Effect of Copper Chelators via the TGF-ß Signaling Pathway on Glioblastoma Cell Invasion.

Glioblastoma multiforme (GBM) is a fast-growing and aggressive type of brain cancer. Unlike normal brain cells, GBM cells exhibit epithelial-mesenchymal transition (EMT), which is a crucial biological process in embryonic development and cell metastasis, and are highly invasive. Copper reportedly plays a critical role in the progression of a variety of cancers, including brain, breast, and lung cancers. However, excessive copper is toxic to cells. D-penicillamine (DPA) and triethylenetetramine (TETA) are well-known copper chelators and are the mainstay of treatment for copper-associated diseases. Following treatment with copper sulfate and DPA, GBM cells showed inhibition of proliferation and suppression of EMT properties, including reduced expression levels of N-cadherin, E-cadherin, and Zeb, which are cell markers associated with EMT. In contrast, treatment with copper sulfate and TETA yielded the opposite effects in GBM. Genes, including TGF-ß, are associated with an increase in copper levels, implying their role in EMT. To analyze the invasion and spread of GBM, we used zebrafish embryos xenografted with the GBM cell line U87. The invasion of GBM cells into zebrafish embryos was markedly inhibited by copper treatment with DPA. Our findings suggest that treatment with copper and DPA inhibits proliferation and EMT through a mechanism involving TGF-ß/Smad signaling in GBM. Therefore, DPA, but not TETA, could be used as adjuvant therapy for GBM with high copper concentrations.

Antioxidant Activity of Sprouts Extracts Is Correlated with Their Anti-Obesity and Anti-Inflammatory Effects in High-Fat Diet-Fed Mice.

Obesity is closely associated with oxidative stress and chronic inflammation leading to related metabolic diseases. Some natural extracts or polyphenols reportedly possess anti-obesity and anti-inflammatory effects as well as antioxidant activity. In this study, we assessed the correlations between the antioxidant, anti-obesity, and anti-inflammatory activities of plant extracts with potent antioxidant activity in diet-induced obese mice. Sprouts of Cedrela sinensis (CS) and Oenothera biennis L. (OB) were selected as the most potent antioxidant plant based on analysis of in vitro antioxidant activity of the extracts of ten different edible plants. C57BL/6 mice were fed with a high-fat diet (HFD) and orally treated with 50% ethanol extract of CS or OB at 50 or 100 mg/kg body weight 5 days a week for 14 weeks. Body weight gain, weight of adipose tissue, adipocyte size, and levels of lipid metabolism, inflammation, and oxidative stress markers were investigated. The CS or OB extract reduced body weight gain, visceral adipose tissue weight, adipocyte size, and plasma leptin levels, and expressions of adipogenic genes (PPARγ and fatty acid synthase) in the adipose tissue and liver of HFD-fed mice. Both extracts also reduced mRNA levels of pro-inflammatory cytokines (IL-6 and TNF-α) and oxidative stress-related genes (heme oxygenase- (HO-) 1 and p40phox). Body weight gain of mice was significantly correlated with visceral adipose tissue weight and adipocyte size. Body weight gain and adipocyte size were significantly correlated with plasma total cholesterol and 8-epi PGF2α levels, mRNA levels of leptin, HO-1, p40phox, and CD-11 in the adipose tissue, and mRNA levels of TNF-α in the adipose tissue and liver. These results suggest that the CS and OB extracts with potent antioxidant activity may inhibit fat deposition in adipose tissue and subsequent inflammation.

S-nitrosylation of transglutaminase 2 impairs fatty acid-stimulated contraction in hypertensive cardiomyocytes.

