Plasma-activated medium ameliorates the chemoresistance of human lung adenocarcinoma cells mediated via downregulation of claudin-2 expression.

Plasma-activated medium (PAM) has various biological activities including anticancer and antimicrobial. However, the effect on chemoresistance in cancer cells has not been clarified in detail. Solid cancer cells form a microenvironment in the body and acquire resistance against anticancer drugs. So far, we reported that claudin-2 (CLDN2), a component of tight junctions, suppresses the anticancer drug-induced cytotoxicity of spheroids that mimic in vivo tumors. Here, we found that the protein level of CLDN2 is downregulated by the sublethal concentration of PAM in human lung adenocarcinoma-derived A549 and PC-3 cells. A cycloheximide pulse-chase assay showed that PAM accelerates the degradation of CLDN2 protein. The PAM-induced reduction of CLDN2 protein was inhibited by a lysosome inhibitor, indicating PAM may enhance the lysosomal degradation of CLDN2. The paracellular permeability to doxorubicin (DXR), an anthracycline antitumor drug, was enhanced by PAM. In the spheroids, the accumulation and toxicity of DXR were enhanced by PAM. In addition, oxidative stress and the expression of nuclear factor erythroid 2-related factor 2, one of the key factors for the acquisition of chemoresistance, were attenuated by PAM. The improvement effect of PAM on chemoresistance was suppressed by the exogenous CLDN2 overexpression. These results indicate that PAM has the ability to downregulate CLDN2 expression and may become an adjuvant drug against lung adenocarcinoma.

Panobinostat, a Histone Deacetylase Inhibitor, Reduces LPS-Induced Expression of Inducible Nitric Oxide Synthase in Rat Immortalized Microglia HAPI Cells.

Microglia, resident immune cells in the central nervous system (CNS), play a critical role in maintaining CNS homeostasis. However, microglia activated in response to brain injury produce various inflammatory mediators, including nitric oxide (NO) and proinflammatory cytokines, leading to considerable neuronal damage. NO generated by inducible NO synthase (iNOS) rapidly reacts with superoxide to form a highly toxic product, peroxynitrite. Therefore, iNOS is considered to be a putative therapeutic target for cerebral ischemia. Here, we examined the effects of panobinostat (Pano), a histone deacetylase inhibitor, on lipopolysaccharide (LPS)-induced iNOS expression using rat immortalized microglia HAPI cells. Pano inhibited LPS-induced expression of iNOS mRNA and NO production in a dose-dependent manner; however, it had little effect on the LPS-induced activation of c-Jun N-terminal kinase (JNK) and p38 or nuclear translocation of nuclear factor-κB (NF-κB). The interferon-ß (IFN-ß)/signal transducer and activator of transcription (STAT) pathway is essential for LPS-induced iNOS expression in macrophages/microglia. We also examined the effects of Pano on LPS-induced IFN-ß signaling. Pano markedly inhibited LPS-induced IFN-ß expression and subsequent tyrosine phosphorylation of STAT1. However, the addition of IFN-ß restored the decreased STAT1 phosphorylation but not the decreased iNOS expression. In addition, Pano inhibited the LPS-increased expression of octamer binding protein-2 and interferon regulatory factor 9 responsible for iNOS expression, but IFN-ß addition also failed to restore the decreased expression of these factors. Thus, we conclude that the inhibitory effects of Pano are due not only to the inhibition of the IFN-ß/STAT axis but also to the downregulation of other factors not involved in this axis.

Sebacic acid, a royal jelly-containing fatty acid, decreases LPS-induced IL-6 mRNA expression in differentiated human THP-1 macrophage-like cells.

