Molecular Interaction Between Boron Tracedrug UTX-51 Derivatives and Bovine Serum Albumin: Application to an Analytical Model of AGEs Destruction by Thermal Neutron Irradiation

Boron tracedrugs possess global molecular tracking abilities and localized destructive power. We investigated the molecular properties of synthesized boron tracedrugs, including UTX-51, and their interactions with the advanced glycation end-product (AGE)-related protein bovine serum albumin (BSA). A conformational analysis of the compounds used in the present study was performed using CAChe (Fujitsu Inc., Tokyo, Japan) and the degree of stereo-hydrophobicity of the conformers obtained was verified using Mopac (Fujitsu Inc.). The interactive properties of global minimum conformers of the derivatives tested with BSA were assessed using Molegro Virtual Docker (CLC bio., Aarhus, Denmark). Among the compounds investigated, UTX-51 was confirmed to interact with BSA based on the formation of hydrogen bonds between BSA and UTX-51. UTX-51 is a promising boron tracedrug and can be used as the lead structure for developing a therapeutic agent for AGE-related diseases, including cancer.


Effect of Isomerization of TX-2036 Derivatives on the Interaction With Tyrosine Kinase Domain of EGF Receptor

From the design and synthesis of enantiomers, we can expect to obtain two compounds with different pharmacokinetics and pharmacological activities at the same time, which is thought to lead to the development of efficient anticancer agents. Chiral-2-nitroimidazole TX-2036 derivatives exhibit stereo-configuration (R- and S-configuration)-dependent tyrosine kinase inhibitory activity, and the activity of the tyrosine kinase domain of EGF receptor (EGFR-tyk) is suppressed. In order to clarify the reason why the effects on EGFR-tyk activity differ depending on stereoisomers, we tried to analyze the interaction between each TX-2036 derivative and EGFR-tyk. The 2-nitroimidazole-based radiosensitizer TX-2036 series were synthesized and their molecular features were examined using protein kinase inhibition assay and molecular structural analysis. R-configured TXs (TX-2043, -2030, and -2036) exhibited more potent protein kinase inhibitory activity than S-configured TXs (TX-2044, - 2031, and -2037), and the IC value of TX-2036 was 1.8 μM. R-configured TXs interacted with Lys and Thr of EGFR-tyk. The combinations of amino acid residues targeted by the S-configured TXs were different from each other (Ile and Thr (TX-2044), Ser, Thr, and Thr (TX-2031), Gly, Cys, and Thr (TX-2037)). Preparing a series of isomers with different target sites was considered beneficial when the target was mutated.


Correlation Between Radiosensitizing Activity and the Stereo-structure of the TX-2036 Series of Molecules

The stereo-configuration (R-, S-configuration) of chiral-2-nitroimidazole derivatives alters their radiosensitizing activity. This study aimed at examining the molecular features of these enantiomers by molecular simulation techniques. A series of 2-nitroimidazole-based radiosensitizer TX-2036 molecules were synthesized, and their profiles were examined using molecular structural analysis such as conformation analysis, molecular orbital analysis, and electrostatic potential analysis. R-configured TXs (TX-2043, -2030, -2036) had a weaker radiosensitizing activity than S-configured TXs (TX-2044, -2031, -2037), and R-compounds had a small minus electrostatic potential (ESP) field in the cyclopentene-1,3-dione region. S-configured TX-2046 had weaker radiosensitizing activity than R-configured TX-2045, and TX-2046 had a small minus ESP field as well as R-configured TX-2043, -2030, - 2036. The cyclopentene-1,3-dione involved in the small minus ESP field affected the radiosensitizing activity of the TX-2036 series of molecules.


Structure-associated Functional Control of TX-1877 Series by Glyco-conjugation

Sugar molecules are often used as a tool to structurally modify chemical compounds. The features of synthesized sugar-conjugated TX-1877 derivatives were herein examined. The molecular stabilities (reactivity) and hydrophobicities of sugar (e.g., monosaccharide and tetra-O-acetylated monosaccharide)-conjugated TXs were analyzed using a molecular simulation (e.g. molecular mechanics (MM) and molecular orbital (MO) analysis). The hydrophobicities of TX-1877 derivatives were increased by tetra-O-acetylation, and TX-2244 exhibited the most potent radiosensitizing activity (enhancement ratio: ER=2.30). The conformations and hydrophobicities of chemical compounds may be controlled by adding monosaccharide- and tetra-O-acetyl-conjugated sugars to TX-1877.


Interactive Analysis of TX-1123 with Cyclo-oxygenase: Design of COX2 Selective TX Analogs.

To date, two cyclo-oxygenase (COX) isoforms, COX1 and COX2, have been identified. In the present study, the COX-inhibitory activities of TX-1123 derivatives with the 2-hydroxyarylidene-4-cyclopentene-1,3-dione structure were examined, and the binding profiles of TX-1123 to COXs were analyzed using docking simulations. X-Ray data on COX1 [protein data bank (PDB) ID=1PGG] and COX2 (PDB ID=3LN1) were used for molecular interactive simulations. The interactive profiles of TX-1123 derivatives with COXs were examined using a molecular simulation technique with Molegro Virtual Docker (CLC bio, Aarhus, Denmark). TX-1123 exhibited COX1-inhibitory activity [half-maximal-inhibitory concentration (IC)=1.57×10 M]. The COX2 inhibitory activity of TX-1123 was potent (IC=1.16×10 M), and the ratio of COX1/COX2 inhibition was 13.5. TX-1123 bound to the COX2 molecule, and the oxygen atom of the 4-cyclopentene-1,3-dione region of TX-1123 interacted with Cys and Gln of COX2. The TX-1123-binding pocket of COX2 differs from that of the COX2-selective celecoxib-binding pocket. TX-1123 exhibited a different COX2-interactive mechanism from that of celecoxib.


Latent pH-responsive ratiometric fluorescent cluster based on self-assembled photoactivated SNARF derivatives

We have developed a self-assembled fluorescent cluster comprising a seminaphthorhodafluor (SNARF) derivative protected by a photoremovable o-nitrobenzyl group. Prior to UV irradiation, a colorless and nonfluorescent cluster was spontaneously assembled in aqueous solution. After UV irradiation, the self-assembled cluster remained intact and showed a large enhancement in pH-responsive fluorescence. The unique pH responsive fluorescent cluster could be used as a dual-emissive ratiometric fluorescent pH probe not only in the test tube but also in HeLa cell cultures.

Science and Technology of Advanced Materials


Structural Insight Into Protein Binding of Boron Tracedrug UTX-97 Revealed by the Co-Crystal Structure With Lysozyme at 1.26 Å Resolution

Boron neutron capture therapy (BNCT) is one of the numbers of radiotherapies for treatment of certain cancers. The ability of low-dose irradiation with neutrons or radioactive beams to provide an acceptable quality of life is an objective which has not yet been achieved; therefore it will be necessary to increase the efficiency of the neutron capture reaction by lower dose irradiation and by achieving higher drug concentrations in living cells. Drug selectivity in targeting the affected cellular compartment is most important. Molecular design and synthesis of drugs should be based on high resolution structures and analysis of specific compounds and host molecules; however, it is necessary to obtain crystals for X-ray structural analysis. Because compounds containing bulky functional groups are difficult to crystalize due to their flexibility, the method described here makes it possible to stabilize these compounds by complexing them with protein molecules. We have first solved the three-dimensional structure of a BNCT drug-protein molecule combination at 1.26 Å resolution, and discuss the nature of the interaction between a BNCT drug and the protein molecule residues.


An Antitumor 2-Hydroxyarylidene-4-cyclopentene-1,3-Dione as a Protein Tyrosine Kinase Inhibitor: Interaction Between TX-1123 Derivatives and Src Kinase

Protein tyrosine kinases (PTKs) play major roles in signal transduction during cell proliferation and apoptosis. Tyrphostin AG17 was previously shown to be a potent tumor growth inhibitor, while AG17 induced apoptosis and inhibited activity of cyclin-dependent kinase 2. We herein describe the binding features of tyrphostin AG17 analogs, such as TX-1123, with Src kinase (Src-K).MATERIALS AND METHODS:Structural data for Src-K were obtained from a protein data bank (ID=2SRC), and the molecular interactions between Src-K and TX-1123 derivatives were examined.RESULTS:TX-1123 exihibited potent Src-K inhibitory activity (half maximal-inhibitory concentration=2.2 M), and fit into the pocket of the Src-K molecule as well as c-AMP did.CONCLUSION:The binding profiles of TX-1123 derivatives differed from each other, while their Src-K inhibitory activities were affected by their fit in the Src-K molecule.


Molecular cloning and characterization of a geranyl diphosphate-specific aromatic prenyltransferase from lemon

Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.


Radiosensitivity Uncertainty Evaluation for the In Vitro Biophysical Modeling of EMT6 Cells

The aims of this study were to evaluate the cell survival uncertainty distribution of radiation and to assess the accuracy of predictions of tumor response by using three different in vitro experimental cell cultures with radiosensitizers (including etanidazole). Using EMT6 cells and X-rays, the cell survival fraction was obtained from 15, 34, and 21 different experiments under normoxic, hypoxic, and hypoxic-plus-radiosensitizer culture, respectively. The α coefficients were 0.257 ± 0.188, 0.078 ± 0.080, and 0.182 ± 0.116 Gy(-1), respectively. The β coefficients were 0.0159 ± 0.0208, 0.0076 ± 0.0113, and 0.0062 ± 0.0077 Gy(-2), respectively. The α coefficient and the dose that killed half of the clonogens population (D50) were significantly different between normoxic cell and hypoxic cell cultures (p<0.01), respectively. The use of radiosensitizers under hypoxic conditions improved radiosensitivity. Our results suggest that parameter value distributions are required for biophysical modeling of applications for radiotherapy.


Case Report: A Breast Cancer Patient Treated with GcMAF, Sonodynamic Therapy and Hormone Therapy

Gc protein-derived macrophage-activating factor (GcMAF) occurs naturally in the human body. It has various functions, such as macrophage activation and antitumor activities. Recently, immunotherapy has become an attractive new strategy in the treatment of cancer. GcMAF-based immunotherapy can be combined with many other therapies. Sonodynamic therapy (SDT) using low-intensity ultrasound is a novel therapeutic modality. Ultrasound has been demonstrated to activate a number of sonosensitive agents allowing for the possibility of non-invasive targeted treatment for both superficial and deep-seated tumors. The current case study demonstrates that GcMAF and SDT can be used in combination with conventional therapies in patients with metastatic cancer, especially where treatment options are limited due to factors such as toxicity. This case study also suggests a new concept of cancer treatment using local destruction of cancer tissue, in this case conducted with SDT, to be used in combination with GcMAF immunotherapy as a systemic treatment.


