Hepatitis C Virus Downregulates Ubiquitin-Conjugating Enzyme E2S Expression To Prevent Proteasomal Degradation of NS5A, Leading to Host Cells More Sensitive to DNA Damage.

Hepatitis C virus (HCV) infection may cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV exploits cellular machineries to establish persistent infection. We demonstrate here that ubiquitin-conjugating enzyme E2S (UBE2S), a member of the ubiquitin-conjugating enzyme family (E2s), was downregulated by endoplasmic reticulum stress caused by HCV in Huh7 cells. UBE2S interacted with domain I of HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. Overexpression of UBE2S suppressed viral propagation, while depletion of UBE2S expression increased viral infectivity. Enzymatically inactive UBE2S C95A mutant exerted no antiviral activity, suggesting that ubiquitin-conjugating enzymatic activity was required for the suppressive role of UBE2S. Chromatin ubiquitination plays a crucial role in the DNA damage response. We showed that the levels of UBE2S and Lys11 chains bound to the chromatin were markedly decreased in the context of HCV replication, rendering HCV-infected cells more sensitive to DNA damage. These data suggest that HCV counteracts antiviral activity of UBE2S to optimize viral propagation and may contribute to HCV-induced liver pathogenesis.IMPORTANCE Protein homeostasis is essential to normal cell function. HCV infection disturbs the protein homeostasis in the host cells. Therefore, host cells exert an anti-HCV activity in order to maintain normal cellular metabolism. We showed that UBE2S interacted with HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. However, HCV has evolved to overcome host antiviral activity. We demonstrated that the UBE2S expression level was suppressed in HCV-infected cells. Since UBE2S is an ubiquitin-conjugating enzyme and this enzyme activity is involved in DNA damage repair, HCV-infected cells are more sensitive to DNA damage, and thus UBE2S may contribute to viral oncogenesis.

Anti-atherogenic effect of Humulus japonicus in apolipoprotein E-deficient mice.

Humulus japonicus (HJ) is used as a traditional medicine in Korea owing to its multiple properties including anti-mycobacterial, antioxidant and antihypertensive effects. The present study aimed to examine the anti­inflammatory and anti-atherogenic effects of a methanol extract of HJ. In lipopolysaccharide-stimulated RAW 264.7 cells, HJ significantly suppressed the mRNA expression and secretion of pro-inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1ß and IL-6)], and the release of inflammatory mediators such as nitrite and prostaglandin E2, together with a concomitant decrease in the mRNA levels of inducible nitric oxide synthase and cyclooxygenase-2. To examine whether HJ is capable of inhibiting experimental atherogenesis in an animal model, we randomly divided apolipoprotein E-deficient (apoE-/-) mice into three groups: mice fed an atherogenic diet plus vehicle (0.5% carboxymethyl cellulose) as the control vehicle group, and mice fed an atherogenic diet plus either 100 (HJ100) or 500 mg/kg (HJ500) of HJ as the experimental groups. After 12 weeks of HJ administration, lipid accumulation and the formation of atherosclerotic lesions in the aorta (en face) and the aortic sinus markedly decreased in the HJ500 group compared with the corresponding values in the vehicle control group. Moreover, monocyte and macrophage infiltration in the aortic sinus was markedly reduced in the HJ500 group. Reverse transcription-quantitative polymerase chain reaction analysis of the whole aorta showed that the mRNA levels of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, CD68 and IL-18 were significantly decreased in the HJ500 group. Collectively, these findings suggest that HJ may suppress atherosclerosis by inhibiting lipid accumulation and the expression of pro-atherogenic factors, and it may be effective at preventing the development of atherosclerosis.

Inhibitory effect of withaferin A on Helicobacter pylori­induced IL­8 production and NF­κB activation in gastric epithelial cells.

