Tyrosinase‐catalyzed oxidation of rhododendrol produces 2‐methylchromane‐6,7‐dione,the putative ultimate toxic metabolite: implications for melanocyte toxicity

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a skin‐whitening agent until it was reported to induce leukoderma in July 2013. To explore the mechanism underlying its melanocyte toxicity, we characterized the tyrosinase‐catalyzed oxidation of RD using spectrophotometry and HPLC. Oxidation of RD with mushroom tyrosinase rapidly produced RD‐quinone, which was quickly converted to 2‐methylchromane‐6,7‐dione (RD‐cyclic quinone) and RD‐hydroxy‐p‐quinone through cyclization and addition of water molecule, respectively. RD‐quinone and RD‐cyclic quinone were identified as RD‐catechol and RD‐cyclic catechol after NaBH₄ reduction. Autoxidation of RD‐cyclic catechol produced superoxide radical. RD‐quinone and RD‐cyclic quinone quantitatively bound to thiols such as cysteine and GSH. These results suggest that the melanocyte toxicity of RD is caused by its tyrosinase‐catalyzed oxidation through production of RD‐cyclic quinone which depletes cytosolic GSH and then binds to essential cellular proteins through their sulfhydryl groups. The production of ROS through autoxidation of RD‐cyclic catechol may augment the toxicity.     

A novel andrographolide derivative AL‐1 exerts its cytotoxicity on K562 cells through a ROS‐dependent mechanism

Andrographolide‐lipoic acid conjugate (AL‐1) is a new in‐house synthesized chemical entity, which was derived by covalently linking andrographolide with lipoic acid. However, its anti‐cancer effect and cytotoxic mechanism remains unknown. In this study, we found that AL‐1 could significantly inhibit cell viability of human leukemia K562 cells by inducing G2/M arrest and apoptosis in a dose‐dependent manner. Thirty‐one AL‐1‐regulated protein alterations were identified by proteomics analysis. Gene ontology and ingenuity pathway analysis revealed that a cluster of proteins of oxidative redox state and apoptotic cell death‐related proteins, such as PRDX2, PRDX3, PRDX6, TXNRD1, and GLRX3, were regulated by AL‐1. Functional studies confirmed that AL‐1 induced apoptosis of K562 cells through a ROS‐dependent mechanism, and anti‐oxidant, N‐acetyl‐l ‐cysteine, could completely block AL‐1‐induced cytotoxicity, implicating that ROS generation played a vital role in AL‐1 cytotoxicity. Accumulated ROS resulted in oxidative DNA damage and subsequent G2/M arrest and mitochondrial‐mediated apoptosis. The current work reveals that a novel andrographolide derivative AL‐1 exerts its anticancer cytotoxicity through a ROS‐dependent DNA damage and mitochondrial‐mediated apoptosis mechanism.     

Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via FUT8 knockout CHO‐S cells

To engineer a host cell line that produces defucosylated mAbs with superior antibody‐dependent cellular cytotoxicity, we disrupted α‐1, 6 fucosyltransferase (FUT8 ) gene in CHO‐S (CHO is Chinese hamster ovary) cells by clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9. The gene knockout cell line was evaluated for growth, stability, and product quality. The growth profile of FUT8 gene knockout CHO‐S (FUT8 ^?/?) cells was comparable with wild type CHO‐S cells. FUT8 catalyzes the transfer of a fucose residue from GDP‐fucose to N‐glycans residue. Defucosylated IgG1 antibodies produced by FUT8 ^?/? cells showed increased binding affinities to human FcγRIIIa and higher activities in mediating antibody‐dependent cellular cytotoxicity, comparing with conventional fucosylated IgG1. Our results demonstrated the potential of using the clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9 technology in cell line engineering for biopharmaceutical industrial applications.     

