CD98 regulates the phosphorylation of HER2 and a bispecific anti-HER2/CD98 antibody inhibits the growth signal of human breast cancer cells.

Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.

Dual-targeting therapy against HER3/MET in human colorectal cancers.

BACKGROUND: Colorectal cancer (CRC) is the most common malignancy in the world, and novel molecular targeted therapies for CRC have been vigorously pursued. We searched for novel combination therapies based on the expression patterns of membrane proteins in CRC cell lines. RESULTS: A positive correlation was observed between the expression of human pidermal growth factor receptor (HER) 3 and mesenchymal-to-epithelial transition factor (MET) on the cell surface of CRC cell lines. The brief stimulation of HER3/MET-high SW1116 CRC cells with both neuregulin-1 (NRG1) and hepatocyte growth factor enhanced ERK phosphorylation and cell proliferation more than each stimulation alone. In addition, a prolonged NRG1 stimulation resulted in the tyrosine phosphorylation of MET. In this context, the Forkhead Box protein M1 (FOXM1)-regulated tyrosine phosphorylation of MET by NRG1 was demonstrated, suggesting the existence of a signaling pathway mediated by FOXM1 upon the NRG1 stimulation. Since the co-expression of HER3 and MET was also demonstrated in in vivo CRC tissues by immunohistochemistry, we investigated whether the co-inhibition of HER3 and MET could be an effective therapy for CRC. We established HER3-and/or MET-KO SW1116 cell lines, and HER3/MET-double KO resulted in the inhibition of in vitro cell proliferation and in vivo tumor growth in nude mice by SW1116 cells. Furthermore, the combination of patritumab, an anti-HER3 fully human mAb, and PHA665752, a MET inhibitor, markedly inhibited in vitro cell proliferation, 3D-colony formation, and in vivo tumor growth in nude mice by SW1116 cells CONCLUSION: The dual targeting of HER3/MET has potential as CRC therapy.

Diverse and divergent functions of IL-32ß and IL-32γ isoforms in the regulation of malignant pleural mesothelioma cell growth and the production of VEGF-A and CXCL8.

In this study, we sought to elucidate the roles of the interleukin (IL)-32ß and IL-32γ in mesothelioma cell growth, and vascular endothelial growth factor (VEGF)-A and C-X-C motif chemokine ligand 8 (CXCL8) expression. IL-32 elicited a growth-promoting effect against one of the six mesotheliomas lines and exerted diverse regulatory functions in VEGF-A and CXCL8 secretion from mesotheliomas stimulated with or without IL-17A. Retroviral-mediated transduction of mesothelioma lines with IL-32γ resulted in enhanced IL-32ß expression, which facilitated or suppressed the in vitro growth, and VEGF-A and CXCL8 expression. Overexpressed IL-32ß-augmented growth and VEGF-A and CXCL8 production were mainly mediated through the phosphatidylinositol-3 kinase (PI3K) signaling pathway. On the other hand, overexpressed IL-32ß-deceased growth was mediated through mitogen-activated protein kinase (MAPK) pathway. NCI-H2373IL-32γ tumors grew faster than NCI-H2373Neo tumors in a xenograft model, which was associated with increased vascularity. These findings indicate that IL-32 are involved in the regulation of growth and angiogenic factor production in mesotheliomas.

Esterification promotes the intracellular accumulation of roxadustat, an activator of hypoxia-inducible factors, to extend its effective duration.

Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.

Altered binding avidities and improved growth inhibitory effects of novel anti-HER3 mAb against human cancers in the presence of HER1-or HER2-targeted drugs.

HER1-and HER2-targeted drugs are effective in cancer therapy, especially against lung, breast and colon malignancies; however, resistance of cancer cells to HER1-and HER2-targeted therapies is becoming a serious problem. The avidity/affinity constant (KA) and growth inhibitory effect of anti-HER3 rat monoclonal antibodies (mAb, Ab1∼Ab6) in the presence of therapeutic mAb or low-molecular-weight inhibitors against HER family proteins were analyzed by flow cytometry-based Scatchard plots (Splot) and cell proliferation assay. The KA of Ab3 and Ab6, but not Ab1 or Ab4, split into dual (high and low) modes of KA, and Ab6 exhibited greater anti-proliferative effects against LS-174T colon cancer cells in the presence of Pertuzumab (anti-HER2 mAb). A high KA by Ab6 and Ab6-mediated increased growth inhibition were observed against NCI-H1838 lung or BT474 breast cancer cells, respectively, in the presence of Panitumumab (anti-HER1 mAb) or Perutuzumab. A high KA by Ab6 and Ab6-mediated increased anti-proliferative effects against NCI-H1838 or BT474 were also respectively observed in the presence of Erlotinib (HER1 inhibitor) or Lapatinib (HER1/HER2 inhibitor). In HER1-knockout (KO) NCI-H1838, the reactivity and KA of Ab4 increased compared with in parent NCI-H1838. In HER1-KO or HER3-KO SW1116 colon cancer cells, dual modes of KA with Pertuzumab were noted, and the combination Ab6 and Pertuzumab promoted growth inhibition of HER1-KO, but not of parent SW1116.

