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Abstract Conjugated fatty acids (CFAs) have been known for their anti-tumor activity. However, the mechanism of action remains unclear. Here, we identify CFAs as inducers of glutathione peroxidase 4 (GPX4) degradation through chaperone-mediated autophagy (CMA). CFAs, such as (10E,12Z)-octadecadienoic acid and α-eleostearic acid (ESA), induced GPX4 degradation, generation of mitochondrial reactive oxygen species (ROS) and lipid peroxides, and ultimately ferroptosis in cancer cell lines, including HT1080 and A549 cells, which were suppressed by either pharmacological blockade of CMA or genetic deletion of LAMP2A, a crucial molecule for CMA. Mitochondrial ROS were sufficient and necessary for CMA-dependent GPX4 degradation. Oral administration of an ESA-rich oil attenuated xenograft tumor growth of wild-type, but not that of LAMP2A-deficient HT1080 cells, accompanied by increased lipid peroxidation, GPX4 degradation and cell death. Our study establishes mitochondria as the key target of CFAs to trigger lipid peroxidation and GPX4 degradation, providing insight into ferroptosis-based cancer therapy.

Abstract Glioblastoma (GBM) is one of the most aggressive and deadly brain tumors; however, its current therapeutic strategies are limited. Selenoprotein P (SeP; SELENOP, encoded by the SELENOP gene) is a unique selenium-containing protein that exhibits high expression levels in astroglia. SeP is thought to be associated with ferroptosis sensitivity through the induction of glutathione peroxidase 4 (GPX4) via selenium supplementation. In this study, to elucidate the role of SeP in GBM, we analyzed its expression in GBM patients and found that SeP expression levels were significantly higher when compared to healthy subjects. Knock down of SeP in cultured GBM cells resulted in a decrease in GPX1 and GPX4 protein levels. Under the same conditions, cell death caused by RSL3, a ferroptosis inducer, was enhanced, however this enhancement was canceled by supplementation of selenite. These results indicate that SeP expression contributes to preserving GPX and selenium levels in an autocrine/paracrine manner, i.e., SeP regulates a dynamic cycling-selenium storage system in GBM. We also confirmed the role of SeP expression in ferroptosis sensitivity using patient-derived primary GBM cells. These findings indicate that expression of SeP in GBM can be a significant therapeutic target to overcome anticancer drug resistance.

Abstract Selenoprotein P (SeP) is a major selenoprotein in serum predominantly produced in the liver. Excess SeP impairs insulin secretion from the pancreas and insulin sensitivity in skeletal muscle, thus inhibition of SeP could be a therapeutic strategy for type 2 diabetes. In this study, we examine the effect of sulforaphane (SFN), a phytochemical of broccoli sprouts and an Nrf2 activator, on SeP expression in vitro and in vivo. Treatment of HepG2 cells with SFN decreases inter- and intra-cellular SeP levels. SFN enhances lysosomal acidification and expression of V-ATPase, and inhibition of this process cancels the decrease of SeP by SFN. SFN activates Nrf2 in the cells, while Nrf2 siRNA does not affect the decrease of SeP by SFN or lysosomal acidification. These results indicate that SFN decreases SeP by enhancing lysosomal degradation, independent of Nrf2. Injection of SFN to mice results in induction of cathepsin and a decrease of SeP in serum. The findings from this study are expected to contribute to developing SeP inhibitors in the future, thereby contributing to treating and preventing diseases related to increased SeP.

