging effects in a number of biological systems. We reported earlier that pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene), a structurally related stilbene found in blueberries, was effective in reducing the incidence and multiplicity of aberrant crypt foci formation in the colon of rats injected with azoxymethane (AOM). Our present study was to identify the chemopreventive potential of pterostilbene with colonic tumor formation as an end point, and further to evaluate the mechanistic action of pterostilbene during colon carcinogenesis. F344 rats were given two AOM injections subcutaneously when they were 7 and 8 weeks old, and continuously fed the control or 40 ppm pterostilbene diet for 45 weeks. Overall analyses indicated that pterostilbene reduced colon tumor multiplicity of non-invasive adenocarcinomas, lowered proliferating cell nuclear antigen, and down-regulated the expression of beta-catenin and cyclin D1. Pterostilbene decreased mucosal levels of the pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-4. Colon tumors from pterostilbene fed animals showed reduced expression of inflammatory markers as well as nuclear staining for phospho-p65, a key molecule in the NF-kappaB pathway. In HT-29 cells, pterostilbene reduced the protein levels of beta-catenin, cyclin D1 and c-MYC, altered the cellular localization of beta-catenin, and inhibited the phosphorylation of p65. Our data with pterostilbene in suppressing colon tumorigenesis, cell proliferation as well as key inflammatory markers in vivo and in vitro suggest the potential use of pterostilbene for colon cancer prevention.

J Surg Res. 2010 Jun 15;161(2):195-201. Epub 2009 Aug 18.

Alosi JA, McDonald DE, Schneider JS, Privette AR, McFadden DW.

University of Vermont, Burlington, Vermont, USA.

Abstract

BACKGROUND: Epidemiologic studies suggest that diets high in fruits and vegetables reduce cancer risk. Resveratrol, a compound present in grapes, has been shown to inhibit a variety of primary tumors. Pterostilbene, an analogue of resveratrol found in blueberries, has both antioxidant and antiproliferative properties. We hypothesized that pterostilbene would induce apoptosis and inhibit breast cancer cell growth in vitro. METHODS: Breast cancer cells were treated with graduated doses of pterostilbene. Cell viability was measured by MTT assay. Apoptosis was evaluated via DNA fragmentation assay and TUNEL assay. Apo-ONE caspase-3/7 assay was used to evaluate caspase activity. Flow cytometry was used to evaluate mitochondrial depolarization, superoxide formation, and cell cycle. Student’s t-test and two-way ANOVA with Bonferroni posttests were utilized for statistical analysis. RESULTS: Pterostilbene decreased breast cancer cell viability in a concentration- and time-dependent manner. Pterostilbene treatment increased caspase-3/7 activity and apoptosis in both cell lines. Caspase-3/7 inhibitors completely reversed pterostilbene’s effects on cell viability. Pterostilbene treatment triggered mitochondrial depolarization, increased superoxide anion, and caused alteration in cell cycle. CONCLUSIONS: Pterostilbene treatment inhibits the growth of breast cancer in vitro through caspase-dependent apoptosis. Mitochondrial membrane depolarization and increased superoxide anion may contribute to the activation downstream effector caspases. Caspase inhibition leads to complete reversal of pterostilbene’s effect on cell viability. Further in vitro mechanistic studies and in vivo experiments are warranted to determine its potential for the treatment of breast cancer. Copyright 2010 Elsevier Inc. All rights reserved.

Banji, D., Banji, O., Dasaroju, S. & Annamalai, A. Piperine and curcumin exhibit synergism in attenuating d-galactose induced senescence in rats. European Journal of Pharmacology 703, 91–9 (2013).

Link to article in: European Journal of Pharmacology
PMID: 23200897

Key Points:

Piperine is the main alkaloid in the fruits of black pepper, long pepper, and other piper species. Piperine has been shown to prevent oxidative stress, depression, and inflammation. It can both stimulate the immune system and serve to protect cells against environmental stresses. Piperine is currently being researched for its potential in the management of central nervous system disorders such as Alzheimer’s disease. Curcumin, a yellow pigment present in the Indian spice turmeric (Curcuma longa), has been accepted as an antioxidant and anti-inflammatory agent. Curcumin has been studied for its ability to potentially prevent neurological diseases, reduce focal brain ischemia, minimize methotrexate induced oxidative stress, and protect the liver from various damage. Curcumin can cross the blood brain barrier if it is adequately absorbed within the body. In combination with piperine, there is a profound increase in neuroprotection as compared with either chemical alone. This indicates that piperine might have increased the bioavailability of curcumin. Observations in this research collaborates with previous reports suggesting the role of piperine as a bioenhancer of curcumin.

Huaping Chen^a, Charles N Landen^b, Yuanyuan Li^a, Ronald D Alvarez^b, Trygve O Tollefsbol

The cellular development of resistance to chemotherapy contributes to the high mortality noted in patients affected by ovarian cancer. Novel compounds that specifically target cellular drug resistance in ovarian cancer are therefore highly desired. Previous epidemiological studies indicate that consumption of green tea and cruciferous vegetables is inversely associated with occurrence of ovarian cancer. Therefore revealing the effects and mechanisms of major components of green tea (epigallocatechin gallate, EGCG) and cruciferous vegetables (sulforaphane, SFN) on ovarian cancer cells will provide necessary knowledge for developing potential novel treatments for the disease. In this study, EGCG or SFN was used to treat both paclitaxel-sensitive (SKOV3-ip1) and -resistant (SKOV3TR-ip2) ovarian cancer cell lines alone or in combination. We found that SFN inhibits cell viability of both ovarian cancer cell lines time- and dose-dependently and that EGCG potentiates the inhibiting effect of SFN on ovarian cancer cells. Cell cycle analysis indicates SFN can arrest ovarian cancer cells in G2/M phase, while EGCG and SFN co-treatment can arrest cells in both G2/M and S phase. Combined EGCG and SFN treatment increases apoptosis significantly in paclitaxel-resistant SKOV3TR-ip2 cells after 6 days of treatment, while reducing the expression of hTERT, the main regulatory subunit of telomerase. Western blotting also indicates that SFN can down-regulate Bcl-2 (a gene involved in anti-apoptosis) protein levels in both cell types. Cleaved poly(ADP-ribose) polymerase (PARP) becomes up-regulated by 6 days of treatment with SFN and this is more pronounced for combination treatment indicating induction of apoptosis. Furthermore, phosphorylated H2AX is up-regulated after 6 days of treatment with SFN alone, and EGCG can potentiate this effect, suggesting that DNA damage is a potential cellular mechanism contributing to the inhibiting effect of EGCG and SFN combination treatment. Taken together, these results indicate that EGCG and SFN combination treatment can induce apoptosis by down-regulating of hTERT and Bcl-2 and promote DNA damage response specifically in paclitaxel-resistant ovarian cancer cell lines and suggest the use of these compounds for overcoming paclitaxel resistance in ovarian cancer treatment.