Nanosilver is being used increasingly in medical and healthcare products due to its strong antimicrobial activity. The toxicity of polyvinylpyrrolidone coated nanosilver (PVP-NAg, 20 nm in diameter) was evaluated by using human hepatocellular carcinoma cells (HepG2). PVP-NAg was dispersed by culture solution. MTT assay was used to examine the proliferation activities of cells. The influence of PVP-NAg on cell cycle, ratio of apoptosis and production of reactive oxygen species (ROS) was analyzed by flow cytometry. MTT assay revealed that PVP-NAg significantly inhibited the proliferation of HepG2 in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50) after 24h exposure was 923.5µg/mL. The nanoparticle treatment caused cell cycle arrest in G2/M phase and the changes were detected in a dose-dependent manner; Cells were double-stained with Annexin V-FITC and propidium iodide, the ratio of apoptosis for 24h exposure was detected to be 3.9, 13.6, and 19.9% at the concentrations of PVP-NAg of 20, 80, and 320µg/mL, respectively. The production of ROS after 24h exposure was 19.0, 26.4, and 33.8% at the concentrations of PVP-NAg of 20, 80, and 160µg/mL, respectively, with cells stained with DCF-DA. Nanosilver could induce apoptosis and increase ROS production in a dose-dependent manner. The results suggest that nanosilver possess cytotoxic effects on HepG2 cells in a dose dependent manner. A possible mechanism of cytotoxicity could be involved in the interruption of the function of mitochondrial respiratory chain by nanosilver.

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Superparamagnetic iron oxide nanoparticles (IONP) have been used in clinical as contrast agents to enhance magnetic resonance imaging. Previous studies reported that intranasal exposure to IONPs caused microglial proliferation and activation in mice. It remains unclear if the function of microglia against pathogens is affected by IONP. The present study investigated the effect of IONP on the functionality of microglia activated by lipopolysaccharide (LPS). Confocal imaging revealed that IONPs were rapidly engulfed by microglia. IONP exposure enhanced the expression of lysosomal membrane protein ectodermal dysplasia-1 (ED-1) and the permeability and alkalinity in lysosomes. In contrast, IONP suppressed the expression of ionized calcium-binding adaptor molecule-1 (Iba-1), a microglial activation marker, phagocytic activity, and the production of proinflammatory cytokines, including IL-1β and IL-6. Taken together, these results demonstrated that IONP interfered with the functionality and lysosomal activity of LPS-stimulated microglia, suggesting an impaired activity against Gram-negative bacteria.

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The present study investigated suitable dispersion methods of nanoparticles for intratracheal instillation study. Direct probe-type and indirect cup-type sonicator were used. Nanoparticles were dispersed in a biocompatible dispersion medium following Poter's protocol. Using dynamic light scattering, dispersion status was examined at several different output powers, sonication durations and sample concentrations. We found macroscopic contamination of titanium broken away from the tip of the probe into the suspension with output power higher than 20W or duration longer than 20min. Size of agglomerated TiO2 decreased with the increase of output power or suspension duration. TiO2 was dispersed into agglomerated particles of 479.7±5.5nm with the cup-type sonicator at 220W for 30min when cooled by circulating 30% ethanol kept at -10°C, but the size decreased to 153.9±1.0nm at 100W and 5°C for 10min, and the size of the latter agglomerates only increased by 5.51% at 1day and by 8.45% at 3days after the dispersion. When dispersed at the concentration of 0.5ug/ml, the average diameters of TiO2 and ZnO agglomerates were 173.0±2.7nm and 160.5±0.7nm, respectively, while they were 420.5±2.1nm and 149.4±1.2nm at 2.5ug/ml, respectively. The study suggests that the temperature plays an important role in nanoparticle dispersion in terms of both size distribution and suspension stability. Dispersion concentration should also be taken into consideration when specific particles are dispersed at different concentrations. Cup-type sonicator might be useful as an alternative to probe-type to keep sample purity.

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In this study, the long term effect of titanium dioxide (TiO₂ nanoparticles (NPs) on O.mossambicus has been attempted. The chronic toxicity of TiO(2) NPs on freshwater fish, tilapia via., concentration-dependent and time-dependent has been achieved. The decline of growth and liver weight ratio of tilapia was substantially confirmed. Like wise the disturbances in the metabolism in carbohydrate , protein and lipid content of fish is also highlighted. Meanwhile histopathology changes including thickening of oedema and lamellae in gills of fish has been exposed. The bioaccumulation observation has demonstrated that TiO(2) NPs are largely accumulated in gill, liver, heart and brain. This work focuses that TiO(2) NPs could translocate among organs and pass through the blood-brain and the blood-heart barrier after long-term exposure. The wide use of TiO(2) NPs in variety of application needs a systematic, coherent and authentic information about the safety of nanoparticles.

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Nanomaterials usually have high reactivity and different physical properties compared with their bulk condition. Toxicity of nanomaterials has recently been reported, and an issue of the effects of prenatal exposure to TiO₂ nanoparticle is emerging. The aim of the present study is to investigate the effects of prenatal exposure to TiO₂ nanoparticle on collagen expression in the kidney. Pregnant ICR mice were exposed to five doses of 100 μg TiO₂ nanoparticle by subcutaneous injection on gestational days 5−17. Kidney tissues were obtained from 3-, 6- and 12-week-old male offspring. Expression of mRNAs and proteins related to collagen was analyzed by quantitative RT-PCR and western blotting, respectively. Matrix metalloprotease (MMP) activity was examined by zymography. Histological analysis was conducted by light microscopy with hematoxylin-eoxin and Masson trichrome staining. The presence of TiO₂ particles in the tissues was determined by field emission-scanning electron microscope/energy dispersive X-ray spectroscopy. TiO₂ nanoparticles, approximately 50−60 nm in diameter, were found in the renal interstitium and perivascular cells of offspring of the exposure group. Col4a1 mRNA expression was increased in the kidney of 6-week-old offspring and Col1a1 and Col3a1 were decreased in 12-week-old offspring of the exposure group. The result showed that prenatal TiO₂ exposure induced accumulated stress on the basement membrane of renal microvasculatures of offspring mice. The protein levels of collagens, the MMP activity and histology were not altered by prenatal TiO₂ exposure. We concluded that prenatal exposure to TiO₂ nanoparticle altered gene expression change of collagen but not induced any histologically detectable abnormality.

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We recently clarified that the side-scatter(ed) light (SS) of flow cytometry (FCM) could be used as a guide to measure the uptake potential of nanoparticles. In this study, the method was improved so as to be able to determine simultaneously the uptake potential of nanoparticles and the production of reactive oxygen species (ROS), and correlations with genotoxicity were evaluated. In the FCM analysis, SS and fluorescence of DCFH-DA based on ROS production were concurrently detected after treatments with ZnO, CuO, Fe₃O₄, TiO₂ and Ag nanoparticles. The ZnO and CuO nanoparticles caused high ROS production, which was more significant in the cells with higher SS intensity. The increase of SS intensity was more significant for TiO₂ than ZnO and CuO, whereas ROS production was higher for ZnO and CuO than TiO₂, suggesting that the extent of ROS production based on the uptake of nanoparticles differed with each kind of nanoparticle. ROS production was correlated with generation of the phosphorylated histone H2AX (γ-H2AX), a marker of DNA damage, and an antioxidant, n-acetylcysteine, could partially suppress the γ-H2AX. This method makes it possible to predict not only uptake potential but also genotoxicity.

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The carbon nanotube is a cylindrical molecule consisting of hexagonally arranged carbon atoms (grapheme sheet) and is a key promising material in nanotechnology. However, elucidation of the effects of carbon nanotube on the respiratory tract is urgently needed. The aim of the present study was to clarify the effects of single-walled carbon nanotube (SWCNT) on genes involved in stress response, by using various human respiratory tract cells; primary cultured normal human bronchial epithelial (NHBE) cells; diseased HBE (DHBE) cells isolated from asthma and chronic obstructive pulmonary disease (COPD) patients, mimicking the inflammatory status of the lung; and the human carcinoma cell lines A549 and FaDu. Cells were exposed to SWCNT manufactured by an arc discharge method with nickel and yttrium as catalysts at concentrations of 0.1 and 1.0 mg/ml for 6 h, and 9 stress-responsive genes were analyzed by real-time polymerase chain reaction. SWCNT down-regulated interleukin-6 expression in all cell types. However, the changes in the expression of metallothionein 2A and tumor necrosis factor-α differed among the cells used in this study. BCL2-associated X protein and BCL2-like1 involved in apoptosis; cyclin D1 involved in the cell cycle; cytochrome P450 1A1 and cytochrome P450 oxidoreductase related to metabolism were also down-regulated in NHBE cells. Primary cultured cells seemed to be more susceptible to SWCNT exposure. SWCNT-evoked down-regulation of stress-responsive genes in both normal and inflamed lung may be involved in the mechanism by which stress response protects against lung injury.