Evaluation of the Sonosensitizing Activities of 5-Aminolevulinic Acid and Sn(IV) Chlorin e6 in Tumor-bearing Chick Embryos

Recently, 5-aminolevulinic acid (5-ALA), precursors of protoporphyrin IX (PpIX), and Sn(IV) chlorin e6 (SnCe6) have been proposed as possible sonosensitizers for sonodynamic therapy of cancer. Therefore, we evaluated the pharmacokinetic properties and sonosensitizing activities of 5-ALA and SnCe6 in vivo by using the EMT6/KU tumor-bearing chick embryos. The concentration of PpIX in tumor and liver tissues and serum increased in a time-dependent manner after the i.v. administration of 5-ALA; PpIX concentrations reached their peak level after 5-7 h. The concentration of SnCe6 reached its maximum value in the tumor tissue and serum immediately after i.v. administration. The combined treatment of 5-ALA or SnCe6 with ultrasound irradiation showed a significant antitumor effect towards EMT6/KU solid tumors. We evaluated the pharmacokinetic properties and sonosensitizing activities of 5-ALA and SnCe6 in a chick embryo model and found that 5-ALA might be more suitable as a sonosensitizer than SnCe6.


A Neutron Dynamic Therapy with a Boron Tracedrug UTX-51 Using a Compact Neutron Generator

We are developing a neutron dynamic therapy (NDT) with boron tracedrugs for a new mechanical-clearance treatment of pathotoxic misfolded, aggregated, and self-propagating prion-associated disease proteins. We present a compact neutron generator-based NDT using a boron tracedrug UTX-51. Our NDT is based on the weak thermal neutron-bombarded destructive action of UTX-51 on bovine serum albumin (BSA) using the neutron beams produced from a compact inertial electrostatic confinement fusion (IECF) neutron generator. BSA as an NDT molecular target was subjected to thermal neutron irradiation for eight hours using a compact neutron generator. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern showed no protein band when 2 nmoles of BSA were irradiated with more than 100 nmoles of UTX-51, while BSA was not affected when irradiated without UTX-51. For the first time, we have succeeded in the molecular destruction of a prion-disease model protein, BSA, by NDT with a boron tracedrug, UTX-51, using a compact neutron generator.


An NDT Study of a Boron Tracedrug UTX-51 for Glycated BSA as an AGE Model

Conventional therapies for diseases that are associated with protein aggregation typically prevent rather than clear protein aggregates. We have proposed neutron dynamic therapy (NDT) as a physical clearance therapy for protein aggregates. Advanced glycation end-products (AGEs), which are aggregated proteins, have been implicated in diabetes, Alzheimer's, and heart disease. Herein, we report the use of the boron tracedrug UTX-51, under thermal neutron irradiation, as an NDT for the targeted clearance of glycated bovine serum albumin (Gly-BSA), a model of AGEs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to detect Gly-BSA decomposition by thermal neutron irradiation treated with UTX-51. The combination of UTX-51 with neutron irradiation showed a decrease in band intensity of Gly-BSA. We present our NDT strategy, which has been used for the targeted clearance of Gly-BSA, suggesting that NDT with boron tracedrugs can be used for the treatment of AGEs-related disease.


Evaluation of the in vivo antioxidative activity of redox nanoparticles by using a developing chicken egg as an alternative animal model

Antioxidants have been demonstrated to exert beneficial effects as pharmacotherapies for cardiovascular diseases. The in vitro systems generally employed to evaluate antioxidants, however, are limited by having no appreciable in vivo redox status of the antioxidants. Therefore, we used our developing chicken egg model to evaluate the in vivo antioxidative activity of a redox nanoparticle possessing 2,2,6,6-tetramethylpiperidine-1-oxyl (RNP(O)). The 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) elicited strong oxidative stress and its LD50 value for chick embryos was 3.5±0.9mg/egg. The low molecular weight nitroxide compound, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), which is known to have the highest level of antioxidant activity, showed no significant protective effect against AAPH-induced embryo lethality. On the contrary, RNP(O) had potent protective effects against AAPH-induced embryo lethality. Moreover, RNP(O) could significantly suppress the production of lipid peroxides in chick serum induced by hydrocortisone. Since RNP(O) has a longer retention time in blood than TEMPOL, RNP(O) may protect the embryo against lethal oxidative stress by suppressing lipid peroxidation. The validity of in vivo experiments using developing chicken eggs was supported by our data, where RNP(O) was determined to elicit strong antioxidative activity in vivo, irrespective of the lack of a significant difference in the in vitro activity between low-molecular weight TEMPOL and RNP(O). Our results support the use of the developing chicken egg model to evaluate the potential in vivo antioxidative activity of RNP(O).


In Vivo Experimental Study of Nanosecond Pulsed Electric Field Effects on Solid Tumors

IEEE Transactions on Dielectrics and Electrical Insulation


Antitumor Effect of Degalactosylated Gc-Globulin on Orthotopic Grafted Lung Cancer in Mice

Group-specific component (Gc)-globulin-derived macrophage-activating factor (GcMAF) generated by a cascade of catalytic reactions with deglycosidase enzymes exerts antitumor activity. We hypothesized that degalactosyl Gc-globulin (DG3), a precursor of GcMAF, also plays a role in recovery from cancer as well as GcMAF due to progression of deglycosylation by generally resident sialidases and mannosidases. We prepared the subtypes of DG3, such as 1f1f and 1s1s and its 22 homodimers, by using vitamin D3-binding Sepharose CL-6B and examined their antitumor activity in mice bearing Lewis lung carcinoma cells, by counting the number of nodules formed in their lungs. Antitumor activity of DG3 was observed regardless of its subtype, being equivalent to that of GcMAF. The injection route of DG3 affected its antitumor activity, with subcutaneous and intramuscular administration being more favorable than the intraperitoneal or intravenous route. In order to obtain significant antitumor activity, more than 160 ng/kg of DG3 were required. DG3 proved to be promising as an antitumor agent, similarly to GcMAF.


Clinical Experience of Integrative Cancer Immunotherapy with GcMAF


Degalactosylated/Desialylated Human Serum Containing GcMAF Induces Macrophage Phagocytic Activity and In Vivo Antitumor Activity

The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.


Lysophosphatidic Acid Produced by Hen Egg White Lysophospholipase D Induces Vascular Development on Extraembryonic Membranes

Lysophosphatidic acid (lysoPtdOH), a lysophospholipid mediator, exerts diverse physiological effects, including angiogenesis, through its specific G-protein-coupled receptors. Previously, we showed that unfertilized hen egg white contains polyunsaturated fatty acid-rich lysoPtdOH and lysophospholipase D (lysoPLD). Here, we examined whether lysoPtdOH was produced by lysoPLD in the presence and absence of a hen fertilized ovum and what the physiological role of lysoPtdOH in hen egg white is. Mass spectrometry showed that fertilized hen egg white contained about 8 μM lysoPtdOH before incubation with an ovum, mainly comprised of 18:1- (12.6 %), 18:2- (37.8 %) and 20:4-molecular species (41.5 %). In an early gestation period, the lysoPtdOH was increased up to 9.6 μM, concomitant with a decrease in the level of polyunsaturated lysophosphatidylcholine (lysoPtdCho). Moreover, lysoPtdOH-degrading activities were found in egg white and the vitelline membrane, showing that these enzymes control lysoPtdOH levels in egg white. In an egg yolk angiogenesis assay, two lysoPtdOH receptor antagonists, Ki16425 and N-palmitoyl serine phosphoric acid (NASP), inhibited blood vessel formation induced by exogenously added 18:1-lysoPtdOH and its precursor lysoPtdCho on the hen yolk sac. Ki16425 and NASP also inhibited blood vessel formation in the chorioallantoic membrane (CAM). Furthermore, the relatively higher levels of LPA1, LPA2, LPA4 and LPA6 mRNA were present in the yolk sac and CAM. These results suggest that lysoPtdOH produced from lysoPtdCho by the action of lysoPLD in hen egg white is involved in the formation of blood vessel networks through several lysoPtdOH receptors on various extraembryonic membranes, including the yolk sac membrane and CAM.


Kinetics and Mechanism for the Scavenging Reaction of the 2,2-Diphenyl-1-picrylhydrazyl Radical by Synthetic Artepillin C Analogues

Bulletin of the Chemical Society of Japan


The novel hypoxic cytotoxin, TX-2098 has antitumor effect in pancreatic cancer; possible mechanism through inhibiting VEGF and hypoxia inducible factor-1 targeted gene expression

Tumor hypoxia has been considered to be a potential therapeutic target, because hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. In the present study, we investigated the antitumor effect of a novel hypoxic cytotoxin, 3-[2-hydroxyethyl(methyl)amino]-2-quinoxalinecarbonitrile 1,4-dioxide (TX-2098) in inhibiting the expression of hypoxia inducible factor-1α (HIF-1α), and consequently vascular endothelial cell growth factor (VEGF) expression in pancreatic cancer. The antitumor effects of TX-2098 under hypoxia were tested against various human pancreatic cancer cell lines using WST-8 assay. VEGF protein induced pancreatic cancer was determined on cell-free supernatant by ELISA. Moreover, nude mice bearing subcutaneously (s.c.) or orthotopically implanted human SUIT-2 were treated with TX-2098. Tumor volume, survival and expression of HIF-1 and associated molecules were evaluated in treatment versus control groups. In vitro, TX-2098 inhibited the proliferation of various pancreatic cancer cell lines. In s.c model, tumors from nude mice injected with pancreatic cancer cells and treated with TX-2098 showed significant reductions in volume (P<0.01 versus control). Quantitative real-time reverse transcription-PCR analysis revealed that TX-2098 significantly inhibited mRNA expression of the HIF-1 associated molecules, VEGF, glucose transporter 1 and Aldolase A (P<0.01 versus control). These treatments also prolong the survival in orthotopic models. These results suggest that the effect of TX-2098 in pancreatic cancer might be correlated with the expression of VEGF and HIF-1 targeted molecules.


Characterization of Coumarin-Specific Prenyltransferase Activities in Citrus limon Peel

Bioscience, Biotechnology, and Biochemistry


Beta-Galactosidase treatment is a common first-stage modification of the three major subtypes of Gc protein to GcMAF

The 1f1f subtype of the group-specific component (Gc) protein is converted into Gc protein-derived macrophage-activating factor (GcMAF) by enzymatic processing with β-galactosidase and sialidase. We previously demonstrated that preGc(1f1f)MAF, a full Gc(1f1f) protein otherwise lacking a galactosyl moiety, can be converted to GcMAF by treatment with mouse peritoneal fluid. Here, we investigated the effects of the β-galactosidase-treated 1s1s and 22 subtypes of Gc protein (preGc(1s1s)MAF and preGc22MAF) on the phagocytic activation of mouse peritoneal macrophages. We demonstrated the presence of Gal-GalNAc disaccharide sugar structures in the Gc(1s1s) protein by western blotting using peanut agglutinin and Helix pomatia agglutinin lectin. We also found that preGc(1s1s)MAF and preGc22MAF significantly enhanced the phagocytic activity of mouse peritoneal macrophages in the presence and absence of mouse peritoneal fluid. We demonstrate that preGc(1s1s)MAF and preGc22MAF proteins can be used as effective macrophage activators.