Macrophages produce many inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in innate immune responses. However, excess production of these mediators by activated macrophages triggers deleterious effects, leading to disorders associated with inflammation. Royal jelly (RJ), a milky-white substance secreted by worker bees, contains unique fatty acids, including 10-hydroxy-2-decenoic acid (10H2DA) and sebacic acid (SA). 10H2DA has been reported to have various biological functions, such as anti-inflammation. However, the anti-inflammatory effect of SA is not fully understood. In this study, we investigated the effects of SA on lipopolysaccharide (LPS)-induced cytokine expression using differentiated human THP-1 macrophage-like cells. SA dose-dependently decreased LPS-induced mRNA expression of IL-6, but not TNF-α and IL-1ß. SA suppressed the phosphorylation of signal transducers and activators of transcription 1 (STAT1) and STAT3, but hardly affected the activation of JNK, p38, or NF-κB. In addition, SA decreased LPS-induced interferon-ß (IFN-ß) expression, and the addition of IFN-ß restored the inhibition by SA of LPS-induced STAT activation and IL-6 expression. Furthermore, SA suppressed LPS-induced nuclear translocation of interferon regulatory factor 3 (IRF3), a transcription factor responsible for IFN-ß expression. Taken together, we conclude that SA selectively decreases LPS-induced expression of IL-6 mRNA through inhibition of the IRF3/IFN-ß/STAT axis.

Inhibition of N-glycosylation by glucosamine hydrochloride inhibits TGF-ß1-induced LOXL2 secretion.

Kidney fibrosis is closely associated with the progression of chronic kidney disease (CKD). Furthermore, copper-containing secretory amine oxidases, such as lysyl oxidase (LOX) and LOX-like 1-4 (LOXL1-4), play pivotal roles in the regulation of extracellular components and facilitate fibrosis. In this study, we investigated the regulation of LOX enzymes in human tubular epithelial HK2 cells to help clarify the role of LOX enzymes in kidney fibrosis. Among 5 LOX enzymes, LOXL2 expression is abundantly expressed in HK2 cells. LOX enzymes inhibitor, ß-aminopropionitrile, suppressed transforming growth factor-ß1 (TGF-ß1)-promoted epithelial-to-mesenchymal transition processes in HK2 cells, indicating that LOX enzymes are involved in TGF-ß1-mediated fibrotic processes. Recent studies suggest that LOX enzymes are secreted into the extracellular spaces by exosomes and promote fibrotic processes. Similar to the previous reports, we observed that exosomes secreted from HK2 cells carry LOXL2 into the extracellular spaces. Furthermore, we determined that N-glycosylation on the asparagine residues plays a key role in LOXL2 secretion. Amino acid mutations in three asparagine residues, which can be glycosylated, suppressed the secretion of mutated LOXL2. Moreover, N-acetylglucosaminyltransferase 5, an enzyme used for the biosynthesis of ß1,6N-acetylglucosamine-branched N-glycans, participated in LOXL2 secretion, and the N-glycosylation inhibitor, glucosamine hydrochloride (GS), inhibited TGF-ß1-mediated LOXL2 secretion and fibrotic processes. Overall, TGF-ß1 promotes LOXL2 secretion and may participate in kidney fibrosis. Our results provide novel insight into the antifibrotic properties of GS that contribute to the inhibition of CKD progression.

Copper-induced diurnal hepatic toxicity is associated with Cry2 and Per1 in mice.

BACKGROUND: This study aimed to investigate diurnal variations in copper-induced hepatic toxicity and the molecular mechanisms underlying this chronotoxicity. METHODS: Male C57BL/6J mice were intraperitoneally injected with copper chloride (CuCl2) at zeitgeber time 2 (ZT2) or 14 (ZT14), twice per week for 5 or 8 weeks. Seventy-two hours after the final CuCl2 injection, the mice were euthanized, and plasma samples were collected. The livers and kidneys were collected and weighed. In vitro experiments were performed to assess cell viability and fluctuations in clock gene expression levels in Hepa1-6 cells after CuCl2 treatment. We examined copper homeostasis- and apoptosis-related genes under clock genes overexpression. RESULTS: Repeated CuCl2 administration for 8 weeks resulted in more severe toxicity at ZT14 compared to ZT2. CuCl2 administration at ZT14 elevated plasma aspartate aminotransferase, hepatic tumor necrosis factor-α, and interleukin-6 for 5 weeks, whereas the toxic effects of CuCl2 administration at ZT2 were weaker. Moreover, CuCl2 treatment inhibited Hepa1-6 cell viability in a dose-dependent manner. We observed increased expression of three clock genes (Ciart, Cry2, and Per1) after CuCl2 treatment. Among them, overexpression of Cry2 and Per1 accelerated CuCl2-induced inhibition of Hepa1-6 cell viability. Moreover, we found that the overexpression of Cry2 and Per1 regulates cleaved caspase-3 by modulating the copper transporter genes ATP7B and CTR1. CONCLUSION: These results suggest that CuCl2-induced diurnal toxicity is associated with Cry2 and Per1 expression through the regulation of copper transporter genes in mice.

