The Role of Lysosome-Associated Membrane Protein 2 in Prostate Cancer Chemopreventive Mechanisms of Sulforaphane
Hahm ER, Singh KB, Kim SH, Powolny AA and Singh SV
Prostate cancer chemoprevention by sulforaphane (SFN), which is a metabolic by-product of glucoraphanin found in broccoli, in preclinical models is associated with induction of both apoptosis and autophagy. However, the molecular mechanism underlying SFN-mediated autophagy, which is protective against apoptotic cell death by this phytochemical, is still poorly understood. The present study demonstrates a role for lysosome-associated membrane protein 2 (LAMP2) in SFN-mediated autophagy and apoptosis. Western blotting revealed dose-dependent induction of LAMP2 protein after treatment with SFN as well as its naturally-occurring analogs in PC-3 and 22Rv1 human prostate cancer cell lines that was confirmed by microscopy (SFN). The mRNA level of LAMP2 was also increased upon treatment with SFN in both cell lines. SFN-mediated increase in the level of autophagy marker microtubule-associated protein light-chain 3B (LC3BII) was augmented by RNA interference of LAMP2 in PC-3 and 22Rv1 cells. Apoptosis induction by SFN treatment was also increased significantly by knockdown of the LAMP2 protein in PC-3 and 22Rv1 cells. Augmentation of SFN-mediated apoptosis by RNA interference of LAMP2 was accompanied by induction and activation of pro-apoptotic protein Bak. Oral administration of SFN to TRAMP mice also resulted in induction of LAMP2 protein expression. Targeted microarray in SFN-treated PC-3 cells revealed induction of many autophagy-related genes (e.g., HSP90AA1, NRF2, etc.) and their expression positively correlated with that of LAMP2 in prostate cancer TCGA. In conclusion, the present study reveals that induction of LAMP2 by SFN inhibits its ability to induce apoptotic cell death at least in human prostate cancer cells.
An integrative investigation on significant mutations and their down-stream pathways in lung squamous cell carcinoma reveals CUL3/KEAP1/NRF2 relevant subtypes
Liu Z, Deng M, Wu L and Zhang S
Molecular mechanism of lung squamous cell carcinoma (LUSC) remains poorly understood, hampering effective targeted therapies or precision diagnosis about LUSC. We devised an integrative framework to investigate on the molecular patterns of LUSC by systematically mining the genomic, transcriptional and clinical information.
Tau accumulates in Crohn's disease gut
Prigent A, Chapelet G, De Guilhem de Lataillade A, Oullier T, Durieu E, Bourreille A, Duchalais E, Hardonnière K, Neunlist M, Noble W, Kerdine-Römer S, Derkinderen P and Rolli-Derkinderen M
A sizeable body of evidence has recently emerged to suggest that gastrointestinal (GI) inflammation might be involved in the development of Parkinson's disease (PD). There is now strong epidemiological and genetical evidence linking PD to inflammatory bowel diseases and we recently demonstrated that the neuronal protein alpha-synuclein, which is critically involved in PD pathophysiology, is upregulated in inflamed segments of Crohn's colon. The microtubule associated protein tau is another neuronal protein critically involved in neurodegenerative disorders but, in contrast to alpha-synuclein, no data are available about its expression and phosphorylation patterns in inflammatory bowel diseases. Here, we examined the expression levels of tau isoforms, their phosphorylation profile and truncation in colon biopsy specimens from 16 Crohn's disease (CD) and 6 ulcerative colitis (UC) patients and compared them to samples from 16 controls. Additional experiments were performed in full thickness segments of colon of five CD and five control subjects, in primary cultures of rat enteric neurons and in nuclear factor erythroid 2-related factor (Nrf2) knockout mice. Our results show the upregulation of two main human tau isoforms in the enteric nervous system (ENS) in CD but not in UC. This upregulation was not transcriptionally regulated but instead likely resulted from a decrease in protein clearance via an Nrf2 pathway. Our findings, which provide the first detailed characterization of tau in CD, suggest that the key proteins involved in neurodegenerative disorders such as alpha-synuclein and tau, might also play a role in CD.
