Raman Spectroscopic and DFT Study of COA-Cl and Its Analogues.

COA-Cl is a newly synthesized adenosine analogue that exhibits various physiological activities. Its angiogenic, neurotropic, and neuroprotective potencies make it promising for the development of medicines. In this study, we show Raman spectroscopic study of COA-Cl to elucidate molecular vibrations and related chemical properties. Density functional theory calculations were combined with the Raman spectroscopic data to understand the details of each vibrational mode. Comparative analysis with adenine, adenosine, and other nucleic acid analogues enabled identification of unique Raman peaks originating from the cyclobutane moiety and chloro group of COA-Cl. This study provides fundamental knowledge and crucial insights for further development of COA-Cl and related chemical species.

Synthesis and Evaluation of Novel Cyclopropane Nucleoside as Potential Tube Formation Agents.

Five novel nucleoside analogs with mono or bis-hydroxymethylated cyclopropane rings at the N9-position of the 2-chloroadenine moiety (2-chloro-carbocyclic oxetanocin A [COA-Cl] analog) were synthesized and evaluated using human umbilical vein endothelial cells. All the prepared compounds (2a-e) showed good to moderate activity with angiogenic potency. cis-2'-(Hydroxymethyl)cycloprop-1'-yl derivative (2b) at 100 µM had greater angiogenic activity than the other compounds did, with relative tube areas of 2.71±0.45 (mean±standard deviation (S.D.)), which was superior to the potency of COA-Cl (2.30±0.59).

COA-Cl, a Novel Synthesized Nucleoside Analog, Exerts Neuroprotective Effects in the Acute Phase of Intracerebral Hemorrhage.

BACKGROUND: A previous study in our laboratory showed the neuroprotective effects of COA-Cl, a novel synthesized adenosine analog, in a rat cerebral ischemia model. The purpose of the present study was to evaluate the neuroprotective effects of COA-Cl in intracerebral hemorrhage (ICH), another common type of stroke, and investigate potential mechanisms of action. METHODS: Adult Sprague-Dawley rats received an injection of 100 µl autologous whole blood into the right basal ganglia. COA-Cl (30 µg/kg) was injected intracerebroventricularly 10 minutes after ICH. A battery of motor deficit tests were performed at 1 day, 3 days, 5 days, and 7 days after ICH. To investigate the mechanism of action, brain water content, TUNEL staining and 8-OHdG immunostaining, and ELISA (to assess oxidative stress) were used. RESULTS: COA-Cl treatment significantly attenuated sensorimotor deficits and reduced brain edema 1 day after ICH. Furthermore, the numbers of perihematomal TUNEL- and 8-OHdG-positive cells were significantly decreased in COA-Cl treated ICH rats. CONCLUSIONS: These results indicate that COA-Cl has neuroprotective effects in ICH. Furthermore, our study provides evidence that COA-Cl may reduce oxidative stress, which may be one mechanism underlying its neuroprotective effects.

Synthesis and Evaluation of Novel Carbocyclic Oxetanocin A (COA-Cl) Derivatives as Potential Tube Formation Agents.

Six novel carbocyclic oxetanocin A analogs (2-chloro-C.OXT-A; COA-Cl) with various hydroxymethylated or spiro-conjugated cyclobutane rings at the N(9)-position of the 2-chloropurine moiety were synthesized and evaluated using human umbilical vein endothelial cells. All prepared compounds (2a-f) showed good to moderate activity with angiogenic potency. Among these compounds, 100 µM cis-trans-2',3'-bis(hydroxymethyl)cyclobutyl derivative (2b), trans-3'-hydroxymethylcyclobutyl analog (2d), and 3',3'-bis(hydroxymethyl)cyclobutyl derivative (2e) had greater angiogenic activity, with relative tube areas of 3.43±0.44, 3.32±0.53, and 3.59±0.83 (mean±standard deviation (S.D.)), respectively, which was comparable to COA-Cl (3.91±0.78). These data may be important for further development of this class of compounds as potential tube formation agents.