Stimulator of interferon genes (STING) is an ER-localized transmembrane protein and the receptor for 2',3'-cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), which is a second messenger produced by cGAMP synthase (cGAS), a cytosolic double-stranded DNA sensor. The cGAS-STING pathway plays a critical role in the innate immune response to infection of a variety of DNA pathogens through the induction of the type I interferons. Pharmacological activation of STING is a promising therapeutic strategy for cancer, thus the development of potent and selective STING agonists has been pursued. Here we report that mouse STING can be activated by phenylarsine oxide (PAO), a membrane permeable trivalent arsenic compound that preferentially reacts with thiol group of cysteine residue (Cys). The activation of STING with PAO does not require cGAS or cGAMP. Mass spectrometric analysis of the peptides generated by trypsin and chymotrypsin digestion of STING identifies several PAO adducts, suggesting that PAO covalently binds to STING. Screening of STING variants with single Cys to serine residues (Ser) reveals that Cys88 and Cys291 are critical to the response to PAO. STING activation with PAO, as with cGAMP, requires the ER-to-Golgi traffic and palmitoylation of STING. Our results identify a non-nucleotide STING agonist that does not target the cGAMP-binding pocket, and demonstrate that Cys of STING can be a novel target for the development of STING agonist. Key words: STING agonist, cysteine modification, innate immunity, phenylarsine oxide

Abstract We recently found that tumor necrosis factor-α (TNF-α) may be involved in neuronal cell death induced by methylmercury in the mouse brain. Here, we examined the cells involved in the induction of TNF-α expression by methylmercury in the mouse brain by in situ hybridization. TNF-α-expressing cells were found throughout the brain and were identified as microglia by immunostaining for ionized calcium binding adaptor molecule 1 (Iba1). Methylmercury induced TNF-α expression in mouse primary microglia and mouse microglial cell line BV2. Knockdown of apoptosis signal-regulating kinase 1 (ASK1), an inflammatory cytokine up-regulator that is responsible for reactive oxygen species (ROS), decreased methylmercury-induced TNF-α expression through decreased phosphorylation of p38 MAP kinase in BV2 cells. Suppression of methylmercury-induced reactive oxygen species (ROS) by antioxidant treatment largely abolished the induction of TNF-α expression and phosphorylation of p38 by methylmercury in BV2 cells. Finally, in mouse brain slices, the TNF-α antagonist (WP9QY) inhibited neuronal cell death induced by methylmercury, as did the p38 inhibitor SB203580 and liposomal clodronate (a microglia-depleting agent). These results indicate that methylmercury induces mitochondrial ROS that are involved in activation of the ASK1/p38 pathway in microglia and that this is associated with induction of TNF-α expression and neuronal cell death.

Abstract Anticancer drug gefitinib causes inflammation-based side effects, such as interstitial pneumonitis. However, its mechanisms remain unknown. Here, we provide evidence that gefitinib elicits pro-inflammatory responses by promoting mature-interleukin-1β (IL-1β) and high-mobility group box 1 (HMGB1) release. Mitochondrial reactive oxygen species (mtROS) driven by gefitinib stimulated the formation of the NLRP3 (NACHT, LRR and PYD-containing protein 3) inflammasome, leading to mature-IL-1β release. Notably, gefitinib also stimulated HMGB1 release, which is, however, not mediated by the NLRP3 inflammasome. On the other hand, gefitinib-driven mtROS promoted the accumulation of γH2AX, a hallmark of DNA damage, leading to the activation of poly (ADP-ribose) polymerase-1 (PARP-1) and subsequent active release of HMGB1. Together our results reveal the potential ability of gefitinib to initiate sterile inflammation via two distinct mechanisms, and identified IL-1β and HMGB1 as key determinants of gefitinib-induced inflammation that may provide insights into gefitinib-induced interstitial pneumonitis.

Abstract Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6.

Although homeobox protein B13 (HOXB13) is an oncogenic transcription factor, its role in stress response has rarely been examined. We previously reported that knockdown of HOXB13 reduces the cytotoxicity caused by various oxidative stress inducers. Here, we studied the role of HOXB13 in cytotoxicity caused by hydrogen peroxide in human lung epithelial A549 cells. The knockdown of HOXB13 reduced hydrogen peroxide-induced cytotoxicity; however, this phenomenon was largely absent in the presence of antioxidants (Trolox or N-acetyl cysteine (NAC)). This suggests that HOXB13 may be involved in the cytotoxicity caused by hydrogen peroxide via the production of reactive oxygen species (ROS). Hydrogen peroxide also increased both the mRNA and protein levels of HOXB13. However, these increases were rarely observed in the presence of a transcriptional inhibitor, which suggests that hydrogen peroxide increases protein levels via increased transcription of HOXB13. Furthermore, cell death occurred in A549 cells that highly expressed HOXB13. However, this cell death was mostly inhibited by treatment with antioxidants. Taken together, our findings indicate that HOXB13 may be a novel factor involved in the induction of oxidative stress, which causes cell death via intracellular ROS production.