Combined Peptide and Small-Molecule Approach toward Nonacidic THIQ Inhibitors of the KEAP1/NRF2 Interaction
Ontoria JM, Biancofiore I, Fezzardi P, Ferrigno F, Torrente E, Colarusso S, Bianchi E, Andreini M, Patsilinakos A, Kempf G, Augustin M, Steinbacher S, Summa V, Pacifici R, Muñoz-Sanjuan I, Park L, Bresciani A, Dominguez C, Sherman LT and Harper S
The NRF2-ARE pathway is an intrinsic mechanism of defense against oxidative stress. Inhibition of the interaction between NRF2 and its main negative regulator KEAP1 is an attractive strategy toward neuroprotective agents. We report here the identification of nonacidic tetrahydroisoquinolines (THIQs) that inhibit the KEAP1/NRF2 protein-protein interaction. Peptide SAR at one residue is utilized as a tool to probe structural changes within a specific pocket of the KEAP1 binding site. We used structural information from peptide screening at the P2 pocket, noncovalent small-molecules inhibitors, and the outcome from an explorative SAR at position 5 of THIQs to identify a series of neutral THIQ analogs that bind to KEAP1 in the low micromolar range. These analogs establish new H-bond interactions at the P3 and P2 pockets allowing the replacement of the carboxylic acid functionality by a neutral primary carboxamide. X-ray crystallographic studies reveal the novel binding mode of these molecules to KEAP1.
Therapeutic mechanism of human neural stem cell-derived extracellular vesicles against hypoxia-reperfusion injury in vitro
Liu Q, Tan Y, Qu T, Zhang J, Duan X, Xu H, Mu Y, Ma H and Wang F
This study aimed to explore that the human neural stem cell derived extracellular vesicles (hNSC-EVs) have therapeutic effect on neuronal hypoxia-reperfusion (H/R) injured neurons in vitro by mediating the nuclear translocation of NF-E2-related factor 2 (Nrf2) to regulate the expression of downstream oxidative kinases.
ZJ01, a Small Molecule Inhibitor of the Kelch-Like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1-Nrf2) Protein-Protein Interaction, Reduces Hyperoxic Acute Lung Injury in a Mouse Model
Wan J, Lin S, Huang X, Li Q, Zeng L and Du S
BACKGROUND Hyperoxic acute lung injury (ALI) is a complication of ventilation in patients with respiratory failure. Nuclear factor erythroid-2-related factor 2 (Nrf2) has an important role in ALI. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2. ZJ01 is a small molecule inhibitor of Keap1-Nrf2 protein-protein interaction (PPI) that can reduce Keap1-induced inhibition of Nrf2. This study aimed to investigate the effects of ZJ01 and the heme oxygenase-1 (HO-1) inhibitor, zinc protoporphyrin IX (ZnPP IX), in a mouse model of hyperoxic ALI. MATERIAL AND METHODS C57BL/6J mice included five study groups: the room air+vehicle-treated group; the room air+ZJ01 group; the hyperoxia+vehicle-treated group; the hyperoxia+ZJ01 group; and the hyperoxia+ZJ01+ZnPP IX group. ZJ01, ZnPP IX, or vehicle were given 1 h after the hyperoxia challenge. The lungs from the mice were harvested at 72 h following the hyperoxia challenge. RESULTS Hyperoxia exposure for 72 h increased the activity of myeloperoxidase, the lung water content, the levels of tumor necrosis factor-alpha (TNF-alpha), and matrix metalloprotease-9 (MMP-9) in the vehicle-treated mice. ZJ01 treatment reduced hyperoxia-induced inflammation and increased the activation of Nrf2 and HO-1 compared with the vehicle-treated mice. Histology of the lungs showed that ZJ01 treatment reduced the changes of hyperoxia-induced ALI. Pretreatment with ZnPP IX reversed the beneficial effect of ZJ01. CONCLUSIONS ZJ01, a Keap1-Nrf2 PPI inhibitor, reduced hyperoxic ALI in a mouse model through the Nrf2/HO-1 pathway.