The myocardium in hypertensive heart exhibits decreased fatty acid utilization and contractile dysfunction, leading to cardiac failure. However, the causal relationship between metabolic remodeling and cardiomyocyte contractility remains unestablished. Transglutaminase 2 (TG2) has been known to promote ATP production through the regulation of mitochondrial function. In this study, we investigated the involvement of TG2 in cardiomyocyte contraction under fatty acid supplementation. Using TG2 inhibitor and TG2-deficient mice, we demonstrated that fatty acid supplementation activated TG2 and increased ATP level and contractility of cardiac myocyte from the normal heart. By contrast, in cardiac myocytes from angiotensin-II-treated rats and mice, the effects of fatty acid supplementation on TG2 activity, ATP level, and myocyte contraction were abolished. We found that TG2 was inhibited by S-nitrosylation and its level increased in hypertensive myocytes. Treatment with inhibitor for neuronal NOS restored fatty acid-induced increase of TG2 activity and myocyte contraction. Moreover, intracellular Ca2+ levels were increased by fatty acid supplementation in both normal and hypertensive myocytes, showing that S-nitrosylation of TG2 but not alteration of intracellular Ca2+ levels is responsible for contractile dysfunction. These results indicate that TG2 plays a critical role in the regulation of myocyte contractility by promoting fatty acid metabolism and provide a novel target for preventing contractile dysfunction in heart with high workload.

Cystamine induces AIF-mediated apoptosis through glutathione depletion.

Cystamine and its reduced form cysteamine showed protective effects in various models of neurodegenerative disease, including Huntington's disease and Parkinson's disease. Other lines of evidence demonstrated the cytotoxic effect of cysteamine on duodenal mucosa leading to ulcer development. However, the mechanism for cystamine cytotoxicity remains poorly understood. Here, we report a new pathway in which cystamine induces apoptosis by targeting apoptosis-inducing factor (AIF). By screening of various cell lines, we observed that cystamine and cysteamine induce cell death in a cell type-specific manner. Comparison between cystamine-sensitive and cystamine-resistant cell lines revealed that cystamine cytotoxicity is not associated with unfolded protein response, reactive oxygen species generation and transglutaminase or caspase activity; rather, it is associated with the ability of cystamine to trigger AIF nuclear translocation. In cystamine-sensitive cells, cystamine suppresses the levels of intracellular glutathione by inhibiting γ-glutamylcysteine synthetase expression that triggers AIF translocation. Conversely, glutathione supplementation completely prevents cystamine-induced AIF translocation and apoptosis. In rats, cysteamine administration induces glutathione depletion and AIF translocation leading to apoptosis of duodenal epithelium. These results indicate that AIF translocation through glutathione depletion is the molecular mechanism of cystamine toxicity, and provide important implications for cystamine in the neurodegenerative disease therapeutics as well as in the regulation of AIF-mediated cell death.

Enhancement of radiation response with combined Ganoderma lucidum and Duchesnea chrysantha extracts in human leukemia HL-60 cells.

We previously demonstrated that combined treatment with extracts of the medicinal mushroom Ganoderma lucidum and the herb Duchesnea chrysantha (GDE) significantly suppresses cell growth and selectively induces apoptosis in human leukemia HL-60 cells, but not in normal cells. GDE?s mechanism of action and its activity against HL-60 cells suggest that it could be suitable for the combined-modality treatment of hematological malignancies. In the present study, we examined whether treatment with a combination of GDE and ionizing radiation enhances the therapeutic effect. We demonstrated that, when used in combination with radiation at a clinically relevant dose of 2 Gy, GDE further suppressed cell proliferation and induced apoptosis as well as micronuclei formation in HL-60 cells, leading to increased cell death. Furthermore, GDE pretreatment not only reduced radiation-induced G2/M-phase arrest, but also induced G1-phase arrest. These events are associated with the inhibition of cyclin-dependent kinase 1 (CDK1) phosphorylation and the dephosphorylation of retinoblastoma protein (pRB). Collectively, these data show that combined treatment with GDE and radiation enhances radiation-induced apoptosis and overall cell death. These findings may be clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of radiotherapy.

Cystamine prevents ischemia-reperfusion injury by inhibiting polyamination of RhoA.