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There are no clear regulations for safety assessment on nanomaterials, and systematic assessment methods are now examined in many countries. In Japan, NEDO project activities discussed extrapolation of 4-week inhalation into 13-week inhalation in rats given the results obtained by intratracheal administration on representative nanomaterials, and proposed determination of period-limited OEL. In the EU, a recommendation on the definition of nanomaterials was adopted by the European Commission in October 2011 while the debate over relation to REACH revision scheduled in 2012 is ongoing. In the US, CNTs and fullerene are regarded as new chemical substances under TSCA Sec. 5, and those which are not listed in the TSCA inventory require submission of a premanufacture notification at least 90 days prior to manufacture or import. Thus, the trend to regulate nanomaterials is intensifying; however, the activities in each country are not harmonized. Given those gaps we examined utility of our newly developed multidimensional evaluation scheme for safety assessment on nanomaterials. This is a scheme for evaluating industrial materials in consideration of manufacturing processes and assessment of nanomaterials included in a finished product. Evaluations on CNTs, fullerene, titanium oxide, silver, and cerium oxide were simulated. The results showed that the scheme’s final stage could be reached in all cases; however, this scheme is conditional upon verification of intratracheal dosing, accumulation of inhalation background data, and development of environmental risk assessment. Therefore, it is considered that technology enhancement on those concerns will allow the scheme to evolve into a more sophisticated design.

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Carbone black particles (CB), which are employed in rubber products and in laser toner, may produce discomfort to the upper respiratory tract. The current IARC evaluation is Group 2B. The present study was conducted to detect carcinogenic activity of carbon black administered by a novel intrapulmonary spraying (IPS). Male and female F344 rats were treated first with DHPN in the drinking water for 2 weeks. The CB suspended in rock candy solution was administered by IPS once every week from week 4 to week 24. Microscopic observation in both male and female showed scattered inflammatory lesions with infiltration of numerous macrophages mixed with a few neutrophils and lymphocytes. Clusters of alveolar macrophages with CB were observed throughout the lung alveoli. Alveolar epithelium cells surrounding the clusters were swollen. This alveolar hyperplasia like lesions (AHLL) were observed in the rats treated with CB regardless of DHPN treatment and these AHLL were located independently of DHPN-induced lesions such as alveolar hyperplasias, adenomas and adenocarcinomas. The average numbers of AHLL in the lung were higher than that of DHPN-induced lesions. Although IPS of CB did not increased the multiplicity of DHPN- induced lesions, this IPS treatment increased the average number of the combined lesions (AHLL + DHPN-induced). These results suggest that it is important for the evaluation of the carcinogenic activity of carbon black to determine whether the AHLL are preneoplastic or not.

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Quantum dots (QDs) is one of most utilized nano-material in colloidal nanocrystalline semi-conductors. It was suggested that QDs potentially have applications in multimodal contrast agents in drug deliver and bioimaging. Since QDs have potential benefits in drug targeting and biomedical imaging, the toxicities of QDs shall be carefully evaluated. QD705 emitted near-infrared fluorescence at 705 nm and can be applied as a probe for detecting vasculature and imaging in biological system. In our present study, we elucidated the cytotoxic mechanisms of QD705-COOH in a mouse renal adenocarcinoma cell line RAG. QD705-COOH increased intracellular reactive oxygen species (ROS) levels, and autophagy formation at 6 hrs, with subsequent cell death at 24 hrs in RAG cells. While P38 MAPK phosphorylation was elevated at 6h after treatment, AKT and ERK phosphorylations remained unchanged. QD705-COOH entered cells and distributed to the endoplastic reticulum (ER) at 6 hrs and re-distributed to the mitochondria at 24 hrs. However, QD705-COOH only affected mitochondrial function, instead of inducing ER stress. N-acetylcysteine (NAC), an antioxidant agent, reduced intracellular ROS levels, decreased QD705-COOH-induced autophagy formation, but enhanced QD705-COOH-induced cell death. NAC also prevented QD705-COOH-induced P38 phosphorylation. It appears that ROS has essential role of regulating QD705-COOH-induced autophagy formation, which subsequently enhanced cell survival. These results suggest that oxidative stress-induced autophagy is a defense (survival) mechanism against cytotoxicity of QD705.

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Nanotechnology and the nano-scale materials were widely used for the development of biomedical products in recent years. Some products, like superparamagnetic iron oxide nanoparticles (SPION), have got the on-market approval from the national authorities after reviewing under current regulatory system. However, many studies showed that the materials will change their physical-chemical characteristics when they become into nano-scale. We are interested to ensure the safety of the biomedical products with nanotechnology. In this study, we used two hepatocarcinoma cell lines, the HepG2 and Huh-7, to establish a cytotoxicity platform for evaluating the physiological and toxicological responses of the cells treated with the SPION. Our results showed growth inhibitions in both HepG2 and Huh-7 cells treated with SPION. The results also showed that the cells treated with 20-nm SPION could induce a higher level of growth inhibition than treated with 10- or 5-nm SPION. The mouse embryonic stem cells (ESC) were also used to evaluate the SPION induced response. The results showed SPION could inhibit the normally differentiating process of mouse ESC. In addition, the results of MTT assay indicated that the SPION could inhibit the cellular mitochondria activities in both HepG2 and Huh-7 cells. The DNA fragmentation analysis also showed the size-dependent DNA ladder phenomena in SPION treated HepG2 or Huh-7 cells. Taken together, we speculated the SPION could induce cellular toxicological events in hepatocarcinoma cells through the damage of mitochondria. However, the mechanism of how the SPION induced toxicological responses in HepG2 and Huh-7 cells needs further studies to elucidate.

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Physicochemical properties of nanomaterials are crucial factors to affect their biological responses. However, some properties are still relatively lack of concern, especially in the surface characteristics of nanoparticles (NPs) which modified with various functional groups for the application of environmental usage or drug delivery. Therefore, in this study we evaluated cytotoxicity of ZnO NPs with diameters of approximately 9 nm, containing different surface groups (triethanolamine (TEA), polyethylene glycol 2000 (PEG), sodium dodecyl sulfate (SDS) and oleic acid (OC)). Infrared spectroscopy (IR) and X-ray photoelectron spectroscopy (XPS) were used to confirm their bindings on the NPs. The effects of NPs coated with or without these surface groups on mitochondrial activity, release of pro-inflammatory factor, intracellular reactive oxygen species (ROS), and DNA damage in A549 cell line were determined by MTT assay, Interleukin-8 (IL-8), 2’,7’-dichlorodihydrofluorescein (DCF) and 8-hydroxy-2’-deoxyguanosine (8-OHdG), respectively. The cytotoxicity (24 h EC₅₀ value, μg/mL) in decreasing order were uncoated ZnO (38), PEG-ZnO (44), TEA-ZnO (45), SDS-ZnO (52), OC-ZnO (56). This could be attributed to the significant decrease in surface charge (especially for hydrophobic SDS- and OC-ZnO) and then the secondary size of ZnO NPs in medium become larger. Uncoated NPs induced the most amounts of intracellular ROS and also 8-OHdG, while the modified NPs showed no significant differences in these oxidative effects. In addition, high correlations between ROS and both cytotoxicity and genotoxicity indicate that NPs-induced ROS might be the main factor to cause the biotoxicity. Interestingly, different from cytotoxicity results, hydrophilic ZnO NPs (TEA, PEG) elicited the most amount of IL-8, which suggested NPs with surface groups might modulate different biological pathways in cells.