Systemically Administered D-allose Inhibits the Tumor Energy Pathway and Exerts Synergistic Effects With Radiation.

BACKGROUND/AIM: The present study investigated the anticancer effects of intraperitoneally administered D-allose in in vivo models of head and neck cancer cell lines. MATERIALS AND METHODS: To assess the direct effects of D-allose, its dynamics in blood and tumor tissues were examined. RESULTS: D-allose was detected in blood and tumor tissues 10 min after its intraperitoneal administration and then gradually decreased. In vivo experiments revealed that radiation plus D-allose was more effective than either treatment alone. Thioredoxin-interacting protein (TXNIP) mRNA over-expression was detected after the addition of D-allose in in vitro and in vivo experiments. D-allose inhibited cell growth, which was associated with decreases in glycolysis and intracellular ATP levels and the prolonged activation of AMPK. The phosphorylation of p38-MAPK was also observed early after the administration of D-allose and was followed by the activation of AMPK and up-regulated expression of TXNIP in both in vitro and in vivo experiments. CONCLUSION: Systemically administered D-allose appears to exert antitumor effects. Further studies are needed to clarify the appropriate dosage and timing of the administration of D-allose and its combination with other metabolic agents.

Dexamethasone induces caveolin-1 in vascular endothelial cells: implications for attenuated responses to VEGF.

Steroids exert direct actions on cardiovascular cells, although underlying molecular mechanisms remain incompletely understood. We examined if steroids modulate abundance of caveolin-1, a regulatory protein of cell-surface receptor pathways that regulates the magnitudes of endothelial response to vascular endothelial growth factor (VEGF). Dexamethasone, a synthetic glucocorticoid, induces caveolin-1 at both levels of protein and mRNA in a time- and dose-dependent manner in pharmacologically relevant concentrations in cultured bovine aortic endothelial cells. Aldosterone, a mineralocorticoid, but not the sex steroids 17ß-estradiol, testosterone, or progesterone, elicits similar caveolin-1 induction. Caveolin-1 induction by dexamethasone and that by aldosterone were abrogated by RU-486, an inhibitor of glucocorticoid receptor, and by spironolactone, a mineralocorticoid receptor inhibitor, respectively. Dexamethasone attenuates VEGF-induced responses at the levels of protein kinases Akt and ERK1/2, small-G protein Rac1, nitric oxide production, and migration. When induction of caveolin-1 by dexamethasone is attenuated either by genetically by transient transfection with small interfering RNA or pharmacologically by RU-486, kinase responses to VEGF are rescued. Dexamethasone also increases expression of caveolin-1 protein in cultured human umbilical vein endothelial cells, associated with attenuated tube formation responses of these cells when cocultured with normal fibroblasts. Immunohistochemical analyses revealed that intraperitoneal injection of dexamethasone induces endothelial caveolin-1 protein in thoracic aorta and in lung artery in healthy male rats. Thus steroids functionally attenuate endothelial responses to VEGF via caveolin-1 induction at the levels of signal transduction, migration, and tube formation, identifying a novel point of cross talk between nuclear and cell-surface receptor signaling pathways.

Effects of monosaccharides including rare sugars on proliferation of Entamoeba histolytica trophozoites in vitro.

Entamoeba histolytica is a parasitic protozoan with roles in pathogenicity of intestinal amoebiasis. E. histolytica trophozoites lack functional mitochondria and their energy production depends mostly on glycolysis. D-Glucose has a pivotal role in this process and trophozoites store this sugar as glycogen in glycogen granules. Rare sugars, which are defined as sugars present in nature in limited amounts, are of interest as natural low-calorie sweeteners for improving physical conditions of humans. One such rare sugar, D-allose, can be absorbed by a sodium-dependent glucose cotransporter as a substitute for D-glucose, and some rare sugars are known to inhibit growth of cancer cells, Caenorhabditis elegans and Tritrichomonas foetus. Based on these observations, we examined the effects of rare sugars on growth of E. histolytica trophozoites, together with those of D-galactose and D-fructose. The results indicate that treatment with D-allose or D-psicose (D-allulose) alone inhibits proliferation of E. histolytica trophozoites, but that these sugars enhance proliferation of trophozoites in the presence of D-glucose or D-galactose. The trophozoites could take up D-glucose and D-galactose, but not D-fructose, D-allose or D-psicose. Cell sizes of the trophozoites also differed depending on the culture medium.