Klotho ameliorates the onset and progression of cataract via suppressing oxidative stress and inflammation in the lens in streptozotocin-induced diabetic rats
Ma Z, Liu J, Li J, Jiang H and Kong J
Increased oxidative stress and inflammation play an important role in the pathogenesis of diabetic cataract. Klotho, known as an anti-ageing protein, has antioxidative and anti-inflammatory properties. Klotho is expressed in limited tissues including the lens. Here we examined whether klotho expression is decreased in diabetic lens and, if so, whether klotho treatment can prevent diabetic cataract formation. Streptozotocin (STZ)-induced diabetic rats and age-matched control rats were treated with vehicle or klotho protein, starting at 1 week after STZ injection. Twelve weeks after treatment, cataract formation was observed in diabetic rats but not control rats. Cataract formation and scores were significantly less in klotho-treated diabetic rats than vehicle-treated diabetic rats. Levels of klotho in plasma, aqueous humor and lens were significantly decreased in vehicle-treated diabetic rats, compared with control rats, but were restored in klotho-treated diabetic rats. Additionally, vehicle-treated diabetic rats had increased oxidative stress and inflammation in the lens, which were associated with decreased antioxidant transcriptional master regulator Nrf2 activity and increased transcription factor NF-κB activity. All of these findings were ameliorated in klotho-treated diabetic rats. Notably, klotho treatment did not alter blood glucose in diabetic rats. These results indicate that klotho reduction may increase susceptibility of the lens to oxidative and inflammatory insults, promoting cataract formation under diabetic conditions. Klotho treatment can ameliorate the onset and progression of diabetic cataract via enhancing Nrf2-mediated antioxidant defense and suppressing NF-κB-mediated inflammatory responses. Klotho in the lens may be a novel therapeutic target for prevention of cataract formation in diabetes.
Chemical characteristics and cytoprotective activities of polysaccharide fractions from Athyrium Multidentatum (Doll.) Ching
Jing L, Sheng J, Jiang J, Wang Y, Shen X, Liu D, Zhang W and Mao S
Five polysaccharide fractions (PS-1, PS-2, PS-3, PS-4 and PS-5) were successfully isolated from Athyrium Multidentatum (Doll.) Ching by anion-exchange column chromatography. Their in vitro cytoprotective activities and the underlying mechanisms were explored in this paper. Chemical analysis suggested that the five polysaccharide fractions were heteropolysaccharides with different molecular weights and monosaccharide compositions. Treatment with these polysaccharide fractions could increase cell viabilities, superoxide dismutase/catalase activities, nitric oxide contents, mitochondrial membrane potential levels and Bcl-2/Bax ratios, and reduce cell apoptosis, intracellular reactive oxygen species production and malondialdehyde contents in HO-damaged cells. Moreover, these polysaccharide fractions enhanced the mRNA expression levels of PI3K, Akt, FOXO3a, Nrf2 and HO-1 and PS-4 exhibited the most powerful effects on the mRNA expression of these genes. Current findings suggested that the polysaccharide fractions decreased HO-induced apoptosis of HUVECs. The activation of PI3K/Akt/FOXO3a and Nrf2/HO-1 signaling pathways might be involved in the protective mechanisms of the active fractions. The polysaccharides might be one of the key bioactive ingredients of Athyrium Multidentatum (Doll.) Ching for the treatment of oxidative damage.
Luteolin alleviates ochratoxin A induced oxidative stress by regulating Nrf2 and HIF-1α pathways in NRK-52E rat kidney cells
Liu M, Cheng C, Li X, Zhou S, Hua J, Huang J, Li Y, Yang K, Zhang P, Zhang Y and Tian J
Ochratoxin A (OTA) is a ubiquitous food contaminant and a critical food safety concern due to its nephron toxic effects, which impacts all parts of the world. Luteolin (LUT) had gained increasing interest as a health-promoting food antioxidant component. However, the preventative effect of LUT against OTA induced oxidative stress was not yet clear and the elucidation of which would provide critical information to develop dietary LUT as a control strategy for OTA. In the current study, the cytoprotective effect of LUT against OTA induced oxidative stress and the mechanism(s) behind was examined in NRK-52E rat kidney cells. The results showed that LUT exerted its preventative effect via restoring cell viability and preventing LDH release. It alleviated the OTA-induced oxidative stress and lipid peroxidation by reducing ROS accumulation, ameliorating the mitochondrial membrane potential reduction and reversing the activities of antioxidant enzymes to the control levels. The regulating roles of Nrf2 and HIF-1α in this process were evaluated by cell immunofluorescence assay, reporter plasmids transfection assay and qRT-PCR analysis. The results showed that LUT activated Nrf2 pathway and increased the antioxidant defense capacities of OTA treated cells. Additionally, LUT also modulated HIF-1α pathway to initiate the angiogenesis and epithelial restitution process.