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Although the whole body inhalation is the most important route of exposure for the toxicological testing of multi-walled carbon nanotube (MWCNT), inhalation studies are still limited primarily because of technical difficulties in controlling the amount of free single MWCNT fibers and aggregated in the air. We took a new approach to resolve the problems by developing an inhalation exposure system using the Acoustical Dust Generator, originally developed by the National Institute for Occupational Safety and Health. For this system, we have developed unique exposure chambers which can load 16 mice/chamber. The exposure concentration was maintained at a constant value by feedback control to the dust generator’s excitation voltage based on relative concentration (cpm) readout of a particle counter (minimum detectable diameter: 0.3µm) monitoring the chamber air. First, we used MWCNT bulk material (MWNT-7, Mitsui) and obtained weight concentration of 0.3 mg/m³ when the relative concentration was set 200,000cpm/cf. Particle distribution and morphology of the MWCNT aerosol of the exposure chamber were measured by scanning electron microscope (SEM); 80% was dispersed single fibers, the length E10µm accounted for 60% and F20µm was 15%. We developed a method to treat lung samples for easier SEM detection of MWCNT. We also developed a novel dispersion method (Taquann Method). In our second preliminary inhalation trial, the MWCNT dispersed with Taquann method were hardly detected by the original particle counter because of very high dispersivity. We are now equipping our system with a ultrafine particle detector for precise feedback control of the Taquann-dispersed MWCNT.

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Nano material attracts attention by the application to electronic products, medical supplies, cosmetics but adverse effect is not fully examined scientifically. Our laboratory has been taking the part of responsibility for adverse effect evaluation of nano materials, and has reported that intraperitoneal injection of the multi-walled carbon nanotube (MWCNT) induced mesothelioma in rat. Here we report the teratogenic study of MWCNT as part of the nano material safety assessment in our laboratory. MWCNT (Mitsui MWCNT-7, Lot No. 060125-01k) was suspended in 2% carboxymethylcellulose sodium solution and was given intraperitoneally at 2, 3, 4 and 5mg/kg body weight or intratracheally at 3, 4 and 5mg/kg body weight to the ICR mouse on day 9 of gestation. In the intraperitoneal experiment, the increase in the number of early death of fetus and reduction of survival fetus were seen dose-dependently, and malformations, such as reduction deformities of limb, cleft palate or fusion of vertebrae and rib, occurred in all dosed groups. In the intratracheal experiment, which was conducted supposing the exposure path in humans, the same malformations occurred in 4 and 5mg/kg groups but statistically significant malformation was not seen in the 3 mg/kg group. Although the amount of human exposure of MWCNT is not yet clear, there is a report which presumed the amounts of occupational exposure to be 0.58-6.20 μg/kg body weight / day. The 3 mg/kg body weight of the no observed adverse effect level (NOAEL) obtained by the intratracheal experiment of this study corresponds to 480 to 5170 times high. Although it became clear that teratogenicity was observed under the certain conditions, it does not need to be anxious about the direct teratogenicity to the humans of MWCNT immediately.

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Silver nanoparticles (AgNPs) are being increasingly used in commercial products (e.g., cosmetics, textiles, and food containers) due to their antimicrobial activities, and thus, more information is needed on their toxicologic properties. In this study, we investigated the toxic effects of AgNPs (7.5, 30, and 60 nm) in combination with manganese (II) chloride or zinc sulfate. The sizes and morphologies of AgNPs were characterized by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy. For cytotoxicity evaluations, MTT assays were conducted using the human carcinoma cell lines (A375p, A549, HepG2, and ACHN). In addition, MnCl₂ or ZnSO₄ was co-treated with AgNPs to determine whether the toxicities of the AgNPs were modified. Intracellular reactive oxygen species (ROS) generation and oxidative stress were determined using a DCF-DA assay, a GSH assay, and a catalase assay. Cell cycle analysis was carried out using flow cytometry and propidium iodide (PI) staining. The genotoxicity of AgNPs was evaluated using a chromosomal aberration test and CHL/IU cells. MTT assays showed that 7.5 nm AgNPs were more toxic to the human carcinoma cell lines than 30 or 60 nm AgNPs, and that 30 nm AgNPs were more toxic than 60 nm AgNPs. Cell viability after treatment with AgNPs was lower for HepG2 cells than the other cell lines. In terms of manganese co-exposure, the cytotoxicity of AgNPs was increased in all four cancer cell lines examined. However, its cytotoxicity was decreased in the presence of zinc in the A375p and HepG2 cell lines. ROS levels and oxidative stress were increased in A375p cells by AgNPs, and oxidative stress induced by AgNPs was increased in the presence of manganese, but decreased in the presence of zinc. Cell cycle delays in the S and G2/M phases were observed in A375p cells treated with AgNPs, but these delays were ameliorated by manganese and zinc. In the chromosomal aberration test, AgNPs induced numerical chromosome aberration and endoreduplication, and these were prevented by zinc and by manganese. Furthermore, the depletion rates of tubulin treated with AgNPs were lower than in controls. In addition, the addition of Mn or Zn to tubulin treated with AgNPs restored tubulin depletion rates to control levels. This study shows that the toxicity of AgNPs is due to the induction of oxidative stress and depleted tubulin following cell cycle changes. Furthermore, these toxic effects appeared to be reduced by zinc but not by manganese due to their effects on ROS generation.

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Zinc oxide nanoparticles (ZnO) are used in an increasing number of industrial products such as paint, coating, cosmetics and other biological application. However, there is not enough information regarding the safety issue. Therefore, this study was designed to investigate the potential harmful effects of ZnO in normal skin using murine normal cell (JB6 Cl 41-5a). Our results showed that ZnO induced reactive oxygen species (ROS). Such increased ROS was closely associated with autophagy induction together with mitochondrial damage. We strongly suggested that ZnO nanoparticle may cause toxicity, thus, careful regulation for ZnO production as well as application should be needed.

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To assess the repeated-dose toxicity of fullerene C60 by oral exposure, an OECD guideline test (TG 407) was conducted with administration of fullerene C60 at 0 (vehicle: corn oil), 1, 10, 100, or 1,000 mg/kg/day for 29 days in rats. No changes were observed in clinical observations, body weights, and food consumption in any treatment groups. Moreover, no treatment-related histopathological changes were found in any organs examined. Blackish feces and black contents of the stomach and large intestine were observed in males and females at 1,000 mg/kg/day in the treatment group. No significant changes in any organ weights were observed at the end of the administration period, but the only organ weights in liver and spleen in the 1,000 mg/kg/day group of male were increased at the end of the recovery period (14 days after the treatment period). The causal relationships between the possible absorption of fullerene C60 and those weight changes were suggested, but the pathological findings as indirect influences, such as swelling and congestion, were not also observed in the those organs. Using LC-MS/MS spectrometry analysis, fullerene C60 were not detected in the liver, spleen or kidney at the end of the administration period and also at the end of the recovery period. In conclusion, the present study revealed no toxicological effects of fullerene C60; however, with the prospective exposure by increased uses in future because of low toxic substance, more long-term exposure study is necessary to clarify the effects of fullerene C60 via oral exposure.

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Viral-induced infectious diseases represent a major health threat and their control remains an unachieved goal, duein part to the limited availability of effective anti-viral drugs and measures. The use of natural products in drug manufacturing is an ancient and well-established practice Acer tegmentosum is a type of deciduous tree that grows in Korea. It has been used in traditional medicine for the treatment of hepatic disorders, such as hepatitis, hepatic cancer, and hepatic cirrhosis. In this study, we report on the antiviral potency of an aqueous or ethanol extract of stem of Acer tegmentosum (At-AE or At-EE) against herpes simplex virus (HSV-1 and HSV-2) and varicella zoster virus (VZV) in cell culture (Vero and MRC-5 cells) using a plaque reduction and MTT assay. Hydroalcoholic extracts of Acer tegmentosum were phytochemically characterized by HPLC-MS analyses. Pretreatment of Vero cells with At-AE or At-EE significantly reduced HSV-induced cell death. At-AE or At-EE exhibited high levels of antiviral activity against both types of herpesvirus in a dose-dependent manner. At maximum noncytotoxic concentrations of the extract, plaque formation was significantly reduced by At-AE or At-EE for HSV-1 and HSV-2, and VZV in a concentration-dependent manner. Hence this extract is capable of exerting an antiviral effect on herpes simplex virus and might be suitable for topical therapeutic use as antiviral drug both in labial and genital herpes infection.
Keywords: Acer tegmentosum; Antiviral; herpes simplex virus; varicella zoster virus