Increased expression of ELOVL7 contributes to production of inflammatory cytokines in THP-1 cell-derived M1-like macrophages.

The elevation of intracellular very long-chain fatty acids (VLCFAs) augments pro-inflammatory activity of macrophages. VLCFAs are considered to function as regulators in macrophage inflammatory responses; however, the precise mechanism of regulating the production of VLCFAs is unclear. In this study, we focused on elongation of the very­long­chain fatty acid protein (ELOVL) family, rate-determining enzymes for VLCFA synthesis, in macrophages. ELOVL7 mRNA was upregulated in human monocytic THP-1 cell-derived M1-like macrophages. Metascape analysis using the RNA-seq data set showed the involvement of NF-κB and STAT1 in transcriptional regulation of ELOVL7 highly correlated genes. Gene ontology (GO) enrichment analysis suggested that ELOVL7 highly correlated genes were closely associated with multiple pro-inflammatory responses, including response to virus and positive regulation of NF-κB signaling. Consistent with RNA-seq analysis, the NF-κB inhibitor BAY11-7082, but not the STAT1 inhibitor fludarabine, canceled ELOVL7 upregulation in M1-like macrophages. ELOVL7 knockdown decreased interleukin (IL)-6 and IL-12/IL-23 p40 production. Moreover, RNA-seq analysis of plasmacytoid dendritic cells (pDCs) revealed that ELOVL7 was upregulated in pDCs treated with TLR7 and TLR9 agonists. In conclusion, we propose that ELOVL7 is a novel pro-inflammatory gene that is upregulated by inflammatory stimuli, and regulates M1-like macrophage and pDC functions.

Royal jelly fatty acids downregulate ANGPTL8 expression through the decrease in HNF4α protein in human hepatoma HepG2 cells.

Royal jelly (RJ) intake has been reported to be effective for reducing serum lipids; however, the mechanism is not fully understood. Angiopoietin-like protein 8 (ANGPTL8), a secreted protein, plays a key role in lipid metabolism. In this study, we investigated the effects of specific fatty acids included in RJ (RJ fatty acids), such as 10-hydroxy-2-decenoic acid, 10-hydroxydecanoic acid, and sebacic acid (SA), on expression of ANGPTL8 in human hepatoma HepG2 cells. SA markedly reduced the expression of ANGPTL8. Reporter assay revealed that SA suppressed ANGPTL8 promoter activity. In addition, we identified a functional binding site of hepatocyte nuclear factor-4α (HNF4α), a liver-enriched transcription factor, in the ANGPTL8 promoter. SA reduced the levels of HNF4α protein and the binding of HNF4α to the ANGPTL8 promoter. Moreover, siRNA knockdown of HNF4α suppressed the expression of ANGTPL8 mRNA. Taken together, we conclude that SA downregulates ANGPTL8 expression via the decrease in HNF4α protein.

Regulation of lysyl oxidase expression in THP-1 cell-derived M2-like macrophages.