Molecular Profiles of Cholesterol-dependent Cytolysin Family-derived 11mer Regions

Cholesterol-dependent cytolysins (CDCs) are secreted from various types of bacteria and are involved in various diseases (e.g. abscess formation). Traditional CDCs has a conserved 11mer region, which is a key structure in membrane recognition. Based on the X-ray data of traditional CDC perfringolysin O (PFO), molecular models of intermedilysin (ILY), pyolysin (PLO), vaginolysin (VLY), and Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) were constructed. The 11mer regions of these models were extracted, and their molecular features were analyzed. The dipole moments of these 11mer regions were classified into four types, and their stereo-hydrophobicity (dGW) was different. It was thought that these results influenced the species specificity and membrane recognition of each cytolysin. Traditional CDCs, ILY, PLO, and VLY consisted of four domains (domains 1 to 4). Domain 0 existed on the N-terminal side in Sm-hPAF in addition to these four domains. The 11mer sequence of Sm-hPAF is the same as that of VLY, but Sm-hPAF has slightly different characteristics (e.g. species specificity, membrane recognition, cholesterol dependency) compared to VLY. Dynamic structure analysis of domain 0 might clarify these differences.


Boron tracedrug: design, synthesis, and pharmacological activity of phenolic BODIPY-containing antioxidants as traceable next-generation drug model

Advances in Experimental Medicine and Biology


Prenylated acylphloroglucinol derivatives: isoprenomics-based design, syntheses and antioxidative activities


Vitamin D Binding Protein-Macrophage Activating Factor Inhibits HCC in SCID Mice

A high incidence of recurrence after treatment is the most serious problem in hepatocellular carcinoma (HCC). Therefore, a new strategy for the treatment of the disease is needed. The aim of the present study was to investigate whether vitamin D binding protein-macrophage activating factor (DBP-maf) is able to inhibit the growth of HCC. The effects of DBP-maf on endothelial cells and macrophage were evaluated by WST-1 assay and phagocytosis assay, respectively. Human HCC cells (HepG2) were implanted into the dorsum of severe combined immunodeficiency (SCID) mice. These mice were divided into control and DBP-maf treatment groups (n = 10/group). The mice in the treatment group received 40 ng/kg/d of DBP-maf for 21 d. DBP-maf showed anti-proliferative activity against endothelial cells and also activated phagocytosis by macrophages. DBP-maf inhibited the growth of HCC cells (treatment group: 126 ± 18mm(3), untreated group: 1691.5 ± 546.9mm(3), P = 0.0077). Histologic examinations of the tumors revealed the microvessel density was reduced and more macrophage infiltration was demonstrated in the tumor of mice in the treatment group. DBP-maf has at least two novel functions, namely, an anti-angiogenic activity and tumor killing activity through the activation of macrophages. DBP-maf may therefore represent a new strategy for the treatment of HCC.


Efficiency of Antimicrobial Defense: Molecular Flexibility of Natural Defensin and Artificial Bis-quaternary Ammonium Compound

Human α-defensins (such as HD5 and HD6) are typical bactericidal peptides. We examined the molecular features of HD5 and HD6 by molecular dynamics (MD) analysis. Molecular features of natural defensins and artificial bis-quaternary ammonium compounds (e.g. 1,6-polymethylenedithio)bis(1-octylpyridinium iodide: 4DTBP-m,8) were analyzed using molecular simulation techniques. HD5 and HD6 had different electrostatic potential profiles, which indicated the region-dependent hydrophobicity. 4DTBP-m,8 derivatives were significantly flexible, and many conformers existed. HD5 and HD6 indicated antimicrobial activity by restricted conformation.


Design, Synthesis and Destructive Dynamic Effects of BODIPY-containing and Curcuminoid Boron Tracedrugs for Neutron Dynamic Therapy

We previously designed the boron tracedrugs UTX-42, UTX-43, and UTX-44, which possess antioxidant potency. In order to explore their destructive dynamic effects when bombarded by weak thermal neutrons, we performed thermal neutron irradiation of bovine serum albumin (BSA) treated with the boron tracedrugs. Boron tracedrugs, including the boron dipyrromethene (BODIPY)-containing compounds UTX-42, UTX-44, and UTX-47 and the curcuminoid compounds UTX-50 and UTX-51, were designed for neutron dynamic therapy based on their molecular orbital calculation. Newly designed UTX-47, UTX-50, and UTX-51 were synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to detect decomposition by thermal neutron irradiation of BSA treated with these boron tracedrugs. The combination of 1.0 μM BSA with 100 μM of each of the boron tracedrugs showed a decrease in band intensity after irradiation. All boron tracedrugs tested caused destructive dynamic damage of BSA during thermal neutron irradiation, suggesting that boron tracedrugs could be used as dynamic drugs for neutron dynamic therapy.


Effect of the Gc-derived Macrophage-activating Factor Precursor (preGcMAF) on Phagocytic Activation of Mouse Peritoneal Macrophages

The 1f1f subtype of the Gc protein (Gc(1f1f) protein) was converted into Gc-derived macrophage-activating factor (GcMAF) by enzymatic processing in the presence of β-galactosidase of an activated B-cell and sialidase of a T-cell. We hypothesized that preGc(1f1f)MAF, the only Gc(1f1f) protein lacking galactose, can be converted to GcMAF in vivo because sialic acid is cleaved by residual sialidase. Hence, we investigated the effect of preGc(1f1f)MAF on the phagocytic activation of mouse peritoneal macrophages. We examined the sugar moiety of preGc(1f1f)MAF with a Western blot using peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) lectin. We also found that preGc(1f1f)MAF significantly enhanced phagocytic activity in mouse peritoneal macrophages but only in the presence of the mouse peritoneal fluid; the level of phagocytic activity was the same as that observed for GcMAF. PreGc(1f1f)MAF can be used as an effective macrophage activator in vivo.


Evaluation of the In vivo Radiosensitizing Activity of EtanidazoleUsing Tumor-bearing Chick Embryo

Chick embryos have been used as alternative experimental animals in various research fields, including virology, immunology, toxicology, oncology, and embryology. Until now, there have been no in vivo models using chick embryo to evaluate radiosensitizing activity. Here, the in vivo radiosensitizing activity of etanidazole, a well-known hypoxic cell radiosensitizer, was evaluated using tumor-bearing chick embryo. On the basis of tumor growth, drug administration and X-ray irradiation were performed on day 15 chick embryo, with the endpoint being day 18 chick embryo. In day 15 chick embryo, an X-ray irradiation dose of equal or less than 10 Gy did not cause significant tumor growth suppression. Intravenous administration of equal or less than 1.0 mg of etanidazole did not cause tumor growth suppression. Neither doses of equal or less than 8 Gy of irradiation nor 1.0 mg of etanidazole caused fatality of the chick embryo. On the basis of these results, we evaluated the radiosensitizing effect of a combination treatment with 8 Gy of irradiation and 1.0 mg of etanidazole. As noted above, 1.0 mg of etanidazole alone and 8 Gy of irradiation alone did not show tumor growth suppression. In contrast, a combination treatment with 8 Gy of irradiation and 1.0 mg of etanidazole showed 35% of significant tumor growth suppression. Thus, we succeeded in evaluating the in vivo radiosensitizing activity of etanidazole using tumor-bearing chick embryo. These results suggest that the use of tumor-bearing chick embryo may be part of a promising system for evaluating radiosensitizing activity.


Formation and regulation of fullerene-incorporation in liposomes under the phase transition temperature

The fullerene-exchange reaction from a cyclodextrin cavity to liposomes represents one of the best methods to prepare lipid membrane-incorporated [70]fullerenes (C(70)). The C(70)-exchange reaction occurred completely at temperatures above the phase transition temperature (T(m)) of the liposomes; however, lowering the temperature to below the T(m) led to C(70) aggregation outside the liposomes. This observation has limited the development of more functional LMIC(70) using a variety of liposome compositions. In this paper, this reaction was found to occur efficiently by the addition of small amounts of lipids bearing a ϖ-moiety. The ϖ-moieties act as a gate when hydrophobic C(70) migrates into the hydrophilic liposome surface. Therefore, the ϖ-moieties should exist in the polar head groups of the lipids and the C(70)-exchange reaction can be controlled by pH.


A retrospective study of a calcium agent (E-Ca) using data on bone mineral density obtained by DXA method

We performed a retrospective analysis of a calcium hydroxide-containing calcium agent (TACHIKAWA DENKAI CALCIUM™: E-Ca) based on data of bone mineral density obtained by dual-energy X-ray absorptiometry (DXA) to clarify the relationship between bone mineral density and E-Ca intake on an empty stomach in those who regularly use E-Ca. We found the percentage of volunteers with their age-matched (AM) values of above 100% to be 89%, and also a moderate positive correlation between AM values and the intake period of E-Ca. Our findings demonstrate that AM values can be used as an effective indicator assessing osteogenesis by regularly administrated calcium agents which exert their effects after long-term use.


N-Acetyl Transferase 2 Polymorphisms Associated with Isoniazid Pharmacodynamics: Molecular Features for Ligand Interaction

Isoniazid is mainly metabolized by arylamine N-acetyltransferase 2 (NAT2). Rapid acetylator types have NAT2*4/*4 alleles. Intermediate acetylator types have any of the following alleles: NAT2*4/*5, *4/*6, or *4/*7. Slow acetylator types do not have the NAT2*4 allele. We examined molecular features of these NAT2 molecules. Structures of NAT2*5, *6, and *7 were constructed based on X-ray data of human NAT2*4 using a molecular modeling technique. The NAT2*4 molecule mostly occupied a positive electrostatic potential field. Ile(114) and Arg(197), which are mutation sites of NAT2*4 to NAT2*5 and NAT2*6, were located in the peripheral part of the positive field. Gly(286), the mutation site from NAT2*4 to NAT2*7, was located near coenzyme A (CoA) in the boundary of the positive and negative fields. Nonbinding energies between NAT2s and isoniazid were larger than those of CoA. Molecular polymorphism appears to influence the reactivity between NAT2 and the external ligand.


Design of a SNARF-based Ratiometric Fluorescent Probe for Esterase

A novel ratiometric fluorescent probe for esterase has been developed based on the SNARF scaffold. The new SNARF derivative was used to monitor the activity of porcine liver esterase by ratiometric emission spectrum change.


A newly designed cell-permeable SNARF derivative as an effective intracellular pH indicator.

