ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.

Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.

Beneficial Effects of Dietary Fiber in Young Barley Leaf on Gut Microbiota and Immunity in Mice.

The health benefits of young barley leaves, rich in dietary fiber, have been studied for several decades; however, their beneficial effects on the intestinal microenvironment remain to be elucidated. To investigate the effects of young barley leaf-derived dietary fiber (YB) on the gut microbiota and immunity, mice were fed an AIN-93G diet containing cellulose or YB and subjected to subsequent analysis. The population of MHC-II-positive conventional dendritic cells (cDCs) and CD86 expression in the cDCs of Peyer's patches were elevated in the YB-fed mice. MHC-II and CD86 expression was also elevated in the bone marrow-derived DCs treated with YB. 16S-based metagenomic analysis revealed that the gut microbiota composition was markedly altered by YB feeding. Among the gut microbiota, Lachnospiraceae, mainly comprising butyrate-producing NK4A136 spp., were overrepresented in the YB-fed mice. In fact, fecal butyrate concentration was also augmented in the YB-fed mice, which coincided with increased retinaldehyde dehydrogenase (RALDH) activity in the CD103+ cDCs of the mesenteric lymph nodes. Consistent with elevated RALDH activity, the population of colonic IgA+ plasma cells was higher in the YB-fed mice than in the parental control mice. In conclusion, YB has beneficial effects on the gut microbiota and intestinal immune system.

Increase in Paracellular Leakage of Amino Acids Mediated by Aging-Induced Reduction of Claudin-4 Expression.

BACKGROUND: Claudins (CLDNs), major components of tight junctions, control paracellular permeabilities of mineral ions and wastes. The absorption of nutrients including glucose and amino acids (AAs) is regulated by intestinal epithelial cells. However, the role of CLDNs is not fully understood. OBJECTIVES: The purpose of this study was to clarify the effect of AA deprivation on the expression of AA transporters and CLDNs, as well as the role of CLDNs in the regulation of paracellular AA fluxes. METHODS: The messenger RNA and protein expression of various CLDNs were examined by real-time quantitative polymerase chain reaction and Western blot analyses, respectively. The AA selectivity of CLDNs was estimated using liquid chromatography-tandem mass spectrometry (LC-MS) analysis. RESULTS: The expression levels of some AA transporters, CLDN4, and CLDN15 were increased by AA deprivation in normal mouse colon-derived MCE301 cells. The expression of AA transporters and CLDN15 in the mouse colon was positively correlated with aging but the expression of CLDN4 was not. The AA deprivation-induced elevation of CLDN4 expression was inhibited by MHY1485, a mammalian target of rapamycin (mTOR) activator. Furthermore, CLDN4 expression was increased by rapamycin, an mTOR inhibitor. mTOR may be involved in the transcriptional activation of CLDN4. The fluxes of AAs from the basal to apical compartments were decreased and increased by CLDN4 overexpression and silencing, respectively. LC-MS analysis showed that the fluxes of all AAs, especially Lys, His, and Arg, were enhanced by CLDN4 silencing. CONCLUSIONS: CLDN4 is suggested to form a paracellular barrier to AAs, especially alkaline AAs, which is attenuated with aging.

Physiological consequences of space flight, including abnormal bone metabolism, space radiation injury, and circadian clock dysregulation: Implications of melatonin use and regulation as a countermeasure.

Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-h light-dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight.

Effect of Wheat-Derived Arabinoxylan on the Gut Microbiota Composition and Colonic Regulatory T Cells.

The health benefits of wheat-derived arabinoxylan, a commonly consumed dietary fiber, have been studied for decades. However, its effect on the gut microenvironment and inflammatory bowel disease remains unclear. The objective of this study was to understand the effect of wheat-derived arabinoxylan on gut microbiota, colonic regulatory T cells (Tregs), and experimental colitis. In this study, healthy and chronic colitis model mice were fed chow containing cellulose or wheat-derived arabinoxylan for 2-6 weeks and subjected to subsequent analysis. A 16S-based metagenomic analysis of the fecal DNA revealed that Lachnospiraceae, comprising butyrate-producing and Treg-inducing bacteria, were overrepresented in arabinoxylan-fed mice. In line with the changes in the gut microbiota, both the fecal butyrate concentration and the colonic Treg population were elevated in the arabinoxylan-fed mice. In a T cell transfer model of chronic colitis, wheat-derived arabinoxylan ameliorated body weight loss and colonic tissue inflammation, which may, in part, be mediated by Treg induction. Moreover, wheat-derived arabinoxylan suppressed TNFα production from type 1 helper T cells in this colitis model. In conclusion, wheat-derived arabinoxylans, by altering the gut microenvironment, may be a promising prebiotic for the prevention of colitis.