Rare sugar D-psicose protects pancreas ß-islets and thus improves insulin resistance in OLETF rats.

Rare sugar D-psicose has cropped up as a non-toxic and effective compound to protect and preserve pancreatic ß-islets in the growing type 2 diabetes mellitus (T2DM) rats through the regulation of glucose and fat metabolism. The present study was undertaken to examine the effect of rare sugar D-psicose on the protection of pancreatic ß-islets using Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a T2DM model. Treated rats were fed with 5% D-psicose or 5% D-glucose supplemented drinking water, and only water in the control for 13 weeks. A non-diabetic Long-Evans Tokushima Otsuka (LETO), fed with water served as a counter control of OLETF. D-Psicose significantly attenuated progressive ß-islet fibrosis and preserved islets, evaluated by hematoxylin-eosin staining, Masson's trichrome staining and immunostainings of insulin and α-smooth muscle actin (SMA). D-Psicose significantly reduced increase in body weight and abdominal fat deposition. Oral glucose tolerance test (OGTT) showed reduced blood glucose levels suggesting the improvement of insulin resistance. All these data suggests that D-psicose protected and preserved pancreatic ß-islets through the maintenance of hyperglycemia and by the prevention of fat accumulation in OLETF rats.

COA-Cl Evokes Protective Responses Against H2O2-and 6-OHDA-Induced Toxic Injury in PC12 Cells.

COA-Cl, a novel adenosine-like nucleic acid analog, has recently been shown to exert neuroprotective effects and to increase dopamine levels both in vivo and in vitro. Therefore, we hypothesized that COA-Cl could protect dopaminergic neurons against toxic insults. Thus, the present study aimed to investigate the protective effects of COA-Cl against hydrogen peroxide (H2O2)- and 6-hydroxydopamine (6-OHDA)-induced toxicity in PC12 cells and to elucidate the possible mechanisms. PC12 cells were incubated with COA-Cl (100 µM) with or without H2O2 or 6-OHDA (200 µM) for 24 h. Treatment with COA-Cl attenuated the decrease in cell viability, SOD activity and the Bcl-2/Bax ratio caused by H2O2. In addition, COA-Cl attenuated the increase in LDH release, ROS production, caspase-3 activity, and apoptosis induced by H2O2. Further, COA-Cl enhanced the protection of PC12 cells against the toxicity caused by 6-OHDA, as evidenced by an increase in cell viability and the Bcl-2/Bax ratio, and a decrease in LDH release. Our results are the first to demonstrate that COA-Cl potentially protects PC12 cells against toxicity induced by H2O2 and 6-OHDA, implying that COA-Cl could be a promising neuroprotective agent for the treatment of Parkinson's disease.

Rare sugar D-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

A rare sugar, D-psicose has progressively been evaluated as a unique metabolic regulator of glucose and lipid metabolism, and thus represents a promising compound for the treatment of type 2 diabetes mellitus (T2DM). The present study was undertaken to examine the underlying effector organs of D-psicose in lowering blood glucose and abdominal fat by exploiting a T2DM rat model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Rats were fed 5% D-psicose or 5% D-glucose supplemented in drinking water, and only water in the control for 13 weeks and the protective effects were compared. A non-diabetic Long-Evans Tokushima Otsuka (LETO), fed with water served as a counter control of OLETF. After 13 weeks feeding, D-psicose treatment significantly reduced the increase in body weight and abdominal fat mass. Oral glucose tolerance test (OGTT) showed the reduced blood glucose and insulin levels suggesting the improvement of insulin resistance in OLETF rats. Oil-red-O staining elucidated that D-psicose significantly reduced lipid accumulation in the liver. Immunohistochemical analysis showed D-psicose induced glucokinase translocation from nucleus to cytoplasm of the liver which enhances glucokinase activity and subsequent synthesis of glycogen in the liver. D-psicose also protected the pathological change of the ß-cells of pancreatic islets. These data demonstrate that D-psicose controls blood glucose levels by reducing lipotoxicity in liver and by preserving pancreatic ß-cell function.