Transglutaminase2 (TGase2) activates Rho-associated kinase (ROCK), an important mediator of ischemia-reperfusion (IR) injury, through polyamination of RhoA. Cystamine, an oxidized dimer of cysteamine inhibits the transamidation activity of TGase2. We examined whether addition of cystamine to an organ preservation solution protects rat cardiomyocyte cells (H9C2) from cell death in IR injury. H9C2 cells were stored under hypoxic conditions at 4 degrees C in laboratory-made preservation solution (SNU) or SNU solution supplemented with cystamine (SNU-C1), and cell preservation in the two solutions was compared by measuring the release of lactate dehydrogenase. The cells were preserved more effectively in SNU-C1 than in SNU solution. Cystamine inhibited the intracellular activity of TGase2 which increased during cold storage or reoxygenation. The inhibition of TGase2 by cystamine reduced the polyamination of RhoA, the interaction between RhoA and ROCK2, and F-actin formation. Cystamine also prevented the activation of caspases during cold storage. These results suggest that addition of cystamine to the organ preservation solution significantly enhances cardiomyocytes preservation apparently by inhibiting TGase2-mediated RhoA-ROCK pathway and that TGase2 may play an important role in IR injury by regulating ROCK.

Screening of estrogenic and antiestrogenic activities from medicinal plants.

The medicinal plant extracts commercially used in Asia were screened for their estrogenic and antiestrogenic activities in a recombinant yeast system featuring both a human estrogen receptor (ER) expression plasmid and a reporter plasmid. Pueraria lobata (flower) had the highest estrogenic relative potency (RP, 7.75×10(-3); RP of 17ß-estradiol=1), followed by Amomum xanthioides (1.25×10(-3)). Next potent were a group consisting of Glycyrrhiza uralensis, Zingiber officinale, Rheum undulatum, Curcuma aromatica, Eriobotrya japonica, Sophora flavescens, Anemarrhena asphodeloides, Polygonum multiflorum, and Pueraria lobata (root) (ranging from 9.5×10(-4) to 1.0×10(-4)). Least potent were Prunus persica, Lycoppus lucidus, and Adenophora stricta (ranging from 9.0×10(-5) to 8.0×10(-5)). The extracts exerting antiestrogenic effects, Cinnamomum cassia and Prunus persica, had relative potencies of 1.14×10(-3) and 7.4×10(-4), respectively (RP of tamoxifen=1). The solvent fractions from selected estrogenic or antiestrogenic herbs had higher estrogenic relative potencies, with their RP ranging from 9.3×10(-1) to 2.7×10(-4) and from 8.2×10(-1) to 9.1×10(-3), respectively. These results support previous reports on the efficacy of Oriental medicinal plants used or not used as phytoestrogens for hormone replacement therapy.

Enhanced induction of mitochondrial damage and apoptosis in human leukemia HL-60 cells by the Ganoderma lucidum and Duchesnea chrysantha extracts.

Combined treatment with the medicinal mushroom Ganoderma lucidum and the herb Duchesnea chrysantha extracts (GDE) causes a synergistic induction of mitochondrial damage and apoptosis in HL-60 cells. GDE treatment is selectively toxic to HL-60 leukemia cells whereas no cytotoxic effect is observed in normal peripheral blood mononuclear cells. GDE-induced apoptosis is associated with Bcl-2 down-regulation, Bax translocation, mitochondrial cytochrome c release and caspase-3 activation, suggesting that apoptosis by this combination occurs through the mitochondria-dependent pathway. The present findings suggest that this combination merits further investigation as a potential therapeutic agent for the treatment of cancer.

Effects of Tiron, 4,5-dihydroxy-1,3-benzene disulfonic acid, on human promyelotic HL-60 leukemia cell differentiation and death.

Tiron, 4,5-dihydroxy-1,3-benzene disulfonic acid, has been known to be a widely used antioxidant to rescue ROS-evoked cell death and a non-toxic chelator to alleviate an acute metal overload. In this study, we showed that Tiron is a potent inducer of cell differentiation and apoptotic cell death in human promyelotic HL-60 leukemia cell. At a low level of concentration (<0.5mM), Tiron caused HL-60 cells to induce differentiation-related alterations such as the increase of CD11b and CD14 expression or chromatin condensation. Hypoxia inducible factor-1alpha (HIF-1alpha) was also increased at mRNA and protein level, and thus the CCAAT/enhancer-binding protein alpha, which is a downstream target of HIF-1alpha and acts as a critical factor for granulocytic differentiation was increased. High dose of Tiron (>0.5mM) induced severe DNA damage in HL-60 cells, as measured by the cytokinesis-block micronucleus test and the comet assay. Consequently, high dose of Tiron led to apoptotic cell death, which showed the DNA fragmentation, the caspase activation and the unbalance between antiapoptotic (Bcl-2) and proapoptotic proteins (Bax). However, an exogenous supplement of iron (FeCl(3)) reversed all of these effects, the cell differentiation and the apoptotic cell death. Therefore, these results suggest that Tiron-mediated differentiation and cell death result from the disturbance of iron metabolism.

