Ultravist Induces the Expression of MCP-1 and VCAM-1 in IL-4-Stimulated HUVECs.

The goal of the present study focused on the adverse reaction of contrast medium (CM) via the induction of inflammatory molecules in human umbilical vein endothelial cells (HUVECs). Ultravist-induced monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) gene expression was markedly increased in interleukin-4 (IL-4)-pretreated HUVECs in a time- and dose-dependent manner and was paralleled by concomitant production of MCP-1 and VCAM-1 proteins. MCP-1 and VCAM-1 gene expression by Ultravist in combination with IL-4 was mediated by the c-Jun N-terminal kinases (JNK1/2) signaling pathway. IL-4-pretreated Ultravist-stimulated HUVECs showed greatly increased migration and adhesion of THP-1 cells. Cell migration was decreased by treatment of CCR2 antagonist, and cell adhesion was also decreased by VCAM-1 blocking antibody. Furthermore, when tested in vivo under similar conditions, MCP-1 protein was significantly increased in Ultravist combined with IL-4-injected mice. Taken together, our findings suggest that MCP-1 blocking may be crucial in preventing the endothelial dysfunction induced by contrast medium in patients with inflammatory disease and atherosclerosis.

A molecular mechanism of nickel (II): reduction of nucleotide excision repair activity by structural and functional disruption of p53.

Nickel is a major carcinogen that is implicated in tumor development through occupational and environmental exposure. Although the exact molecular mechanisms of carcinogenesis by low-level nickel remain unclear, inhibition of DNA repair is frequently considered to be a critical mechanism of carcinogenesis. Here, we investigated whether low concentrations of nickel would affect p53-mediated DNA repair, especially nucleotide excision repair. Our results showed that nickel inhibited the promoter binding activity of p53 on the downstream gene GADD45A, as a result of the disturbance of p53 oligomerization by nickel. In addition, we demonstrated that nickel exposure trigger the reduction of GADD45A-mediated DNA repair by impairing the physical interactions between GADD45A and proliferating cell nuclear antigen or xeroderma pigmentosum G. Notably, in the GADD45A-knockdown system, the levels of unrepaired DNA photoproducts were higher than wild-type cells, elucidating the importance of GADD45A in the nickel-associated inhibition of DNA repair. These results imply that inhibition of p53-mediated DNA repair can be considered a potential carcinogenic mechanism of nickel at low concentrations.

Identification of female-specific blood stains using a 17ß-estradiol-targeted aptamer-based sensor.

Blood stain evidence obtained from a violent crime scene provides decisive clues that can enable a case to be solved through forensic analyses such as genetic identification. However, collected samples usually contain a mixture of biological material from different sources, making genetic identification difficult. To address this issue, we developed an activatable aptamer sensor targeting 17ß-estradiol for detection of female-specific blood in mixed samples. With the sensor, we were able to detect blood originating from females using a variable light source (495 nm). The sensor was especially sensitive to blood from young females (10-40 years) but not to blood from older females (≥ 50 years). Genomic DNA was extracted from the female blood specimens identified by this method and used for quantification and short tandem repeat genotyping. We confirmed that there was no fluorescence interference from the aptamer sensor. These results indicate that this novel aptamer sensor can be used to analyze evidentiary blood samples and thereby facilitate subsequent genetic identification.

A Protective Mechanism of Visible Red Light in Normal Human Dermal Fibroblasts: Enhancement of GADD45A-Mediated DNA Repair Activity.

The phototherapeutic effects of visible red light on skin have been extensively investigated, but the underlying biological mechanisms remain poorly understood. We aimed to elucidate the protective mechanism of visible red light in terms of DNA repair of UV-induced oxidative damage in normal human dermal fibroblasts. The protective effect of visible red light on UV-induced DNA damage was identified by several assays in both two-dimensional and three-dimensional cell culture systems. With regard to the protective mechanism of visible red light, our data showed alterations in base excision repair mediated by growth arrest and DNA damage inducible, alpha (GADD45A). We also observed an enhancement of the physical activity of GADD45A and apurinic/apyrimidinic endonuclease 1 (APE1) by visible red light. Moreover, UV-induced DNA damages were diminished by visible red light in an APE1-dependent manner. On the basis of the decrease in GADD45A-APE1 interaction in the activating transcription factor-2 (ATF2)-knockdown system, we suggest a role for ATF2 modulation in GADD45A-mediated DNA repair upon visible red light exposure. Thus, the enhancement of GADD45A-mediated base excision repair modulated by ATF2 might be a potential protective mechanism of visible red light.