Therapeutic potential of glutathione-enhancers in stress-related psychopathologies
Zalachoras I, Hollis F, Ramos-Fernández E, Trovo L, Sonnay S, Geiser E, Preitner N, Steiner P, Sandi C and Morató L
The mammalian brain has high energy demands, which may become higher in response to environmental challenges such as psychogenic stress exposure. Therefore, efficient neutralization of reactive oxygen species that are produced as a by-product of ATP synthesis is crucial for preventing oxidative damage and ensuring normal energy supply and brain function. Glutathione (GSH) is arguably the most important endogenous antioxidant in the brain. In recent years, aberrant GSH levels have been implicated in different psychiatric disorders, including stress-related psychopathologies. In this review, we examine the available data supporting a role for GSH levels and antioxidant function in the brain in relation to anxiety and stress-related psychopathologies. Additionally, we identify several promising compounds that could raise GSH levels in the brain by either increasing the availability of its precursors or the expression of GSH-regulating enzymes through activation of Nuclear factor erythroid-2-related factor 2 (Nrf2). Given the high tolerability and safety profile of these compounds, they may represent attractive new opportunities to complement existing therapeutic manipulations against stress-related psychopathologies.
Vaccine adjuvant activity of a TLR4-activating synthetic glycolipid by promoting autophagy
Chou YJ, Lin CC, Dzhagalov I, Chen NJ, Lin CH, Lin CC, Chen ST, Chen KH and Fu SL
Toll-like receptors (TLRs) play crucial roles in host immune defenses. Recently, TLR-mediated autophagy is reported to promote immune responses via increasing antigen processing and presentation in antigen presenting cells. The present study examined whether the synthetic TLR4 activator (CCL-34) could induce autophagy to promote innate and adaptive immunity. In addition, the potential of CCL-34 as an immune adjuvant in vivo was also investigated. Our data using RAW264.7 cells and bone marrow-derived macrophages showed that CCL-34 induced autophagy through a TLR4-NF-κB pathway. The autophagy-related molecules (Nrf2, p62 and Beclin 1) were activated in RAW264.7 cells and bone marrow-derived macrophages under CCL-34 treatment. CCL-34-stimulated macrophages exhibited significant antigen-processing activity and induced the proliferation of antigen-specific CD4+T cells as well as the production of activated T cell-related cytokines, IL-2 and IFN-γ. Furthermore, CCL-34 immunization in mice induced infiltration of monocytes in the peritoneal cavity and elevation of antigen-specific IgG in the serum. CCL-34 treatment in vivo did not cause toxicity based on serum biochemical profiles. Notably, the antigen-specific responses induced by CCL-34 were attenuated by the autophagy inhibitor, 3-methyladenine. In summary, we demonstrated CCL-34 can induce autophagy to promote antigen-specific immune responses and act as an efficient adjuvant.
Photoprotective Properties of Vitamin D and Lumisterol Hydroxyderivatives
Slominski AT, Chaiprasongsuk A, Janjetovic Z, Kim TK, Stefan J, Slominski RM, Hanumanthu VS, Raman C, Qayyum S, Song Y, Song Y, Panich U, Crossman DK, Athar M, Holick MF, Jetten AM, Zmijewski MA, Zmijewski J and Tuckey RC
We have previously described new pathways of vitamin D3 activation by CYP11A1 to produce a variety of metabolites including 20(OH)D3 and 20,23(OH)D3. These can be further hydroxylated by CYP27B1 to produce their C1α-hydroxyderivatives. CYP11A1 similarly initiates the metabolism of lumisterol (L3) through sequential hydroxylation of the side chain to produce 20(OH)L3, 22(OH)L3, 20,22(OH)L3 and 24(OH)L3. CYP11A1 also acts on 7-dehydrocholesterol (7DHC) producing 22(OH)7DHC, 20,22(OH)7DHC and 7-dehydropregnenolone (7DHP) which can be converted to the D3 and L3 configurations following exposure to UVB. These CYP11A1-derived compounds are produced in vivo and are biologically active displaying anti-proliferative, anti-inflammatory, anti-cancer and pro-differentiation properties. Since the protective role of the classical form of vitamin D3 (1,25(OH)D3) against UVB-induced damage is recognized, we recently tested whether novel CYP11A1-derived D3- and L3-hydroxyderivatives protect against UVB-induced damage in epidermal human keratinocytes and melanocytes. We found that along with 1,25(OH)D3, CYP11A1-derived D3-hydroxyderivatives and L3 and its hydroxyderivatives exert photoprotective effects. These included induction of intracellular free radical scavenging and attenuation and repair of DNA damage. The protection of human keratinocytes against DNA damage included the activation of the NRF2-regulated antioxidant response, p53-phosphorylation and its translocation to the nucleus, and DNA repair induction. These data indicate that novel derivatives of vitamin D3 and lumisterol are promising photoprotective agents. However, detailed mechanisms of action, and the involvement of specific nuclear receptors, other vitamin D binding proteins or mitochondria, remain to be established.