Withaferin A (WA), a withanolide purified from Withania somnifera, has been known to exert anti-inflammatory effects. The present study sought to determine the effects of WA on Helicobacter (H.) pylori-mediated inflammation in the AGS gastric epithelial cell line. Cellular production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) was measured by ELISA. Western blot analysis was performed to determine the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) as well as hypoxia-inducible factor 1α stabilization. Bacterial growth was also examined by measuring the optical density. Pre-treatment or co-treatment with WA efficiently reduced IL-8 production by AGS cells in response to H. pylori infection. H. pylori-induced activation of NF-κB, but not MAPKs, was also inhibited by pre-treatment of WA in the cells. However, WA did not affect VEGF production and HIF-1α stabilization induced by H. pylori in AGS cells. In addition, WA did not influence the growth of H. pylori, suggesting that the anti-inflammatory effect of WA was not due to any bactericidal effect. These findings indicate that WA is a potential preventive or therapeutic agent for H. pylori-mediated gastric inflammation.

Stephania delavayi Diels. inhibits breast carcinoma proliferation through the p38 MAPK/NF-κB/COX-2 pathway.

The nuclear factor κB (NF-κB)/inhibitor of κ kinase-ß (IKKß) signaling pathway is important in tumor promotion and progression. MDA-MB-231 human breast carcinoma cells express COX-2 and show a constitutive phosphorylation of NF-κB. Many non-specific inhibitors of IKKß and NF-κB are used to inhibit tumor promotion and progression. The Stephania delavayi Diels. (S. delavayi Diels.) extract has been reported to safely activate B cell immunity and there is evidence suggesting that it may be a promising new anticancer therapeutic agent. S. delavayi Diels. extract suppressed proliferation of the breast cancer cell lines MDA-MB-231 and MCF-7 by inducing cell death. To aid in the development of the S. delavayi Diels. extract as a therapeutic agent, its mechanisms of action were investigated, in particular its effects on p38 MAPK, NF-κB and COX-2, which play important roles in inflammation and cancer. S. delavayi Diels. stimulated p38 MAPK phosphorylation but reduced NF-κB phosphorylation and COX-2 expression in a dose- and time-dependent manner. Thus, S. delavayi Diels., which appears to act primarily through p38 MAPK/NF-κB/COX-2 signaling in breast carcinomas, may be a potent anticancer agent with target specificity and low toxicity.

Evaluation of the toxic potentials of a new camptothecin anticancer agent CKD-602 on fertility and early embryonic development in rats.

This study examined the potential adverse effects of a new camptothecin anticancer agent, CKD-602, on the fertility and early embryonic development of Sprague-Dawley rats. Ninety-six rats of each gender were divided into four groups: three treatment groups and a control group. CKD-602 was administered intravenously to male rats at 0, 4.7, 14, and 42 microg/kg from 63 days prior to mating until the end of the mating period, and to female rats from 14 days before mating until day 6 of gestation. All the males were sacrificed after the end of the 14-day mating period, while all the females were subjected to a caesarean section on day 15 of gestation. In the high dose group, a high incidence of hair loss was observed in both genders. A decrease in the level of food consumption, followed by a decrease in body weight gain was also observed in both genders. At the scheduled necropsy, the gross postmortem examinations revealed an increase in the incidence of thymic atrophy, paleness of the thoracic and abdominal organs in both genders and an increase in the serum testosterone concentration. In addition, there was a decrease in the thymus weight of the males and an increase in the liver, spleen, kidneys, lung, and heart weights of the females. There was an increase in the number of fetal deaths and post-implantation losses as well as a decrease in the litter size found at the caesarean section of the dams. No treatment-related effect on the histopathological findings, sexual cycle, pre-coital time, mating index, fertility index, pregnancy index, and sperm parameters was observed. There were no adverse effects on the general findings and reproductive performance of the parent animals and early embryonic development in the low and medium dose groups. Overall, the no-observed-adverse-effect levels (NOAELs) of CKD-602 are believed to be 14 microg/kg for both general toxicity and early embryonic development, and more than 42 microg/kg for the reproductive performance of the parent animals.

Characteristics of home cage locomotion in cynomolgus monkeys.

