Inhibition of glutamine metabolism increases sensitivity to plasma-activated medium-induced cytotoxicity.

Non-thermal atmospheric pressure plasma (NTP), an ionized gas containing electrons, ions, radicals, and photons, has various biological effects, including wound healing and anticancer effects. Plasma-activated medium (PAM), which is prepared by irradiating medium with NTP, preferentially kills cancer cells. Large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) included in PAM are closely related to its anticancer effects. The precise mechanism of PAM-induced cytotoxicity is not fully understood; however, PAM exposure has been reported to reduce cellular energy metabolism. Glutamine (Gln) is an important amino acid as an energy source in cancer cells. Gln is converted to glutamate by glutaminase (GLS), and is utilized to synthesize ATP and glutathione (GSH). Expression levels of GLS have been shown to be higher in certain types of cancers. In this study, we examined the effects of GLS inhibition on PAM cytotoxicity using breast cancer MDA-MB-231 cells. Pretreatment with BPTES, a glutaminase 1 (GLS1) inhibitor, dose-dependently enhanced PAM-induced cell death. PAM-induced ROS production and γ-H2AX formation, a DNA damage marker, were increased in cells pretreated with BPTES compared with PAM alone. BPTES pretreatment enhanced a PAM-induced decrease in intracellular GSH, indicating the possibility that BPTES reduces the antioxidant capacity of MDA-MB-231 cells. In addition, BPTES pretreatment enhanced PAM-induced loss of the mitochondrial membrane potential and reduction of ATP production. Moreover, GLS1 knockdown promoted PAM-induced cell death. Taken together, the combination of GLS1 inhibitors such as BPTES is considered to be useful for enhancing the cytotoxic effects of PAM against cancer cells.

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.

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 in the tumor microenvironment and tumor metastasis.

Copper (Cu), an essential micronutrient, plays an essential role in several physiological processes, including cell proliferation and angiogenesis; however, its dysregulation induces oxidative stress and inflammatory responses. Significant Cu accumulation is observed in several tumor tissues. The bioavailability of intracellular Cu is tightly controlled by Cu transporters, including Cu transporter 1 (CTR1) and Cu-transporting P-type ATPase α and ß (ATP7A and ATP7B), and Cu chaperones, including Cu chaperone for superoxide dismutase 1 (CCS) and antioxidant-1 (Atox-1). In several tumor tissues, these abnormalities that induce intra-cellular Cu accumulation are involved in tumor progression. In addition, functional disturbance in Cu-containing secretory enzymes, such as superoxide dismutase 3 (SOD3), and lysyl oxidase enzymes (LOX and LOXL1-4) with abnormal Cu dynamics plays a key role in tumor metastasis. For example, the loss of SOD3 in tumor tissues induces oxidative stress, which promotes neovascularization and epithelial-to-mesenchymal transition (EMT). LOX promotes collagen crosslinking, which functions in the metastatic niche formation. Accordingly, restricted Cu regulation may be a novel strategy for the inhibition of tumor metastasis. However, it is unclear how these Cu disturbances occur in tumor tissues and the exact molecular mechanisms underlying Cu secretory enzymes. In this review article, I discuss the role of Cu transporters, Cu chaperones, and Cu-containing secretory enzymes in tumor progression to better understand the role of Cu homeostasis in tumor tissues.

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.

Combined action of FOXO1 and superoxide dismutase 3 promotes MDA-MB-231 cell migration.

Superoxide dismutase 3 (SOD3), one of SOD isozymes, maintains extracellular redox homeostasis through the dismutation reaction of superoxide. Loss of SOD3 in tumor cells induces oxidative stress and exacerbates tumor progression; however, interestingly, overexpression of SOD3 also promotes cell proliferation through the production of hydrogen peroxide. In this study, we investigated the functional role of SOD3 in human breast cancer MDA-MB-231 cell migration and the molecular mechanisms involved in high expression of SOD3 in MDA-MB-231 cells and human monocytic THP-1 cells. The level of histone H3 trimethylation at lysine 27 (H3K27me3), a marker of gene silencing, was decreased in 12-O-tetra-decanoylphorbol-13-acetate (TPA)-treated THP-1 cells. Also, that reduction was observed within the SOD3 promoter region. We then investigated the involvement of H3K27 demethylase JMJD3 in SOD3 induction. The induction of SOD3 and the reduction of H3K27me3 were inhibited in the presence of JMJD3 inhibitor, GSK-J4. Additionally, it was first determined that the knockdown of the transcription factor forkhead box O1 (FOXO1) significantly suppressed TPA-elicited SOD3 induction. FOXO1-mediated SOD3 downregulation was also observed in MDA-MB-231 cells, and knockdown of FOXO1 and SOD3 suppressed cell migration. Our results provide a novel insight into epigenetic regulation of SOD3 expression in tumor-associated cells, and high expression of FOXO1 and SOD3 would participate in the migration of MDA-MB-231 cells.

Two new methoxylated flavones isolated from Casimiroa edulis La Llave and their MMP-9 inhibitory activity.