Oncogenic transformation of NIH/3T3 cells by the overexpression of L-type amino acid transporter 1, a promising anti-cancer target.

L-type amino acid transporter 1 (LAT1)/SLC7A5 is the first identified CD98 light chain disulfide linked to the CD98 heavy chain (CD98hc/SLC3A2). LAT1 transports large neutral amino acids, including leucine, which activates mTOR, and is highly expressed in human cancers. We investigated the oncogenicity of human LAT1 introduced to NIH/3T3 cells by retrovirus infection. NIH/3T3 cell lines stably expressing human native (164C) or mutant (164S) LAT1 (naLAT1/3T3 or muLAT1/3T3, respectively) were established. We confirmed that endogenous mouse CD98hc forms a disulfide bond with exogenous human LAT1 in naLAT1/3T3, but not in muLAT1/3T3. Endogenous mouse CD98hc mRNA increased in both naNIH/3T3 and muLAT1/3T3, and a similar amount of exogenous human LAT1 protein was detected in both cell lines. Furthermore, naLAT1/3T3 and muLAT1/3T3 cell lines were evaluated for cell growth-related phenotypes (phosphorylation of ERK, cell-cycle progression) and cell malignancy-related phenotypes (anchorage-independent cell growth, tumor formation in nude mice). naLAT1/3T3 had stronger growth- and malignancy- related phenotypes than NIH/3T3 and muLAT1/3T3, suggesting the oncogenicity of native LAT1 through its interaction with CD98hc. Anti-LAT1 monoclonal antibodies significantly inhibited in vitro cell proliferation and in vivo tumor growth of naLAT1/3T3 cells in nude mice, demonstrating LAT1 to be a promising anti-cancer target.

In lysate RNA digestion provides insights into the angiogenin's specificity towards transfer RNAs.

Under adverse conditions, tRNAs are processed into fragments called tRNA-derived stress-induced RNAs (tiRNAs) by stress-responsive ribonucleases (RNases) such as angiogenin (ANG). Recent studies have reported several biological functions of synthetic tiRNAs lacking post-transcriptional modifications found on endogenous tiRNAs. Here we describe a simple and reproducible method to efficiently isolate ANG-cleaved tiRNAs from endogenous tRNAs. Using this in vitro method, more than 50% of mature tRNAs are cleaved into tiRNAs which can be enriched using complementary oligonucleotides. Using this method, the yield of isolated endogenous 5'-tiRNAGly-GCC was increased about fivefold compared to when tiRNAs were obtained by cellular treatment of ANG. Although the non-specific ribonuclease activity of ANG is much lower than that of RNase A, we show that ANG cleaves physiologically folded tRNAs as efficiently as bovine RNase A. These results suggest that ANG is highly specialized to cleave physiologically folded tRNAs. Our method will greatly facilitate the analysis of endogenous tiRNAs to elucidate the physiological functions of ANG.

Identification of novel biomarkers of hepatocellular carcinoma by high-definition mass spectrometry: Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry and desorption electrospray ionization mass spectrometry imaging.