Endocrine-disrupting Chemicals: Review of Toxicological Mechanisms Using Molecular Pathway Analysis.

Endocrine disruptors are known to cause harmful effects to human through various exposure routes. These chemicals mainly appear to interfere with the endocrine or hormone systems. As importantly, numerous studies have demonstrated that the accumulation of endocrine disruptors can induce fatal disorders including obesity and cancer. Using diverse biological tools, the potential molecular mechanisms related with these diseases by exposure of endocrine disruptors. Recently, pathway analysis, a bioinformatics tool, is being widely used to predict the potential mechanism or biological network of certain chemicals. In this review, we initially summarize the major molecular mechanisms involved in the induction of the above mentioned diseases by endocrine disruptors. Additionally, we provide the potential markers and signaling mechanisms discovered via pathway analysis under exposure to representative endocrine disruptors, bisphenol, diethylhexylphthalate, and nonylphenol. The review emphasizes the importance of pathway analysis using bioinformatics to finding the specific mechanisms of toxic chemicals, including endocrine disruptors.

NAA10 controls osteoblast differentiation and bone formation as a feedback regulator of Runx2.

Runt-related transcription factor 2 (Runx2) transactivates many genes required for osteoblast differentiation. The role of N-α-acetyltransferase 10 (NAA10, arrest-defective-1), originally identified in yeast, remains poorly understood in mammals. Here we report a new NAA10 function in Runx2-mediated osteogenesis. Runx2 stabilizes NAA10 in osteoblasts during BMP-2-induced differentiation, and NAA10 in turn controls this differentiation by inhibiting Runx2. NAA10 delays bone healing in a rat calvarial defect model and bone development in neonatal mice. Mechanistically, NAA10 acetylates Runx2 at Lys225, and this acetylation inhibits Runx2-driven transcription by interfering with CBFß binding to Runx2. Our study suggests that NAA10 acts as a guard ensuring balanced osteogenesis by fine-tuning Runx2 signalling in a feedback manner. NAA10 inhibition could be considered a potential strategy for facilitating bone formation.

Human AP endonuclease 1: a potential marker for the prediction of environmental carcinogenesis risk.

Human apurinic/apyrimidinic endonuclease 1 (APE1) functions mainly in DNA repair as an enzyme removing AP sites and in redox signaling as a coactivator of various transcription factors. Based on these multifunctions of APE1 within cells, numerous studies have reported that the alteration of APE1 could be a crucial factor in development of human diseases such as cancer and neurodegeneration. In fact, the study on the combination of an individual's genetic make-up with environmental factors (gene-environment interaction) is of great importance to understand the development of diseases, especially lethal diseases including cancer. Recent reports have suggested that the human carcinogenic risk following exposure to environmental toxicants is affected by APE1 alterations in terms of gene-environment interactions. In this review, we initially outline the critical APE1 functions in the various intracellular mechanisms including DNA repair and redox regulation and its roles in human diseases. Several findings demonstrate that the change in expression and activity as well as genetic variability of APE1 caused by environmental chemical (e.g., heavy metals and cigarette smoke) and physical carcinogens (ultraviolet and ionizing radiation) is likely associated with various cancers. These enable us to ultimately suggest APE1 as a vital marker for the prediction of environmental carcinogenesis risk.

Branched-chain amino acids ameliorate fibrosis and suppress tumor growth in a rat model of hepatocellular carcinoma with liver cirrhosis.