We have successfully developed a new cell-permeable SNARF derivative that is activated inside the cell by ester hydrolysis and is notable for having reduced background fluorescence.


Design of Novel Hypoxia-Targeting IDO Hybrid Inhibitors Conjugated with an Unsubstituted L-TRP as an IDO Affinity Moiety

We presented here design, syntheses and inhibitory activities of novel hypoxia-targeting IDO hybrid inhibitors conjugated with an unsubstituted L-Trp as an IDO affinity moiety without inhibitor 1MT, such as L-Trp-TPZ hybrids 1 (TX-2274), 2 (UTX-3), 3 (UTX-4), and 4 (UTX-2). TPZ-monoxide hybrids 1 and 3 were good competitive IDO inhibitors, while TPZ hybrids 2 and 4 were uncompetitive IDO inhibitors. Among them TPZ-monoxide hybrid 1 have the strongest IDO inhibitory activity. It suggests that TPZ-monoxide hybrids 1 and 3 are able to bind the active site of IDO, TPZ hybrids 2 and 4 are able to bind the enzyme-substrate complex. We proposed the possible mechanism of action of TPZ hybrid 2 that may first affect as a hypoxic cytotoxin, and then metabolized to TPZ-monoxide hybrid 1, which may do as an IDO inhibitor more effectively than its parent TPZ hybrid 2.

Advances in Experimental Medicine and Biology


Structural Investigation of the Binding of 5-Substituted Swainsonine Analogues to Golgi -Mannosidase II

Golgi alpha-mannosidase II (GMII) is a key enzyme in the N-glycosylation pathway and is a potential target for cancer chemotherapy. The natural product swainsonine is a potent inhibitor of GMII. In this paper we characterize the binding of 5alpha-substituted swainsonine analogues to the soluble catalytic domain of Drosophila GMII by X-ray crystallography. These inhibitors enjoy an advantage over previously reported GMII inhibitors in that they did not significantly decrease the inhibitory potential of the swainsonine head-group. The phenyl groups of these analogues occupy a portion of the binding site not previously seen to be populated with either substrate analogues or other inhibitors and they form novel hydrophobic interactions. They displace a well-organized water cluster, but the presence of a C(10) carbonyl allows the reestablishment of important hydrogen bonds. Already approximately tenfold more active against the Golgi enzyme than the lysosomal enzyme, these inhibitors offer the potential of being extended into the N-acetylglucosamine binding site of GMII for the creation of even more potent and selective GMII inhibitors.


Role of C-terminal region of yeast ADP/ATP carrier 2 protein: dynamics of flexible C-terminal arm.

The ADP/ATP carrier catalyzes the exchange of ADP and ATP across the inner mitochondrial membrane. The molecular dynamics of modeled yeast type 2 AAC (yAAC2) was analyzed and molecular parameters were determined. The yAAC2 C-terminal moved flexibly and a negative electrostatic potential field (ESP) was located in the C-terminal region. The ESP field is always located in the C-terminal area during C-terminal truncation (d1-d9). Further C-terminal truncation occurred on field invagination into the core region (d11, d14, d16). The 2-6 C-terminal amino acid truncation did not affect the biological activity. The d7-d9 truncated mutants lost their biological function. A critical point in yAAC2 function was shown between d6 and d7 C-terminal truncation. The C-terminal structure of yAAC2 is thought to be involved in biological function control.


Downregulation of matrix metalloprotease-9 and urokinase plasminogen activator by TX-1877 results in decreased tumor growth and metastasis on xenograft model of rectal cancer

Purpose It is well known that hypoxic milieu is the primary cancer environment. Therefore, tumor hypoxia is considered to be a potential therapeutic target. In the present study, we investigated the antitumor and antimetastatic effect of hypoxic cell radiosensitizer, TX-1877 on xenograft model of rectal cancer. Methods Nude mice bearing subcutaneously or orthotopically implanted human colon cancer cell lines HCT-116 and HT-29 were treated with TX-1877, irradiation or TX-1877 with irradiation. Tumor volume, survival, expression of matrix metalloproteinase (MMP)-2, MMP-7, MMP-9 and urokinase-type plasminogen activator (uPA) and incidence of lymph node metastasis were evaluated in treatment versus control group. Results In subcutaneous model, tumor treated with TX-1877 and irradiation showed significant reductions in volume (P < 0.05 vs. control, TX-1877 or irradiation group). Quantitative real-time reverse transcription-PCR and immunohistochemical analysis revealed that TX-1877 significantly inhibited expression of the MMP-9 and uPA. These treatments also inhibited the para-aortic lymph node metastasis, however, did not prolong the survival in orthotopic model. Conclusions These data show that the treatment of TX-1877 with irradiation decreased growth of human rectal cancer and, furthermore, suppressed lymph node metastasis.


Design of a bioreductively-activated fluorescent pH probe for tumor hypoxia imaging

We have designed and evaluated UTX-12 as a novel fluorescent pH probe for tumor hypoxia imaging. UTX-12 consists of a p-nitro benzyl moiety, which is a latent hypoxia-selective leaving group activated by nitro reduction, directly linked to SNARF. Although UTX-12 itself is colorless and non-fluorescent in aqueous solution, nitro reduction triggers the release of SNARF which has well-characterized long wavelength absorption and fluorescence that is sensitive to pH. The resultant SNARF, released intracellularly by enzymatic reduction of UTX-12, allows measurement of pH by pH-dependent dual emission shifts. UTX-12 showed clear differences in fluorescence behavior between hypoxic and aerobic conditions in liver microsomes and inside V79 cells. These data are confirmation that UTX-12 is biologically reduced inside tumor cells and the released SNARF should monitor intracellular pH of tumor cells selectively with reduced background signal.


Correlation between antiangiogenic activity and antioxidant activity of various components from propolis

Propolis possesses various physiological activities. In this study, we examined the antiangiogenic and antioxidant activities of various components from propolis: acacetin, apigenin, artepillin C, caffeic acid phenethyl ester, chrysin, p-coumaric acid, galangin, kaempferol, pinocembrin, and quercetin. The effects of these components were tested on in vitro models of angiogenesis, tube formation and growth of human umbilical vein endothelial cells (HUVECs). Furthermore, these components were evaluated for their antioxidant activities by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging and ferric reducing/antioxidant power (FRAP) assays. Two propolis components, caffeic acid phenethyl ester, and quercetin, possessed strong inhibitory effects on tube formation and on endothelial cell proliferation and, coincidentally, showed strong antioxidant activity. Artepillin C, galangin, and kaempferol also possessed strong antiangiogenic and antioxidant activities to a slightly less degree. In contrast, acacetin, apigenin, and pinocembrin possessed a considerable degree of antiangiogenic activities, although they showed very low antioxidant activities. From these results, we propose that components from propolis such as artepillin C, caffeic acid phenethyl ester, galangin, kaempferol, and quercetin might represent a new class of dietary-derived antioxidative compounds with antiangiogenic activities. These propolis components may have the potential to be developed into pharmaceutical drugs for the treatment of angiogenesis-dependent human diseases such as tumors.


Artepillin C (ARC) in Brazilian green propolis selectively blocks oncogenic PAK1 signaling and suppresses the growth of NF tumors in mice

There are mainly three types of propolis whose major anticancer ingredients are entirely different: (1) CAPE (caffeic acid phenethyl ester)-based propolis in Europe, Far East and New Zealand, (2) artepillin C (ARC)-based Brazilian green propolis and (3) Brazilian red propolis. It was shown previously that NF (neurofibromatosis)-associated tumors require the kinase PAK1 for their growth, and CAPE-based propolis extracts such as Bio 30 suppress completely the growth of NF tumors in vivo by blocking PAK1 signaling. Also it was demonstrated that ARC suppresses angiogenesis, suggesting the possibility that ARC also blocks oncogenic PAK1 signaling. Here it is shown for the first time that both ARC and green propolis extract (GPE) indeed block the PAK1 signaling selectively, without affecting another kinase known as AKT. Furthermore, it was confirmed that ARC as well as GPE suppress almost completely the growth of human NF tumor xenografts in mice, as does Bio 30. These results suggest that both CAPE-based and ARC-based propolis extracts are natural anti-PAK1 remedies and could be among the first effective NF therapeutics available on the market. Since more than 70% of human cancers such as breast and prostate cancers require the kinase PAK1 for their growth, it is quite possible that GPE could be potentially useful for the treatment of these cancers, as is Bio 30.


Flexible Structure of Cytochrome P450: Promiscuity of Ligand Binding in the CYP3A4 Heme Pocket

CYP3A4 is the most abundant xenobiotic-metabolizing cytochrome P450 isoform. We examined the structural features of the CYP3A4 molecule with regard to ligand access. The deleted amino acid sequences of X-ray data sets of CYP3A4s were complemented by molecular modeling techniques. Molecular features of the ligand accessible regions in CYP3A4 were analyzed and their molecular parameters (e.g. dipole moment, solvation free energy, electrostatic potential fields) were determined. Three ligand accessible regions (region 1-3) were present in erythromycin-bound CYP3A4, and these dipole moments indicated the same features as ketoconazole- or metyrapone-bound CYP3A4 molecules. In progesterone-bound CYP3A4, four candidate ligand accessible regions were observed and progesterone could be bound by two selected ligand accessible regions. The heme pocket of CYP3A4 is very flexible and is able to interact with various types of substrate.


A chemical biosynthesis design for an antiatherosclerosis drug by acyclic tocopherol intermediate analogue based on "isoprenomics"

Phytyl quinols, namely acyclic tocopherols, are key intermediates of tocopherol biosynthesis, but their biological activities remain unclear. We therefore investigated the structure-activity relationship of phytyl quinols to apply a chemical biosynthesis design for an antiatherosclerosis drug based on isoprenomics. We have achieved the biosynthesis-oriented design and synthesis of alpha- (TX-2254) and beta-(TX-2247) phytyl quinol as an unnatural intermediate, other gamma- (TX-2242) and delta-(TX-2231) phytyl quinol as a natural one. Geometry optimization and Molecular orbital (MO) calculation of TX-2254 showed a unique right-angle structure; however, MO energy of TX-2254 and d-alpha-tocopherol were very similar. Radical reactivity of TX-2231 was equal to dl-alpha-tocopherol, whereas TX-2254, TX-2247, and TX-2231 showed lower reactivity than dl-alpha-tocopherol. All four phytyl quinols showed almost the same moderate inhibitory activity against low-density lipoprotein (LDL) oxidation instead of their different degree of C-methylation with character different from tocopherols. In vivo toxicities of phytyl quinols against chick embryo chorioallantoic membrane (CAM) vasculature were hardly observed. We proposed phytyl quinols were possible antioxidants in plants and animals, like vitamin E.