RATIONALE: Hepatocellular carcinoma (HCC) is a highly malignant disease for which the development of prospective or prognostic biomarkers is urgently required. Although metabolomics is widely used for biomarker discovery, there are some bottlenecks regarding the comprehensiveness of detected features, reproducibility of methods, and identification of metabolites. In addition, information on localization of metabolites in tumor tissue is needed for functional analysis. Here, we developed a wide-polarity global metabolomics (G-Met) method, identified HCC biomarkers in human liver samples by high-definition mass spectrometry (HDMS), and demonstrated localization in cryosections using desorption electrospray ionization MS imaging (DESI-MSI) analysis. METHODS: Metabolic profiling of tumor (n = 38) and nontumor (n = 72) regions in human livers of HCC was performed by an ultrahigh-performance liquid chromatography quadrupole time-of-flight MS (UHPLC/QTOFMS) instrument equipped with a mixed-mode column. The HCC biomarker candidates were extracted by multivariate analyses and identified by matching values of the collision cross section and their fragment ions on the mass spectra obtained by HDMS. Cryosections of HCC livers, which included both tumor and nontumor regions, were analyzed by DESI-MSI. RESULTS: From the multivariate analysis, m/z 904.83 and m/z 874.79 were significantly high and low, respectively, in tumor samples and were identified as triglyceride (TG) 16:0/18:1(9Z)/20:1(11Z) and TG 16:0/18:1(9Z)/18:2(9Z,12Z) using the synthetic compounds. The TGs were clearly localized in the tumor or nontumor areas of the cryosection. CONCLUSIONS: Novel biomarkers for HCC were identified by a comprehensive and reproducible G-Met method with HDMS using a mixed-mode column. The combination analysis of UHPLC/QTOFMS and DESI-MSI revealed that the different molecular species of TGs were associated with tumor distribution and were useful for characterizing the progression of tumor cells and discovering prospective biomarkers.

The guanylate cyclase C agonist linaclotide ameliorates the gut-cardio-renal axis in an adenine-induced mouse model of chronic kidney disease.

BACKGROUND: Cardiorenal syndrome is a major cause of mortality in patients with chronic kidney disease (CKD). However, the involvement of detrimental humoral mediators in the pathogenesis of cardiorenal syndrome is still controversial. Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine or carnitine derived by the gut microbiota, has been linked directly with progression of cardiovascular disease and renal dysfunction. Thus, targeting TMAO may be a novel strategy for the prevention of cardiovascular disease and chronic kidney disease. METHODS: Linaclotide, a guanylate cyclase C agonist, was administered to adenine-induced renal failure (RF) mice and changes in renal function and levels of gut-derived uremic toxins, as well as the gut microbiota community, were analyzed using metabolomic and metagenomic methods to reveal its cardiorenal effect. RESULTS: Linaclotide decreased the plasma levels of TMAO at a clinically used low dose of 10 µg/kg in the adenine-induced RF mouse model. At a high concentration of 100 µg/kg, linaclotide clearly improved renal function and reduced the levels of various uremic toxins. A reduction in TMAO levels following linaclotide treatment was also observed in a choline-fed pro-atherosclerotic model. Linaclotide ameliorated renal inflammation and fibrosis and cardiac fibrosis, as well as decreased the expression of collagen I, transforming growth factor-ß, galectin-3 (Gal-3) and ST2 genes. Plasma levels of Gal-3 and ST2 were also reduced. Because exposure of cardiomyocytes to TMAO increased fibronectin expression, these data suggest that linaclotide reduced the levels of TMAO and various uremic toxins and may result in not only renal, but also cardiac, fibrosis. F4/80-positive macrophages were abundant in small intestinal crypts in RF mice, and this increased expression was decreased by linaclotide. Reduced colonic claudin-1 levels were also restored by linaclotide, suggesting that linaclotide ameliorated the 'leaky gut' in RF mice. Metagenomic analysis revealed that the microbial order Clostridiales could be responsible for the change in TMAO levels. CONCLUSION: Linaclotide reduced TMAO and uremic toxin levels and could be a powerful tool for the prevention and control of the cardiorenal syndrome by modification of the gut-cardio-renal axis.

An agonistic anti-Toll-like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy.

Immune-checkpoint blockade antibodies have been approved for the treatment of cancer. However, poorly immunogenic tumours are less responsive to such therapies. Agonistic anti-Toll-like receptor 4 (TLR4) monoclonal antibodies (mAbs) activate only cell-surface TLR4; in contrast, lipopolysaccharide (LPS) activates both TLR4 and intracellular inflammatory caspases. In this study, we investigated the adjuvant activity of an anti-TLR4 mAb in T-cell-mediated antitumour immunity. The anti-TLR4 mAb induced the activation of antigen-specific T-cells in adoptive transfer studies. The growth of ovalbumin (OVA)-expressing tumours was significantly suppressed by administration of OVA and the anti-TLR4 mAb in combination, but not individually. The antitumour effect of anti-PD-1 mAb was enhanced in mice administered with OVA plus the anti-TLR4 mAb. The OVA-specific IFN-γ-producing CD8 T-cells were induced by administration of OVA and the anti-TLR4 mAb. The suppression of tumour growth was diminished by depletion of CD8, but not CD4, T-cells. The inflammatory response to the anti-TLR4 mAb was of significantly lesser magnitude than that to LPS, as assessed by NF-κB activation and production of TNF-α, IL-6 and IL-1ß. Administration of LPS (at a dose that elicited levels of proinflammatory cytokines comparable to those by the anti-TLR4 mAb) plus OVA induced no or less-marked activation of OVA-specific T-cells and failed to suppress tumour growth in mice. In conclusion, the agonistic anti-TLR4 mAb induces potent CD8 T-cell-dependent antitumour immunity and an inflammatory response of lesser magnitude than does LPS. The agonistic anti-TLR4 mAb has potential as an adjuvant for use in vaccines against cancer.