PURPOSE: Recent studies have revealed that branched-chain amino acids (BCAA) reduce the development of hepatocellular carcinoma (HCC) in patients with obesity and hepatitis C virus infection by improving insulin resistance (IR). The aim of this study was to examine the anti-cancer and anti-fibrotic effects of BCAA on the development of diethylnitrosamine (DEN)-induced HCC and liver cirrhosis in a rat model. METHODS: Male SD rats received weekly intraperitoneal injections of DEN (50 mg/kg of body weight) for 16 weeks to induce HCC. They were fed a diet containing 3% casein, 3% or 6% BCAA for 13 weeks beginning 6 weeks after DEN administration. DEN was used to induce HCC through stepwise development from cirrhosis to HCC. The effect of BCAA was evaluated in tumor tissues by histopathologic analyses, reverse transcription-polymerase chain reaction, and Western blotting. RESULTS: The mean area and number of dysplastic nodules (DNs) and tumors in the casein group tended to be larger than those in the BCAA group 16 weeks after DEN administration. The mean fibrotic area in the BCAA group was smaller than that in the casein group. The BCAA group showed decreased mRNA levels for markers of fibrosis, angiogenesis, and apoptosis inhibition. Compared with the casein group, the BCAA group had lower levels of α-smooth muscle actin, vascular endothelial growth factor, p-ß-catenin, p-p38 mitogen-activated protein kinase, proliferating cell nuclear antigen, and caspase-3 protein expression, as well as a higher level of cleaved caspase-3 protein expression. CONCLUSIONS: BCAA supplementation of the diet ameliorated liver fibrosis and HCC development in a DEN-induced rat model of HCC with liver cirrhosis, but not in the IR model. These results provide a rationale for anti-fibrosis and chemoprevention using BCAA treatment for HCC with liver cirrhosis, as well as decreasing the ammonia level.

Enhanced redox factor 1 (REF1)-modulated p53 stabilization and JNK1 dissociation in response to selenomethionine.

AIM: p53 is reportedly activated without any genotoxicity through redox modulation of redox factor 1 (REF1). REF1 is documented to modulate the redox status under selenomethionine (SeMet). In this study, we investigated the mechanism of p53 stabilization by SeMet. MATERIALS AND METHODS: We mainly used ubiquitination assay and immunoprecipitation to determine the potential role of REF1 and c-jun N-terminal kinase 1 (JNK) in modulation of p53 stabilization by SeMet. RESULTS: The amount of ubiquitinated p53 decreased significantly under SeMet treatment, suggesting that SeMet might inhibit the proteasome-dependent degradation of p53. In addition, we observed that JNK was considerably associated with p53 in REF1 siRNA-treated cells, implying a possible role for SeMet-induced REF1 activity in modulation of the interaction between JNK and p53 via changes in p53 redox status. CONCLUSION: Our results suggest that the alternate mechanism of p53 stabilization by SeMet might provide an important clue in elucidating the molecular mechanism of chemopreventative compounds against various oxidative stresses.

A novel chemopreventive mechanism of selenomethionine: enhancement of APE1 enzyme activity via a Gadd45a, PCNA and APE1 protein complex that regulates p53-mediated base excision repair.

Organic selenium compounds have been documented to play a role in cancer prevention. Our previous study showed that selenomethionine (SeMet) induces p53 activation without genotoxic effects including apoptosis and cell cycle arrest. In this study, we investigated the mechanism by which organic selenium compounds promote p53-mediated base excision repair (BER) activity. Our data demonstrated for the first time that the interaction between growth arrest and DNA damage-inducible protein 45A (Gadd45a), which is a p53-activated downstream gene, and two BER-mediated repair proteins, proliferating cell nuclear antigen (PCNA) and apurinic/apyrimidinic endonuclease (APE1/Ref-1), was significantly increased in a p53-dependent manner following treatment with organic selenium compounds. Furthermore, we observed that the activity of APE1 was significantly increased in a p53-dependent manner in response to the organic selenium compounds. These results suggest that BER activity is dependent on wild-type p53 activity and is mediated by the modulation of protein interactions between Gadd45a and repair proteins in response to organic selenium compounds. We propose that p53-dependent BER activity is a distinct chemopreventive mechanism mediated by organic selenium compounds, and that this may provide insight into the development of effective chemopreventive strategies against various oxidative stresses that contribute to a variety of human diseases, particularly cancer.