Advances in Experimental Medicine and Biology


Possible involvement of radical intermediates in the inhibition of cysteine proteases by allenyl esters and amides

In order to investigate crystallographically the mechanism of inhibition of cysteine protease by alpha-methyl-gamma,gamma-diphenylallenecarboxylic acid ethyl ester 3, a cysteine protease inhibitor having in vivo stability, we synthesized N-(alpha-methyl-gamma,gamma-diphenylallenecarbonyl)-L-phenylalanine ethyl ester 4. Reaction of 4 with thiophenol, the SH group of which has similar pK(a) value to that of cysteine protease, produced oxygen-mediated radical adducts 6 and 7 in ambient air but did not proceed under oxygen-free conditions. Catalytic activities of two thiol enzymes including cathepsin B were also lowered in the absence of oxygen. These results suggest that cysteine protease can act through an oxygen-dependent radical mechanism.

Bioorganic & Medicinal Chemistry Letters


The novel hypoxic cell radiosensitizer, TX-1877 has antitumor activity through suppression of angiogenesis and inhibits liver metastasis on xenograft model of pancreatic cancer.

Tumor hypoxia has been considered to be a potential therapeutic target, because hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. In the present study, we investigated the antitumor effect of hypoxic cell radiosensitizer, TX-1877 in inhibiting angiogenesis and liver metastasis on pancreatic cancer xenograft model. The antitumor effects of TX-1877 were tested against various human tumor cell lines using cell proliferation assay. Nude mice bearing s.c. or orthotopically implanted human SUIT-2 were treated with TX-1877 alone, irradiation alone or TX-1877 and irradiation. Tumor volume, survival, expression of angiogenic molecules and liver metastasis were evaluated in treatment versus control groups. In vitro, TX-1877 inhibited the proliferation and potentiated the radiosensitivity of various pancreatic cancer cell lines. In an orthotopic model, tumors from nude mice injected with pancreatic cancer cells and treated with TX-1877 and irradiation showed significant reductions in volume (p<0.05 versus control, TX-1877 alone or irradiation alone). Quantitative real-time reverse transcription-PCR and immunohistochemical analysis revealed that treatment with TX-1877 alone or with TX-1877 and irradiation inhibited expression of the angiogenic molecules, vascular endothelial growth factor; basic fibroblast growth factor, interleukin-8 and matrix metalloproteinase 9 more than control or did treatment with irradiation alone. These treatments also induced apoptosis in cancer cells. These data show that treatment of TX-1877 and irradiation decreased growth of human pancreatic cancer, suppressed angiogenesis and inhibited liver metastasis, leading to prolonged survival.


Synthesis and biological activity of 1-methyl-tryptophan-tirapazamine hybrids as hypoxia-targeting indoleamine 2,3-dioxygenase inhibitors

We have designed and synthesized new hypoxic-neoplastic cells-targeted indoleamine 2,3-dioxygenase (IDO) inhibitors. 1-Methyl-tryptophan (1MT)-tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-dioxide) hybrid inhibitors including 1 (TX-2236), 2 (TX-2235), 3 (TX-2228), and 4 (TX-2234) were prepared. All of these compounds were uncompetitive IDO inhibitors. TPZ-monoxide hybrids 1 and 3 showed higher IDO inhibitory activities than TPZ hybrids 2 and 4. Among these hybrids, hybrid 1 was the most potent IDO inhibitor. TPZ hybrids 2 and 4 showed stronger hypoxia-selective cytotoxicity than TPZ to EMT6/KU cells. These data suggest that TPZ hybrids 2 and 4 may act through their dual biological functions: first, they function as hypoxic cytotoxins in hypoxic cells, and then are metabolized to their TPZ-monoxide (3-amino-1,2,4-benzotriazine 1-oxide) hybrids, which function as IDO inhibitors.


TX-2152: A conformationally rigid and electron-rich diyne analogue of FTY720 with in vivo antiangiogenic activity

We designed FTY720 analogues with conformationally rigid and electron-rich acetylenic chains as antiangiogenic agents (the monoyne 1: TX-2148, the diyne 2: TX-2152, the triyne 3: TX-2256). Molecular orbital (MO) calculations of our designed acetylenic analogues and FTY720 showed that the localization of the lowest unoccupied MO and the highest occupied MO increased from phenyl ring to acetylenic chain compared with that of FTY720. These acetylenic analogues were synthesized from p-hydroxyphenylethanol as a starting material. The construction of the acetylenic chain was carried out by an iterative strategy using a Sonogashira cross-coupling reaction and desilylative bromination in two steps. The corresponding overall yields of the monoyne 1, the diyne 2, and the triyne 3 were 27% (11 steps), 13% (13 steps), and 10% (15 steps). The in vivo antiangiogenic activities of these acetylenic analogues and FTY720 were evaluated by the chick embryo chorioallantoic membrane (CAM) assay and compared to the activities of the known antiangiogenic agent TNP-470. The diyne 2 showed more potent antiangiogenic activity (90% inhibition) than FTY720 (77% inhibition) and other acetylenic analogues (the monoyne 1: 42% inhibition, the triyne 3: 60% inhibition), and TNP-470 (82% inhibition) at a dose of 10 μg/CAM, without showing toxicity. The diyne 2 also had potent inhibitory activity at a dose of 5 and 2.5 μg/CAM. These results indicate that the flexibility of C8 alkyl chain of FTY720 is not required for its antiangiogenic activity. We suggest that the diyne 2 (TX-2152) may be a promising candidate as an antiangiogenic agent for antineoplastic drug discovery.


Design of antiangiogenic hypoxic cell radiosensitizers: 2-Nitroimidazoles containing a 2-aminomethylene-4-cyclopentene-1,3-dione moiety

We designed chiral 2-nitroimidazole derivatives containing a 2-aminomethylene-4-cyclopentene-1,3-dione moiety as antiangiogenic hypoxic cell radiosensitizers. Based on results of molecular orbital calculations, the 2-aminomethylene-4-cyclopentene-1,3-dione moiety is expected to show high electrophilicity comparable to that of the 2-methylene-4-cyclopentene-1,3-dione moiety included in TX-1123 and tyrphostin AG17. We evaluated the antiangiogenic and radiosensitizing effects of the new compounds, along with other biological properties including their activities as hypoxic cytotoxicities and protein tyrosine kinase (PTK) inhibitory activities. Among the compounds tested, 5 (TX-2036) proved to be the strongest antiangiogenic hypoxic cell radiosensitizer. All the other chiral 2-nitroimidazole derivatives having 2-aminomethylene-4-cyclopentene-1,3-dione moiety tested were also antiangiogenic hypoxic cell radiosensitizers. The PTK inhibitory activity of 5 (TX-2036) showed this to be a promising and potent EGFR kinase inhibitor, having an IC50 value of lower than 2 μM. This compound also was an Flt-1 kinase inhibitor having an IC50 value of lower than 20 μM. Our results show that these chiral 2-nitroimidazole derivatives that contain the 2-aminomethylene-4-cyclopentene-1,3-dione moiety as a potent antiangiogenic pharmacophoric descriptor are promising lead candidates for the development of antiangiogenic hypoxic cell radiosensitizers.


Anti-inflammatory effects of a bioavailable compound, Artepillin C, in Brazilian propolis

Artepillin C is the major compound in the Brazilian green propolis from Baccharis dracunculifolia. Our aim in this study was to investigate the anti-inflammatory effects, absorption, and bioavailability of Artepillin C in mice. The animals used were male Swiss mice subjected to: paw oedema by carrageenan (300 μg/paw), carrageenan-induced peritonitis, and prostaglandin E2 determination. We also measured in vitro nitric oxide production by RAW 264.7 cells and NF-κB activity in HEK 293 cells. Finally, we measured the absorption and bioavailability of Artepillin C in plasma from mice by means of GC-MS after a single oral dose (10 mg/kg). In vivo, Artepillin C produced a maximal inhibition of 38% after 360 min on paw oedema. Artepillin C also decreased the number of neutrophils during peritonitis (IC50: 0.9 (0.5 1.4) mg/kg). Treatment with Artepillin C decreased prostaglandin E2 by 29 ± 3% and 58 ± 5% at 1 and 10 mg/kg, respectively, with a mean ID50 of 8.5 (8.0 8.7)mg/kg). Similarly, in in vitro models, Artepillin C (3, 10, or 100 μM) decreased nitric oxide production by RAW 264.7 cells with a mean IC50 of 8.5 (7.8 9.2) μM. In HEK 293 cells, Artepillin C reduced NF-κB activity with a mean IC50 of 26 (22 30) μg/ml), suggesting anti-inflammatory activity, particularly during acute inflammation. Lastly, Artepillin C was absorbed after an oral dose (10 mg/kg) with maximal peaks found at 1 h (22 μg/ml). Collectively, Artepillin C showed anti-inflammatory effects mediated, at least in part, by prostaglandin E2 and nitric oxide inhibition through NF-κB modulation, and exhibited bioavailability by oral administration.


Design, synthesis, and radiosensitizing activities of sugar-hybrid hypoxic cell radiosensitizers

We have designed sugar-hybrid TX-1877 derivatives conjugated with sugar moieties including β-glucose (β-Glc), β-galactose (β-Gal), α-mannose (α-Man) and N-acetyl-β-galactosamine (β-GalNAc). Compound 1 (TX-1877) was glycosylated with appropriate peracetylated sugars using BF3-OEt2 to give acetylated sugar-hybrids, 5 (TX-2244), 6 (TX-2245), 7 (TX-2246), and 10 (TX-2243). Removal of the acetyl groups afforded the sugar-hybrids having free hydroxyl groups, 11 (TX-2141), 12 (TX-2218), 13 (TX-2217) and 14 (TX-2068). We evaluated their radiosensitizing activities by an in vitro radiosensitization assay. All free hydroxyl hybrids have lower enhancement ratio (ER) values (ER 1.43) and lower n-octanol/water partition coefficient (Poct) values (Poct < 1.00 × 10-2) than does 1 (TX-1877, ER = 1.75, Poct: 5.60 × 10-2). All acetylated hybrids have similar Poct values (3.55 × 10-2 1.05 × 10-1) to 1 (TX-1877) and have improved ER values (ER 1.47) compared to the hybrids having free hydroxyl groups. Among these, 5 (TX-2244) is the most active radiosensitizer (ER = 2.30). We found a good correlation (r = 0.866) between the magnitude of Poct (logPoct) and the ER value of 5 (TX-2244), 6 (TX-2245), 7 (TX-2246), 10 (TX-2243) and 1 (TX-1877), suggesting that increasing the hydrophobicity is reflected in increased in vitro radiosensitizing activity. In the present study, we have succeeded in producing sugar-hybrid hypoxic cell radiosensitizers that have an increased radiosensitizing activity that does not depend on increased hydrophobicity.