Identification, evolution, and regulation of expression of Guinea pig trappin with an unusually long transglutaminase substrate domain.

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.

LPS-induced CD53 expression: a protection mechanism against oxidative and radiation stress.

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. Its biological role remains unknown. Using microarrays, we identified CD53 as one of the principal genes up-regulated by exposure of macrophages to LPS. Northern blot analysis confirmed the induction of CD53 in RAW264.7 macrophages treated with LPS or SNAP (a nitric oxide donor). Cells stably transfected with sense CD53 cDNA had increased levels of intracellular GSH and lower levels of peroxide, and were more resistant to H2O2 and to UVB irradiation. Cells harboring antisense CD53 had the opposite properties. We propose that the induction of CD53 is a major mechanism by which macrophages protect themselves against LPS-induced oxidative stress and UVB irradiation.

Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation: implications of transglutaminase 2 in age-related cataractogenesis.

Transglutaminase (TGase) 2 is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamination. An aberrant activity of TGase 2 has implicated its possible roles in a variety of diseases including age-related cataracts. However, the molecular mechanism by which TGase 2 is activated has not been elucidated. In this report, we showed that oxidative stress or UV irradiation elevates in situ TGase 2 activity. Neither the expression level nor the in vitro activity of TGase 2 appeared to correlate with the observed elevation of in situ TGase 2 activity. Screening a number of cell lines revealed that the level of TGase 2 activation depends on the cell type and also the environmental stress, suggesting that unrecognized cellular factor(s) may specifically regulate in situ TGase 2 activity. Concomitantly, we observed that human lens epithelial cells (HLE-B3) exhibited about 3-fold increase in in situ TGase 2 activity in response to the stresses. The activated TGase 2 catalyzed the formation of water-insoluble dimers or polymers of alphaB-crystallin, betaB(2)-crystallin, and vimentin in HLE-B3 cells, providing evidence that TGase 2 may play a role in cataractogenesis. Thus, our findings indicate that in situ TGase 2 activity must be evaluated instead of in vitro activity to study the regulation mechanism and function of TGase 2 in biological and pathological processes.

Thiamine protects against paraquat-induced damage: scavenging activity of reactive oxygen species.

To demonstrate the superoxide anion (O(2)(-)) scavenging activity of thiamine, we comparatively investigated the inhibition of cell growth reduction and repression of the oxidative stress-inducible gene expression (soxS, sodA, zwf and soi-19::lacZ) triggered by paraquat, intracellular O(2)(-) generator, using an Escherichia coli system. When thiamine (>1 µM) was added to the culture, a decrease of growth rate caused by paraquat was significantly recovered. Paraquat treatment (1 µM) to aerobically grown E. coli highly increased the expression of soxS and its regulons sodA and zwf, genes for manganese-containing superoxide dismutase (Mn-SOD) and glucose-6-phosphate dehydrogenase (G6PDH) to cope with the oxidative stress. However, the induction of Mn-SOD and G6PDH was suppressed by the thiamine supplement. The induction of the soi-19::lacZ gene, whose expression was dependent on paraquat, was also repressed by more than 10 µM of the thiamine addition to the culture. To characterize the role of thiamine, which challenges the paraquat toxicity, an in vitro experiment of nitroblue tetrazolium (NBT) reduction was performed. The NBT reduction by O(2)(-) generated in the xanthine/hypoxanthine system was inhibited by the thiamine supplement in a dose-dependent manner. Moreover, it competed with the 2-deoxy-d-ribose in absorbing the hydroxyl radical (OH) generated by γ-irradiation (800 Gy) and thus inhibited the formation of malondialdehyde in vitro. In conclusion, this evidence suggests that thiamine may partly act as an antioxidant to scavenge O(2)(-) (or OH) directly and thus affect the cellular response to oxidative stress induced by reactive oxygen species.