Homeobox protein B13 (HOXB13), a transcription factor, is related to methylmercury toxicity; however, the downstream factors involved in enhancing methylmercury toxicity remain unknown. We performed microarray analysis to search for downstream factors whose expression is induced by methylmercury via HOXB13 in human embryonic kidney cells (HEK293), which are useful model cells for analyzing molecular mechanisms. Methylmercury induced the expression of oncostatin M (OSM), a cytokine of the interleukin-6 family, and this was markedly suppressed by HOXB13 knockdown. OSM knockdown also conferred resistance to methylmercury in HEK293 cells, and no added methylmercury resistance was observed when both HOXB13 and OSM were knocked down. Binding of HOXB13 to the OSM gene promoter was increased by methylmercury, indicating the involvement of HOXB13 in the enhancement of its toxicity. Because addition of recombinant OSM to the medium enhanced methylmercury toxicity in OSM-knockdown cells, extracellularly released OSM was believed to enhance methylmercury toxicity via membrane receptors. We discovered tumor necrosis factor receptor (TNF) receptor 3 (TNFR3) to be a potential candidate involved in the enhancement of methylmercury toxicity by OSM. This toxicity mechanism was also confirmed in mouse neuronal stem cells. We report, for the first time, that HOXB13 is involved in enhancement of methylmercury toxicity via OSM-expression induction and that the synthesized OSM causes cell death by binding to TNFR3 extracellularly.

Methylmercury (MeHg) is selectively toxic to the central nervous system, but mechanisms related to its toxicity are poorly understood. In the present study, we identified the chemokine, C-C motif Chemokine Ligand 4 (CCL4), to be selectively upregulated in the brain of MeHg-administered mice. We then investigated the relationship between CCL4 expression and MeHg toxicity using in vivo and in vitro approaches. We confirmed that in C17.2 cells (a mouse neural stem cell line) and the mouse brain, induction of CCL4 expression occurs prior to cytotoxicity caused by MeHg. We also show that the addition of recombinant CCL4 to the culture medium of mouse xprimary neurons attenuated MeHg toxicity, while knockdown of CCL4 in C17.2 cells resulted in higher MeHg sensitivity compared with control cells. These results suggest that CCL4 is a protective factor against MeHg toxicity and that induction of CCL4 expression is not a result of cytotoxicity by MeHg but is a protective response against MeHg exposure.

Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing selenium instead of sulfur). SeP functions for the transport of selenium to specific tissues in a receptor-dependent manner. Apolipoprotein E receptor 2 (ApoER2) has been identified as a SeP receptor. However, diverse variants of ApoER2 have been reported, and the details of its tissue specificity and the molecular mechanism of its efficiency remain unclear. In the present study, we found that human T lymphoma Jurkat cells have a high ability to utilize selenium via SeP, while this ability was low in human rhabdomyosarcoma cells. We identified an ApoER2 variant with a high affinity for SeP in Jurkat cells. This variant had a dissociation constant value of 0.67 nM and a highly glycosylated O-linked sugar domain. Moreover, the acidification of intracellular vesicles was necessary for selenium transport via SeP in both cell types. In rhabdomyosarcoma cells, SeP underwent proteolytic degradation in lysosomes and transported selenium in a Sec lyase–dependent manner. However, in Jurkat cells, SeP transported selenium in Sec lyase–independent manner. These findings indicate a preferential selenium transport pathway involving SeP and highaffinity ApoER2 in a Sec lyase–independent manner. Herein, we provide a novel dynamic transport pathway for selenium via SeP. [ABSTRACT FROM AUTHOR]