Lysyl oxidase (LOX) is a copper-containing enzyme and its overexpression in tumor tissues promote tumor metastasis through the crosslinking of extracellular matrix. Our previous report demonstrated that LOX expression is significantly increased in human leukemic THP-1 cell-derived M2-like macrophages, and histone modification plays a key role in its induction. However, the rigorous mechanism of LOX regulation remains unclear. In this study, we investigated the role of functional transcription factors, hypoxia-inducible factor 1α (HIF1α), signal transducer and activator of transcription 3 (STAT3) and forkhead box O1 (FOXO1) in LOX regulation in M2-like macrophages. HIF1α expression was significantly increased in M2-like macrophages, and HIF1α inhibitor, TX402, suppressed LOX induction. The significant STAT3 activation was also observed in M2-like macrophages. Additionally, LOX induction was canceled in the presence of STAT3 inhibitor, S3I-201, suggesting that HIF1α and STAT3 pathways play a critical role in LOX induction. On the other hand, our ChIP results clearly indicated that the enrichment of FOXO1 within the lox promoter region was dramatically decreased in M2-like macrophages. In this context, knockdown of FOXO1 further enhanced LOX induction. LOX induction and HIF1α binding to the lox promoter region were suppressed in FOXO1-overexpressed cells, suggesting that the FOXO1 binding to the lox promoter region counteracted HIF1α binding to that region. Overall, the present data suggested that both of HIF1α and STAT3 were required for LOX induction in M2-like macrophages, and loss of FOXO1 within the lox promoter region facilitated HIF1α binding to that region which promoted LOX induction.

Hyperthermia synergistically enhances cancer cell death by plasma-activated acetated Ringer's solution.

Applications of non-thermal plasma (NTP) discharges in medicine, particularly cancer therapy, have increased in recent years. The aim of the present study was to investigate the advantages of the combined application of NTP-irradiated acetated Ringer's solution (PAA) and hyperthermia, a heat treatment at 42 °C, on A549 cancer cell death and elucidate the underlying mechanisms. Cell death was enhanced more by the above combined treatment and was accompanied by increases in intracellular calcium ([Ca2+]i). The activation of transient receptor potential melastatin 2 (TRPM2) may enhance cell death because the addition of TRPM2 inhibitors and knockdown of TRPM2 significantly abrogated the above phenomena. TRPM2 is a temperature-sensitive, Ca2+-permeable, non-elective cation channel and hydrogen peroxide (H2O2) and ADP ribose are its main agonists. PAA functioned as a donor of reactive oxygen species, mainly H2O2, and a treatment with PAA under hyperthermia induced both mitochondrial and nuclear damage with DNA breaks. The activation of poly(ADP-ribose) polymerase-1 as the DNA repair mechanism induced TRPM2 activation because this enzyme accumulates ADP ribose. The sensitivity of fibroblasts as normal cells to PAA was less than that of A549 cells. These results suggest that hyperthermia synergistically induces the sensitivity of cancer cells to PAA.

Ability of plasma-activated acetated Ringer's solution to induce A549 cell injury is enhanced by a pre-treatment with histone deacetylase inhibitors.

Non-thermal plasma (NTP) is applicable to living cells and has emerged as a novel technology for cancer therapy. NTP affect cells not only by direct irradiation, but also by an indirect treatment with previously prepared plasma-activated liquid. Histone deacetylase (HDAC) inhibitors have the potential to enhance susceptibility to anticancer drugs and radiation because these reagents decondense the compact chromatin structure by neutralizing the positive charge of the histone tail. The aim of the present study was to demonstrate the advantage of the combined application of plasma-activated acetated Ringer's solution (PAA) and HDAC inhibitors on A549 cancer cells. PAA maintained its ability for at least 1 week stored at any temperature tested. Cell death was enhanced more by combined regimens of PAA and HDAC inhibitors, such as trichostatin A (TSA) and valproic acid (VPA), than by a single PAA treatment and was accompanied by ROS production, DNA breaks, and mitochondria dysfunction through a caspase-independent pathway. These phenomena induced the depletion of ATP and elevations in intracellular calcium concentrations. The sensitivities of HaCaT cells as normal cells to PAA were less than that of A549 cells. These results suggest that HDAC inhibitors synergistically induce the sensitivity of cancer cells to PAA.

Lysyl oxidase expression is regulated by the H3K27 demethylase Jmjd3 in tumor-associated M2-like macrophages.