Cardiac glycosides stimulate endocytosis of GLUT1 via intracellular Na+ ,K+ -ATPase α3-isoform in human cancer cells.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na+ ,K+ -ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na+ ,K+ -ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [3 H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na+ ,K+ -ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca2+ -releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca2+ chelator, a Ca2+ -ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca2+ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells.

Overexpression of D-dopachrome tautomerase increases ultraviolet B irradiation-induced skin tumorigenesis in mice.

Ultraviolet irradiation (UV) exposure is the leading factor underlying the development of skin malignancies. D-dopachrome tautomerase (D-DT), a functional homolog of macrophage migration inhibitory factor (MIF), has functional similarities to MIF. However, its role, unlike the role of MIF in photocarcinogenesis, is unknown. We therefore explored the role of D-DT in photocarcinogenesis by developing D-DT transgenic (D-DT Tg) mice and provided a research model for future studies targeting D-DT. Chronic UVB exposure accelerated tumor development in D-DT Tg mice compared with wild-type (WT) mice, with a higher incidence of tumors observed in D-DT Tg mice than in WT mice. In D-DT Tg irradiated mouse keratinocytes, the p53, PUMA, and Bax expression was lower than that in WT mice. These results indicate that D-DT Tg overexpression confers prevention against UVB-induced apoptosis in keratinocytes. Taken together, these findings support D-DT as a functionally important cytokine in photocarcinogenesis and potential therapeutic target for the prevention of photocarcinogenesis.

Osteoclastic and Osteoblastic Responses to Hypergravity and Microgravity: Analysis Using Goldfish Scales as a Bone Model.

It is known that the bone matrix plays an important role in the response to physical stresses such as hypergravity and microgravity. In order to accurately analyze the response of bone to hypergravity and microgravity, a culture system under the conditions of coexistence of osteoclasts, osteoblasts, and bone matrix was earnestly desired. The teleost scale is a unique calcified organ in which osteoclasts, osteoblasts, and the two layers of bone matrix, i.e., a bony layer and a fibrillary layer, coexist. Therefore, we have developed in vitro organ culture systems of osteoclasts and osteoblasts with the intact bone matrix using goldfish scales. Using the scale culture system, we examined the effects of hypergravity with a centrifuge and simulated ground microgravity (g-µG) with a three-dimensional clinostat on osteoclasts and osteoblasts. Under 3-gravity (3G) loading for 1 day, osteoclastic marker mRNA expression levels decreased, while the mRNA expression of the osteoblastic marker increased. Upon 1 day of exposure, the simulated g-µG induced remarkable enhancement of osteoclastic marker mRNA expression, whereas the osteoblastic marker mRNA expression decreased. In response to these gravitational stimuli, osteoclasts underwent major morphological changes. By simulated g-µG treatments, morphological osteoclastic activation was induced, while osteoclastic deactivation was observed in the 3G-treated scales. In space experiments, the results that had been obtained with simulated g-µG were reproduced. RNA-sequencing analysis showed that osteoclastic activation was induced by the down-regulation of Wnt signaling under flight-microgravity. Thus, goldfish scales can be utilized as a bone model to analyze the responses of osteoclasts and osteoblasts to gravity.

Hydroxylated benzo[c]phenanthrene metabolites cause osteoblast apoptosis and skeletal abnormalities in fish.