Selenoprotein P (SeP), encoded by the SELENOP gene, is the major selenium-containing protein in human plasma. SeP has 10 residues of selenocysteine (Sec, cysteine analog in which the sulfur is replaced by selenium), and Sec plays a significant role in the multifunctional properties of SeP. The one Sec residue on the N-terminal side functions for the redox reaction that reduces lipid hydroperoxides, while the 9 Sec residues on the C-terminal side are responsible for the selenium supplying activity. In the middle of SeP, the domain rich in basic amino acids containing consecutive histidine is present. SeP has been reported to have multiple metal-binding abilities such as Hg, Cd, Cu, Ni, Zn, and Co; however, its physiological significance and the effects on SeP functions remain unclear. In this review, the findings to date on the metal-binding properties of SeP and its structural relevance are summarized, particularly for methylmercury. The binding of other selenoproteins to metals is also described. Finally, the interactions of selenoproteins with various metals and its significance for biological defense are discussed. [ABSTRACT FROM AUTHOR]

Redox regulation of proteins via cysteine residue oxidation is involved in the control of various cellular signal pathways. Pyruvate kinase M2 (PKM2), a rate-limiting enzyme in glycolysis, is critical for the metabolic shift from glycolysis to the pentose phosphate pathway under oxidative stress in cancer cell growth. The PKM2 tetramer is required for optimal pyruvate kinase (PK) activity, whereas the inhibition of inter-subunit interaction of PKM2 induced by Cys358 oxidation has reduced PK activity. In the present study, we identified three oxidation-sensitive cysteine residues (Cys358, Cys423 and Cys424) responsible for four oxidation forms via the thiol oxidant diamide and/or hydrogen peroxide (H2O2). Possibly due to obstruction of the dimer-dimer interface, H2O2-induced sulfenylation (-SOH) and diamide-induced modification at Cys424 inhibited tetramer formation and PK activity. Cys423 is responsible for intermolecular disulfide bonds with heterologous proteins via diamide. Additionally, intramolecular polysulphide linkage (-Sn-, n ≧ 3) between Cys358 and an unidentified PKM2 Cys could be induced by diamide. We observed that cells expressing the oxidation-resistant PKM2 (PKM2C358,424A) produced more intracellular reactive oxygen species (ROS) and exhibited greater sensitivity to ROSgenerating reagents and ROS-inducible anti-cancer drugs compared with cells expressing wild-type PKM2. These results highlight the possibility that PKM2 inhibition via Cys358 and Cys424 oxidation contributes to eliminating excess ROS and oxidative stress. [ABSTRACT FROM AUTHOR]

Homeobox protein B13 (HOXB13), a transcription factor, is related to methylmercury toxicity; however, the downstream factors involved in enhancing methylmercury toxicity remain unknown. We performed microarray analysis to search for downstream factors whose expression is induced by methylmercury via HOXB13 in human embryonic kidney cells (HEK293), which are useful model cells for analyzing molecular mechanisms. Methylmercury induced the expression of oncostatin M (OSM), a cytokine of the interleukin-6 family, and this was markedly suppressed by HOXB13 knockdown. OSM knockdown also conferred resistance to methylmercury in HEK293 cells, and no added methylmercury resistance was observed when both HOXB13 and OSM were knocked down. Binding of HOXB13 to the OSM gene promoter was increased by methylmercury, indicating the involvement of HOXB13 in the enhancement of its toxicity. Because addition of recombinant OSM to the medium enhanced methylmercury toxicity in OSM-knockdown cells, extracellularly released OSM was believed to enhance methylmercury toxicity via membrane receptors. We discovered tumor necrosis factor receptor (TNF) receptor 3 (TNFR3) to be a potential candidate involved in the enhancement of methylmercury toxicity by OSM. This toxicity mechanism was also confirmed in mouse neuronal stem cells. We report, for the first time, that HOXB13 is involved in enhancement of methylmercury toxicity via OSM-expression induction and that the synthesized OSM causes cell death by binding to TNFR3 extracellularly. [ABSTRACT FROM AUTHOR]