Generation and Characterization of Anti-phenyl Sulfate Monoclonal Antibodies and a Potential Use for Phenyl Sulfate Analysis in Human Blood.

Patients with chronic kidney disease (CKD) have increased blood levels of phenyl sulfate (PS), a circulating uremic toxin. In this study, we produced anti-PS monoclonal antibodies (mAbs) and characterized their cross-reactivity to structural PS analogs. To induce PS-specific mAbs, we synthesized 4-mercaptophenyl sulfate with a sulfhydryl group at the para-position of PS and conjugated it to carrier proteins via bifunctional linkers. Using these PS conjugates as immunogens and as antigens for enzyme-linked immunosorbent assay (ELISA) screening, we produced by a hybridoma method two novel mAbs (YK33.1 and YKS19.2) that react with PS conjugates independent of carrier and linker structures. Although all of the PS analogs tested, with the exception of indoxyl sulfate, were cross-reactive to both mAbs in phosphate buffered saline (PBS), PS specificity for YKS19.2 was enhanced in human plasma and serum. YKS19.2 mAb was cross-reactive only with o-cresyl sulfate, which is absent in human blood. PS sensitivity for YKS19.2 mAb increased to an IC50 of 10.4 µg/mL when 0.1% Tween 20 was added in a primary competitive reaction. To explore potential clinical applications, we determined concentrations of PS in serum samples from 19 CKD patients by inhibition ELISA using YKS19.2 mAb and compared them to those found using an LC-MS/MS method. A good correlation was observed between each value (R2=0.825). Therefore, the unique antigen specificity of YKS19.2 mAb could be useful for prescreening of patients with accumulated PS or for comprehensive analysis of uremic toxins that have a PS-like structure.

Mitochonic Acid 5 (MA-5) Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases.

Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model "Mitomouse" (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial.

A novel indole compound MA-35 attenuates renal fibrosis by inhibiting both TNF-α and TGF-ß1 pathways.

Renal fibrosis is closely related to chronic inflammation and is under the control of epigenetic regulations. Because the signaling of transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) play key roles in progression of renal fibrosis, dual blockade of TGF-ß1 and TNF-α is desired as its therapeutic approach. Here we screened small molecules showing anti-TNF-α activity in the compound library of indole derivatives. 11 out of 41 indole derivatives inhibited the TNF-α effect. Among them, Mitochonic Acid 35 (MA-35), 5-(3, 5-dimethoxybenzyloxy)-3-indoleacetic acid, showed the potent effect. The anti-TNF-α activity was mediated by inhibiting IκB kinase phosphorylation, which attenuated the LPS/GaIN-induced hepatic inflammation in the mice. Additionally, MA-35 concurrently showed an anti-TGF-ß1 effect by inhibiting Smad3 phosphorylation, resulting in the downregulation of TGF-ß1-induced fibrotic gene expression. In unilateral ureter obstructed mouse kidney, which is a renal fibrosis model, MA-35 attenuated renal inflammation and fibrosis with the downregulation of inflammatory cytokines and fibrotic gene expressions. Furthermore, MA-35 inhibited TGF-ß1-induced H3K4me1 histone modification of the fibrotic gene promoter, leading to a decrease in the fibrotic gene expression. MA-35 affects multiple signaling pathways involved in the fibrosis and may recover epigenetic modification; therefore, it could possibly be a novel therapeutic drug for fibrosis.

Tyrosine hydroxylase gene expression is facilitated by alcohol followed by the degradation of the protein by ubiquitin proteasome system.