To study the toxicity of 3-hydroxybenzo[c]phenanthrene (3-OHBcP), a metabolite of benzo[c]phenanthrene (BcP), first we compared it with its parent compound, BcP, using an in ovo-nanoinjection method in Japanese medaka. Second, we examined the influence of 3-OHBcP on bone metabolism using goldfish. Third, the detailed mechanism of 3-OHBcP on bone metabolism was investigated using zebrafish and goldfish. The LC50s of BcP and 3-OHBcP in Japanese medaka were 5.7 nM and 0.003 nM, respectively, indicating that the metabolite was more than 1900 times as toxic as the parent compound. In addition, nanoinjected 3-OHBcP (0.001 nM) induced skeletal abnormalities. Therefore, fish scales with both osteoblasts and osteoclasts on the calcified bone matrix were examined to investigate the mechanisms of 3-OHBcP toxicity on bone metabolism. We found that scale regeneration in the BcP-injected goldfish was significantly inhibited as compared with that in control goldfish. Furthermore, 3-OHBcP was detected in the bile of BcP-injected goldfish, indicating that 3-OHBcP metabolized from BcP inhibited scale regeneration. Subsequently, the toxicity of BcP and 3-OHBcP to osteoblasts was examined using an in vitro assay with regenerating scales. The osteoblastic activity in the 3-OHBcP (10-10 to 10-7 M)-treated scales was significantly suppressed, while BcP (10-11 to 10-7 M)-treated scales did not affect osteoblastic activity. Osteoclastic activity was unchanged by either BcP or 3-OHBcP treatment at each concentration (10-11 to 10-7 M). The detailed toxicity of 3-OHBcP (10-9 M) in osteoblasts was then examined using gene expression analysis on a global scale with fish scales. Eight genes, including APAF1, CHEK2, and FOS, which are associated with apoptosis, were identified from the upregulated genes. This indicated that 3-OHBcP treatment induced apoptosis in fish scales. In situ detection of cell death by TUNEL methods was supported by gene expression analysis. This study is the first to demonstrate that 3-OHBcP, a metabolite of BcP, has greater toxicity than the parent compound, BcP.

Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis.

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C- cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c-/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.

Neurotoxicity of a pyrethroid pesticide deltamethrin is associated with the imbalance in proteolytic systems caused by mitophagy activation and proteasome inhibition.

Pyrethroids are one of the most commonly used classes of synthetic pesticides in the world. Recent laboratory and epidemiological evidence suggested that pyrethroids have potential adverse effects in the mammalian brain; however, the underlying mechanisms of the neurotoxic effects of pyrethroids have not been fully elucidated. In the present study, we investigated the mechanisms of effects of a type II pyrethroid deltamethrin (DM) in a neuronal cell model focusing on the proteolytic function, including autophagy and the ubiquitin-proteasome system. We confirmed that a micromolar concentration of DM dose-dependently decreased the cell viability and induced apoptotic cell death. Our results showed that DM enhanced autophagy in association with an accumulation of autophagosomes and increase in the levels of autophagy markers LC3-II/LC3-I ratio and p62 which were much elevated in the presence of lysosomal inhibitors bafilomycin A1 and chloroquine. We also found that DM caused a dysfunction of mitochondria with a decrease of mitochondrial membrane potential and mitochondrial DNA copy number as well as colocalization with autophagosomes. Moreover, a decrease in the activities of three major proteasomal enzymes and an accumulation of ubiquitinated proteins were observed by the exposure to DM. Transcriptome analysis revealed that up-regulated genes supported the activation of autophagy with induction of cellular stress responses including oxidative stress and endoplasmic reticulum stress, while down-regulated genes related to the cell cycle and DNA replication. These findings provide novel insights into the neurotoxicity of DM which underlie the imbalance in proteolytic function caused by mitophagy activation and proteasome inhibition.

Upregulation of Claudin-7 Expression by Angiotensin II in Colonic Epithelial Cells of Mice Fed with NaCl-Depleted Diets.