Copper is one of the essential micronutrients, and copper-containing enzymes contribute to crucial functions in the body. Lysyl oxidase is a copper-containing enzyme that remodels the extracellular matrix by cross-linking collagen and elastin. The overexpression of lysyl oxidase was recently shown to promote tumor metastasis. M2-like macrophages were also found to significantly accumulate in the tumor microenvironment, and correlated with a poor patient's outcome. We speculate that M2-like macrophages promote tumor progression via lysyl oxidase expression. Epigenetics, a mitotically heritable change in gene expression without any change in DNA sequencing, is also associated with tumor progression. However, the relationship between lysyl oxidase expression in M2-like macrophages and epigenetics remains unclear. Lysyl oxidase expression was significantly induced in human leukemic THP-1 cell-derived M2-like macrophages. Furthermore, the level of histone H3 tri-methylation at lysine 27 was decreased, and a pre-treatment with a H3K27 demethylase inhibitor notably suppressed lysyl oxidase expression in M2-like macrophages. Lysyl oxidase derived from M2-like macrophages also enhanced breast cancer cell migration, and this was suppressed by a H3K27 demethylase inhibitor. The present results suggest the mechanism of lysyl oxidase expression in M2-like macrophages as an aspect of epigenetics, particularly histone methylation.

Inhibition of NAMPT markedly enhances plasma-activated medium-induced cell death in human breast cancer MDA-MB-231 cells.

Plasma-activated medium (PAM), which is prepared by non-thermal atmospheric pressure plasma (NTP) irradiation of cell-free medium, has been shown to exhibit tumor-specific cytotoxicity. Since PAM contains reactive oxygen species (ROS) and reactive nitrogen species (RNS), its anticancer effects are considered to be responsible for oxidative stress induced by these reactive molecules. We previously reported that PAM-induced cell death is closely related to energy failure associated with a decrease in intracellular nicotinamide adenine dinucleotide (NAD+) and ATP levels. Nicotinamide phosphoribosyltransferase (NAMPT), which is a rate-limiting enzyme for NAD+ synthesis in the salvage pathway, was shown to be overexpressed in many types of cancer cells. The NAMPT inhibitor FK866 significantly depletes NAD+ and subsequently suppresses cancer cell proliferation. In this study, we examined the effects of FK866 on PAM-induced cytotoxicity using human breast cancer MDA-MB-231 cells. FK866 dose-dependently enhanced PAM-induced cell death in MDA-MB-231 cells. The combination of PAM and FK866 markedly induced intracellular NAD+ and ATP depletion. Knockdown of NAMPT by siRNA increased the cytotoxicity of PAM. The addition of NAD+ mitigated PAM-induced cell death. In addition, cotreatment with PAM and FK866 augmented ROS production and the decrease in intracellular reduced glutathione (GSH) compared to treatment with PAM alone. FK866 had little effect on PAM-induced mitochondrial dysfunction. Furthermore, the combination of PAM and FK866 decreased the level of NADPH, which is required for GSH metabolism, compared with PAM alone. Taken together, we conclude that cotreatment with NAMPT inhibitors is beneficial for anticancer therapy using PAM.

Sublethal treatment with plasma-activated medium induces senescence-like growth arrest of A549 cells: involvement of intracellular mobile zinc.

Plasma-activated medium (PAM) is a solution produced by exposing a liquid medium to non-thermal atmospheric pressure plasma (NTAPP). A number of reactive molecules, such as reactive oxygen species and reactive nitrogen species, are contained in PAM. Therefore, exposure to high doses of PAM results in cell death. We previously demonstrated that intracellular zinc (Zn2+) serves as an important mediator in PAM-induced cell death; however, the effects of sublethal treatment with PAM on cell functions are not fully understood. In the present study, we found that sublethal PAM treatment suppressed cell proliferation and induced senescence-like changes in lung adenocarcinoma A549 cells. Cell cycle analysis revealed that PAM induced cell cycle arrest at the G2/M phase. PAM increased the level of intracellular free Zn2+ and the Zn2+ chelator TPEN counteracted PAM-induced growth suppression, suggesting that Zn2+ functions in PAM-induced growth suppression. In addition, sublethal treatment with PAM induced phosphorylation of ATM kinase, accumulation of p53 protein, and expression of p21 and GADD45A, which are known p53 target genes, in a Zn2+-dependent manner. These results suggest that the induction of growth arrest and cellular senescence by sublethal PAM treatment is mediated by Zn2+-dependent activation of the ATM/p53 pathway.