The profiles of home cage locomotor activity and its rhythmicity were investigated using a camera system for 4 consecutive days in cynomolgus monkeys. Nine male and nine female cynomolgus monkeys were used in this study, and were found to have the same profile in nocturnal behavior in that they were mostly inactive during the night. The locomotor activity of both sexes showed a normal activity and inactivity rhythm, which had an inverse relationship between normal activity and inactivity, showing a fluctuation profile of being higher in the light cycle and lower in the dark cycle. The four-day locomotion time of the females was higher than that of the males in the dark period. The females also showed a higher locomotion count and D/L ratio in the dark period than the males during the 4 consecutive days. These findings indicate that the females have greater locomotor activity in the dark period than that of the males. The present findings may serve as useful basic data for the behavioral assessment of overall animal movements in toxicological studies using monkeys.

Embryotoxic effects of CKD-602, a new camptothecin anticancer agent, in rats.

CKD-602 is a newly developed camptothecin anticancer agent. Preclinical studies suggest that it may have greater antitumor activity and lower toxicity than other camptothecin anticancer agents. The potential of CKD-602 to induce embryotoxicity was investigated in the Sprague-Dawley rat. One hundred mated females (sperm in vaginal lavage=day 0) were distributed among three treatment groups and a control group. CKD-602 was administered intravenously at dose levels of 0, 5, 20 and 80 microg/kg/d to pregnant rats from days 6 to 15 of gestation. The vehicle control rats received an equivalent volume of 1 ml distilled water with d-mannitol 50mg and tartaric acid 0.06 mg. All dams were subjected to the caesarean section on day 20 of gestation. There were no signs of maternal toxicity or embryotoxicity at 5 microg/kg/d, but at 20 microg/kg/d, there was an increase in relative brain weight. At 80 microg/kg/d, reduced food intake, suppressed body weight and increased weight of spleen were observed in dams. An increase in the resorptions and dead fetuses, a decrease in litter size, fetal and placental weights were also found. In addition, various types of external, visceral and skeletal malformations occurred. Characteristic malformations included absent eye bulge, agnathia, dilated cerebral ventricle, anophthalmia, absent thoracic centrum, fused vertebral arch, fused rib, among others. Visceral and skeletal variations were observed. Retarded ossification of several skeletal districts and delayed ossification of sternebrae, metatarsals and sacrocaudal vertebrae were also observed. The results show that CKD-602 is embryotoxic and teratogenic at a minimally maternally toxic dose, i.e. at 80 microg/kg/d in rats. The no-observed-adverse-effect level (NOAEL) of CKD-602 for developmental toxicity was considered to be 20 microg/kg/d, however, the NOAEL for maternal toxicity was 5 microg/kg/d.

Effects of CKD-602, a new camptothecin anticancer agent, on pregnant does and embryo-fetal development in rabbits.

CKD-602 is a newly developed camptothecin anticancer agent. Preclinical studies suggest that it may have greater antitumor activity and lower toxicity than other camptothecin anticancer agents. The potential of CKD-602 to induce developmental toxicity was investigated in the New Zealand White rabbit. Seventy-two artificially inseminated females (artificial insemination=day 0) were distributed among three treatment groups and a control group. CKD-602 was at dose levels of 0, 0.024, 0.048, or 0.096 mg x kg(-1) x day(-1) administered intravenously to pregnant does from days 6 to 18 of gestation. All does were subjected to caesarean section on day 28 of gestation. At 0.096 mg x kg(-1) x day(-1), 2 cases of abortion and 3 cases of death in pregnant rabbits were found in late gestation. In addition, an increase in the embryonic resorptions and a decrease in the litter size were found. At 0.048 mg x kg(-1) x day(-1), a single doe aborted on gestational day 26. An increase in the embryonic resorptions and fetal morphological alterations and a decrease in the litter size were also found. There were no signs of maternal toxicity or developmental toxicity at 0.024 mg x kg(-1) x day(-1). The results show that 13-day repeated intravenous dose of CKD-602 during the major organogenetic period in rabbits produces increased incidence of abortion and death, increased number of embryonic resorptions and fetal morphological alterations, and decreased litter size at dose levels of above 0.048 mg x kg(-1) x day(-1). In the current experimental conditions, the no-observed-adverse-effect levels (NOAELs) of CKD-602 are considered to be 0.048 mg x kg(-1) x day(-1) for does and 0.024 mg x kg(-1) x day(-1) for embryo-fetal development.