OBJECTIVES: Alcohol intake induces brief periods of euphoria; however, its continuous consumption can lead the development of alcohol tolerance. The euphoria, an intense feeling of wellbeing, is deeply associated with dopamine. Dopamine biosynthesis is strictly regulated by tyrosine hydroxylase (TH), a rate-limiting enzyme of dopamine. The aim of this study was to examine the transient or chronic effects of ethanol treatment on TH protein level in vitro. METHODS: Cultured primary mesencephalic neurons were prepared and exposed to 100 mM ethanol for 48 hours or 168 hours. TH and cAMP-responsive element (CRE)-mediated transcriptional activity was measured by reporter gene assay using pTH9.0kb-Luc and pCRE-Luc reporter plasmid. TH protein expression and TH phosphorylation was analyzed by Western blot analysis. Dopamine content was measured by high-performance liquid chromatography (HPLC). RESULTS: Ethanol treatment for 48 hours facilitates TH transcriptional activity and TH protein expression in a cAMP-dependent protein kinase A (PKA) and MAPK/Erk kinase (MEK)-dependent manner in cultured mesencephalic neurons. Ethanol also facilitated TH phosphorylation, which resulted in the elevation of dopamine content. On the other hand, treatment with ethanol for 168 hours did not show significant elevation of TH gene expression and dopamine biosynthesis. Intriguingly, simultaneous treatment with MG-132, a 26S proteasomal inhibitor, recovered the ethanol-induced increase of TH protein expression and dopamine biosynthesis. CONCLUSION: Transient ethanol-treatment facilitates TH gene expression and its phosphorylation in a PKA- and MEK-dependent manner to elevate dopamine biosynthesis, whereas continuous exposure to ethanol abolishes its potent effects on the dopaminergic function to reduce dopamine content. This reduction seems to originate from the decrease of TH protein level by degradation of the protein. Our current data may contribute to the better understanding of alcohol tolerance associated with degradation of TH protein to reduce total-TH level and dopamine biosynthesis.

Licochalcones extracted from Glycyrrhiza inflata inhibit platelet aggregation accompanied by inhibition of COX-1 activity.

Licochalcones extracted from Glycyrrhiza inflata are known to have a variety of biological properties such as anti-inflammatory, anti-bacterial, and anti-tumor activities, but their action on platelet aggregation has not yet been reported. Therefore, in this study we investigated the effects of licochalcones on platelet aggregation. Collagen and U46619, a thromboxane A2 receptor agonist, caused rabbit platelet aggregation, which was reversed by pretreatment with licochalcones A, C and D in concentration-dependent manners. Among these compounds, licochalcone A caused the most potent inhibitory effect on collagen-induced platelet aggregation. However, the licochalcones showed marginal inhibitory effects on thrombin or ADP-induced platelet aggregation. In addition to rabbit platelets, licochalcone A attenuated collagen-induced aggregation in human platelets. Because licochalcone A also inhibited arachidonic acid-induced platelet aggregation and production of thromboxane A2 induced by collagen in intact platelets, we further examined the direct interaction of licochalcone A with cyclooxygenase (COX)-1. As expected, licochalcone A caused an inhibitory effect on both COX-1 and COX-2 in vitro. Regarding the effect of licochalcone A on COX-1 enzyme reaction kinetics, although licochalcone A showed a stronger inhibition of prostaglandin E2 synthesis induced by lower concentrations of arachidonic acid, Vmax values in the presence or absence of licochalcone A were comparable, suggesting that it competes with arachidonic acid at the same binding site on COX-1. These results suggest that licochalcones inhibit collagen-induced platelet aggregation accompanied by inhibition of COX-1 activity.

A Heterodimeric Cytokine, Consisting of IL-17A and IL-17F, Promotes Migration and Capillary-Like Tube Formation of Human Vascular Endothelial Cells.

The interleukin (IL)-17 family, consisting of six homodimeric cytokines IL-17A, IL-17B, IL-17C, IL-17D, IL-17E/IL-25, and IL-17F, mediates a variety of biological activities including regulation of chemokine secretion and angiogenesis. Among the IL-17 family members, IL-17A and IL-17E/IL-25 are angiogenesis stimulators, while IL-17B and IL-17F are angiogenesis inhibitors. Recently, IL-17A/F heterodimer, comprised of the IL-17A and IL-17F subunits, was found as another member of the IL-17 cytokine family. However, to date, it has been unknown whether IL-17A/F has biological actions to affect the angiogenesis-related vascular endothelial functions. Therefore, in this study, we investigated the biological effects of IL-17A/F on the growth, migration and capillary-like tube formation of vascular endothelial cells. Recombinant IL-17A/F protein had no direct effects on the growth of human dermal microvascular endothelial cells (HMVECs), whereas, after 4-hour incubation in a modified Boyden Chemotaxicell chamber, IL-17A/F significantly induced migration of HMVECs over a wide range of doses via the phosphatidylinositol-3 kinase (PI3K) signaling pathway. We further investigated the biological effect of IL-17A/F on capillary-like tube formation using a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs), which mimicked the in vivo microenvironment. In this co-culture system, IL-17A/F significantly promoted capillary-like endothelial tube formation in a dose-dependent fashion via the PI3K and extracellular signal-regulated kinase (ERK) signaling pathways. Additionally, IL-17A/F up-regulated secretion of angiogenic growth factors such as IL-8 and growth-related oncogene (GRO)-α by HDFs. These findings identify a novel biological function for IL-17A/F as an indirect angiogenic agent.