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Are reactive oxygen species (ROS) toxic to human? ROS have been recognized to be “toxic/harmful” substances because of their potent oxidant property capable of damaging cellular components including DNA and protein. However, accumulating experimental evidence indicates that ROS may play important roles in regulation of cell signal transduction processes. We reported that (–)-xanthatin, a naturally occurring sesquiterpene lactone found in Cocklebur, can selectively induce GADD45γ, a novel tumor suppressor gene, in human breast cancer cells (Takeda et al., Chem. Res. Toxicol., 24:855–865, 2011). We have recently found that (–)-xanthatin inhibits topoisomerase IIα (Topo IIα), which may lead to induction of GADD45γ. Here, these studies have been further extended to reveal an association between (–)-xanthatin-mediated Topo IIα inhibition and GADD45γ induction. By performing experiments comparing (–)-xanthatin and its related structural analogs, (–)-xanthatin exclusively up-regulated intracellular ROS levels with concomitant Topo IIα inhibition. Given the reported very short half-life of GADD45γ mRNA (~60 min), the observation that (–)-xanthatin-mediated induction of GADD45γ was detected even after 48 h exposures, led us to hypothesize that ROS may be involved in (–)-xanthatin-mediated induction of GADD45γ. The results obtained demonstrated that (–)-xanthatin-derived ROS substantially stabilized GADD45γ expression at both mRNA and protein levels. Taken together, the data strongly suggest that (–)-xanthatin exerts anti-cancer effects via “ROS-assisted” stabilization of the tumor suppressor, GADD45γ, subsequent to its initial induction by Topo IIα inhibition, and that this ROS signal is a “positive” mediator of the anti-cancer process.

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Angiogenesis, the formation of new capillaries from preexisting blood vessels, plays an important role in physiologic and pathologic processes, such as the embryonic development, wound healing, tumor growth, metastasis, and various inflammatory disorders. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis induction of these angiogenic processes. Thus, inhibition of these critical angiogenic steps is a practicable therapeutic strategy for these diseases. Antrodia cinnamomea in the Polyporaceae, Basidiomycotine family, is a native mushroom parasitic on the endemic perennial tree, Cinnamomum kanehirai Hay in Taiwan. 4AAQB is an ubiquinone-derivate isolated from antrodia cinnamomea which have been shown to antioxidation, anti-inflammation and antitumor activities. In this study, 4AAQB showed an inhibitory activity on migration and tube formation of human umbilical vascular endothelial cells in a concentration-dependent manner (0.3-10 µg/ml). Using aortic ring sprouting, and mouse matrigel implant models, 4AAQB significantly inhibited VEGF-induced neovascularization (0.3-10 µg/ml). In addition, VEGF-induced ERK 1/2 and PI3K p85α tyrosine phosphorylation were blocked by 4AAQB. These results indicate that 4AAQB is a potential candidate for the treatment of angiogenesis related-diseases.

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Despite the growing popularity of herbal medicines and food supplements, their adverse effects on cardiovascular homeostasis remain largely unknown, especially regarding their pro-thrombotic risks. Here, through screening of the extracts from 21 herbal teas widely consumed, we discovered that Dipsacus asper (DA), previously known to have analgesic and anti-inflammatory efficacy may induce procoagulant activity in platelets, a critical promoter of thrombosis. Dipsacus saponin C (DSC) was identified as the key active ingredient for DA-induced procoagulant activities through activity-guided purification.
DSC- and DA-induced procoagulant activities were achieved by the exposure of PS and MP generation that were caused from the alteration in activities of scramblase and flippase. These events were initiated by increased intracellular calcium and ATP depletion. Notably, DSC induced a series of apoptotic events including the disruption of ΔΨ_m, mitochondrial translocation of Bax, cytochrome c release, and caspase-3 activation. Key roles of apoptosis and caspase activation were demonstrated by the reversal of DSC-induced PS exposure and procoagulant activities with the pretreatment of caspase inhibitors. These results were also confirmed in vivo where the rats exposed to DSC exhibited ΔΨ_m dissipation and PS exposure in platelets. Most importantly, DSC or DA treatment led to increased thrombus formation in rat venous thrombosis model, demonstrating that herbal medicines or natural products like DA or DSC might have prothrombotic risks through procoagulant activation.

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Immune system is our body’s natural defense system to protect us from invaders such as viruses, bacteria and other agents that have the potential to make trouble in body. The present studies evaluated that WK4, herbal extract, could enhance immunostimulating activity such as the phagocytic uptake and release of cytokine secretion in macrophage. Western blot was used to detect the expression of protein related with macrophage activation and examined defense effects of WK4 on bacterial infected mice model. We observed that WK4 enhances phagocytic activity in peritonral macrophages. During the phagocytotic process, production of NO and TNF-a were increase. WK4 significantly induced the phosphorylation of signal molecules of MAPK (ERK, JNK, p38). WK4 was very effective for preventing pathogenic S. Typhimurium infection in mice. Animal weight loss by infection was monitored. The body weight of infectious mice was significantly decreased, but WK4 treated mice exhibited significant improvement in maintaining body weight. When the mice were treated with WK4 for 5 days, the release of TNF-α, IFN-γ and IL-12 were increased significantly compared to the untreated group. After infection, plasma level of IFN-γ , IL-12, TNF-α in WK-4 treated group were higher than infection control group. Higher TNF-α level in treated mice could enhance bactericidal activity synergistically with IFN-γ and trigger nitric oxide production. In conclusion, WK4 enhanced non-specific immune activities and resistance to an experimental challenge with S. Typhimurium infection.

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Breast cancer is one of the most common cancers in the world, and its occurrence has been increasing in recent years. Although surgical resection and chemotherapy are common options for carcinoma, surgery is the treatment choice for only the small fraction of patients with localized disease. While Cytotoxic chemotherapy agents are minimally effective at improving patient survival, chemotherapy drugs can damage normal cells. Thus, it is very important to find a new agent for cancer treatment. Lantana camara L. is a species of flowering plant in the verbena family. Verbenaceae is native to the American tropics. Until now, there is no study on the antitumor effect of Lantana camara L.. To find the antitumor effect Lantana camara L., the anti-proliferative activity was assayed using MTT assay. Cell cycle analysis was done using flow cytometry and apoptosis detection was done using by fluorescent staining and Annexin V-propidium iodide double staining. Western blot was used to detect the expression of protein related with apoptosis. Lantana camara L. have the effect of anti proliferation with IC50 value of 63.90 μg/mL. It induced G2/M phase arrest through the modulation of cell cycle related proteins and apoptosis activation. These finding suggest that Lantana camara L. should be useful as a chemotherapeutic agent for breast cancer patients.

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The risk of bleeding is one of the major clinical problems of anti-platelet agents such as aspirin and clopidogrel which modulate platelet aggregation induced by traditional chemical agonists. Targeting shear stress-induced platelet aggregation, primarily occurring under abnormally high shear stress in atherosclerotic lesion, may be an attractive alternative for the development of anti-thrombotic agents with reduced bleeding risk. Here we demonstrated novel anti-platelet and anti-thrombotic activities of protocatechuic acid (PCA), a bioactive component from herbal materials, through its selective inhibition of shear-induced platelet activation and aggregation. In isolated human platelets, PCA decreased high shear stress-induced platelet change including intracellular calcium mobilization, granular secretion, adhesion receptor expression, and aggregation. The inhibitory effect of PCA was mediated through blocking of the initial interaction between von Willerbrand factor (vWF) and platelet receptor GP Ib. Notably, PCA did not affect platelet aggregation induced by chemical agonists, demonstrating its selective protection against shear-induced platelet activation. In vivo relevancy was examined using rat arterial thrombosis model, where PCA showed significant anti-thrombotic activity. While aspirin and clopidogrel prolonged bleeding time in rat tail transection model, PCA did not change bleeding time suggesting the reduced risk of bleeding. With this study, we believe that novel therapeutic potential of PCA was elucidated, supporting its possible application as an anti-platelet and anti-thrombotic drug candidate.