Enhanced ability of plasma-activated lactated Ringer's solution to induce A549 cell injury.

Non-thermal plasma (NTP) is applicable to living cells and has emerged as a novel technology for cancer therapy. Plasma-activated medium (PAM), which is prepared by the irradiation of culture medium with NTP, induces cell death in cancer cells. However, difficulties are associated with applying PAM to the clinical phase because culture media cannot be used for medical treatments. The objectives of the present study were to demonstrate the inhibitory effects of plasma-activated lactated Ringer's solution (PAL) on the viability of the A549 cancer cell line and elucidate the underlying mechanisms. The anti-tumor activity of PAL was significantly stronger than that of PAM, whereas their concentrations of H2O2 and nitrite were similar. Lactated Ringer's solution (Lac-R) consists of lactate and three types of inorganic salts. The results showing that NTP irradiation of the lactate solution rather than the inorganic salt solution induced the inactivation of catalase were dependent on the presence or absence of nitrite in these solutions. We detected nitrotyrosine in A549 cells treated with PAM or PAL, and the addition of catalase to PAM rather than to PAL reduced its production. The PAL treatment of A549 cells led to mitochondrial dysfunction with the down-regulation of NF-κB-Bcl2 signaling.

Non-thermal atmospheric pressure plasma-induced IL-8 expression is regulated via intracellular K+ loss and subsequent ERK activation in human keratinocyte HaCaT cells.

Non-thermal atmospheric pressure plasma (NTAPP) has recently emerged as a novel medical therapy for skin wounds. Interleukin-8 (IL-8) is thought to play a critical role in wound healing. NTAPP irradiation has been reported to promote production of IL-8; however, the mechanism is not fully understood. The aim of this study was to elucidate the underlying mechanism of NTAPP-induced IL-8 expression in human keratinocyte HaCaT cells. NTAPP irradiation of HaCaT cells increased IL-8 mRNA expression in an irradiation time-dependent manner. Although hydrogen peroxide (H2O2) was generated in culture medium irradiated with NTAPP, treatment of HaCaT cells with H2O2 itself failed to induce the expression. In addition, we found that NTAPP irradiation of HaCaT cells decreased intracellular K+ levels. High intracellular K+ concentrations suppressed NTAPP-induced IL-8 mRNA expression, and the K+ ionophore valinomycin (Val) enhanced the induction of IL-8 mRNA. Moreover, NTAPP stimulated activation of ERK MAP kinase and the ERK inhibitor prevented NTAPP-induced IL-8 mRNA expression. NTAPP-induced ERK activation was inhibited in the presence of high concentrations of extracellular K+ and enhanced in the presence of Val. Taken together, these findings suggest that NTAPP irradiation stimulates intracellular K+ loss and subsequent ERK activation, leading to the induction of IL-8 expression.

Copper chaperone antioxidant-1, Atox-1, is involved in the induction of SOD3 in THP-1 cells.

Superoxide dismutase (SOD) 3, a copper (Cu)-containing anti-oxidative enzyme, plays a key role in extracellular redox homeostasis. Cu chaperone antioxidant-1 (Atox-1) not only delivers Cu ions to SOD3 at the trans-Golgi network, it also functions as a transcription factor of SOD3; however, the role of Atox-1 in the regulation of SOD3 during the monocytic differentiation of THP-1 cells has not yet been elucidated. A treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the expression of the Cu transport protein ATP7A in THP-1 cells. On the other hand, the nuclear translocation of Atox-1 was detected in TPA-treated THP-1 cells, and was suppressed in the presence of the Cu chelator, bathocuproinedisulfonic acid. Furthermore, Atox-1 bound to the SOD3 promoter region in TPA-treated THP-1 cells. The overexpression of Atox-1 in THP-1 cells significantly enhanced TPA-elicited SOD3 expression, whereas its knockdown suppressed this induction. The present results demonstrate that Atox-1 functions as a key molecule in TPA-elicited SOD3 expression.

CAPE increases the expression of SOD3 through epigenetics in human retinal endothelial cells.