Effect of molecular chirality and side chain bulkiness on angiogenesis of haloacetylcarbamoyl-2-nitroimidazole compounds

BACKGROUND: Angiogenesis is required for tumor growth and metastasis, and is an exciting target for cancer treatment. We designed and synthesized antiangiogenetic TX agent (TX-1898, -1900), and analyzed their structural features. TXs have a chiral center and S- and R-enantiomers. Conformation analysis and molecular dynamics simulation were undertaken. Materials and METHODS: Molecular models of TXs were constructed using InsightlI-Discover. Conformation analysis was performed with CONFLEX, and z-matrix data were extracted to calculate molecular orbital (MO) parameters (i.e. solvation free energy (dGW)). Their molecular dynamics were simulated with the Discover3 module, and the total energy and dihedral angles were estimated. RESULTS: The methyl-including TXs (Group 1: TX-1863, -1878, -1866, -1879) had 130-229 conformers (-1.26-14.6 kcal/mol). The t-butyl-including group (Group 2: TX-1880, -1881, -1882, -1883) had 244 - 294 conformers (3.69 - 16. 76 kcal/mol), and the p-t-butylphenyl-containing TXs (Group 3: TX-1897, -1899, -1898, -1900) had 584 - 711 conformers (-7.48 -5.18 kcal/mol). The dGWprofile of nine samples, which were extracted from these conformers, were examined and one minimum dGW point was observed in the haloacetylcarbamoyl-2-nitroimidazole TXs (Group 1 -3). CONCLUSION: TX-1898 exhibited significant antiangiogenic activity. The order of antiangiogenic activity was as follows: TX-1898 (93% at 5 microg/pellet) > TX-1900 (82% at 5 microg/pellet) > TX-1897 (64% at 10 microg/pellet) > TX-1899 (58% at 10 microg/pellet). The chiral center has an important role for orienting the molecular characteristics.


Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-D-ribose-5-phosphate aldolase.

Genes encoding 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25 degrees C), although both enzymes showed much less 2-deoxy-d-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25 degrees C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 A. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.


The usefulness of mild temperature hyperthermia combined with continuous tirapazamine administration under reduced dose-rate irradiation with gamma-rays

We clarified the usefulness of mild temperature hyperthermia (MTH) in combination with the continuous administration of tirapazamine (TPZ) under reduced dose-rate irradiation (RDRI) using gamma-rays. SCC VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a 24 h continuous subcutaneous infusion of TPZ either with or without MTH under high dose-rate irradiation (HDRI) or RDRI using gamma-rays. After the irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in non-proliferating tumour cells without BrdU labeling (= quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cell populations was determined using tumours that were not pretreated with BrdU. The sensitivity of both the total and Q cell populations, especially the latter, was significantly reduced with RDRI compared with HDRI. TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Further, MTH combined with TPZ raised the sensitivity of both the total and Q cell populations, especially the latter, under RDRI more markedly than under HDRI. From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially in combination with MTH, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.


Dependency of the effect of a vascular disrupting agent on sensitivity to tirapazamine and gamma-ray irradiation upon the timing of its administration and tumor size, with reference to the effect on intratumor quiescent cells

The effect of vascular disrupting agent ZD6126 with time on the sensitivity to the hypoxic cytotoxin tirapazamine (TPZ) and gamma-rays was examined in large and small solid tumors. Mice bearing SCC VII tumors 1 or 1.5 cm in diameter received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells, followed by injection with or without ZD6126. In the absence of ZD6126, or 1 or 24 h following ZD6126 injection, the response to TPZ or gamma-ray irradiation in quiescent (Q) cells was assessed in terms of induced micronucleus (MN) frequency using immunofluorescence staining for BrdU. The MN frequency in the total cell population was determined from the tumors not pretreated with BrdU. Another group of tumor-bearing mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors. One hour after ZD6126 injection, both small and large tumors showed lower and higher sensitivity, and 24 h after, higher and lower sensitivity, to gamma-rays and TPZ, respectively, than the tumors not treated with ZD6126. Further, they showed larger and smaller HFs 1 and 24 h after ZD6126 injection, respectively. Without ZD6126 and 1 h after injection, small tumors were more sensitive to gamma-rays and less sensitive to TPZ than large tumors, probably due to the smaller HFs than large tumors. In contrast, 24 h after the injection, these differences in sensitivity and the HF between small and large tumors were reversed. The changes in sensitivity and the size of the HF were more marked in the total cell population than in Q cells. Following ZD6126 treatment, in terms of tumor control, especially large tumors and total tumor cell population, administering TPZ 1 h later and gamma-ray irradiation 24 h later were effective. Intratumor physiologic factors such as the size of the HF, depending on the time after ZD6126 injection, have to be taken into account when combining another treatment with ZD6126.

Journal of Cancer Research and Clinical Oncology


The usefulness of a continuous administration of tirapazamine combined with reduced dose-rate irradiation using gamma-rays or reactor thermal neutrons

We clarified the usefulness of the continuous administration of tirapazamine (TPZ) in combination with reduced dose-rate irradiation (RDRI) using gamma-rays or reactor thermal neutrons. Squamous cell carcinoma (SCC) VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a single intraperitoneal injection or 24 h continuous subcutaneous infusion of TPZ in combination with conventional dose-rate irradiation (CDRI) or RDRI using gamma-rays or thermal neutrons. After irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cells was determined using tumours that were not pre-treated with BrdU. The sensitivity of both total and Q cells, especially of Q cells, was significantly reduced with RDRI compared with CDRI. Combination of TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Furthermore, the continuous administration of TPZ raised the sensitivity of both total and Q cell populations, especially the former, more markedly than the single administration, whether combined with CDRI or RDRI using gamma-rays or thermal neutrons. From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially when administered continuously, combined with RDRI, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.


The Role of Gc Protein Oligosaccharide Structure as a Risk Factor for COPD

The risk of chronic obstructive pulmonary disease (COPD) is related to Gc protein allele type, such as Gc*1F, Gc*IS, Gc*2. It has been reported that Gc*1F increased COPD risk, while Gc*2 suppressed the risk. Thus, the allele type of Gc protein is an important factor in COPD. These Gc proteins differ in sugar composition at Thr418 or Thr420. In this study, features of the sugar structure of modeled Gc proteins were investigated. Gc protein (GclF, Gc1S, Gc2) models were constructed based on X-ray data of vitamin D binding protein (ID=1J7E) using InsightII-Discover with the Homology module, and the molecular orbital (MO) parameters [e.g., dipole moment, solvation free energy (dGW)] of the oligosaccharide were analyzed. The MO parameter of the sugar moiety was different for each Gc protein model. In beta-1,4 bond models, the dipole moment of Gc2 protein was larger (56.6 debye) than Gcl type (GclF: 21.9, Gc1S: 29.8 debye) protein, and it was directed towards the intermolecular space. The Gc2 oligosaccharide region was the most hydrophobic (dGW=-999.4 KI) among the Gc proteins analyzed in this study. The electrostatic potential (ESP) field of beta-1,4 type Gc2 protein was similarly distributed to beta-1,4 linked Gcl-type proteins (GclF, GclS). In the beta-1,3 type Gc protein models, the results of these parameters (i.e., dipole moment, dGW and ESP) were similar to those of beta-1,4 type models. Conclusion: The relationship between COPD risk and the features of the sugar structure in Gc proteins was examined, and it appeared that the active factors (i.e., dipole moment, dGW) might be risk factors for COPD, but passive factors (i.e., ESP) did not affect COPD risk. The bond type (beta-1,4 or beta-1,3) between galactose and N-acetylgalactosamine did not affect the molecular features.


Hypoxia accelerates cancer invasion of hepatoma cells by upregulating MMP expression in an HIF-1alpha-independent manner

Tumor hypoxia has been reported to induce tumor progression in several carcinomas. Current studies have shown that hypoxia inducible factor-1alpha (HIF-1alpha) is stabilized under hypoxic conditions and transactivates various genes related to cancer aggressiveness. In the present study, we examined whether hypoxia affects cancer invasion in hepatocellular carcinoma. We aimed to solve the molecular mechanism of tumor invasion under the hypoxic condition. We showed that tumor hypoxia accelerated cancer invasion in two hepatoma cell lines. Using Western blot and RT-PCR analyses we demonstrated striking evidence that the expression of HIF-1alpha, ETS-1, MMP-7 and MT1-MMP was strongly upregulated by hypoxic stimulation. To examine whether these invasion-related genes are regulated by HIF-1alpha, we treated hepatoma cells with TX-402, which was reported to repress HIF-1alpha expression. HIF-1alpha expression was strongly repressed by the TX-402 treatment. In contrast, the expression of ETS-1, MMP-7 and MT1-MMP mRNA was not affected by TX-402 treatment. We further established stable transfectants in which HIF-1alpha dominant negative vector was introduced into Hep3B cells (pHIF-1alphaDN). In the pHIF-1alphaDN cells, the expression of ETS-1, MMP-7 and MT1-MMP was not repressed. Moreover, the invasion activity of pHIF-1alphaDN was not altered, compared with that of the mock. In hepatoma cells, we provided evidence that hypoxic stress accelerates cancer invasion by upregulating ETS-1 and the MMP family by an HIF-1alpha-independent pathway.


Molecular dynamics of human-specific cytolysin: analysis of membrane binding motif for therapeutic application

Intermedilysin (ILY) is a human-specific cytolysin secreted from Streptococcus intermedius. In this study, the dynamic structure of ILY, StreptolysinO (SLO) and their 12mer substituted mutants for 500 ps was analyzed. Several parameters, such as dipole moment and electrostatic potential, were determined to elucidate the molecular mechanism of membrane binding. Molecular models of lLY, SLO and their mutants were constructed using Insightll-Discover with the Homology module. Their molecular dynamics were simulated with the Discover3 (Insight module), and z-matrix data of the membrane-binding 12mer region were extracted to calculate the molecular orbital (MO) parameters (i.e., dipole moment, solvation free energy (dGW)). Cytolysins vibrated like a bow, and the dipole moment direction of ILY 12mer region was different from that of SLO. Certain ILY mutants indicated the SLO-like dipole properties, which had an SLO-type limer cysteine motif amino acid sequence. The ILY 11mer region was more hydrophobic than that of SLO, and seemed to interact easily with the cell membrane without cholesterol. The electrostatic potential field distribution of ILY differed from that of SLO, especially in the 11mer region. In the 11mer region, the dipole moment directions of these cytolysins were constant during molecular movement, and ready to interact with membrane components (i.e., cholesterol, phospholipid).