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Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is latent but constitutively activated in many types of cancers. It is well known that STAT3 plays a key role in inflammation-associated tumorigenesis. Curcumin, a natural compound isolated from the traditional Indian spice Curcuma longa (Zingiberaceae), have anti-tumor, anti-proliferative and anti-inflammatory properties. This compound has been extensively used as a traditional medicine over the centuries mainly due to its pronounced therapeutic effects. Many studies suggest that curcumin regulates various cancer-related intracellular signaling without harming normal cells and tissues. Here, we report that curcumin effectively inhibits activation of STAT3 in dose- and time-dependent manners and causes apoptosis of H-Ras transformed breast epithelial cells (H-Ras MCF10A). Specific cysteine residues present in STAT3 appear to be critical for the activity as well as conformation of STAT3. We identified cysteine residue 259 as a putative target of curcumin. We found that α,β-unsaturated carbonyl moiety of curcumin appears to be essential in its direct binding with STAT3. Tetrahydrocurcumin that lacks the electrophilic α,β-unsaturated carbonyl moiety failed to inhibit STAT3 activation and to induce apoptosis in the same cell line. Therefore, our findings suggest that curcumin can abrogate persistently activated STAT3 through direct interaction, thereby inhibiting STAT3-associated carcinogenesis.

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Purpose: Many post-menopausal women experience some symptoms such as hot-flush, insomnia and osteoporosis, because of the decreased production of estrogens. To relief such symptoms, natural and synthetic estrogens have been subscribed for patients as the hormone replacement therapy. However, many epidemiological studies have revealed long-term treatment of estrogens increased the risk of several cancers. Therefore, alternative estrogens, which are estrogenic but no carcinogenic, are needed. One candidate of them would be phytoestrogens, some of which are reported to reduce breast cancer risk. Propolis, a natural product derived by honey bees from plants, is a mixture of several hundred-kinds of chemicals including phytoestrogens. In this study, we determined estrogenic activity of propolis using several in vitro and in vivo assays.
Methods: Ethanolic extract of Blazilian propolis (EEP) was used for assays directly or after fractionation using HPLC. Estrogenic activity was determined using following assays, 1) estrogen receptor (ER) competitive binding assay, 2) estrogen-dependent luciferase reporter gene assay, and 3) epithelial cell proliferation of mammary gland and uterus in ovariectmized (OVX) rats.
Results and discussion: EEP induced epithelial cell proliferation in mammary gland and uterus in OVX rats. This effect was diminished by pretreatment of ICI182,780 (ICI). EEP induced ER binding and reporter gene expression in in vitro assays, and five fractions of EEP isolated by preparative HPLC showed unequivocal ER binding. One fraction contained kempferol and an unknown estrogenic compound. These results suggest the daily intake of EEP would relief post-menopausal symptoms.

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Objective: We aim at developing a new method to decoct traditional medicine with a microwave oven for patients’ convenience. We previously investigated the effective components in Kakkonto to confirm the effectiveness of this new decoction method. In this study, we investigated toxic components in Keishikabushito to confirm the safety of this method.
Method: According to preliminary experiments, we decocted a daily dose of Keishikabushito containing 1 g of processed Aconite root (Bushi) by a microwave oven in 600mL water at 500 W for 30 min and conventional decoction device in 500mL water at 600 W for 40 min, respectively. Then, the contents of toxic components from Bushi (aconitine, mesaconitine, hypaconitine), and three other effective as well as toxic components (benzoylaconine, benzoylmesaconine, benzoylhypaconine), the hydrolysates of the three toxic components in heating process, were analyzed with HPLC method.
Result: In both decoctions obtained by the new and conventional methods, aconitine, mesaconitine and hypaconitine with strong toxicity couldn’t be observed because of hydrolysis in heating process. Benzoylaconine, also not observed, may be further hydrolyzed. The contents of benzoylmesaconine and benzoylhypaconine were 0.68±0.06 mg, 4.75±0.15 mg in decoction decocted by a microwave oven, and 0.70±0.01 mg, 4.89±0.12 mg in decoction decocted by the conventional method (n=5).
Conclusion: There was no significant difference of the contents of toxic components between the new and conventional methods. Decocting Keishikabushito with a microwave oven is as safe as the conventional method. In addition, the decoction time can be saved. This new decoction method may be applied widely.

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Garlic extracts have been reported to show hepatoprotective activities. In this study, we investigated the protective effects of thiacremonone, a sulfur compound isolated from garlic, on acetaminophen (APAP)-induced hepatotoxicity in C57BL/6. After pretreatment of thiacremonone (50, 100 and 200 mg/kg) for 5 days, mice was fasted then given 500 mg/kg APAP via intraperitoneal injection. We measured GSH/GSSG ratio, survival rate, aspartate aminotransferase (AST) level, alanine aminotransferase (ALT) levels and performed histologic analysis. GSH/GSSG ratio was decreased from 12.9 to 8.7 in APAP–treated mice, but decrease slightly from 12.9 to 10.75 in thiacremonone-pretreated mice. AST level was increased from 8.6 to 11754 in APAP treated mice, but decreased dose-dependently to 4264 in 200mg/kg thiacremonone treated mice. ALT level also was increased from 3 to 18631 in APAP treated mice, but decreased dose-dependently to 6185 in 200mg/kg thiacremonone treated mice. APAP treated mice showed around 30% survival rate after 48hr, however 200mg/kg thiacremonone treated mice showed 80% survival rate. Furthermore histological analysis of liver sections revealed considerably decreased liver injury in thacremonmone-pretreated mice. APAP-induced activation (increase of kupffer cells and NKT cells number) of kupffer cells and NKT cells was prevented in Thiacremonone-pretreated mice. In cytokine array, we found that I-309, IL-7, IL-13, IL-17, M-CSF, MIG, and MIP-1 α, β was induce by APAP but recovered by thiacremonone. There results demonstrate that thiacremonone prevent APAP-induced hepatotoxicity via reduction of activations of NKT cells and kupffer cells through reduction of hepatotoxicity-inducing cytokine, IL-17, M-CSF, MIG, MIP-1α, and MIP-1β.

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Intestinal microflora (IM) plays a considerable role in the metabolism of endogenous and exogenous substances in the diet, and is thought to have both beneficial and deleterious effects on human health. IM are able to produce toxic or carcinogenic metabolites and induced more potent cytotoxicity against cells than non-metabolites. This study was conducted to investigate the cytotoxic responses of geniposide and its metabolites (genipin) to determine the metabolism of cytotoxic activities. Genipin, geniposide metabolites, increases strong cytotoxic effect in hepatoma cells, but not with geniposide. Western blot analysis revealed that the cleaved-caspase-3 and bax protein significantly increased in a dose-dependent manner after treatment of activated GS for 24 h. However, GS alone did not induce cytotoxicity as well as caspase-3. In addition, activated GS-induced apoptosis was confirmed by apoptosis assays. In this study, GP or activated GS caused reactive oxygen species (ROS) generation and the activated GS-induced cell death was prevented by antioxidants N-acetylcysteine (NAC) suggesting involvement of ROS generation in GP or activated GS-induced cell death. GP or activated GS induced a sustained activation of the phosphorylation of JNK, but not p38 and ERK. Moreover, activated GS-induced cell death was reversed by inhibitor of JNK, but not by p38 or by ERK inhibitor. Taken together, these findings suggest that the human IM is capable to metabolize GS to GP and its related biological activities and induced apoptosis through a ROS/JNK signaling.

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Curcumin (Cur), a naturally occurring polyphenol, is one of the most extensively investigated photochemicals with chemopreventive effect. Quantum Dots (QDs), a promising nanoparticle as a photosensitizer, have been used in photodynamic therapy (PDT). Although the cytotoxicity of QDs has been studied, little is known about the genotoxic effect of QDs on human health. Moreover, the effect of co-treatment with QDs and Cur under UVA has not been studied yet. In this study, we examined the cytotoxic and genotoxic effect of CdSe/ZnS QDs and/or Cur under the UVA (2J/cm²) to investigate the selective effect on HL-60 cells and normal human lymphocytes.
The cell viability was measured by WST assay and the apoptotic cell death was investigated by flow cytometry. We performed comet assay and micronuclei assay to examine genotoxic effect. Treatment with QDs under the UVA decreased cell viability and increased apoptotic cell death in HL-60 cells and normal human lymphocytes. Cur and QDs/UVA decreased cell viability and induced apoptosis compared with the QDs/UVA in HL-60 cells, whereas no significant cell death was observed in normal human lymphocytes. When HL-60 cells were exposed to Cur and QDs/UVA, the frequency of micronuclei and induction of olive tail moment were increased.
In conclusion, Cur and QDs/UVA enhanced more cell death than QDs/UVA in HL-60 cells. This suggests that a treatment of Cur could be useful to increase the efficacy in PDT.