Dopamine or biopterin deficiency potentiates phosphorylation at (40)Ser and ubiquitination of tyrosine hydroxylase to be degraded by the ubiquitin proteasome system.

The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists.

Mitochonic Acid 5 (MA-5), a Derivative of the Plant Hormone Indole-3-Acetic Acid, Improves Survival of Fibroblasts from Patients with Mitochondrial Diseases.

Mitochondria are key organelles implicated in a variety of processes related to energy and free radical generation, the regulation of apoptosis, and various signaling pathways. Mitochondrial dysfunction increases cellular oxidative stress and depletes ATP in a variety of inherited mitochondrial diseases and also in many other metabolic and neurodegenerative diseases. Mitochondrial diseases are characterized by the dysfunction of the mitochondrial respiratory chain, caused by mutations in the genes encoded by either nuclear DNA or mitochondrial DNA. We have hypothesized that chemicals that increase the cellular ATP levels may ameliorate the mitochondrial dysfunction seen in mitochondrial diseases. To search for the potential drugs for mitochondrial diseases, we screened an in-house chemical library of indole-3-acetic-acid analogs by measuring the cellular ATP levels in Hep3B human hepatocellular carcinoma cells. We have thus identified mitochonic acid 5 (MA-5), 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid, as a potential drug for enhancing ATP production. MA-5 is a newly synthesized derivative of the plant hormone, indole-3-acetic acid. Importantly, MA-5 improved the survival of fibroblasts established from patients with mitochondrial diseases under the stress-induced condition, including Leigh syndrome, MELAS (myopathy encephalopathy lactic acidosis and stroke-like episodes), Leber's hereditary optic neuropathy, and Kearns-Sayre syndrome. The improved survival was associated with the increased cellular ATP levels. Moreover, MA-5 increased the survival of mitochondrial disease fibroblasts even under the inhibition of the oxidative phosphorylation or the electron transport chain. These data suggest that MA-5 could be a therapeutic drug for mitochondrial diseases that exerts its effect in a manner different from anti-oxidant therapy.

Conformational change in transfer RNA is an early indicator of acute cellular damage.

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

A validated quantitative liquid chromatography-tandem quadrupole mass spectrometry method for monitoring isotopologues to evaluate global modified cytosine ratios in genomic DNA.

5-Hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) represent important epigenetic modifications to DNA, and a sensitive analytical method is required to determine the levels of 5hmC in the genomic DNA of tumor cells or cultured cell lines because 5hmC is present at particular low levels in these cells. We have developed a sensitive liquid chromatography-tandem quadrupole mass spectrometric method for quantifying 5-hydroxymethyldeoxycytidine (5hmdC), 5-methyldeoxycytidine (5mdC), and deoxyguanosine (dG) levels using stable isotope labeled internal standards, and used this method to estimate the global level of 2 modified cytosines in genomic DNA prepared from small number of cells. The quantification limits for 5hmdC, 5mdC and dG were 20pM, 2nM and 10nM, respectively. MRM transitions for isotopologue (isotopologue-MRM) were used to quantify the 5mdC and dG levels because of the abundance of these nucleosides relative to 5hmdC. The use of isotopologue-MRM for the abundant nucleosides could also avoid the saturation of the detector, and allow for all three nucleosides to be analyzed simultaneously without the need for the dilution and re-injection of samples into the instrument. The global ratios of modified cytosine nucleosides to dG were estimated following the quantification of each nucleoside in the hydrolysate of genomic DNA. The limit of estimation for the global 5hmC level was less than 0.001% using 200ng of DNA. Using this method, we found that MLL-TET1, which a fusion protein in acute myelogenous leukemia, did not produce 5hmC, but interfered with TET1 activity to produce 5hmC in cells. Our analytical method is therefore a valuable tool for further studies aiming at a deeper understanding of the role of modified cytosine in the epigenetic regulation of cells.