Dietary NaCl depletion increases Na+ and Cl- absorption in the colon, but the mechanisms are not fully understood. So far, we reported that the expression of claudin-7 (CLDN7), a tight junction (TJ) protein, was upregulated in the mice fed with NaCl-depleted diets, but the regulatory mechanism has not been clarified. Here, we found that angiotensin II (ANGII) increases the mRNA level of CLDN7, which was inhibited by losartan, a type 1 ANGII (AT1) receptor antagonist. Immunofluorescence measurement showed that CLDN7 is colocalized with zonula occludens-1 at the TJ in untreated and ANGII-treated cells. ANGII decreased transepithelial electrical resistance (TER) and increased permeability to C1- without affecting permeability to lucifer yellow, a paracellular flux marker. In contrast, TER was increased by CLDN7 knockdown in the absence and presence of ANGII. ANGII increased the nuclear distribution of phosphorylated p65 subunit of NF-κB, which was inhibited by losartan. The ANGII-induced elevation of CLDN7 expression was blocked by BAY 11-7082 (BAY), an NF-κB inhibitor. Luciferase reporter assay showed that ANGII increases promoter activity of CLDN7, which was inhibited by the treatment with losartan or BAY, and introduction of mutations in κB-binding motifs in the promoter. The binding of p65 on the promoter region of CLDN7 was increased by ANGII, which was inhibited by losartan and BAY in chromatin immunoprecipitation assay. Our data suggest that ANGII acts on AT1 receptor and increases paracellular permeability to Cl- mediated by the elevation of CLDN7 expression in the colon.

CDKN2A, CDK1, and CCNE1 overexpression in sebaceous gland carcinoma of eyelid.

PURPOSE: To investigate the overexpression of genes in sebaceous gland carcinoma (SGC) of the eyelid compared to sebaceous adenoma of the eyelid in order to elucidate the molecular mechanism underlying pathogenesis. METHODS: We performed histopathological examination of eyelid tissues surgically removed from four patients diagnosed with SGC (cases 1-3) and sebaceous adenoma (case 4) of the eyelid. Next, we performed global gene expression analysis of surgical tissue samples using a GeneChip® system and the Ingenuity Pathways Knowledge Base. The results of the GeneChip® analysis were explored with quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RESULTS: In the SGC samples, we found that 211, 199, and 199 genes, respectively, showed ≥ 2.0-fold higher expression than those in the sebaceous adenoma sample (case 4); 194 genes were common to all three SGC samples. For the 194 genes with upregulated expression, functional category analysis showed that SGC of the eyelid employed a unique gene network, including cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase 1 (CDK1), and cyclin E1 (CCNE1), which are related to cell cycle progression, incidence of tumor, and cell viability. Furthermore, qRT-PCR analysis showed that the expression levels of CDKN2A, CDK1, and CCNE1 were significantly upregulated in all SGC cases compared to those in the sebaceous adenoma case. These data were similar to the results of microarray analysis. CONCLUSION: Overexpression of cell cycle-related genes CDKN2A, CDK1, CCNE1, and their gene network may help elucidate the pathogenic pathway of SGC of the eyelid at the molecular level.

Growth and neurite stimulating effects of the neonicotinoid pesticide clothianidin on human neuroblastoma SH-SY5Y cells.

Neonicotinoids are one of most widely used pesticides targeting nicotinic acetylcholine receptors (nAChRs) of insects. Recent epidemiological evidence revealed increasing amounts of neonicotinoids detected in human samples, raising the critical question of whether neonicotinoids affect human health. We investigated the effects of a neonicotinoid pesticide clothianidin (CTD) on human neuroblastoma SH-SY5Y cells as in vitro models of human neuronal cells. Cellular and functional effects of micromolar doses of CTD were evaluated by changes in cell growth, intracellular signaling activities and gene expression profiles. We examined further the effects of CTD on neuronal differentiation by measuring neurite outgrowth. Exposure to CTD (1-100 µM) significantly increased the number of cells within 24 h of culture. The nAChRs antagonists, mecamylamine and SR16584, inhibited this effect, suggesting human α3ß4 nAChRs could be targets of neonicotinoids. We observed a transient intracellular calcium influx and increased phosphorylation of extracellular signal-regulated kinase 1/2 shortly after exposure to CTD. Transcriptome analysis revealed that CTD down-regulated genes involved in neuronal function (e.g., formation of filopodia and calcium ion influx) and morphology (e.g., axon guidance signaling and cytoskeleton signaling); these changes were reflected by a finding of increased neurite length during neuronal differentiation. These findings provide novel insight into the potential risks of neonicotinoids to the human nervous system.

Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

RANKL, Ephrin-Eph and Wnt10b are key intercellular communication molecules regulating bone remodeling in autologous transplanted goldfish scales.