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The oral adminstration of five leaf extracts namely Cathranthus roseus, Datura metal, Delonix regia, Eucalyptus globules and Pongamia glabra on efficiency of total free amino acids in the hemolymph of Pericallia ricini, a serious lepidopteran pest on castor Ricinus communis, was assessed. The total amino acids showed quantitative difference in the hemolymph of normal and treated larvae. HPLC Analysis of the haemolymph of P. ricini revealed the presence of 17 aminoacids. Serine, Histidine, Glutamic acid, Aspartic acid, Arginine and Methionine exhibited quantitative decrease while Alanine, Phenylalanine, Isoleucine, Leucine showed an obvious increase after treatment of the plant extracts. Some amino acids such as Asparagine, Glutamine, Threonine, Valine, Ornithine were found in plant extract treated sample but it was absent in control insects. Along with the depletion of amino acids, there was also a drastic change in the protein profile of the hemolymph of P.ricini. It is evident that the plant extracts induced significant reduction in protein content and amino acids. The depletion in amino acids adversely affected the protein turnover in P. ricini. The depletion of aminoacids and protein due to plant extract treatment caused an upset in the balance of set patterns of growth in treated insects. It is suggested that plant origin insecticides isolated from C. roseus, D. metal, D. regia, E. globules and P. glabra may be used as an alternatives in the management of insect pest P. ricini because they will be highly economical and less hazardous to the environment.

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In an attempt to dissect the mechanism of Strychnos nux-vomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, on HepG2 cells were screened by MTT assay. Brucine, among the four alkaloids, exhibited the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucine-treated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular [Ca²⁺], which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of [Ca²⁺]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca²⁺ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis.

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Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes plays a critical role in the development of these inflammatory lesions. The present study was conducted to evaluate the antimicrobial property of honeybee (Apis mellifera L.) venom (BV) against the etiologic agents of acne vulgaris and the pharmacological activities of honeybee (Apis melifera L.) venom (BV) have been used in wound healing for centuries. Incubation of the skin bacteria P. acnes, clindamycin-resistant P. acnes, Staphylococcus epidermidis or Streptococcus pyrogenes with BV yielded the minimal inhibitory concentration (MIC). Production of inflammatory cytokines (interleukin-8 (IL-8) and tumor necrosis factor- α (TNF-α)) were examined in THP-1 cells. To study wound healing, full thickness skin defects were produced on the dorsal area of mice. The wound sizes were small in the BV group compared to the Control and Vaseline groups. The BV group demonstrated decreased TGF-β1, fibronectin and VEGF mRNA levels and increased collagen-I mRNA levels. The expressions of TGF-β1, fibronectin and VEGF proteins was significantly lower in the BV group compared to the Control group, while the expression of collagen-I was increased in the BV group as indicated by immunohistochemical staining. These data suggested that BV has effective antimicrobial and anti-inflammatory activity against P. acnes, and we suggest that BV is an alternative treatment for antibiotic therapy of acne vulgaris and significant wound healing activity.

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Ginseng is traditional medicinal herb for physical strength, digestive organs, nervous system, metabolic system and circulatory system. Black ginseng is produced from ginseng by nine repeated steaming-drying cycles. The pharmacological and biological activities of black ginseng are believed to be better than those of ginseng or red ginseng. Red ginseng is the most commonly used health functional food in Korea and its functionality was demonstrated through many studies, while black ginseng is little reported on its functionality scientifically. Therefore we tested the prevention effect of acute hepatitis induced by CCl₄ for 10-day oral-repeated trial of black ginseng in male Sprague-Dawley rats (0, 1, 3, 10 ml/kg/half day). Hepatotoxicity was induced by CCl₄ (1 ml/kg, ip) and positive control was treated with silymarin (100 mg/kg, po). After administration of black ginseng, serum alkaline phosphatase (ALP), alanine aminotransferas (ALT), aspatate aminotransferase (AST), total bilirubin (T-Bil), gamma-glutamyl transferase (γ-GTP) and hepatic histopathologies were examined. Urinary metabolite profiles were obtained by ¹H NMR analysis. Serum enzymes and hepatic histopathologies showed dose-dependent protective effects of black ginseng. Urinary metabolomics profiles might provide mechanistic insight of black ginseng. These results suggest that black ginseng might use for prevention of acute hepatitis.

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Thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are related with AD and are elevated in serum and lesional skin of AD patients. Citrus unshiu (CU) contains various flavonoids and the content is high at premature fruits rather than mature fruits. In present study, we investigated the effect of quercetagetin, a component of premature CU, on the production of TARC and MDC in HaCaT human keratinocytes. As results, quercetagetin showed inhibitory activity on the protein production and mRNA expression of TARC and MDC in IFN-γ and TNF-α-stimulated HaCaT human keratinocytes. Also, quercetagetin inhibited the phosphorylation of STAT1, key transcription factor initiating IFN-γ signaling pathway, in a time- and dose-dependent manner. Moreover, quercetagetin augmented the mRNA expression of TGF-β1 that down-regulates the expression of TARC and MDC. These results suggest that quercetagetin may have an anti-atopic activity by inhibiting the inflammatory chemokines (TARC and MDC) via the regulation of STAT1 and TGF-β1.
Key words : Quercetagetin, Citrus unshiu, Atopic dermatitis, TARC/CCL17, MDC/CCL22, Jak-STAT pathway, TGF-β1

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Ginseng is the most commonly used herbal products in Korea. It has many pharmacological activities, but ginseng is not free from adverse effects, possibly through drug-drug interaction (DDI). Various studies reported that ginseng inhibits CYP450s. The objective of this study was to determine the effect of red ginseng (p. ginseng) extract on the toxicokinetic of deltamethrin in male Sprague Dawley adult rats. Deltamethrin (DLM) [(S)-a-cyano-3-phenoxybenzyl-(1R, cis)-2,2-dimethyl-3-(2,2-dibromovinyl)-cyclopropane-1-carboxylate] is the synthetic type II pyrethroid insecticide and it is one of potent neurotoxicants. The rats were orally administered the red ginseng extract at 0, 10, 100 mg/kg for 2 weeks, followed by oral administration of DLM at 10 mg/kg in glycerol formal. Toxicokinetic parameters of DLM in plasma were not significantly changed by treatment of red ginseng extract. The present results indicated that red ginseng extract has no significant effect on toxicokinetics of DLM.

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Nitric oxide (NO) is an important cellular signaling molecule in inflammatory diseases. It is synthesized from L-arginine by a specific enzyme, NO synthase (NOS) that is composed three types, endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Among them, iNOS is activated by various stimuli such as lipopolysaccharide (LPS), a wall component of gram-negative bacteria, in macrophage. LPS binds a TLR4 on the macrophage and activates a down-stream of TLR4 signaling pathway, including mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB pathways. Sargaquinoic acid isolated from S. siliquastrum is known to have various activities, except anti-inflammatory activity. In this study, we investigated the effect of sargaquinoic acid on NO production and the possible mechanism. The results demonstrated that sargaquinoic acid inhibited the production of NO and the expression of iNOS protein in LPS-stimulated RAW264.7 macrophages. Moreover, sargaquinoic acid inhibited the degradation of IκB-α and the translocation of NF-κB, a key transcription factor for regulation of iNOS expression. However, sargaquinoic acid did not influence on the phosphorylation of MAPKs or STAT signaling pathway by LPS-stimulation. These results suggest that sargaquinoic acid specifically prevents NO production in macrophages via the blockade of NF-κB activation and may thus have therapeutic applications in various inflammatory diseases.
Keywords: Sargaquinoic acid, Inflammation, Nitric Oxide, Nuclear factor-κB, RAW264.7 macrophage