The protective effect and mechanism of COA-Cl in acute phase after spinal cord injury.

Spinal cord injury (SCI) induces severe motor and sensory dysfunction. We previously showed the neuroprotective effects of COA-Cl, a novel synthesized adenosine analog, in a rat stroke model. In this study, we evaluated the neuroprotective effects of COA-Cl in acute phase of SCI. SCI was induced in rats at the T9 vertebra by using a drop device. Rats were divided into acute and subacute groups. A 5-day dose of 6 mg/kg COA-Cl in saline was given to the acute group immediately after SCI and the subacute group 4 days after SCI. Motor function assessed by Basso-Beattie-Bresnahan scoring and inclined plane test improved significantly in the acute group while the subacute group did not. Histological evaluation and TUNEL staining revealed that both the cavity volume and apoptosis were significantly decreased in the acute group compared with the subacute group. In addition, pERK/ERK was increased in the acute group 7 days after SCI. These results suggest that COA-Cl exerts neuroprotective effects via the ERK pathway when administered in the acute phase after SCI, resulting in the recovery of motor function. COA-Cl could be a novel therapeutic agent for the acute phase of SCI.

A novel nucleic acid analogue shows strong angiogenic activity.

A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100muM was stronger than that of vascular endothelial growth factor (VEGF), a positive control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.

COA-Cl prevented TGF-ß1-induced CTGF expression by Akt dephosphorylation in normal human dermal fibroblasts, and it attenuated skin fibrosis in mice models of systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. Although transforming growth factor (TGF)-ß1-induced connective tissue growth factor (CTGF/CCN2) expression has been presented in SSc fibrosis, the therapeutic potential of targeting CTGF in SSc has not been fully explored. COA-Cl is a novel nucleic acid analog, which is reported to have pleiotropic beneficial biologic effects. OBJECTIVE: We examine the effects of COA-Cl on TGF-ß1-induced CTGF expression in normal human dermal fibroblast (NHDF). We also examined the effects of COA-Cl on CTGF expression in a mouse SSc model of angiotensin II (Ang II)-induced skin fibrosis. METHODS: NHDF was cultured for in vitro experiments. For in vivo experiments, C57BL/6J mice were treated with Ang II for 14 days by subcutaneous osmotic pump. Quantitative real-time polymerase chain reaction, western blot analysis, immunohistochemical staining and immunofluorescence staining were performed to examine the expression levels of CTGF and phosphorylation levels of Smad2/3, ERK1/2 and Akt. RESULTS: COA-Cl attenuated the TGF-ß1-induced expression of both CTGF mRNA and protein in NHDF. Although COA-Cl did not alter the TGF-ß1-induced phosphorylation of Smad2/3 or ERK1/2, it reduced the TGF-ß1-induced phosphorylation levels of Akt in NHDF. Notably, COA-Cl dephosphorylated the Akt of lysates of TGF-ß1-treated NHDF. COA-Cl reduced the levels of CTGF mRNA, CTGF protein, dermal thickness, collagen content and Akt phosphorylation in the skin of mice SSc model. CONCLUSION: These results imply that the inhibition of TGF-ß1-induced CTGF expression by COA-Cl may be a therapeutic approach for SSc.

COA-Cl (2-Cl-C.OXT-A) can promote coronary collateral development following acute myocardial infarction in mice.