Brassinin, a brassica-derived phytochemical, regulates monocyte-to-macrophage differentiation and inflammatory responses in human monocytes and murine macrophages
Kang B, Hwang J and Choi HS
The effects and molecular mechanisms of brassinin (BR), an indole phytoalexin from cruciferous vegetables, on monocyte-to-macrophage differentiation and inflammatory responses were investigated in this study.
Therapeutic and Mechanistic effects of Curcumin in Huntington's disease
Labanca F, Ullah H, Khan H, Milella L, Xiao J, Dajic-Stevanovic Z and Jeandet P
Curcumin is a spice derived nutraceutical which gained tremendous attention because of its profound medicinal values. It alters a number of molecular pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), signal transducer and activator of transcription 3 (STAT3), nuclear factor erythroid 2-related factor 2 (Nrf2) and cyclooxygenases-2 (COX-2), which make it potential therapeutic choice in treating multiple disorders. It also possesses potential to prevent protein aggregation and thus protect against degeneration of neurons in neurodegenerative disorders including Huntington's disease (HD). HD is an autosomal dominant disorder linked with altered gene expression which leads to increase in size of cytosine, adenine and guanine (CAG) trinucleotide repeats, aids in protein aggregation throughout the brain and thus damages neurons. Upstream regulation of oxidative stress and inflammatory cascade are two important factors that drive HD progression. Available therapies just suppress the severity of symptoms with a number of side effects. Curcumin targets multiple mechanisms in treating or preventing HD including antioxidant and antiinflammatory potential, metal ion chelation, transcriptional alterations and upregulating activity of molecular chaperons, heat shock proteins (HSPs). Having a favorable safety profile, curcumin can be an alternative therapeutic choice in treating neurodegenerative disorders like HD. This review will focus on mechanistic aspects of curcumin in treating or preventing HD and its potential to arrest disease progression and will open new dimensions for safe and effective therapeutic agents in diminishing HD.
USP9X prevents AGEs-induced upregulation of FN and TGF-β1 through activating Nrf2-ARE pathway in rat glomerular mesangial cells
Huang K and Zhao X
Oxidative stress is a key pathological factor for diabetic renal fibrosis by activating TGF-β/Smad pathway in glomerular mesangial cells (GMCs) to promote the synthesis of extracellular matrix such as fibronectin (FN). Nuclear factor-E2-related factor (Nrf2)- anti-oxidant response element (ARE) anti-oxidative pathway has crucial renoprotective effects, and inhibiting ubiquitin-mediated degradation of Nrf2 delays diabetic renal fibrosis development. Ubiquitin-specific protease 9X (USP9X) has close relationship with oxidative stress and TGF-β/Smad pathway, but whether it regulate diabetic renal fibrosis remains unclarified. Here, we found that advanced glycation-end products (AGEs) dose- and time-dependently reduced the protein expression and deubiquitinase activity of USP9X in GMCs. USP9X overexpression attenuated AGEs-induced upregulation of FN, TGF-β1, and Collagen Ⅳ, three fibrosis-related marker proteins, in a deubiquitinase activity-dependent manner. While USP9X depletion with siRNAs further promoted the expressions of those proteins in AGEs-treated GMCs. Under AGEs treatment conditions, USP9X overexpression markedly increased the total and nuclear levels, ARE-binding ability, and transcriptional activity of Nrf2, upregulated the protein expressions of Nrf2 downstream genes HO-1 and NQO1, and eventually reduced the excessive production of ROS. Overexpression of the deubiquitinase catalytically inactive USP9X-C1556S mutant failed to exert such effects. Silencing Nrf2 abolished the renoprotective effects of USP9X. Further study showed that upon AGEs stimulation, Nrf2 transferred into the nucleus and the interaction between USP9X and Nrf2 was weakened. AGEs also increased Nrf2 ubiquitination level, and overexpression of USP9X, instead of USP9X-C1556S, significantly reduced the ubiquitination level of Nrf2. Taken together, USP9X reduced Nrf2 ubiquitination level and promoted Nrf2-ARE pathway activation to prevent the accumulation of extracellular matrix, eventually alleviated the pathological process of diabetic renal fibrosis.