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We investigated whether the snake venom toxin (SVT) from Vipera lebetina turanica enhances the apoptotic cell death ability of tumor necrosis factor (TNF)-related cell death inducing ligand (TRAIL) in the cancer cells. TRAIL inhibited HCT116 cell growth dose dependently, but not in TRAIL resistant HT-29, A549 and HepG2 cells with even higher dose of TRAIL. SVT enhanced expression of cell death receptor (DR) in TRAIL resistant cancer cells in a dose dependent manner but not by TRAIL. Combination of SVT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29, A549 and HepG2 cells. Consistent with cell growth inhibition, the expression of TRAIL receptors; DR4 and DR5 was significantly increased as well as apoptotic cell death related proteins such as cleaved caspase-3, -8, -9 and Bax. But the expression of survival proteins such as cFLIP, survivin, XIAP and Bcl2 was decreased by the combination treatment of SVT and TRAIL in HCT116 and HT29 cells. Deletion of DR4 or DR5 by small interfering RNA significantly reversed cell growth inhibitory and apoptotic cell death blocking effects of SVT not only in HCT116 but also in HT-29 cells. Pretreatment of JNK inhibitor SP600125 and ROS scavenger N-acetylcysteine reduced the SVT and TRAIL-induced upregulation of DR4 and DR5 expression and apoptotic cell death related protein expression such as caspase-3 and-9 as well as cell growth inhibitory effects. Our results suggested that SVT facilitates TRAIL-induced apoptotic cell death in cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/ JNK pathway signals.

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Regulatory T cells (Tregs) play key roles in the immune response by preventing or suppressing the differentiation, proliferation and function of various immune cells. Recent studies report that the omega-3 polyunsaturated fatty acids (n-3 PUFAs) in fish oil can reduce inflammation in allergic patients. The beneficial effects of natural fish oil (NFO) have been described in many diseases, but the mechanism by which fermented fish oil (FFO) modulates the immune system and the allergic response is poorly understood. In this study, we produced FFO and tested its ability to suppress the allergic inflammatory response and to activate CD4⁺CD25⁺Foxp3⁺ Tregs. The ability of FFO and NFO to modulate the immune system was investigated using a mouse model of AD. Administration of FFO or NFO in the drinking water alleviated the allergic inflammation in the skin, and FFO was more effective than NFO. Neither FFO nor NFO increased forkhead box P3 (Foxp3) expression and/or affected the number of CD4⁺CD25⁺ T cells without any stimulation. However, FFO treatment did increase the expression of the immune-suppressive cytokines transforming growth factor-β (TGF-β) and Interferon-10 (IL-10). In addition, ingestion of FFO increased Foxp3 expression and the number of CD4⁺CD25⁺Foxp3⁺ Tregs in CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 compared with NFO. These results suggest that the anti-allergic effect of FFO is associated with enrichment of CD4⁺CD25⁺Foxp3⁺ T cells at the inflamed sites and that FFO may be effective in treating the allergic symptoms of AD.

Key word : Atopic dermatitis AD, natural fish oil, fermented fish oil, transforming growth factor-β, forkhead box P3, regulatory T cells

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SKP2 is the substrate recognition subunit of the SCF^SKP2 ubiquitin ligase complex. It is implicated in ubiquitin-mediated degradation of the cyclin dependent kinase (CDK) inhibitor p27^KIP1, and positively regulates the G1/S transition. Overexpression of SKP2 has been found in many kinds of tumors. In the present study, we found that SKP2 expression level increased in esophageal squamous cell carcinoma tissues. Elevated expression of SKP2 correlated significantly with tumor stage and positive lymph node metastasis (p<0.05). Moreover, significantly negative correlation was found between SKP2 expression and the survival of patients who received radiotherapy (p<0.05). At the molecular level, forced expression of SKP2 promoted the radio-resistance ability of EC9706 cells. Knockdown of SKP2 expression sensitized cancer cells to radiation and a wobble mutant of SKP2 that was resistant to SKP2 siRNA can rescue this effect. Increased or decreased expression level of SKP2 had effects on Rad51 expression in the condition of radiation. These results demonstrate for the first time that overexpression of SKP2 correlated with the increased radiotherapy resistance of esophageal squamous cell carcinoma. Elevated expression of SKP2 promoted the radio-resistance of cancer cells and this effect was mediated at least in part by Rad51 pathway.

Keywords: SKP2; ESCC; radiation resistance; Rad51

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Purpose: Radiation therapy is an important cancer modality, especially in the treatment of lung cancer. However, recent studies have shown that radiation can actually increase invasion by several cancer types, including glioma, breast cancer, melanoma, pancreatic cancer and hepatocellular carcinoma. The purpose of this study was to investigate the effect of radiation on the invasion of the pulmonary adenocarcinoma cell line, A549.
Methods and Materials: Invasion of A549 cells irradiated with 2 Gy and 4 Gy doses of γ-ray was detected using transwell matrigel invasion assay. The level of matrix metalloproteinase (MMP) 2 and phosphorylated STAT3 was detected by reverse transcription PCR and/or immunoblotting. The enzyme activity of MMP-2 was examined by gelatin zymography.
Results: The invasiveness of A549 cells was significantly enhanced by γ-ray radiation at dose levels of either 2 Gy or 4 Gy. Furthermore, radiation was found to promote the transcription and expression of MMP-2, and increase MMP-2 enzyme activity. Radiation activated the phosphorylation of STAT3 and promoted the nucleic localization of STAT3. Blocking STAT3 phosphorylation using a special inhibitor (AG490) suppressed the radiation-induced elevation of MMP-2 expression, enzyme activity and invasiveness of A549 cells. Finally, we also found that expression of vascular endothelial growth factor (VEGF) could be up-regulated by radiation, which was also associated with the activation of STAT3.
Conclusions: Our results indicated that radiation resulted in the activation of STAT3, which subsequently entered the nucleus and triggered the transcription of VEGF and MMP-2. This in turn led to the increased invasiveness of A549 cells.

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Deinococcus radiodurans ddrO gene have played an important role in the process to withstand radiation and oxygen free radical damage. PCR amplification ddrO gene and cloned ddrO gene into pGEM-T easy Vector . Shuttle express vector pRADZ3(+)ddrO is constructed and identified by using restriction enzyme SpeI and NdeI double digestion for recombinant T(+)ddrO and shuttle vector pRADZ3 respectively,Connect ddrO fragment and big fragment of shuttle vector pRADZ3 through T4 DNA ligase. Identification by PCR and digestion, confirmed that recombination shuttle vector pRADZ3(+)ddrO constructs successfully. Transforms the recombination shuttle vector to E.coli DH5α competence cell, let it to express the DdrO protein under the normal culture condition (not to need Inducer),and through Western blot confirmed that the ddrO gene can express stably in DH5α. Compare with DH5α which transformed empty vector pRADZ3 ,and observing the change of this two kind of E.coli under the ultraviolet exposure and the H₂O₂ oxidation pressure survival ratio, studies the DdrO protein anti-ultraviolet radiation and the against oxidized damage function in E. the coli DH5α. The experimental result demonstrate that ddrO gene express in E. coli be able to strengthen it ability to bears the ultraviolet radiation and the strong oxidant H₂O₂.

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Although conventional clinical treatment with low LET (linear energy transfer) including gamma-ray and X-ray has been widely used for radiotherapy in various cancers, however, ineffective outcomes occur due to radioresistance caused by p53 mutation. High LET has become alternative since it is able to induce apoptosis regardless of p53 status. Indeed, the molecular mechanisms toward high LET have been suggested. Nevertheless, most studies have been done in monolayer culture system which cannot promptly represent solid tumor microenvironment. Here we applied in vivo mimic 3D spheroid to conduct microarray-based genomic expression and molecular signaling pathway analyses under neutron irradiation. As a result, 3D spheroid system was achieved using thermorevesible gel system. An effective apoptosis-inducible dose of neutron was determined by Acridine Orange (AO) staining in 3D spheroid. Differentially expressed genes in both unique and common responses to neutron were identified in the 3D spheroid compared to the monolayer cells. Total 95 and 169 genes were notably altered at transcription level toward neutron in monolayer and 3D spheroid system, respectively. Based on microarray data, putative apoptosis signaling was depicted using Pathway Studio software. In 3D-in vivo mimic model, the molecular networks interacted with ITGB1, MAP4K4, PAPPA, and SGK1 might be suggested as plausible molecular pathways. In conclusion, we demonstrate novel molecular signaling and corresponding targets of in vitro solid tumor following high LET exposure. This result might provide critical clues for clarification of neutron-induced apoptosis mechanism.