Dimethyl sulfide protects against oxidative stress and extends lifespan via a methionine sulfoxide reductase A‐dependent catalytic mechanism

Methionine (Met) sulfoxide reductase A (MsrA) is a key endogenous antioxidative enzyme with longevity benefits in animals. Only very few approaches have been reported to enhance MsrA function. Recent reports have indicated that the antioxidant capability of MsrA may involve a Met oxidase activity that facilities the reaction of Met with reactive oxygen species (ROS). Herein, we used a homology modeling approach to search the substrates for the oxidase activity of MsrA. We found that dimethyl sulfide (DMS), a main metabolite that produced by marine algae, emerged as a good substrate for MsrA‐catalytic antioxidation. MsrA bounds to DMS and promoted its antioxidant capacity via facilitating the reaction of DMS with ROS through a sulfonium intermediate at residues Cys72, Tyr103, and Glu115, followed by the release of dimethyl sulfoxide (DMSO). DMS reduced the antimycin A‐induced ROS generation in cultured PC12 cells and alleviated oxidative stress. Supplement of DMS exhibited cytoprotection and extended longevity in both Caenorhabditis elegans and Drosophila. MsrA knockdown abolished the cytoprotective effect and the longevity benefits of DMS. Furthermore, we found that the level of physiologic DMS was at the low micromolar range in different tissues of mammals and its level decreased after aging. This study opened a new window to elucidate the biological role of DMS and other low‐molecular sulfides in the cytoprotection and aging.     

Hydrogen peroxide regulates glucose‐regulated protein 78 expression via a cyclooxygenase‐2 dependent mechanism

This study was designed to investigate the effect of hydrogen peroxide on the expression of endoplasmic reticulum stress marker glucose‐regulated protein 78 (GRP78) in endothelial cells and reveals the possible role of cyclooxygenase in this effect. The porcine endothelial cell line was cultured in 1640 medium. Western blot and immunocytochemistry were used to detect the expression of GRP78. The caspase‐12 activity was analyzed with the immune fluorescence method. The results showed that after the endothelial cells were incubated with 250 μM of hydrogen peroxide for 12 h, apoptosis increased, which was antagonized by the cyclooxygenase‐2 inhibitor nimesulide or the nonselective cyclooxygenase inhibitor aspirin, but not by the cyclooxygenase‐1 inhibitor piroxicam. The expression of GRP78 was induced in endothelial cells after exposure to hydrogen peroxide for 12 h. The overexpression of GRP78 was inhibited by nimesulide and aspirin, but not by piroxicam. There are no significant differences in caspase‐12 activity among all groups. The present study provides evidence that hydrogen peroxide induced GRP78 overexpression in endothelial cells by a mechanism involving cyclooxygenase‐2‐dependent pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:279–285, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336     

Acacetin inhibits expression of matrix metalloproteinases via a MAPK‐dependent mechanism in fibroblast‐like synoviocytes

It is well known that rheumatoid arthritis (RA) is an autoimmune joint disease in which fibroblast‐like synoviocytes (FLSs) play a pivotal role. In this study, we investigated the anti‐arthritic properties of acacetin in FLSs. The expression of matrix metalloproteinase (MMP)‐1, MMP‐3 and MMP‐13 were investigated by quantitative RT‐PCR and western blot at gene and protein levels. At the same time, the phosphorylation of mitogen‐activated protein kinases (MAPK) was investigated. The DNA‐binding activity of NF‐κB was investigated by electrophoretic mobility shift assay. We found that acacetin inhibits p38 and JNK phosphorylation and reduces MMP‐1, MMP‐3 and MMP‐13 expression in interleukin‐1β‐induced FLSs. Our results suggest that acacetin has antiarthritic effects in FLSs. Thus, acacetin should be further studied for the treatment of arthritis.     