M1-microglia (neurotoxic microglia) regulate neuronal development and cell death and are involved in many pathologies in the brain. Although organotypic brain slice cultures are widely used to study the crosstalk between neurons and microglia, little is known about the properties of microglia in the mouse cerebral cortex slices. Here, we aimed to optimize the mouse cerebral slice cultures that reflect microglial functions and evaluate the effects of neurotoxic metals on M1-microglial activation. Most microglia in the cerebral slices prepared from postnatal day (P) 7 mice were similar to mature microglia in adult mice brains, but those in the slices prepared from P2 mice were immature, which is a conventional preparation condition. The degree of expression of M1-microglial markers (CD16 and CD32) and inflammatory cytokines (tumor necrosis factor-α and interleukin-1β) by lipopolysaccharide, a representative microglia activator, in the cerebral slices of P7 mice were higher than that in the slices of P2 mice. These results indicate that M1-microglial activation can be evaluated more accurately in the cerebral slices of P7 mice than in those of P2 mice. Therefore, we next examined the effects of various neurotoxic metals on M1-microglial activation using the cerebral slices of P7 mice and found that methylmercury stimulated the activation to M1-microglia, but arsenite, lead, and tributyltin did not induce such activation. Altogether, the optimized mouse cerebral slice cultures used in this study can be a helpful tool to study the influence of various chemicals on the central nervous system in the presence of functionally mature microglia. [ABSTRACT FROM AUTHOR]

The Fukushima nuclear accident of March 11, 2011, resulted in soil and water contamination by radioactive cesium. Radioactive cesium transported by rivers was also found in the ocean sludge of Tokyo Bay. Cesium adsorbed on the sludge is not easily removed. One of the authors has developed an ocean sludge decomposition system that employs micro-bubble circulation. The circulation of micro-bubbles creates an aerobic state that activates aerobic bacteria, facilitating decomposition and purification of the sludge. The objective of this study was to investigate the effect of the addition of bacteria to the micro-bubble circulation system on the efficient removal of radioactive cesium from ocean sludge. We tested the water purification effect of the system by adding bacteria directly. Results confirmed that the decomposition of the deposited sludge using our system facilitates the elution of the radioactive cesium. Any cesium eluted in the water can be remedied using existing technology such as zeolites. Purification efficiency seems to be greatest when additional bacteria are added directly to the process. With time, bacterial concentration doubles, and 76% of cesium in the liquid phase (dried sludge) and 51% of cesium in the solid phase (water) is removed. [ABSTRACT FROM AUTHOR]

Following the Fukushima nuclear accident of March 11, 2011, soil and water were contaminated by radioactive cesium. Moreover, radioactive cesium was found in the ocean sludge in Tokyo Bay, carried by rivers flowing into the bay. The cesium adsorbed in the sludge cannot easily be removed. The objective of this study was to investigate the effect of the addition of bacteria to the micro-bubble circulation system on the efficient removal of radioactive cesium from ocean sludge. One of the authors has developed an ocean sludge decomposition system employing circulation of micro-bubbles. Model sludge was prepared using seawater, sea sludge, and cesium chloride. Bacteria were added to the system after 24 h. Dried tangle extract was added as a nutrient at 24 h and 36 h. The decomposition experiment was carried out for 120 h. The circulation of micro-bubbles created an aerobic state that activated aerobic bacteria, facilitating decomposition and purification of the sludge. Thus, decomposition of the deposited sludge using our system renders the elution of the radioactive cesium possible. If the cesium is eluted in the water, we can fix it using existing technology such as zeolites. We identified and isolated the most useful bacteria for sludge decomposition. Effects on purification seem to be greatest when additional bacteria are added directly to the process. The methodology proposed is expected to facilitate decomposition of sludge and removal of radioactive cesium from the environment. [ABSTRACT FROM AUTHOR]