Safety pharmacology of DW-224a, a novel fluoroquinolone antibiotic agent.

To investigate the safety pharmacology of a novel fluoroquinolone antibiotic agent, DW-224a, on the vital functions, we studied its effects on the central nervous system, cardiovascular system and respiratory system. To determine the effects on the central nervous system, we used a modified Irwin's test at each time point after oral administration of DW-224a to mice. In this test, we found that the treatment of test article had no effects on motor activity, behavioral changes, coordination, and sensory/motor reflex responses. The effects of DW-224a on the cardiovascular system were evaluated by the use of a telemetry system in beagle dogs. At 360 min post-DW-224a (100 mg/kg) administration, QT interval prolongation was observed. However, there were no changes in heart rate, blood pressure, and electrocardiogram at all doses and each time points with the exception of QT-interval prolongation as compared to the vehicle treated group. In experiments designed to determine the changes of respiratory function in rats, we found no changes at all doses and time points. We investigated the effects of DW-224a on the human ether-a-go-go-related gene (hERG) mediated potassium currents to evaluate its potential to induce QT interval prolongation. When whole cell patch-clamp electrophysiology was used, DW-224a inhibited hERG currents with IC50 of 218.12 +/- 39.51 microM though its effect was less potent than that of E-4031, a positive control drug. Our data suggested that DW-224a showed no adverse effects on the central nervous system, cardiovascular system, and respiratory system, with the exception of the effect on the QT interval prolongation.

Fine localization of Nefl and Nef3 and its exclusion as candidate gene for lens rupture 2(lr2).

Cataract causing lr2 gene is found in the CXSD mouse, which is a recombinant inbred strain of BALB/c and STS mice. For the process of positional cloning of lr2, several candidate genes were selected in the middle region of chromosome 14, but most of them were excluded by combination of recombination and homozygosity mapping. Components of neurofilament proteins, neurofilament light polypeptide (Nefl) and neurofilament3 medium (Nef3), were linked to D14Mit87 which was not separated from the lr2 locus in the homozygosity mapping. When the expression levels of Nefl and Nef3 in eyes were compared in CXSD and BALB/c mice, there were no differences in expression levels. The cDNA sequences of the two genes from CXSD, BALB/c and STS mice were subsequently compared. Several nucleotide differences in cDNA sequences were detected between the mice strains but the majority of the changes were silent mutations that did not alter the amino acids. The sole amino acid difference, E567K in the glutamate rich region of Nfm, between BALB/c and CXSD was found to be a simple genetic polymorphism because the same substitution existed in STS, a non-cataract mouse strain. Therefore we excluded Nefl and Nef3 from the candidate genes for lr2 based on expression and mutation analyses.

cDNA microarray gene expression profiling of hydroxyurea, paclitaxel, and p-anisidine, genotoxic compounds with differing tumorigenicity results.

The potential application of toxicogenomics to predictive toxicology has been discussed widely, but the utility of the approach remains largely unproven. Using cDNA microarrays, we compared the gene expression profiles produced in mouse lymphoma cells by three genotoxic compounds, hydroxyurea (a carcinogen), p-anisidine (a noncarcinogen), and paclitaxel (carcinogenicity unknown). To minimize the effect of biological variability and technological limitations, quadruplicate observations were made for each compound and a subset of genes yielding reproducible induction/repression was selected for comparison. A method was applied to attach normalized expression data to genes with a low false-discovery rate (<0.1) to yield more confidence regarding differential expression. This analysis identified genotoxicity-specific gene expression. Seven genes were consistently upregulated and 12 downregulated more than 2-fold by the three genotoxic compounds. Using additional genes, the expression pattern induced by the genotoxic noncarcinogen, p-anisidine, was readily distinguished from that associated with the genotoxic carcinogen, hydroxyurea. Comparison of paclitaxel-induced expression data to data for p-anisidine and hydroxyurea suggested that paclitaxel's profile is more similar to the genotoxic noncarcinogen. With further supporting evidence it may be possible to perform large-scale monitoring of gene expression during drug and chemical development that can provide an early warning of potential toxicological responses.