Age‐at‐onset‐dependent effects of sulfur amino acid restriction on markers of growth and stress in male F344 rats

Trade‐offs in life‐history traits are clinically and mechanistically important. Sulfur amino acid restriction (SAAR) extends lifespan. But whether this benefit comes at the cost of other traits including stress resistance and growth is unclear. We investigated the effects of SAAR on growth markers (body weight, IGF1, and IGFBP3) and physiological stresses. Male‐F344 rats were fed control (0.86% Met) and SAAR (0.17% Met) diets starting at 2, 10, and 20 months. Rats were injected with keyhole‐limpet‐hemocyanin (KLH) to measure immune responses (anti‐KLH‐IgM, anti‐KLH‐IgG, and delayed‐type‐hypersensitivity [DTH]). Markers of ER stress (FGF21 and adiponectin), detoxification capacity (glutathione [GSH] concentrations, GSH‐S‐transferase [GST], and cytochrome‐P₄₅₀‐reductase [CPR] activities), and low‐grade inflammation (C‐reactive protein [CRP]) were also determined. SAAR decreased body weight, liver weight, food intake, plasma IGF1, and IGFBP3; the effect size diminished with increasing age‐at‐onset. SAAR increased FGF21 and adiponectin, but stress damage markers GRP78 and Xbp1_s/us were unchanged, suggesting that ER stress is hormetic. SAAR increased hepatic GST activity despite lower GSH, but CPR activity was unchanged, indicative of enhanced detoxification capacity. Other stress markers were either uncompromised (CRP, anti‐KLH‐IgM, and DTH) or slightly lower (anti‐KLH‐IgG). Increases in stress markers were similar across all ages‐at‐onset, except for adiponectin, which peaked at 2 months. Overall, SAAR did not compromise stress responses and resulted in maximal benefits with young‐onset. In survival studies, median lifespan extension with initiation at 52 weeks was 7 weeks (p = .05); less than the 33.5‐week extension observed in our previous study with 7‐week initiation. Findings support SAAR translational studies and the need to optimize Met dose based on age‐at‐onset.     

Synaptic drosophila UNC‐13 is regulated by antagonistic G‐protein pathways via a proteasome‐dependent degradation mechanism

UNC‐13 is a highly conserved plasma membrane‐associated synaptic protein implicated in the regulation of neurotransmitter release through the direct modulation of the SNARE exocytosis complex. Previously, we characterized the Drosophila homologue (DUNC‐13) and showed it to be essential for neurotransmitter release immediately upstream of vesicular fusion (“priming”) at the neuromuscular junction (NMJ). Here, we show that the abundance of DUNC‐13 in NMJ synaptic boutons is regulated downstream of GαS and Gαq pathways, which have inhibitory and facilitatory roles, respectively. Both cAMP modulation and PKA function are required for DUNC‐13 synaptic up‐regulation, suggesting that the cAMP pathway enhances synaptic efficacy via DUNC‐13. Similarly, PLC function and DAG modulation also regulate the synaptic levels of DUNC‐13, through a mechanism that appears independent of PKC. Our results suggest that proteasome‐mediated protein degradation is the primary mechanism regulating DUNC‐13 levels at the synapse. Both PLC‐ and PKA‐mediated pathways appear to regulate synaptic levels of DUNC‐13 through controlling the rate of proteasome‐dependent DUNC‐13 degradation. We conclude that the functional abundance of DUNC‐13 at the synapse, a key determinant of synaptic vesicle priming and neurotransmitter release probability, is primarily regulated by the rate of protein degradation, rather than translocation or transport, convergently controlled via both cAMP and DAG signal transduction pathways. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 417–438, 2003     

Fumonisin B1 actuates oxidative stress‐associated colonic damage via apoptosis and autophagy activation in murine model

In the present study, we investigated the cytotoxic mechanism of Fumonisin B1 (FB1) in mice colonic region in a time course manner. Herein, after consecutive 4 days of exposure to FBI (2.5 mg/kg body weight), we observed disintegration of mice colon, as evidenced by histopathological analysis. FB1 significantly increased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities in serum and plasma, decreased ceramide level, increased sphinganine level, and increased lipid peroxidase level along with the breakdown of the antioxidant system. Further, FB1‐induced ER stress caused apoptosis and autophagy activation in mice colon, evidenced by increased expression of IRE1‐α, p‐JNK, Casp3, and LC3I/II. In addition, we also noticed a reduced protein kinase C expression in mice colon exposed to FB1, suggesting its role in ER stress‐induced cell death. Taken together, study suggests both physiologically and biochemically, FB1 toxicity to mice colon induced by oxidative stress‐associated apoptosis and autophagy activation.     