Artepillin C isoprenomics: design and synthesis of artepillin C isoprene analogues as lipid peroxidation inhibitor having low mitochondrial toxicity

We designed and synthesized isoprene analogues of artepillin C, a major component of Brazilian propolis, and investigated the inhibitory activity on lipid peroxidation of rat liver mitochondria (RLM) and RLM toxicity based on isoprenomics. We succeeded in the synthesis of artepillin C isoprene analogues using regioselective prenylation within the range from 22% to 53% total yield. Reactivity of artepillin C and its isoprene analogues with ABTS (2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonate)) radical cations showed only a slight difference among the molecules. The isoprene side-chain elongation analogues of artepillin C showed almost the same inhibitory activity against RLM lipid peroxidation as artepillin C. Artepillin C and its isoprene analogues had very weak RLM uncoupling activity. Moreover, artepillin C and its isoprene analogues exhibited a lower inhibitory activity against adenosine 5'-triphosphate (ATP) synthesis by about two orders of magnitude than the effective inhibitory activity against RLM lipid peroxidation. From these results we conclude that artepillin C isoprene analogues could be potent lipid peroxidation inhibitors having low mitochondrial toxicity. We also conclude that elongation of the isoprene side chain of artepillin C to increase lipophilicity had little influence on the inhibitory activity toward RLM lipid peroxidation.


Artepillin C isoprenomics: design and synthesis of artepillin C analogues as antiatherogenic antioxidants

Advances in Experimental Medicine and Biology


Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guerin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.


The usefulness of mild temperature hyperthermia combined with a newly developed hypoxia-oriented 10B conjugate compound, TX-2100, for boron neutron capture therapy

To evaluate the usefulness of a new 10B-compound (TX-2100) as a 10B-carrier in boron neutron capture therapy (BNCT), compared with the simultaneous use of its component drugs, sodium borocapate-10B (BSH) and 3-amino-2-quinoxalinecarbonitrile 1,4-dioxide (TX-402). Further, the usefulness of mild temperature hyperthermia (MTH, 40 degrees Celsius, 30 min) combined with TX-2100 was also examined compared with MTH combined with the concurrent administration with its component drugs. TX-2100 is a hybrid compound that has both a hypoxic cytotoxin unit (TX-402) and a thermal neutron-sensitizing unit (BSH). TX-2100 or both TX-402 plus BSH in combination with MTH or not was administered to SCC VII tumour-bearing mice intra-peritoneally. Then, the 10B concentrations in the tumours and normal tissues were measured by gamma-ray spectrometry. Meanwhile, SCC VII tumour-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumours, then treated with TX-2100, TX-402 plus BSH or BSH only, in the same manner as in the biodistribution experiments, either with or without MTH. Right after thermal neutron irradiation during which intra-tumour 10B concentrations remained at similar levels, the tumours were excised, minced and trypsinized. The tumour cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker) and the micronucleus (MN) frequency in cells without BrdU labelling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, the MN frequency in the total (P + Q) tumour cell population was determined from the tumours that were not pre-treated with BrdU. The clonogenic cell survival was also determined in mice given no BrdU. 10B biodistribution analyses in tumours, brain, skin, muscles, blood and liver indicated that the administration of TX-2100 plus MTH is most favourable for concentrating a sufficient amount of 10B in tumours and maintaining a high enough 10B concentration during irradiation. In addition, MTH had a stronger sensitizing effect when combined with TX-2100 than with the concurrent administration of its components TX-402 and BSH on both the total and Q cell populations in solid tumours. MTH was very effective in combination with the newly-developed TX-2100. The sensitizing effect in combination with MTH should be examined when new 10B-carriers are designed.


Evaluation of Hypoxic Cell Radio-sensitizers in Terms of Radio-sensitizing and Repair-inhibiting Potential Dependency on p53 Status of Tumor Cells and the Effects on Intratumor Quiescent Cells

Intratumor quiescent (Q) cells and p53-mutated tumor cells are more difficult to control than intratumor proliferating (P) cells and p53 wild-type tumor cells, respectively. The usefulness of 3 hypoxic cell radio-sensitizers was compared in terms of a radio-sensitizing effect under aerobic and hypoxic conditions and a repair-inhibiting effect following irradiation on both Q and total (P + Q) cell populations in solid tumors. The dependency of these effects on the p53 status of tumor cells was also examined using tumor cell lines with identical genetic backgrounds except for their p53 status. Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. The nude mice bearing the tumors and C3H/He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all the P cells in the tumors. Tumor-bearing mice received gamma-ray irradiation while alive or following tumor clamping after being administered no drug, nimorazole, SR-2514 or misonidazole, or received no drug, nimorazole, SR-2514 or misonidazole straight after gamma-ray irradiation. For the group irradiated after receiving the drug, the tumors were excised immediately following irradiation, while for the group irradiated before receiving the drug, the tumors were excised 24 h after irradiation. The excised tumors were minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in the cells without BrdU labelling (= quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, the MN frequency in the total tumor cell population was determined from the tumors that had not been pretreated with BrdU. The clonogenic cell survival was also determined in the mice given no BrdU. Both the radio-sensitizing effects under aerobic and hypoxic conditions and the repair-inhibiting effects following gamma-ray irradiation increased in the following order: nimorazole < SR-2514 < misonidazole in both total and Q cells in these 3 tumors. Both effects were more marked in the Q cells and p53-mutated tumors than in the total cells and p53-wild tumors, respectively. In terms of controlling radio-resistant Q tumor cells and p53-mutated tumor cells, the combination of radio-sensitizers and conventional radiotherapy is promising both for radio-sensitization and for repair-inhibition, but further study of the toxicity to normal tissues is needed.


Evaluation of Hypoxia-Specific Cytotoxic Bioreductive Agent-Sodium Borocaptate-10B Conjugates, as 10B-Carriers in Boron Neutron,Capture Therary

To evaluate the usefulness of 5 new 10B-compounds (TX-2091, TX-2095, TX-2097, TX-2100, and TX-2110) as 10B-carriers in boron neutron capture therpy (BNCT). They were conjugates that had been synthesized from a hypoxia-specific cytotoxic bioreductive agent, quinoxaline oxide TX-402, and a clinically used 10B-carrier, sodium borocaptate-10B (BSH). The 5 new compounds were hybrid compounds that have both a hypoxic cytotoxin unit and a thermal neutron-sensitizing unit, BSH. These new compounds and BSH were administered intraperitoneally to SCC VII tumor-bearing mice. Then, the 10B concentrations in the tumors and normal tissues were measured by gamma-ray spectrometry. Subsequently, SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumors, then treated with TX-2100, which was chosen based on the results of the above-mentioned biodistribution analyses, or BSH in the same manner as in the biodistribution studies. Right after irradiation, during which intratumor 10B concentrations were kept at levels similar to each other, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling [= quiescent (Q) cells] was determined using immunofluorescence staining for BrdU. Meanwhile, the MN frequency in the total (P+Q) tumor cell population was determined from the tumors that were not pretreated with BrdU. Clonogenic cell survival was also determined in mice given no BrdU. 10B biodistribution analyses in tumors, brain, skin, muscles, blood, and liver indicated that TX-2100 has the most favorable characteristics for concentrating a sufficient amount of 10B in tumors and maintaining a high enough 10B concentration during irradiation. In addition, TX-2100 had a significantly stronger radio-sensitizing effect with reactor thermal neutron beams than BSH on both total and Q cells in solid tumors. Further, TX-2100 clearly exhibited a radio-sensitizing effect with gamma-rays not only on total cells but also on Q and hypoxic tumor cells, which was not achieved by BSH. A 10B-carrier that acts as a hypoxic cytotoxin on tumor cells as well as having the potential to keep 10B in tumors and sensitize tumor cells more markedly than conventional 10B-carriers, such as TX-2100, is a promising candidate for use in BNCT.


Innate-immune Therapy for Lung Carcinoma Based on Tissue-macrophage Activation with lipopolysaccharide

Over the last decade, tumor-specific antigens have been discovered, but so far it has not been possible to use them as part of an effective acquired immunotherapy. This failure may be due to the fact that the expression of the MHC class 1 is low and in lung cancer cells is heterogeneous. Therefore, it may be advantageous to develop techniques that activate the antitumor mechanism of the innate immune system. An experimental model was developed for testing lung cancer therapies that are based on the stimulation of macrophages, which then activate innate immunity. A549, a human lung adenocarcinoma cell line, was co-cultured with a rat macrophage cell line (NR8383), or a human macrophage cell line (THP 1) at the ratios of 1:1 or 1:5. The experiments were performed with lipopolysaccharide (LPS) or in its absence. The cytotoxicity rate to A549 cells was estimated over time using a dye-uptake method and the amount of lactate dehydrogenase released was measured. The amount of nitric oxide (NO) induced in the medium was assayed, because it may be a candidate as a useful cytotoxic factor. High cytotoxicity was observed to A549 cells when co-cultured with NR8383 cells in the presence of LPS. This effect was not observed in the absence of LPS. Similar results, although to a lesser extent, were observed when A549 cells were co-cultured with THP-1 cells. A high concentration of NO was measured in the co-culture medium of A549 cells and NR8383 cells when LPS was present. The induction of cell death in lung cancer cells occurred after contact with macrophages that had been activated by LPS. The NO that was produced by macrophages in response to LPS was responsible for some of this effect.


Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity

Gcタンパク質(ビタミンD結合タンパク質)がpolymorphismによってそのGcMAF前駆体活性に影響を及ぼすことをgenotyping, phenotyping, 酵素学的研究によって明らかにした．


Establishment of an in vitro model using NR8383 cells and mycobacterium bovis calmette-guerin that mimics a chronic infection of mycobacterium tuberculosis.

Mycobacterium tuberculosis infection affects one-third of the world's population and causes the death of three million people each year. To clarify details of M. tuberculosis survival strategies, it is important to establish a suitable in vitro model that mimics a chronic infection in alveolar macrophages by M. tuberculosis. For this reason, we established a new in vitro model using a rat alveolar macrophage cell line, NR8383. Basic characteristics, including phagocytotic ability and production of nitrogen oxide and tumor necrosis factor in response to several stimuli, of NR8383 cells were compared with those of primary alveolar macrophages. The course after phagocytosis of live or killed M. bovis bacilli Calmette-Guerin (BCG) was examined over 21 days using NR8383 cells as the host. The characteristics that have been examined to date were nearly the same for both primary alveolar macrophage and NR8383 cells. Live BCG phagocytosed by NR8383 cells had successfully begun to grow in the cells within 7 days, while killed BCG were almost completely destroyed by 21 days. BCG-infected NR8383 cells are potentially a suitable in vitro model that mimics a chronic infection with M tuberculosis.


