Activation of the HMGB1-RAGE axis upregulates TH expression in dopaminergic neurons via JNK phosphorylation.

The derangement of tyrosine hydroxylase (TH) activity reduces dopamine synthesis and is implicated in the pathogenesis of Parkinson's disease. However, the extracellular modulator and intracellular regulatory mechanisms of TH have yet to be identified. Recently, high-mobility group box 1 (HMGB1) was reported to be actively secreted from glial cells and is regarded as a mediator of dopaminergic neuronal loss. However, the mechanism for how HMGB1 affects TH expression, particularly through the receptor for advanced glycation endproducts (RAGE), has not yet been investigated. We found that recombinant HMGB1 (rHMGB1) upregulates TH mRNA expression via simultaneous activation of JNK phosphorylation, and this induction of TH expression is blocked by inhibitors of RAGE and JNK. To investigate how TH expression levels change through the HMGB1-RAGE axis as a result of MPP+ toxicity, we co-treated SN4741 dopaminergic cells with MPP+ and rHMGB1. rHMGB1 blocked the reduction of TH mRNA following MPP+ treatment without altering cell survival rates. Our results suggest that HMGB1 upregulates TH expression to maintain dopaminergic neuronal function via activating RAGE, which is dependent on JNK phosphorylation.

Exogenous SPARC suppresses proliferation and migration of prostate cancer by interacting with integrin ß1.

BACKGROUND: The matricellular protein secreted protein acidic and rich in cysteine (SPARC) plays an important role on tumor metastasis and progression in several cancers. However, the roles of SPARC in prostate cancer (PCa) remain unclear. METHODS: To identify SPARC protein in prostate tissue, immunohistochemical analysis of SPARC was conducted using human prostate tissue microarray. To detect SPARC expression in prostate cancer (LNCaP, DU145, and PC-3) and stromal cells, RT-PCR, western blot analysis, and ELISA was conducted. To reveal the function of exogenous SPARC in PCa cells, AKT phosphorylation was confirmed by western blot analysis after coculture with stromal cells. Proliferation and migration of PCa cells were examined by addition of SPARC. The interaction between SPARC and integrin ß1 was confirmed by western blot analysis after immunoprecipitation. RESULTS: SPARC protein was expressed well in normal tissue compared with PCa tissue. ELISA showed high secreted SPARC protein in normal prostate-derived stromal cell (PrSC) compared with PCa-derived stromal cell (PCaSC) and PCa. PCa cells cocultured with PrSC showed reduced AKT phosphorylation more than with PCaSC. PCa cells cocultured with PrSC whose SPARC was knocked-down restored AKT phosphorylation. Moreover, PCa cells treated with SPARC led to reduced AKT phosphorylation. Immunoprecipitation with SPARC revealed interaction of SPARC and integrin ß1 in PCa cells. Inhibited proliferation and migration of PCa cells by SPARC was restored by integrin ß1 neutralizing antibody. CONCLUSIONS: Reduced SPARC secretion from stromal cells might affect PCa progression mediating through limiting AKT phosphorylation after interaction with integrin ß1.

CCR7-CCL19/CCL21-regulated dendritic cells are responsible for effectiveness of sublingual vaccination.

Our previous studies demonstrated the potential of the sublingual (s.l.) route for delivering vaccines capable of inducing mucosal as well as systemic immune responses. Those findings prompted us to attempt to identify possible inductive mechanism of s.l. vaccination for immune responses. Within 2 h after s.l. administration with cholera toxin (CT), significantly higher numbers of MHC class II(+) cells accumulated in the s.l. mucosa. Of note, there were brisk expression levels of both CCL19 and CCL21 in cervical lymph nodes (CLN) 24 h after s.l. vaccination with CT. In reconstitution experiments using OVA-specific CD4(+) or CD8(+) T cells, s.l. vaccination elicited strong Ag-specific T cell proliferation mainly in CLN. Interestingly, Ag-specific T cell proliferation completely disappeared in CD11c-depleted and CCR7(-/-) mice but not in Langerin-depleted, macrophage-depleted, and CCR6(-/-) mice. Similar to CD4(+) T cell responses, induction of Ag-specific IgG (systemic) and IgA (mucosal) Ab responses were significantly reduced in CD11c-depleted and CCR7(-/-) mice after s.l. vaccination with OVA plus CT. Although CD8alpha(-) dendritic cells ferried Ag from the s.l. mucosa, both migratory CD8alpha(-) and resident CD8alpha(+) dendritic cells were essential to prime CD4(+) T cells in the CLN. On the basis of these findings, we believe that CCR7 expressed CD8alpha(-)CD11c(+) cells ferry Ag in the s.l. mucosa, migrate into the CLN, and share the Ag with resident CD8alpha(+)CD11c(+) cells for the initiation of Ag-specific T and B cell responses following s.l. challenge. We propose that the s.l. mucosa is one of the effective mucosal inductive sites regulated by the CCR7-CCL19/CCL21 pathway.