A novel role for Gadd45α in base excision repair: modulation of APE1 activity by the direct interaction of Gadd45α with PCNA.

The growth arrest and DNA damage inducible, alpha (Gadd45α) protein regulates DNA repair by interacting with proliferating cell nuclear antigen (PCNA). Our previous study suggested a potential role for Gadd45α in the base excision repair (BER) pathway by affecting apurinic/apyrimidinic endonuclease 1 (APE1) protein in addition to its accepted role in nucleotide excision repair (NER). Here, we investigated whether the interaction of Gadd45α with PCNA affects APE1 activity. To address this issue, we used a siRNA directed to Gadd45α and a form of Gadd45α with a mutation to the predicted site of PCNA binding. There was a reduction of APE1 activity in cells transfected with the Gadd45α siRNA. Furthermore, the interaction of Gadd45α with PCNA and APE1 was lower in cells transfected with mutant Gadd45α compared with cells transfected with wild-type Gadd45α. Indeed, we observed that the APE1 activity in the Gadd45α-interacting complex was significantly lower in cells that overexpress mutant Gadd45α compared with cells that overexpress wild-type Gadd45α. We conclude that the PCNA binding site on Gadd45α plays a critical role in modulating the interaction with PCNA and APE1, affecting BER activity. These results provide novel insights into the mechanisms by which BER activity is modulated, although the interaction of Gadd45α with APE1 needs to be clarified.

Base excision DNA repair defect in thioredoxin-1 (Trx1)-deficient cells.

Thioredoxin-1 (Trx1) is an antioxidant enzyme with a protective role in the removal of oxidative stress. We investigated the mechanism by which the redox modulator Trx1 affects base excision repair (BER) activity to understand the protective role of Trx1. We constructed a Trx1 knockdown system to demonstrate the specific mechanism of Trx1 shRNA cells compared with that in the wild type cells, leading to increased cellular susceptibility to a sublethal dose of BER-inducible toxicant, nitrosomethylurea (NMU). In addition, we observed a modulatory role of Trx1 in the BER pathway via the p53 downstream gene, growth arrest, and DNA-damage-inducible protein 45 α (Gadd45a). The protein level and function of p53, a Trx1 downstream gene, coincidently decreased in the Trx1 shRNA cells. Futhermore, Trx1 shRNA cells showed decreased Gadd45a expression and interaction of Gadd45a with apurinic/apyrimidinic endonuclease 1 (APE1) as well as APE activity. In conclusion, Trx1 might cooperate in the control of APE1 function by modulating the p53-mediated BER via the protein-protein interaction between Gadd45a and APE1, providing insight into the novel role of redox factor Trx1 in modulation of BER.

Molecular and genomic approach for understanding the gene-environment interaction between Nrf2 deficiency and carcinogenic nickel-induced DNA damage.

Nickel (Ⅱ) is a toxic and carcinogenic metal which induces a redox imbalance following oxidative stress. Nuclear factor erythroid-2 related factor 2 (Nrf2) is a redox factor that regulates oxidation/reduction status and consequently mediates cytoprotective responses against exposure to environmental toxicants. In this study, we investigated the protective roles of the Nrf2 gene against oxidative stress and DNA damage induced by nickel at sub-lethal doses. Under nickel exposure conditions, we detected significantly increased intracellular ROS generation, in addition to higher amounts of DNA damage using comet assay and γ-H2AX immunofluorescence staining in Nrf2 lacking cells, as compared to Nrf2 wild-type cells. In addition, we attempted to identify potential nickel and Nrf2-responsive targets and the relevant pathway. The genomic expression data were analyzed using microarray for the selection of synergistic effect-related genes by Nrf2 knockdown under nickel treatment. In particular, altered expressions of 6 upregulated genes (CAV1, FOSL2, MICA, PIM2, RUNX1 and SLC7A6) and 4 downregulated genes (APLP1, CLSPN, PCAF and PRAME) were confirmed by qRT-PCR. Additionally, using bioinformatics tool, we found that these genes functioned principally in a variety of molecular processes, including oxidative stress response, necrosis, DNA repair and cell survival. Thus, we describe the potential biomarkers regarded as molecular candidates for Nrf2-related cellular protection against nickel exposure. In conclusion, these findings indicate that Nrf2 is an important factor with a protective role in the suppression of mutagenicity and carcinogenicity by environmental nickel exposure in terms of gene-environment interaction.