Artepillin C Isoprenomics: Facile Total Synthesis and Discovery of Amphiphilic Antioxidant


Combination of the antivascular agent ZD6126 with hypoxic cytotoxin treatment, with reference to the effect on quiescent tumor cells and the dependency on p53 status of tumor cells

Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received a hypoxic cytotoxin, tirapazamine (TPZ) or TX-402, with or without a vascular targeting agent (VTA), ZD6126. Another group of mice given ZD6126 received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors. After each treatment, the tumor cells were isolated and incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling [quiescent (Q) cells] was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. Both hypoxic cytotoxins showed significantly greater toxicity toward SAS/mp53 and Q than SAS/neo and total tumor cells, respectively. The sensitivity to TX-402 was significantly higher than that to TPZ in both total and Q tumor cells of both tumors. The significant enhancive effect by ZD6126 combined with each hypoxic cytotoxin was similar irrespective of p53 status, and slightly greater for total than Q cells probably because of a more marked increase in the size of the HFs in total than Q cells on the use of ZD6126 in both tumors, resulting in a reduction of the difference in the sensitivity to the hypoxic cytotoxin between total and Q cells. In the treatment of conventional cancer therapy-resistant Q tumor cells or p53-mutated tumor cells, the use of hypoxic cytotoxin was very promising either alone or when VTA was co-administered. TX-402 might be more promising than TPZ, although further study of the toxicity to normal tissue is needed.


Napthalimidobenzamide DB-51630: A Novel DNA binding agent inducing p300 gene expression and exerting a potent anti-cancer activity

Desined napthalimidobenzamide DB-51630 was a novel DNA binding agent inducing p300 gene expression and exerting a potent anti-cancer activity.

分子設計したnapthalimidobenzamide誘導体であるDB-51630がp300遺伝子発現誘導および抗腫瘍活性をもつ新規DNA結合剤であった．


TX-1877, a bifunctional hypoxic cell radiosensitizer, enhances anticancer host response: immune cell migration and nitric oxide production.

TX-1877は マクロファージや樹状細胞などの免疫担当細胞を癌組織に集積させNO産生により制癌作用を示すことを明らかにし，免疫賦活作用をもつ低酸素細胞放射線増感剤であることを報告した．


The usefulness of continuous administration of hypoxic cytotoxin combined with mild temperature hyperthermia, with reference to effects on quiescent tumor cell populations.

To evaluate the usefulness of continuous administration of hypoxic cytotoxins in terms of targeting acute hypoxia in solid tumours and the significance of combination with mild temperature hyperthermia (MTH) (40 degrees C, 60 min), the cytotoxic effects of singly or continuously administered tirapazamine (TPZ) and TX-402 were examined in combination with or without MTH in vivo. Further, the effects were also analysed on total (=proliferating (P)+quiescent (Q)) and Q cell populations in solid tumours with the method for selectively detecting the Q cell response. C3H/He mice bearing SCC VII tumours received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days to label all P cells. The tumour-bearing mice then received a single intra-peritoneal injection or 24 h continuous subcutaneous infusion of hypoxic cytotoxin, TPZ or TX-402, with or without MTH. On the other hand, to detect the changes in the hypoxic fraction (HF) in the tumours by MTH, another group of mice with or without MTH received a series of test doses of gamma-rays while alive or after tumour clamping. After each treatment, the tumour cells were isolated and incubated with a cytokinesis blocker (=cytochalasin-B) and the micronucleus (MN) frequency in cells without BrdU labelling (=Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total tumour cells was determined from the tumours that were not pre-treated with BrdU. The sensitivity to TX-402 was slightly higher than that to TPZ in both total and Q tumour cells. Continuous administration elevated the sensitivity of both total and Q cells, especially total cells. MTH raised the sensitivity of Q cells more remarkably than that of total cells in both single and continuous administrations. It was thought to be probably because of the higher dose distribution of hypoxic cytotoxin in intermediately hypoxic areas derived mainly from chronic hypoxia through MTH. From the viewpoint of tumour control as a whole including both total and Q tumour cells, the continuous administration of hypoxic cytotoxin combined with MTH may be useful for sensitizing tumour cells in vivo.


Utilization of macrophages in anticancer therapy: the macrophage network theory

Appropriate and rational modulation of innate immunity may enhance the therapeutic efficacy of emerging immune therapies for treating cancer. One of the crucial cells of innate immunity is the macrophage. The purpose of this article was to review those issues that suggest ways of exploiting macrophage local functions in immune therapy, and to discuss the suitability of low molecular-weight lipopolysaccharides as potent modulators of macrophage functions for immune therapy of cancer.


Association of the Macrophage Activating Factor (MAF) Precursor Activity with Polymorphism in Vitamin D-binding Protein


Angiogenesis inhibitorTX-1898: synthesis of the enantiomers of sterically diverse haloacetylcarbamoyl-2-nitroimidazole hypoxic cell radiosensitizers

立体多様性のある工学活性ハロアセチルカルバモイル-2-ニトロイミダゾ-ル系低酸素細胞放射線増感剤の分子設計し，新規血管新生阻害活性のある (R)-p-tert-butylphenyl-bromoacetylcarbamoyl-w-nitroimidazoleであるTX―1898を創製した．


The usefulness of 2-nitroimidazole-sodium borocaptate-10B conjugates as10B-carriers in boron neutron capture therapy

BNCTの10Bキャリアーとして2-Nitroimidazole-BSH-10B Conjugate, TX-2041がγ線に対して放射線増感効果，BNCTの10BキャリアーとしてBSHそのものより優れていることを明らかにした．


Applicability of the 2-Nitroimidazole-sodium borocaptate-10B Conjugate, TX-2060, as a 10B-carrier in Boron Neutron Capture Therapy

BNCTの10Bキャリアーとして2-Nitroimidazole-BSH-10B Conjugate, TX-2060がBSHそのものより，薬物動態学的にも，Q細胞および全がん細胞に対する毒性においても優れていることを明らかにした．


Design of Hypoxia-Trageting Protein Tyrosine Kinase Inhibitor using an innovative pharmacophore 2-methylene-4-cyclopentene-1,3-dione

本総説は，低酸素を標的とするPTK阻害剤の開発戦略を紹介し，特にその分子設計におけるファルマコホアとして，2-methylene-4-cyclopentene-1,3-dione構造の重要性に関して，我々の研究成果をもとに論述したものである．

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics


Localization of enolase in synaptic plasma membrane as an alpha gamma heterodimer in rat brain.

Enolase, a glycolytic enzyme, is a multifunctional protein with location diversity. We revealed the intracellular distribution of enolase isozymes, such as alphaalpha-, alphagamma- and gammagamma-enolases, in rat brain synaptic terminals by biochemical and immunoelectron microscopic analyses. Specific activity of enolase of synaptic plasma membrane fraction (SPM2) obtained from synaptosomes was 23.2 +/- 4.4 x 10(-2) micromol/mg protein/min in the presence of 0.25% Triton X-100 and that of synaptosomal cytoplasm (LS) was 67.4 +/- 12.1 x 10(-2) micromol/mg protein/min. About half of enolase activity in synaptosomes was distributed to soluble fraction while the remaining stayed in particulate membrane fractions by ultracentrifugation. Immunoblot analysis of the fractions demonstrated both alpha and gamma subunits were distributed in SPM. In addition, immunoelectron microscopic analysis also revealed that both subunits were immunoreactive on the SPM. Using coimmunoprecipitation assay, we confirmed that the enolase was present not only as a homodimer form but also as an alphagamma hybrid form associated with membrane, where both subunits were coimmunoprecipitated from lysate of SPM2 in the presence of Mg(2+). These findings indicate that all forms (alphaalpha, alphagamma, and gammagamma) of enolase translocate to the plasma membrane and associate with some components in the SPM.

本論文では，解糖系酵素であるエノァーゼのαー及びγーアイソザイムが，ラット脳のシナプス形質膜に会合して存在することを生化学的解析及び免役電子顕微鏡による解析によって証明した．


Synthesis of the new mannosidase inhibitor, diversity-oriented 5-substiruted swainsonine analogues, via stereoselective Mannich reaction

本論文では，免役修飾剤をめざしたswainsonineアナログの分子設計とαーマンノシダーゼ阻害剤活性に関する構造活性相関，及び多様性を指向した医薬品分子設計において有用な立体選択的Mannish反応の開発について報告した．


Gc Protein-derived Macrophage Activating Factor (GcMAF): Isoelectric Focusing Pattern and Tumoricidal Activity

本論文において，等電点電気泳動法によってGcタンパク質の多型を分類し，種々の多型の糖鎖構造の違いによって，これより生成するGcMAFのマクロファージ活性化作用，及びそれを介した殺細胞活性が異なることを明らかにした．


Beneficial effect of macrophage activating agent NK-4 on Thai HIV-infected patients

We examined the effect of NK-4 (4,4'-[3-[2-(1-ethyl-4(1H)-quinolinylidene) ethylidene] propenylene] bis(-ethylquinolinium iodide)) on the clinical course of Thai HIV-infected patients receiving antiretroviral drugs. Twelve subjects with CD4 count < 500/mm3 were enrolled to receive 0.5 mg of NK-4 orally once daily for 8 weeks and then twice daily during the follow-up. Most patients showed vitality, increased appetite, stable body-weight, increased CD4 counts and no serious adverse effects due to viral load. We suggest that the increased CD4 counts are due to the macrophage activating effect of NK-4. The patients with increased CD4 counts showed lower alpha-N-acetylgalactosaminidase (alpha-NaGalase) activity than those with decreased CD4 counts. These results suggest that the macrophage activating agent NK-4, as an inexpensive and safe drug for HIV-infected patients, may play a beneficial role in the clinical treatment of HIV-infected patients.


Antiangiogenic hypoxic cytotoxin TX-402 inhibits hypoxia-inducible factor 1 signaling pathway

本論文で，血管新生阻害作用を有する新規hypoxic cytotoxin TX-402は，低酸素誘導因子(HIF-1)シグナル経路を阻害することによって，血管新生阻害作用などの低酸素応答反応を抑制することを明らかにした．


乳癌の内分療法抵抗性克服を目指した研究:Hypoxic cytotoxinsを用いた実験

本論文において，乳がんホルモン療法におけるホルモン依存性の消失をめざして，hypoxic cytotoxinの併用について検討した．その結果，hhypoxic cytotoxinはホルモン療法抵抗性の獲得を遅延させ，効果を増強する可能性があることを示した．


Efficient radical scavenging ability of artepillin C, a major component of Brazilian propolis, and the mechanism

本論文では，ブラジル産プロポリスの主要な活性成分であるartepillin Cのラジカル消去作用とその機構について解析した．


Characterization of human Gc protein-derived macrophage activation factor (GcMAF) and its functional role in macrophage tumoricidal activity

本論文では，GcMAFの抗腫瘍作用の作用機構として，マクロファージ貪食能と活性酸素生成を活性化することを明らかにした．

Advances in Experimental Medicine and Biology


A novel hypoxia-dependent 2-nitroimidazole KIN-841 inhibits tumour-specific angiogenesis by blocking production of angiogenic factors

本論文において， 我々が分子設計した新規低酸素細胞放射線増感剤KIN-841が強い放射線増感効果のほかに，血管新生因子の生成を阻害することによって強い血管新生阻害作用を示すことを明らかにした．