Acute and Subacute Oral Toxicity of Mumefural, Bioactive Compound Derived from Processed Fruit of Prunus mume Sieb. et Zucc., in ICR Mice.

Mumefural is a bioactive compound derived from the processed fruit of Prunus mume Sieb. et Zucc., a traditional health food; however, its safety has not been evaluated. We investigated the toxicity of mumefural through single and repeated oral administration at doses of 1250, 2500, and 5000 mg/kg in Institute of Cancer Research (ICR) mice. The acute toxicity assessment was not associated with adverse effects or death. Similarly, the subacute (four weeks) toxicity assessment did not reveal any mumefural-associated mortality, abnormal organ damage, or altered clinical signs, body weight, food consumption, or hematological parameters. However, albumin/globulin ratio and chloride ion levels were significantly increased in male mice treated with mumefural at ≥ 2500 mg/kg. Female mice exhibited significantly higher levels of chloride, sodium, and potassium ions, at a dose of 5000 mg/kg. Furthermore, the administration of 2500 and 5000 mg/kg mumefural decreased the absolute weight of spleen in male mice. These findings indicated that the approximate lethal dose of mumefural in ICR mice was > 5000 mg/kg. No significant mumefural toxicity was observed at ≤ 5000 mg/kg. Our findings provide a basis for conducting future detailed studies to evaluate reproductive, neurological, genetic, and chronic toxicity of mumefural.

The Effect of apoM Polymorphism Associated with HDL Metabolism on Obese Korean Adults.

BACKGROUND: Apolipoprotein M (apoM) is a recently identified apolipoprotein associated with high-density lipoprotein (HDL) in coronary artery disease (CAD), but the association between apoM polymorphism and obesity has not been reported. AIM: To investigate the association between apoM polymorphism and obesity prevalence in 584 Korean adults. METHODS: A total of 584 individuals aged between 30 and 80 years were recruited from Yonsei Medical Center in Seoul, Korea, and divided into obese (OB; body mass index, BMI ≥25) and nonobese (non-OB; BMI <25) groups. Anthropometric variables, lipid profiles, insulin-resistant profiles, reverse cholesterol transport (RCT) enzymes, HDL subfraction, and apoM polymorphism were determined. RESULTS: In OB with T-855C polymorphism, TT genotype carriers significantly showed 6.2% higher diastolic blood pressure (DBP), 1.3% lower amount of HDL2b subfraction, and 19.7% higher lecithin-cholesterol acyltransferase (LCAT) mass than TC+CC carriers. OB subjects with the T allele of T-778C polymorphism significantly demonstrated 43% higher plasma insulin, 17.7% higher total cholesterol, 26.7% higher triglyceride, 40.7% higher leptin, 1.6% lower HDL2b, and 12.6% higher LCAT mass than those with the C allele. These results were reversed in non-OB with T-778C polymorphism regarding HDL subfractions and RCT enzymes. CONCLUSION: apoM T-855C and T-778C polymorphisms were found to be associated with obesity by regulating HDL metabolism, and the T alleles of apoM T-778C were shown to be more strongly correlated.

Metabolic characterization of imatinib-resistant BCR-ABL T315I chronic myeloid leukemia cells indicates down-regulation of glycolytic pathway and low ROS production.