Efficacy and safety of metronomic chemotherapy for patients with advanced primary hepatocellular carcinoma with major portal vein tumor thrombosis.

BACKGROUND/AIMS: Low-dose metronomic chemotherapy involves the frequent administration of comparatively low doses of cytotoxic agents with no extended breaks, and it may be as efficient as and less toxic than the conventional maximum tolerated dose therapy. This study evaluated the feasibility and therapeutic efficacy of metronomic chemotherapy in patients with advanced hepatocellular carcinoma (HCC) with major portal vein thrombosis (PVT). METHODS: Thirty consecutive HCC patients with major PVT with or without extrahepatic metastasis were prospectively allocated to metronomic chemotherapy consisting of epirubicin being infused through the correct hepatic artery at a dose of 30 mg/body surface area (BSA) every 4 weeks, and cisplatin (15 mg/BSA) and 5-fluorouracil (50 mg/BSA) every week for 3 weeks, with intervening 1 week breaks. The treatment response was assessed using response evaluation criteria in solid tumors (RECIST). RESULTS: In total, 116 cycles of metronomic chemotherapy were administered to the 30 patients, with a median of 3 cycles given to individual patients (range, 1-15 cycles). Six patients (20.0%) achieved a partial response and six patients (20.0%) had stable disease. The median time to disease progression and overall survival were 63 days (range, 26-631 days) and 162 days (95% confidence interval; range, 62-262 days), respectively. Overall survival was significantly associated with baseline alpha-fetoprotein level (P=0.001) and tumor response (P=0.005). The baseline alpha-fetoprotein level was significantly associated with the disease control rate (P=0.007). Adverse events were tolerable and managed successfully with conservative treatment. CONCLUSIONS: Metronomic chemotherapy may be a safe and useful palliative treatment in HCC patients with major PVT.

Dura mater graft-associated Creutzfeldt-Jakob disease: the first case in Korea.

Since 1987, dura mater graft-associated iatrogenic Creutzfeldt-Jakob disease (dCJD) has been reported in many countries. We report the first case of dCJD in Korea. A 54-yr-old woman, who underwent resection of the meningioma in the left frontal region and received a dura mater graft 23 yr ago presented with dysesthesia followed by psychiatric symptoms and ataxia. Her neurological symptoms rapidly progressed to such an extent that she exhibited myoclonus, dementia, and pyramidal and extrapyramidal signs within 8 weeks. The 14-3-3 protein was detected in her cerebrospinal fluid; however, an electroencephalogram did not reveal characteristic positive sharp wave complexes. Diffusion-weighted magnetic resonance images, obtained serially over 64 days, revealed the rapid progression of areas of high signal intensity in the caudate nucleus and cingulate gyrus to widespread areas of high signal intensity in the cortex and basal ganglia. Pathological examination of brain biopsy specimens confirmed the presence of spongiform changes and deposition of prion protein in the neurons and neuropils.

Hypoxia-inducible factor 1alpha is deregulated by the serum of rats with adjuvant-induced arthritis.

Rheumatoid arthritis (RA) is known to be associated with increased risks of hypoxia-related diseases, whose progresses are critically determined by HIF-1alpha. The authors hypothesized that the hypoxia-related complications of RA are associated with HIF-1alpha deregulation by some factor(s) in RA serum. Arthritis was induced in female Lewis rats by injecting complete Freund's adjuvant. The effects of arthritic rat serum (ARS) on hypoxic responses were investigated by incubating Hep3B cells in ARS. In the presence of ARS, HIF-1alpha was down-regulated and inactivated under hypoxic conditions. ARS inactivated AKT and mTOR, which led to impaired HIF-1alpha protein synthesis. Furthermore, insulin was found to be deficient in ARS and insulin supplementation fully recovered HIF-1alpha synthesis with AKT and mTOR activation. These results suggest that HIF-1alpha deregulation by components in serum is responsible for the RA-associated aggravation of hypoxic diseases in extra-articular tissues.