Extracellular-superoxide dismutase (EC-SOD or SOD3), which catalyzes the dismutation of superoxide anions into hydrogen peroxide, plays a key role in vascular protection against reactive oxygen species (ROS). The excess generation of ROS is closely involved in the pathogenesis of diabetic retinopathy (DR); therefore, the maintenance of SOD3 expression at high levels is important for the prevention of DR. In the present study, we showed that caffeic acid phenethyl ester (CAPE) increased the expression of SOD3 through the acetylation of histone within the SOD3 promoter region in human retinal endothelial cells (HRECs). Histone acetylation within its promoter was focused on the inhibition of histone deacetylase (HDAC), and we examined the involvement of myocyte enhancer factor 2 (MEF2) and HDAC1 in CAPE-elicited SOD3 expression. Our results demonstrate that SOD3 silencing in basal HRECs is regulated by HDAC1 composed with MEF2A/2D hetero dimers. Moreover, phosphorylation of threonine 312 in MEF2A and dissociation of HDAC1 from SOD3 promoter play pivotal roles in CAPE-elicited SOD3 expression. Overall, our findings provide that CAPE may be one of the seed compounds that maintain redox homeostasis.

Tumor necrosis factor-α decreases EC-SOD expression through DNA methylation.

Extracellular-superoxide dismutase (EC-SOD) is a secreted antioxidative enzyme, and its presence in vascular walls may play an important role in protecting the vascular system against oxidative stress. EC-SOD expression in cultured cell lines is regulated by various cytokines including tumor necrosis factor-α (TNF-α). TNF-α is a major mediator of pathophysiological conditions and may induce or suppress the generation of various types of mediators. Epigenetics have been defined as mitotically heritable changes in gene expression that do not affect the DNA sequence, and include DNA methylation and histone modifications. The results of the present study demonstrated that TNF-α significantly decreased EC-SOD level in fibroblasts with an accompanying increase in methylated DNA. In DNA methylation and demethylation, cytosine is methylated to 5-methylcytosine (5mC) by DNA methyltransferase (DNMT), and 5mC is then converted to 5-hydroxymethylcytosine (5hmC) and cytosine in a stepwise manner by ten-eleven translocation methylcytosine dioxygenases (TETs). However, DNMT did not participate in TNF-α-induced DNA methylation within the EC-SOD promoter region. On the other hand, TNF-α significantly suppressed TET1 expression and EC-SOD mRNA levels were decreased by the silencing of TET1 in fibroblasts. These results demonstrate that the down-regulation of EC-SOD by TNF-α is regulated by DNA methylation through reductions in TET1.

Suppression of EC-SOD by oxLDL During Vascular Smooth Muscle Cell Proliferation.

Reactive oxygen species (ROS) produced by endothelial cells and macrophages play important roles in atherogenesis because they promote the formation of oxidized low-density lipoproteins (oxLDL). Extracellular-superoxide dismutase (EC-SOD) is mainly produced by vascular smooth muscle cells (VSMCs), is secreted into the extracellular space, and protects cells from the damaging effects of the superoxide anion. Thus, the expression of EC-SOD in VSMCs is crucial for protecting cells against atherogenesis; however, oxLDL-induced changes in the expression of EC-SOD in VSMCs have not yet been examined. We herein showed that oxLDL decreased EC-SOD mRNA and protein levels by binding to lectin-like oxidized LDL receptor-1 (LOX-1). Moreover, we demonstrated the significant role of mitogen-activated protein kinase (MEK)/extracellular-regulated protein kinase (ERK) signaling in oxLDL-elicited reductions in the expression of EC-SOD and proliferation of VSMCs. The results obtained with the FCS treatment indicate that oxLDL-elicited reductions in the expression of EC-SOD are related to the proliferation of VSMCs. We herein showed for the first time that luteolin, a natural product, restored oxLDL-induced decreases in the expression of EC-SOD and proliferation of VSMCs. Collectively, the results of the present study suggest that oxLDL accelerates the development of atherosclerosis by suppressing the expression of EC-SOD and also that luteolin has potential as a treatment for atherosclerosis. J. Cell. Biochem. 117: 2496-2505, 2016. © 2016 Wiley Periodicals, Inc.