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Osteopontin (OPN) serves as an indicator of resistance to radiotherapy. However, the role of OPN in the development of acquired radioresistance in human lung cancer cells has not yet been fully elucidated. Therefore, the potential importance of OPN as a marker of lung cancer with a potential significant role in the development of radioresistance against repeated radiotherapy has prompted us to define the pathways by which OPN regulates lung cancer cell growth. In addition, autophagy has been reported to play a role in the radiosensitization of cancer cells. Here, we demonstrated that increased OPN expression through induction of nuclear p53 following irradiation was inhibited by exogenous beclin-1 (BECN1). Our results clearly show that BECN1 gene expression led to induction of autophagy and inhibition of cancer cell growth and angiogenesis and the induction of autophagy abrogated the radioresistance of the cancer cells. Interestingly, we found knockdown of OPN by lentivirus-mediated shRNA induced the autophagy of human lung cancer cell. Taken together these results suggest that OPN and BECN1 can be molecular targets for overcoming radioresistance by controlling autophagy.

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It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow cytomery. Recently, the Pig-a mutation assay has been developed to evaluate in vivo genotoxicity in laboratory animals and humans. To date, the assay has been used mainly to assess the mutagenicity of chemicals known to be powerful point mutagens. In this study, we modified the Pig-a gene mutation assay described by Kimoto et al. (Mutat Res 723:36-42, 2011) and used three-color staining with fluorescently labeled anti-CD24, anti-TER-119, and anti-CD71 to detect the Pig-a mutant frequency in total red blood cells (RBCs) and in reticulocytes (RETs) from X-irradiated mice. Evaluating RETs enabled us to determine Pig-a mutant frequency more quickly than using RBCs; however, measuring Pig-a mutant frequencies in total RBCs increased the accuracy and reliability of the assessment. Single exposures to X-irradiation resulted in dose- and time-dependent increases in Pig-a mutant frequency that subsequently were reduced with time until background frequencies were reached. The same total amount of radiation delivered as a single dose or as four repeat doses given at weekly intervals both increased Pig-a mutant frequencies to comparable levels three weeks after the single dose or the last dose of the repeat irradiation; these elevated frequencies subsequently decreased to background levels. Our results indicate that the three-color Pig-a assay will be useful for evaluating the in vivo genotoxicity of radiation.

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Translationally controlled tumor protein (TCTP) has been implicated that are related to cell growth, proliferation, apoptosis, and even the allergic response of the host through interaction proteins. Our data showed that mice continue to highly express TCTP in the brain from embryonic to adult stage. Despite the abundantly expressed TCTP in the brain of mice and the importance of TCTP in the regulation of apoptosis, little is known regarding its role in the neurogenesis of nervous system. We address the function of TCTP in the nervous system using Nestin-cre-driven gene mutated mice. The mice which disrupted TCTP in neuronal progenitors die at perinatal stage. The Nestin-cre; TCTP^f/f pups displayed reduced body size of P0.5 and lack of milk in stomach compared with littermate control. In addition to decrease of cell proliferation, TUNEL and caspase assay reveals that newly committed TCTP deficient cells increase the apoptosis as they commence migration away from the ventricular zone. The mechanisms involved the phenotype of Nestin-cre dependent knockout of TCTP might correlate with the decreased expression of mMcl-1, Bcl-xL, hax-1 and Oct4 in Nestin-cre; TCTP knockout mice. Taken together, our results demonstrate that TCTP is a critical protein of cell survival during early neuronal and glial differentiation. Thus, increased neuronal and functional loss through increasing apoptosis and decreased proliferation may contribute to the perinatal lethality of mutant mice.

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Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR), consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras–specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform–specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

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Objective: Infusion phlebitis is the most common complication and the major problem in peripheral parenteral nutrition (PPN), for which the acidity of PPN solution is an important risk factor. To minimize the risk, most Japanese PPN products are formulated to have no acidic effects, whereas most European counterparts are acidic. We conducted the study to clarify that differences in acidity reflect phlebitic potentials of PPN solutions. Methods: A Japanese PPN solution, AF (pH6.56) and 2 European counterparts, E2-in-1 (pH5.88) and E3-in-1 (pH5.54), were used as test solutions. To make the calorie and osmolality of AF and E2-in-1 (non-lipid solutions) similar to those of E3-in1 (lipid-containing solution), a 1/10 volume of 20% lipid emulsion was supplemented (AF+L, E2+L). Each solution was infused into ear veins of 8 rabbits for 8 hr at 10 mL/kg/hr. The infused veins were sampled 24 hr after the end of infusion and examined histopathologically. The phlebitic findings including “loss of venous endothelial cells” and “inflammatory cell infiltration” were graded on a scale of 0 (no change) to 3 (severe change), and the grades were analyzed with the Wilcoxon rank-sum test. Results: E2-in-1, E2+L, and E3-in-1 caused slight to moderate phlebitic changes, whereas AF and AF+L scarcely did. Of the 3 commercial products, AF showed significantly lower phlebitic potential than E2-in-1 and E3-in-1 did; of the 3 lipid-containing solutions, AF+L significantly lower than E2+L and E3-in-1. Conclusions: These results suggest the acidity of PPN solutions reflects phlebitic potentials and PPN solution with a physiological pH is advantageous for peripheral infusion.

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People working under the manhole and in confined spaces such as tanks and others are at the risk of being exposed to high rate of death due to lack of oxygen. Therefore, we investigated activity change of SD rats and lethal concentrations resulting from the concentration rate of oxygen using inhalation chamber and nitrogen gas as well as oxygen to analyze the causes and prevent accidents by finding out the lethal concentrations resulting from the oxygen concentration rates. SD male rats were divided into 4 groups and each of the group with 10 rats were exposed to a confined space with 4%, 5%, 6% and 7% oxygen concentration rates respectively. We investigated activity change of test animals using ultraviolet detector and identifying when the rats died and what the lethal concentrations were at that moment during 4 hours or 240 minutes before taking the time to recover for the rats for 48 hours. We confirmed that the rats in the space with lack of oxygen got increasingly active but they showed decreased activity after the first fifty minutes. They recover in terms of their activity after being exposed to space with 21% of oxygen rate for four hours. The fatality rate for the rats was 100% at 4% oxygen concentration within fifty minutes; 90% at 5% oxygen concentration within 230 minutes; 20% at 6% oxygen concentration within 240 minutes and 0% at 7% of oxygen concentration rate. In conclusion, we found out through oxygen concentration rate test that the Concentration of deaths was 5 to 6%. Also LC50(rat, 4hr) of Dose-Response concentration was calculated to be 5.5%.

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AIM: To explore the association between runt related transcription factor 3 (RUNX3) gene polymorphisms and gastric cancer prognosis. METHODS: 10 tagging single-nucleotide polymorphisms (tSNPs) were genotyped in RUNX3 using TaqMan method in 944 gastric cancer patients. The Kaplan-Meier method with log-rank test and Cox proportional hazards model were used for survival analyses. RESULTS: Among the 10 tSNPs, SNP10 rs2282718 AA genotype was found to be significantly associated with a poor gastric cancer survival in a recessive model (log-rank P = 0.035), and this effect was more pronounced among subgroups with diffuse-type gastric cancer (HR = 1.45, 95% CI = 1.08-1.94), T4 depth of invasion (HR = 2.48, 95% CI = 1.04-5.92), distant metastasis (HR = 2.22, 95% CI = 1.06-4.64), and stage IV (HR = 2.30, 95% CI = 1.27-4.16). Multivariate Cox regression analysis revealed that the SNP10 rs2282718 was an independent prognostic factor to predict gastric cancer clinical outcome. Moreover, the combined genotypes of these ten tSNPs showed a locus-dosage effect on gastric cancer survival (P _trend = 0.006). CONCLUSIONS: Our results suggested that the genetic variation in RUNX3 may contribute to gastric cancer prognosis and the SNP10 rs2282718 could be an independent marker of survival assessment and individualized clinical therapy for gastric cancer, particularly among the diffuse-type gastric cancer in Chinese populations.

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