This study aimed to investigate the precise data of gene expression, functions, and chronological relationships amongst communication molecules involved in the bone remodeling process with an in vivo model using autologous transplanted scales of goldfish. Autotransplantation of methanol-fixed cell-free scales triggers scale resorption and regeneration, as well as helps elucidate the process of bone remodeling. We investigated osteoclastic markers, osteoblastic markers, and gene expressions of communicating molecules (RANKL, ephrinB2, EphB4, EphA4, Wnt10b) by qPCR, in situ hybridization for Wnt10b, and immunohistochemistry for EphrinB2 and EphA4 proteins to elucidate the bone remodeling process. Furthermore, functional inhibition experiments for the signaling of ephrinB2/Eph, ephrin/EphA4, and Wnt10b using specific antibodies, revealed that these proteins are involved in key signaling pathways promoting normal bone remodeling. Our data suggests that the remodeling process comprises of two successive phases. In the first absorption phase, differentiation of osteoclast progenitors by RANKL is followed by the bone absorption by mature, active osteoclasts, with the simultaneous induction of osteoblast progenitors by multinucleated osteoclast-derived Wnt10b, and proliferation of osteoblast precursors by ehprinB2/EphB4 signaling. Subsequently, during the second formation phase, termination of bone resorption by synergistic cooperation occurs, with downregulation of RANKL expression in activated osteoblasts and Ephrin/EphA4-mediated mutual inhibition between neighboring multinucleated osteoclasts, along with simultaneous activation of osteoblasts via forward and reverse EphrinB2/EphB4 signaling between neighboring osteoblasts. In addition, the present study shows that autologous transplantation of methanol-fixed cell-free scale is an ideal in vivo model to study bone remodeling.

Autophagy impairment by Helicobacter pylori-induced methylation silencing of MAP1LC3Av1 promotes gastric carcinogenesis.

Helicobacter pylori (H. pylori) infection induces methylation silencing of tumor suppressor genes causing gastric carcinogenesis. Impairment of autophagy induces DNA damage leading to genetic instability and carcinogenesis. We aimed to identify whether H. pylori infection induced methylation silencing of host autophagy-related (Atg) genes, impairing autophagy and enhancing gastric carcinogenesis. Gastric mucosae were obtained from 41 gastric cancer patients and 11 healthy volunteers (8 H. pylori-uninfected and 3 H. pylori-infected). Methylation status of Atg genes was analyzed by a methylation microarray and quantitative methylation-specific PCR (qMSP); mRNA expression was assessed by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation, migration and invasion were assessed in normal rat gastric epithelial cells. Gene knock-down was performed by siRNA. Autophagy was assessed by western blotting. Of 34 Atg genes, MAP1LC3A variant 1 (MAP1LC3Av1) and ULK2 were identified by methylation microarray analysis as exhibiting specific methylation in H. pylori-infected mucosae and gastric cancer tissues. Methylation silencing of MAP1LC3Av1 was confirmed by qMSP, qRT-PCR and de-methylation treatment in two gastric cancer cell lines. Knock-down of map1lc3a, the rat homolog of the human MAP1LC3Av1, inhibited autophagy response and increased cell proliferation, migration and invasion in normal rat gastric epithelial cells, despite the presence of map1lc3b, the rat homolog of the human MAP1LC3B gene important for autophagy. Furthermore, MAP1LC3Av1 was methylation-silenced in 23.3% of gastric cancerous mucosae and 40% of non-cancerous mucosae with H. pylori infection. MAP1LC3Av1 is essential for autophagy and H. pylori-induced methylation silencing of MAP1LC3Av1 may impair autophagy, facilitating gastric carcinogenesis.

Checkpoint kinase 2 is dispensable for regulation of the p53 response but is required for G2/M arrest and cell survival in cells with p53 defects under heat stress.