Long-term imatinib treatment induces drug-resistant chronic myeloid leukemia (CML) cells harboring T315I gate keeper mutation of breakpoint cluster region (BCR)-ABL oncogenic kinase. However, although cell proliferation is coupled with cellular energy status in CML carcinogenesis, the metabolic characteristics of T315I-mutant CML cells have never been investigated. Here, we analyzed cell proliferation activities and metabolic phenotypes, including cell proliferation, oxygen consumption, lactate production, and redox state in the KBM5 (imatinib-sensitive) and KBM5-T315I (imatinib-resistant) CML cell lines. Interestingly, KBM5-T315I cells showed decreased cell proliferation, lactate production, fatty acid synthesis, ROS production, and down regulation of mRNA expression related to ROS scavengers, such as SOD2, catalase, GCLm, and GPx1. Taken together, our data demonstrate that the lower growth ability of KBM5-T315I CML cells might be related to the decreased expression of glycolysis-related genes and ROS levels, and this will be used to identify therapeutic targets for imatinib resistance in CML.

Pattern formation through selective chemical transformation of self-assembled benzaldimine monolayer by soft X-ray irradiation.

Benzaldimine monolayer was exposed to soft X-rays, and the involved chemical transformation was investigated using X-ray photoelectron spectra and near-edge X-ray absorption fine structure spectroscopy. The spectroscopy indicated that irradiation of soft X-ray (550 eV)-induced selective transformation of the imine group into a nonhydrolyzable one, i.e., the amine group. Utilizing the selective chemical transformation of the imine group with the soft X-ray irradiation, we were able to generate a micropattern. AFM images showed that the patterning with alternating surface topology was effective. The patterned monolayer was further modified with biotin and Cy3-tagged Streptavidin sequentially. Fluorescence images showed that the above molecules were selectively immobilized onto the amine-terminated region of the patterned surface. The current system is found to be more efficient than the predecessor, 4-nitrobenzaldimine monolayer.

High-capacity glycolytic and mitochondrial oxidative metabolisms mediate the growth ability of glioblastoma.

Among the primary brain tumors, glioblastoma multiforme (GBM) has a radical proliferation ability that complicates the therapeutic modulation of cancer progression. The majority of GBM patients have a low survival rate (<1 year) due to radical tumor growth and late cancer diagnosis. Previous reports have shown that astrocytes have a specific metabolic organization that includes the production of lactate, the storage of glycogen, and use of lactate to support neurons which possess higher capacity of metabolism compared to neurons. We hypothesized that these characteristics of astrocytes could contribute to enhanced proliferation of GBM compared to neuroblastoma (NB). Here, we show that U87MG cells (a model of GBM) proliferate more rapidly than SH-SY5Y cells (a model of NB). A higher extracellular acidification rate and maximal mitochondrial oxygen consumption rate were observed in U87MG cells compared to SH-SY5Y cells. The expression levels of lactate dehydrogenase (LDH)-A and LDH-B were higher in U87MG cells and primary cultured astrocytes than in SH-SY5Y cells and neurons. Furthermore, the mRNA levels of succinate dehydrogenase and peroxisome proliferator-activated receptor-γ were high in U87MG cells, suggesting that these cells have high capacity for mitochondrial metabolism and uptake of fatty acids related to synthesis of the cell membrane, respectively. Taken together, we demonstrate that GBM cells are characterized by activation of the LDH-expression-related glycolytic pathway and mitochondrial metabolic capacity, suggesting two innate properties of astrocytes that could provide a driving force for the growth ability of GBM. Based on these findings, we propose that therapeutic approaches aimed at treating GBM could target LDH for modulating the metabolic properties of GBM cells.

Suppression of mitochondrial respiration with auraptene inhibits the progression of renal cell carcinoma: involvement of HIF-1α degradation.

Renal cell carcinoma (RCC) progression resulting from the uncontrolled migration and enhanced angiogenesis is an obstacle to effective therapeutic intervention. Tumor metabolism has distinctive feature called Warburg effect, which enhances the aerobic glycolysis rapidly supplying the energy for migration of tumor. To manipulate this metabolic change characteristic of aggressive tumors, we utilized the citrus extract, auraptene, known as a mitochondrial inhibitor, testing its anticancer effects against the RCC4 cell line. We found that auraptene impaired RCC4 cell motility through reduction of mitochondrial respiration and glycolytic pathway-related genes. It also strongly disrupted VEGF-induced angiogenesis in vitro and in vivo. Hypoxia-inducible factor 1a (HIF-1a), a key regulator of cancer metabolism, migration and angiogenesis that is stably expressed in RCCs by virtue of a genetic mutation in the von Hippel-Lindau (VHL) tumor-suppressor protein, was impeded by auraptene, which blocked HIF-1a translation initiation without causing cytotoxicity. We suggest that blockade HIF-1a and reforming energy metabolism with auraptene is an effective approach for suspension RCC progression.