Casimiroa edulis La Llave is known to contain unusual 5,6-dimethoxyflavones bearing a variously oxygenated B-ring. Phytochemical investigation of the leaves and the roots of C. edulis achieved the isolation of two new methoxylated flavones, named casedulones A (1) and B (2), together with 12 known analogues. Their unique structures were established with the aid of spectral analyses and total syntheses. Pre-treatment with 20 µM of 1 and 2 suppressed MMP-9 expression in LPS-mediated THP-1 cells, indicating that the characteristic flavonoids in C. edulis could be potential anti-angiogenics for cancer prevention.

6-Hydroxydopamine disrupts cellular copper homeostasis in human neuroblastoma SH-SY5Y cells.

Copper (Cu) is an essential trace element that plays an important role in maintaining neuronal functions such as the biosynthesis of neurotransmitters. In contrast, exposure to excess Cu results in cell injury. Therefore, intracellular Cu levels are strictly regulated by proteins related to Cu-trafficking, including ATP7A. Parkinson's disease (PD) is a neurodegenerative disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra. Recently, the abnormality of Cu homeostasis was demonstrated to be related to the pathogenesis of PD. However, the association between Cu dyshomeostasis and PD remains unclear. In this study, we examined the effects of 6-hydroxydopamine (6-OHDA), a neurotoxin used for the production of PD model animals, on cellular Cu trafficking in human neuroblastoma SH-SY5Y cells. 6-OHDA reduced the protein levels of the Cu exporter ATP7A and the Cu chaperone Atox1, but not CTR1, a Cu importer; however, it did not affect the expression of ATP7A and Atox1 mRNAs. The decreased levels of ATP7A and Atox1 proteins were restored by the antioxidant N-acetylcysteine and the lysosomal inhibitor bafilomycin A1. This suggests that 6-OHDA-induced oxidative stress facilitates the degradation of these proteins. In addition, the amount of intracellular Cu after exposure to CuCl2 was significantly higher in cells pretreated with 6-OHDA than in untreated cells. Moreover, 6-OHDA reduced the protein levels of the cuproenzyme dopamine ß-hydroxylase that converts dopamine to noradrenaline. Thus, this study suggests that 6-OHDA disrupts Cu homeostasis through the dysregulation of cellular Cu trafficking, resulting in the dysfunction of neuronal cells.

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.

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.

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.

Hydrogen sulfide increases copper-dependent neurotoxicity via intracellular copper accumulation.

Copper (Cu) is an essential trace element and acts as a redox cofactor for many enzymes; however, excess Cu is toxic to cells. Hydrogen sulfide (H2S) is a well-known toxic gaseous molecule, but it has various biological effects such as neuromodulation and vasodilation. H2S was recently demonstrated to be involved in the detoxification of heavy metals, including zinc and cadmium, suggesting that H2S helps to maintain the homeostasis of heavy metals in cells. However, it is unclear how H2S impacts cellular Cu dynamics. In this study, we examined the effects of H2S on Cu cytotoxicity. Human neuroblastoma SH-SY5Y cells were exposed to CuSO4 in the presence of the H2S donor NaHS. CuSO4 alone slightly induced cell injury, whereas the combination of CuSO4 and NaHS (Cu/NaHS) increased Cu cytotoxicity. The Cu chelator bathocuproinedisulfonic acid mitigated Cu/NaHS-induced cytotoxicity. Compared with CuSO4 alone, Cu/NaHS markedly promoted ROS generation, mitochondrial dysfunction, and a decrease in ATP production. In addition, reporter assay using the metal responsive element (MRE)-driven reporter plasmid revealed that Cu/NaHS augmented Cu-dependent MRE activation. The amount of intracellular Cu was significantly higher in cells treated with Cu/NaHS than in those treated with CuSO4 alone. Moreover, Cu/NaHS markedly suppressed the level of the Cu exporter ATP7A, but not ATP7B, protein, whereas the combination did not affect that of the Cu importer CTR1 protein. Taken together, we conclude that the marked decrease in the ATP7A protein level by Cu/NaHS promotes intracellular Cu accumulation and leads to increased Cu cytotoxicity.

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.

Inhibitory effects of 4-hydroperoxy-2-decenoic acid ethyl ester on phorbol ester- and TGF-ß1-induced MMPs expression.

Matrix metalloproteinases (MMPs), zinc-containing proteinases, play a critical role in tumour progression by degrading extracellular matrix components. MMP2 and MMP9 are secreted from tumour-associated macrophages as well as tumour cells and have been implicated in the formation of the tumour microenvironment. Therefore, the inhibition of these MMPs may suppress tumour progression and metastasis. 4-Hydroperoxy-2-decenoic acid ethyl ester (HPO-DAEE) is known to cause apoptosis in the human lung cancer cell line A549 by inducing endoplasmic reticulum (ER) stress. However, the effects of HPO-DAEE on tumour progression remain unclear. HPO-DAEE pre-treatment significantly suppressed phorbol 12-myristate 13-acetate (TPA)-triggered MMP activation in human monocytic THP-1 cells. It also enhanced the expression of haem oxygenase-1, an antioxidant enzyme, and suppressed the TPA-triggered intracellular accumulation of reactive oxygen species (ROS). Furthermore, HPO-DAEE suppressed transforming growth factor-ß1-triggered human prostate cancer PC3 cell migration and this was accompanied by the inhibition of MMP expression and activities. The present results indicate that HPO-DAEE may exert inhibitory effects on tumour progression by suppressing MMP expression and activities.