Inhibition of deubiquitination by PR‐619 induces apoptosis and autophagy via ubi‐protein aggregation‐activated ER stress in oesophageal squamous cell carcinoma

ObjectivesTargeting the deubiquitinases (DUBs) has become a promising avenue for anti‐cancer drug development. However, the effect and mechanism of pan‐DUB inhibitor, PR‐619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated.Materials and MethodsThe effect of PR‐619 on ESCC cell growth and cell cycle was evaluated by CCK‐8 and PI staining. Annexin V‐FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca²⁺ concentration was monitored by Fluo‐3AM fluorescence. The accumulation of ubi‐proteins and the expression of the endoplasmic reticulum (ER) stress‐related protein and CaMKKβ‐AMPK signalling were determined by immunoblotting.ResultsPR‐619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR‐619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4‐Noxa axis. Moreover, the ER stress increased cytoplasmic Ca²⁺ and then stimulated autophagy through Ca²⁺‐CaMKKβ‐AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR‐41, could reduce the accumulation of ubi‐proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR‐619‐treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR‐619.ConclusionsOur findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR‐619) and imply that targeting DUBs may be a potential anti‐ESCC strategy.     

Arabidopsis OTU1, a linkage‐specific deubiquitinase,is required for endoplasmic reticulum‐associated protein degradation

Endoplasmic reticulum (ER)‐associated degradation (ERAD) is part of the ER protein quality‐control system (ERQC), which is critical for the conformation fidelity of most secretory and membrane proteins in eukaryotic organisms. ERAD is thought to operate in plants with core machineries highly conserved to those in human and yeast; however, little is known about the plant ERAD system. Here we report the characterization of a close homolog of human OTUB1 in Arabidopsis thaliana, designated as AtOTU1. AtOTU1 selectively hydrolyzes several types of ubiquitin chains and these activities depend on its conserved protease domain and/or the unique N‐terminus. The otu1 null mutant is sensitive to high salinity stress, and particularly agents that cause protein misfolding. It turns out that AtOTU1 is required for the processing of known plant ERAD substrates such as barley powdery mildew O (MLO) alleles by virtue of its association with the CDC48 complex through its N‐terminal region. These observations collectively define AtOTU1 as an OTU domain‐containing deubiquitinase involved in Arabidopsis ERAD.     

Ca2+‐dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid β‐treated astrocytes and in a model of Alzheimer's disease

Neurotoxic effects of amyloid β peptides are mediated through deregulation of intracellular Ca²⁺ homeostasis and signaling, but relatively little is known about amyloid β modulation of Ca²⁺ homeostasis and its pathological influence on glia. Here, we found that amyloid β oligomers caused a cytoplasmic Ca²⁺ increase in cultured astrocytes, which was reduced by inhibitors of PLC and ER Ca²⁺ release. Furthermore, amyloid β peptides triggered increased expression of glial fibrillary acidic protein (GFAP), as well as oxidative and ER stress, as indicated by eIF2α phosphorylation and overexpression of chaperone GRP78. These effects were decreased by ryanodine and 2APB, inhibitors of ryanodine receptors and InsP₃ receptors, respectively, in both primary cultured astrocytes and organotypic cultures of hippocampus and entorhinal cortex. Importantly, intracerebroventricular injection of amyloid β oligomers triggered overexpression of GFAP and GRP78 in astrocytes of the hippocampal dentate gyrus. These data were validated in a triple‐transgenic mouse model of Alzheimer's disease (AD). Overexpression of GFAP and GRP78 in the hippocampal astrocytes correlated with the amyloid β oligomer load in 12‐month‐old mice, suggesting that this parameter drives astrocytic ER stress and astrogliosis in vivo. Together, these results provide evidence that amyloid β oligomers disrupt ER Ca²⁺ homeostasis, which induces ER stress that leads to astrogliosis; this mechanism may be relevant to AD pathophysiology.     

Acute inhibition of PMCA4, but not global ablation,reduces blood pressure and arterial contractility via a nNOS‐dependent mechanism

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild‐type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild‐type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl‐l ‐NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4.