Determination of hexamethylenetetramine in foods by high-performance liquid chromatography (HPLC).

A simple and rapid method was developed and validated for the determination of hexamethylenetetramine (HMT) in foods. Samples were homogenised and extracted with methanol, followed by centrifugation. The resulting solution was filtered and injected into the high-performance liquid chromatograph (HPLC). HMT was separated using a Zorbax SCX-300 column coupled to a photodiode array detector. The calibration curve was linear in the range of 1.0-100 µg ml(-1), with good correlation coefficients (r(2) = 0.9992). The recoveries of HMT from foods spiked at levels of 10, 50 and 100 mg kg(-1) ranged from 91.6% to 103.8%, with relative standard deviations (RSDs) between 0.9% and 5.3%. The limit of detection and the limit of quantification of HMT were 0.3 and 1.0 mg kg(-1) based on three food matrixes (provolone cheese, glass noodle and tofu snack), respectively. Uncertainty associated with accuracy contributed mostly to the expanded uncertainty. No detectable levels of HMT were found in any of the samples retailed in Korea. The method was successful in determining HMT in foods.

SOD3 acts as a tumor suppressor in PC-3 prostate cancer cells via hydrogen peroxide accumulation.

BACKGROUND: The functions of superoxide dismutase-3 (SOD3), which acts on the cell surface and protects cells from oxidative stress, remain uncertain in the progression of prostate cancer. MATERIALS AND METHODS: To verify SOD3 expression in human prostate tissue, immunohistochemistry was performed using tissue microarrays. To investigate the effects of SOD3 on proliferation, migration, and invasion, SOD3 was overexpressed and recombinant SOD3 was employed in PC-3 prostate cancer cells. H2O2 levels, reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, catalase activity, and 8-oxo-2'-deoxyguanosine (8-OHdG) were estimated in SOD3-overexpressing PC-3 cells. RESULTS: Immunohistochemistry revealed reduced expression of SOD3 in prostate cancer tissue. SOD3 overexpression in PC-3 cells inhibited cell proliferation, migration, and invasion. Recombinant SOD3 had the same effect. H2O2 accumulation was increased by SOD3 overexpression, GSH/GSSG ratio was decreased, and catalase activity was decreased. DNA damage in SOD3-overexpressing cells was confirmed by 8-OHdG elevation. CONCLUSION: Since SOD3 acts as a tumor suppressor, SOD3 overexpression and recombinant SOD3 might lead to treatment for prostate cancer.

Dendritic cells in colonic patches and iliac lymph nodes are essential in mucosal IgA induction following intrarectal administration via CCR7 interaction.

This study examined dendritic cells (DC) following intrarectal (IR) vaccination with the mucosal adjuvant cholera toxin (CT). Three rounds of IR vaccination with ovalbumin (OVA) and CT resulted in brisk levels of systemic and mucosal Ig responses. Immunohistochemical studies revealed that CD11c+ MHC class II+ cells accumulated primarily in the colonic patches (CP) and lamina propria of the large intestine (LI-LP), iliac LN (ILN) and MLN following IR vaccination with CT. Adoptively transferred CFSE-labeled OVA-specific CD4+ T cells proliferated significantly, secreting predominantly Th1-type cytokines in the CP (48 h after IR vaccination with CT) and Th2-type cytokines in the ILN (96 h after IR vaccination with CT). Following three IR vaccinations, CP-null mice that were generated by in utero treatment with anti-IL-7R Ab showed reduced levels of serum IgG and fecal IgA antibodies, suggesting a crucial role for CP in the initiation of systemic and mucosal immune responses. Of most interest, IR vaccination reduced IgA levels in fecal extracts significantly more in the CCR7-/- mice than in the wild-type mice. These results indicate that IR vaccination primarily mobilizes CD11c+ cells in the CP and ILN to induce optimal mucosal immune responses by CCR7 interaction.