ROS mediate the hypoxic repression of the hepcidin gene by inhibiting C/EBPalpha and STAT-3.

Hepcidin, a liver peptide, systemically inhibits iron utilization and is downregulated under hypoxic conditions. However, little is known about the mechanism underlying the hypoxic suppression of hepcidin. Here, we tested the possibility that HIF-1 and ROS are involved in hepcidin regulation. Hepcidin mRNA, pre-mRNA, and protein levels were reduced in mouse livers and in HepG2 cells after hypoxic incubation, and HIF-1 overexpression and knock-down studies showed that hepcidin regulation is independent of HIF-1. On the other hand, ROS levels were significantly elevated in hypoxic HepG2 cells, and anti-oxidants prevented the hypoxic down-regulation of hepcidin. Conversely, a prooxidant, H(2)O(2), suppressed hepcidin expression in these cells even in normoxia. Of the various transcription factors examined, C/EBPalpha and STAT-3 were found to dissociate from hepcidin promoter under hypoxia, but to become fully engaged after anti-oxidant treatment. These results suggest that ROS repress the hepcidin gene by preventing C/EBPalpha and STAT-3 binding to hepcidin promoter during hypoxia.

6-Pyruvoyltetrahydropterin synthase orthologs of either a single or dual domain structure are responsible for tetrahydrobiopterin synthesis in bacteria.

6-Pyruvoyltetrahydropterin synthase (PTPS) catalyzes the second step of tetrahydrobiopterin (BH4) synthesis. We previously identified PTPS orthologs (bPTPS-Is) in bacteria which do not produce BH4. In this study we disrupted the gene encoding bPTPS-I in Synechococcus sp. PCC 7942, which produces BH4-glucoside. The mutant was normal in BH4-glucoside production, demonstrating that bPTPS-I does not participate in BH4 synthesis in vivo and bringing us a new PTPS ortholog (bPTPS-II) of a bimodular polypeptide. The recombinant Synechococcus bPTPS-II was assayed in vitro to show PTPS activity higher than human enzyme. Further computational analysis revealed the presence of mono and bimodular bPTPS-II orthologs mostly in green sulfur bacteria and cyanobacteria, respectively, which are well known for BH4-glycoside production. In summary we found new bacterial PTPS orthologs, having either a single or dual domain structure and being responsible for BH4 synthesis in vivo, thereby disclosing all the bacterial PTPS homologs.

A domain responsible for HIF-1alpha degradation by YC-1, a novel anticancer agent.

HIF-1alpha is believed to promote tumor growth and metastasis, and many efforts have been made to develop new anticancer agents based on HIF-1alpha inhibition. YC-1 is a widely used HIF-1alpha inhibitor both in vitro and in vivo, and is being developed as a novel class of anticancer drug. However, little is known about the mechanism by which YC-1 degrades HIF-1alpha. As the first step for understanding the mechanism of action of YC-1, we here identified the HIF-1alpha domain responsible for YC-1-induced protein degradation. YC-1 blocked the HIF-1alpha induction by hypoxia, iron chelation, and proteasomal inhibition and also degraded ectopically expressed HIF-1alpha. In deletion analyses, C-terminal HIF-1alpha was found to be sensitively degraded by YC-1. Using a GFP-fusion method, the YC-1-induced degradation domain was identified as the aa. 720-780 region of HIF-1alpha. We next tested the possible involvement of HDAC7 or OS-9 in YC-1-induced HIF-1alpha degradation. However, their binding to HIF-1alpha was not affected by YC-1, suggesting that they are not involved in the YC-1 action. It is also suggested that YC-1 targets a novel pathway regulating HIF-1alpha stability.