Methylmercury (MeHg) is an environmental electrophile that covalently modifies cellular proteins. In this study, we identified proteins that undergo S-mercuration by MeHg. By combining two-dimensional SDS-PAGE, atomic absorption spectrometry and ultra performance liquid chromatography mass spectrometry (UPLC/MS/MS), we revealed that ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a target for S-mercuration in human neuroblastoma SH-SY5Y cells exposed to MeHg (1 µM, 9 hr). The modification site of UCH-L1 by MeHg was Cys152, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. MeHg was shown to inhibit the catalytic activity of recombinant human UCH-L1 in a concentration-dependent manner. Knockdown of UCH-L1 indicated that this enzyme plays a critical role in regulating mono-ubiquitin (monoUb) levels in SH-SY5Y cells and exposure of SH-SY5Y cells to MeHg caused a reduction in the level of monoUb in these cells. These observations suggest that UCH-L1 readily undergoes S-mercuration by MeHg through Cys152 and this covalent modification inhibits UCH-L1, leading to the potential disruption of the maintenance of cellular monoUb levels. [ABSTRACT FROM AUTHOR]

Although covalent modification of protein thiols by electrophilic metals is implicated in disruption of protein functions associated with toxicity, there are limited methods available to detect such modifications. In the present study, we established a convenient method to assess modification of protein thiols by electrophiles, referred to as a biotin-PEAC5-maleimide (BPM)-labeling assay. In this assay, protein S-modification by electrophiles can be estimated by a decrease in protein modification by BPM, a thiol reactive probe. Using methylmercury (MeHg) as a model electrophilic metal, thiol modification of cellular proteins was detected by the BPM-labeling assay in SH-SY5Y cell lysates and primary mouse hepatocytes. The sensitivity and reliability of the assay was confirmed by atomic absorption spectrome-try with recombinant Keap 1 as a model thiol protein. This assay was applied to not only MeHg but also to other metals such as cadmium and lead. We also established a BPM-precipitation assay with avidin-agar-ose beads to separate BPM-modified cellular proteins followed by detection with the individual antibodies. This assay was available for detecting MeHg-induced S-modification of cellular Keap1 in SH-SY5Y cells. Taken together, we have developed reliable simple methods to estimate protein S-modification by electrophilic metals. [ABSTRACT FROM AUTHOR]

Methylmercury (MeHg) covalently modifies cellular proteins through their SH groups, resulting in cytotoxicity. We report that cystathionine β-synthase (CBS), which catalyzes the production of hydrogen sulfide, contributes to cellular protection against MeHg. Pretreatment with NaHS or overexpression of CBS reduced MeHg cytotoxicity, whereas transfection with CBS small interfering RNA enhanced MeHg toxicity in human neuroblastoma SH-SY5Y cells. Bismethylmercury sulfide ((MeHg)2S) was identified as a metabolite of MeHg in SH-SY5Y cells exposed to MeHg and in the livers of rats treated with MeHg. (MeHg)2S had little chemical protein modification capability and little cytotoxicity compared with MeHg in vitroand in vivo. [ABSTRACT FROM AUTHOR]

A safe, rapid, and environmentally friendly synthesis of iodosylarene (ArIO) has been developed using NaClO under a carbon dioxide (CO2) atmosphere. Exposure of iodoarene to NaClO-5H2O in acetonitrile under CO2 (1 atm) resulted in the clean formation of ArIO within 10 minutes in high yield. The absence of a base in this method enables the direct use of in-situ-generated iodosylarene not only for a variety of oxidative transformations (synthesis of sulfilimine, pentavalent bismuth, benzyne adduct, etc.), but also for the synthesis of iodonium ylide and imino-3-iodane in one pot. [ABSTRACT FROM AUTHOR]