2-Cl-C.OXT-A (COA-Cl) is a novel nucleic acid analogue that promotes tube-forming activity of human umbilical vein endothelial cells (HUVEC) through vascular endothelial growth factor (VEGF). The development of coronary collateral circulation is critical to rescue the ischemic myocardium and to prevent subsequent irreversible ischemic injury. We evaluated whether COA-Cl can promote angiogenesis in ischemic tissue, reduce infarct size and preserve cardiac contractility in vivo. Mice received COA-Cl or placebo daily for three days after myocardial infarction (MI) by coronary ligation. The degree of angiogenesis in ischemic myocardium was assessed by staining endothelial cells and vascular smooth muscle cells, and measuring infarct size/area-at-risk. In mice treated with COA-Cl, enhanced angiogenesis and smaller infarct size were recognized, even given a similar area at risk. We observed increases in the protein expression levels of VEGF and in the protein phosphorylation level of eNOS. In addition, the heart weight to body weight ratio and myocardial fibrosis in COA-Cl mice were decreased on Day 7. Administration of COA-Cl after MI promotes angiogenesis, which is associated with reduced infarct size and attenuated cardiac remodeling. This may help to prevent heart failure due to cardiac dysfunction after MI.

COA-Cl induces dopamine release and tyrosine hydroxylase phosphorylation: In vivo reverse microdialysis and in vitro analysis.

We found that local perfusion of COA-Cl (0.1, 0.4, or 1.0 mM) into the dorsal striatum of living mice produced a significant and dose-dependent increase in extracellular DA levels, with the highest dose of 1.0 mM COA-Cl producing an approximately 5-fold increase in DA. Consistent with in vivo findings, 0.1 and 0.2 mM COA-Cl significantly and dose-dependently enhanced DA release 3.0 to 5.0-fold in PC12 cells, an in vitro model of DA-responsive neurons. Interestingly, the increase in striatal DA levels by COA-Cl in vivo was similar in magnitude to that observed in PC12 cells. Treatment with 0.1 mM COA-Cl significantly increased both Ser31 and Ser40 phosphorylation of tyrosine hydroxylase (TH) in PC12 cells, and Ser40 phosphorylation in iCell neurons, without altering total TH protein levels. Further, we examined whether COA-Cl could stimulate neurite outgrowth in PC12 cells and iCell neurons and found that COA-Cl significantly induced neurite outgrowth in both cell lines. Our results provide the first evidence that COA-Cl can stimulate dose-dependent DA release and activation of TH phosphorylation, suggesting that COA-Cl may be a promising therapeutic candidate for the treatment of neurological dysfunction associated with low DA.

Data on COA-Cl administration to the APP/PS2 double-transgenic mouse model of Alzheimer׳s disease: Improved hippocampus-dependent learning and unchanged spontaneous physical activity.

We herein present behavioral data regarding whether COA-Cl, a novel adenosine-like nucleic acid analog that promotes angiogenesis and features neuroprotective roles, improves cognitive and behavioral deficits in a murine model for Alzheimer׳s disease (AD). COA-Cl induced significant spatial memory improvement in the amyloid precursor protein/presenilin 2 double-transgenic mouse model of AD (PS2Tg2576 mice). Correspondingly, non-spatial novel object cognition test performance also significantly improved in COA-Cl-treated PS2Tg2576 mice; however, these mice demonstrated no significant changes in physical activity or motor performance. COA-Cl did not change the spontaneous activities and cognitive ability in the wild-type mice.

Both Ser361 phosphorylation and the C-arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint.

Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor that is down-regulated in several cancer tissues and tumor cell lines. Overexpression of TXNIP causes cell cycle arrest at the G1/S checkpoint in the hepatocellular carcinoma cell line HuH-7. TXNIP contains putative phosphorylation sites, but the effects of its phosphorylation have not been fully characterized. TXNIP also contains two α-arrestin domains (N-arrestin and C-arrestin) whose functions are not fully understood. Here, we reveal an association between TXNIP and cell cycle regulatory proteins (p27kip1, Jun activation domain-binding protein 1 (JAB1), Cdk2, and cyclin E), suggesting its participation in cell cycle regulation. We observed phosphorylation of TXNIP and used both in vivo and in vitro kinase assays to demonstrate that TXNIP can be phosphorylated by p38 mitogen-activated protein kinase. Furthermore, we also identified Ser361 in TXNIP as one of the major phosphorylation sites. Cell cycle analysis showed that Ser361 phosphorylation participates in TXNIP-mediated cell cycle arrest. In addition, the C-arrestin domain may also play an important role in cell cycle arrest. We also showed that phosphorylation at Ser361 may be important for the association of TXNIP with JAB1 and that the C-arrestin domain is necessary for the nuclear localization of this molecule. Collectively, these studies reveal that TXNIP participates in cell cycle regulation through association with regulatory proteins, especially JAB1, and that C-arrestin-dependent nuclear localization is important for this function. This work may facilitate the development of a new cancer therapy strategy that targets TXNIP as a key molecule inhibiting cancer cell growth via cell cycle blockade at the G1/S checkpoint.

Effect of COA-Cl on transforming growth factor-ß1-induced epithelial-mesenchymal transition in RLE/Abca3 cells.

Transforming growth factor (TGF)-ß1 has received much attention as a major inducer of epithelial-mesenchymal transition (EMT) in pathological conditions such as cancer and organ fibrosis. In this study, we examined the effect of a novel nucleic acid analog, COA-Cl, on TGF-ß1-induced EMT using RLE/Abca3, a cell line having alveolar type II cell-like phenotype. Changes in the cell morphology consistent with EMT were induced by TGF-ß1, whereas, this response was suppressed by co-treatment of the cells with COA-Cl. In addition, co-treatment with COA-Cl abolished TGF-ß1-induced downregulation of cytokeratin 19 and upregulation of α-smooth muscle actin transcripts. In order to delineate the mechanism underlying the inhibitory effect of COA-Cl on TGF-ß1-induced EMT in RLE/Abca3 cells, we examined the role of zinc finger E-box binding homeobox (ZEB) family transcription factors in this phenomenon. Our results demonstrated that the treatment of cells with COA-Cl suppressed the TGF-ß1 mediated increase in the mRNA levels of ZEB2. Overall, it was concluded that COA-Cl may have an inhibitory effect on TGF-ß1-induced EMT-like phenotypical changes in RLE/Abca3 cells via suppression of ZEB2 mRNA expression.

HPLC retention behaviors of pi-electron rich compounds on Ni2+- and Cu2+-phthalocyanine derivatives bound to silica gels in polar eluents.

The effect of the central metal of columns packed with silica gels binding Ni(2+)- and Cu(2+)-phthalocyanine derivatives (Ni-and Cu-PCS(D)s) on the retention behavior of poly-aromatic-hydrocarbons (PAHs) thereof in a polar eluent was examined. The retention factors of PAHs on the Ni- and Cu-PCS(D)s in 80% methanol showed a good linear correlation. The Cu-PCS(D) column exhibited the pi-pi interactions for PAHs, while the Ni-PCS(D) column exhibited the pi-d interactions for PAHs in addition to the pi-pi interaction for PAHs. Further, an investigation of the retention behavior of anthracene derivatives having different substituents revealed that the Ni- and Cu-PCS(D) columns could recognize the differences of substituents only in a polar eluent.

Evaluation of the state of active ingredients in pharmaceutical preparations using fourier transform-Raman difference spectra.

To examine the pharmaceutical application of Fourier transform (FT)-Raman spectroscopy, the state of active pharmaceutical ingredients (APIs) in a preparation of several forms was evaluated by investigating the Raman difference spectra between the preparation and excipient. The difference spectra indicated that APIs in alacepril tablets, caffeine sustained-release granules, and quinidine sulfate granules remained unchanged after the manufacturing process. However, the state of sparfloxacin in nanoparticles changed, although it remained unchanged in tablets or powders. These results show that the FT-Raman difference spectrum is expected to be utilized in the field of quality control of crystalline pharmaceutical preparations.