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.

[Regulation of Extracellular Redox Homeostasis in Tumor Microenvironment].

Excessive generation of reactive oxygen species (ROS) has been implicated in the progression of tumors. Superoxide dismutase 3 (SOD3) is a copper-containing secretory antioxidative enzyme that plays a critical role in redox homeostasis, particularly in extracellular spaces. Considerable evidence suggests that SOD3 protein expression is significantly decreased or lost in several tumor tissues, and this loss results in tumor metastasis. On the other hand, epigenetic disturbances, including DNA hyper-/hypomethylation, histone de/acetylation, and histone de/methylation, may be involved in tumorigenesis and the progression of metastasis. However, regulation of SOD3 in the tumor microenvironment and the involvement of epigenetics in its expression remain unclear. To elucidate the molecular mechanisms underlying SOD3 expression, we investigated the involvement of epigenetics, including DNA methylation and histone modifications, in its regulation in tumor cells and macrophages. SOD3 expression in human monocytic THP-1 cells and human lung cancer A549 cells was silenced by DNA hypermethylation within the SOD3 promoter region. Furthermore, the DNA demethylase, ten-eleven translocation 1, was shown for the first time to play a key role in regulation of DNA methylation within that region. We also demonstrated that myocyte enhancer factor 2 functioned as one of the transcription factors of SOD3 expression in THP-1 cells. Collectively, these novel results will contribute to the elucidation of epigenetic redox regulation, and may provide important insights into tumorigenesis and tumor metastasis.

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.

Induction of Human-Lung-Cancer-A549-Cell Apoptosis by 4-Hydroperoxy-2-decenoic Acid Ethyl Ester through Intracellular ROS Accumulation and the Induction of Proapoptotic CHOP Expression.

Royal jelly, a natural product secreted by honeybees, contains several fatty acids, such as 10-hydroxy-2-decenoic acid (DE), and shows anti- and pro-apoptotic properties. 4-Hydroperoxy-2-decenoic acid ethyl ester (HPO-DAEE), a DE derivative, exhibits potent antioxidative activity; however, it currently remains unclear whether HPO-DAEE induces cancer-cell death. In the present study, treatment with HPO-DAEE induced human-lung-cancer-A549-cell death (52.7 ± 10.2%) that was accompanied by DNA fragmentation. Moreover, the accumulation of intracellular reactive oxygen species (ROS, 2.38 ± 0.1-fold) and the induction of proapoptotic CCAAT-enhancer-binding-protein-homologous-protein (CHOP) expression (18.4 ± 4.0-fold) were observed in HPO-DAEE-treated cells. HPO-DAEE-elicited CHOP expression and cell death were markedly suppressed by pretreatment with N-acetylcysteine (NAC), an antioxidant, by 2.40 ± 1.57-fold and 5.7 ± 1.6%, respectively. Pretreatment with 4-phenylbutyric acid (PBA), an inhibitor of endoplasmic reticulum stress, also suppressed A549-cell death (38.4 ± 1.1%). Furthermore, we demonstrated the involvement of extracellular-signal-regulated protein kinase (ERK) and p38-related signaling in HPO-DAEE-elicited cell-death events. Overall, we concluded that HPO-DAEE induces A549-cell apoptosis through the ROS-ERK-p38 pathway and, at least in part, the CHOP 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.

The MEF2A and MEF2D function as scaffold proteins that interact with HDAC1 or p300 in SOD3 expression in THP-1 cells.

Superoxide dismutase 3 (SOD3) is a SOD isozyme and plays a key role in extracellular redox homeostasis. We previously demonstrated that histone acetylation is involved in 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited SOD3 expression in human monocytic THP-1 cells; however, the molecular mechanisms responsible for its expression have not yet been elucidated in detail. The results of the present study demonstrated that the binding of histone deacetylase 1 (HDAC1) to the SOD3 promoter region contributed to SOD3 silencing in basal THP-1 cells. On the other hand, the dissociation of HDAC1 from the SOD3 promoter region and the enrichment of p300, a histone acetyltransferase (HAT), within that region were observed in TPA-induced THP-1 cells. Myocyte enhancer factor 2 (MEF2) functions as a scaffold protein that interacts with histone deacetylases (HDAC) or HAT and regulates gene expression. The present results showed that the MEF2A and MEF2D function as mediators for TPA-elicited SOD3 expression by interacting with HDAC or p300. Additionally, the knockdown of MEF2A or MEF2D in human skin fibroblasts suppressed SOD3 expression at the mRNA and protein levels. Our results provide an insight into epigenetic regulation of redox gene expression, and may ultimately contribute to suppressing the progression of tumours and vascular diseases.