Safety pharmacology of CKD-602, a novel anticancer agent.

CKD-602 ((20S)-7-[2-(N-isopropylamino)-ethyl]-camptothecin.HCl, CAS 213819-48-8) is a new class of anticancer drug that belongs to the topoisomerase inhibitors. Its effect on the central nervous system (CNS), general behavior, cardiovascular-respiratory system and the other organ systems were studied. When intravenously administered, CKD-602 up to doses of 5 mg/kg caused an increase of body temperature, increase of respiration rate, decrease of gastrointestinal transport, showed analgesic action and produced antisecretory action in pylorus ligated rats. However, CKD-602 showed no effects on general behavior, motor coordination, spontaneous locomotor activity, hexobarbital sleeping time, convulsion, cardiovascular, smooth muscle and urinary tract system. These findings demonstrate that CKD-602 in doses up to 5 mg/kg has minor effects on the CNS in animals. However, CKD-602 does not exert any general pharmacological effects at the dose of 1 mg/kg except the effects on gastric secretion.

Down-regulation of Raf-1 kinase is associated with paclitaxel resistance in human breast cancer MCF-7/Adr cells.

Experiments were carried out to determine the role of Raf-1 kinase in the development of drug resistance and apoptosis induced by paclitaxel. In the present study, paclitaxel sensitivity, Raf-1 activity and mitogen-activated protein kinases activation were compared in two cell lines: parental human breast cancer cells and its drug resistant variant (MCF-7/Adr) cells. Paclitaxel treatment of parental MCF-7 cells caused a marked inhibition of Raf-1 kinase activity, concomitant with its mobility shift after 18 h exposure. In addition, paclitaxel greatly increased c-Jun N-terminal protein kinase (JNK) activity whereas showing a small enhancing effect on extracellular-regulated kinases (ERK) activity. Interestingly, MCF-7/Adr cells have lower basal Raf-1 activity, yet have much higher basal ERK activity than parental cells. However, it appeared that PD 98059, which turns off ERK through mitogen-activated protein kinase kinase (MEK) inhibition, enhanced basal Raf-1 kinase activity in MCF-7/Adr cells. Thus, the findings suggest that paclitaxel-induced apoptosis is mediated by JNK and occurs in parallel with suppression of the Raf-1 kinase activity in parental MCF-7 cells. In addition, down-regulation of Raf-1 kinase, which can be induced through the sustained ERK activation, may contribute to the development of acquired resistance in MCF-7/Adr cells.

Choline phosphate potentiates sphingosine-1-phosphate-induced Raf-1 kinase activation dependent of Ras--phosphatidylinositol-3-kinase pathway.

In NIH3T3 cells, sphingosine-1-phosphate (S1P) caused a significant increase of Raf-1 kinase activity as early as 2 min. Interestingly, choline phosphate (ChoP) produced synergistic increase of S1P-stimulated Raf-1 kinase activation in the presence of ATP while showing additive effect in the absence of ATP. However, Raf-1 kinase activation induced by S1P decreased in the presence of ATP when applied alone. The overexpression of N-terminal fragment of Raf-1 (RfI) to inhibit Raf--Ras interaction caused the inhibition of S1P-induced Raf-1 kinase activation. Also, wortmannin, phosphatidylinositol-3-kinase (PI3K) inhibitor, exhibited inhibitory effects on S1P-induced activation of Raf-1 kinase. In addition, we demonstrated that the chemical antioxidant, N-acetylcysteine attenuated Raf-1 activation induced by S1P, suggesting that H(2)O(2) may be required for the signalling pathway leading to Raf-1 activation. This H(2)O(2)-induced Raf-1 kinase activation was also blocked by inhibition of Ras--PI3K signalling pathway using alpha-hydroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H(2)O(2) stimulation of Ras--PI3K pathway, and is enhanced by ChoP in the presence of ATP.