Hyperthermia induced by heat stress (HS) is known to inhibit proliferation and induce cell death in cancer. We previously demonstrated that checkpoint kinase 1 (Chk1) contributes to G2/M arrest and cell survival under HS; however, the role of Chk2, a functional analog of Chk1, in regulation of the cell cycle and cell death under HS is still unknown. Here, we addressed the role of Chk2 using Molt-4 cells with p53-targeted shRNA (Molt-4/shp53) and parental control cells (Molt-4/V). Chk2 inhibition suppressed C-terminal acetylation of p53 and delayed the induction of p53-target genes in Molt-4/V cells under HS; however, Chk2 inhibition failed to inhibit apoptosis induced by HS, indicating that Chk2 was dispensable for p53-dependent apoptosis under HS. In contrast, Chk2 inhibition abrogated G2/M arrest and promoted cell death induced by HS in HeLa cells and Molt-4/shp53 cells. Thus, we demonstrated for the first time that Chk2 was required for cell cycle arrest and cell survival, particularly in cells with p53 defects under HS. These findings indicated that Chk2 may be a selective target for p53-mutated or -deficient cancer treated with hyperthermia.

Chlorpheniramine Increases Paracellular Permeability to Marker Fluorescein Lucifer Yellow Mediated by Internalization of Occludin in Murine Colonic Epithelial Cells.

Ions, small molecules, and drugs are absorbed in the intestinal epithelium mediated by transcellular and paracellular pathways. The function of various transporters expressing in the apical and basolateral membranes of intestinal epithelial cells has been well characterized. In contrast, claudins and occludin, components of the tight junctions (TJs), determine the paracellular permeability to ions and low molecular weight compounds, but the properties for permeability has not been clarified in detail. In the present study, we examined the effects of anti-histamine drugs, chlorpheniramine and diphenhydramine, on transepithelial electrical resistance (TER) and permeability to lucifer yellow (LY), a marker of paracellular permeability, using murine colonic MCE301 cells. Chlorpheniramine significantly decreased the steady state of TER and increased permeability to LY, whereas the effects of diphenhydramine were not significant. The mRNAs of occludin and claudin-1-claudin-8 except for claudin-5 were expressed in MCE301 cells. Both anti-histamine drugs did not change solubility of claudins to 0.5% Triton X-100 solution. In contrast, the detergent solubility and intracellular localization of occludin were significantly increased by chlorpheniramine. These results indicate that occludin is dissociated from the TJs by chlorpheniramine. Chlorpheniramine increased protein phosphatase-2A (PP-2A) activity, which was inhibited by cantharidin, a potent PP-2A inhibitor. Furthermore, the changes of TER, permeability to LY, and de-phosphorylation and tight junctional localization of occludin caused by chlorpheniramine were recovered by cantharidin. These results suggest that chlorpheniramine could increase paracellular permeability to low molecular weight compounds mediated by the activation of PP-2A and internalization of occludin in the colonic epithelial cells.

Seawater Polluted with Highly Concentrated Polycyclic Aromatic Hydrocarbons Suppresses Osteoblastic Activity in the Scales of Goldfish, Carassius auratus.

We have developed an original in vitro bioassay using teleost scale, that has osteoclasts, osteoblasts, and bone matrix as each marker: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Using this scale in vitro bioassay, we examined the effects of seawater polluted with highly concentrated polycyclic aromatic hydrocarbons (PAHs) and nitro-polycyclic aromatic hydrocarbons (NPAHs) on osteoblastic and osteoclastic activities in the present study. Polluted seawater was collected from two sites (the Alexandria site on the Mediterranean Sea and the Suez Canal site on the Red Sea). Total levels of PAHs in the seawater from the Alexandria and Suez Canal sites were 1364.59 and 992.56 ng/l, respectively. We were able to detect NPAHs in both seawater samples. Total levels of NPAHs were detected in the seawater of the Alexandria site (12.749 ng/l) and the Suez Canal site (3.914 ng/l). Each sample of polluted seawater was added to culture medium at dilution rates of 50, 100, and 500, and incubated with the goldfish scales for 6 hrs. Thereafter, ALP and TRAP activities were measured. ALP activity was significantly suppressed by both polluted seawater samples diluted at least 500 times, but TRAP activity did not change. In addition, mRNA expressions of osteoblastic markers (ALP, osteocalcin, and the receptor activator of the NF-κB ligand) decreased significantly, as did the ALP enzyme activity. In fact, ALP activity decreased on treatment with PAHs and NPAHs. We conclude that seawater polluted with highly concentrated PAHs and NPAHs influences bone metabolism in teleosts.