Chondroitin sulfate is not digested at all in the mouse small intestine but may suppress interleukin 6 expression induced by tumor necrosis factor-α.

Salmon nasal cartilage proteoglycan (PG) was orally administered to mice. The PG digest was recovered from the small intestine, and its sugar chain size and unsaturated disaccharide content were examined. The elution position of the PG digest following Sepharose CL-4B chromatography was consistent with that of actinase-digested PG prior to administration. The PG digest was incubated with chondroitinase ABC, which resulted in the elution pattern of the unsaturated disaccharides being identical to that of the degraded product of actinase-digested PG. The core protein of PG was digested in the mouse small intestine, but chondroitin sulfate, which is the sugar chain of PG, was not degraded at all. Then, the effects of chondroitin 4- and 6-sulfates on human colon cancer cells were examined. These chondroitin sulfates were found to suppress the expression of interleukin-6 induced by TNF-α. Overall, the chondroitin sulfate chain may act on the intestinal epithelium and suppress inflammation of the intestinal tract.

Antimicrobial properties of L-amino acid oxidase: biochemical features and biomedical applications.

Mucus layer that covers the body surface of various animal functions as a defense barrier against microbes, environmental xenobiotics, and predators. Previous studies have reported that L-amino acid oxidase (LAAO), present in several animal fluids, has potent properties against pathogenic bacteria, viruses, and parasites. LAAO catalyzes the oxidative deamination of specific L-amino acids with the generation of hydrogen peroxide and L-amino acid metabolites. Further, the generated hydrogen peroxide is involved in oxidation (direct effect) while the metabolites activate immune responses (indirect effect). Therefore, LAAO exhibits two different mechanisms of bioactivation. Previously, we described the selective, specific, and local oxidative and potent antibacterial actions of various LAAOs as potential therapeutic strategies. In this review, we focus on their biochemical features, enzymatic regulations, and biomedical applications with a view of describing their probable role as biochemical agents and biomarkers for microbial infections, cancer, and autoimmune-mediated diseases. We consider that LAAOs hold implications in biomedicine owing to their antimicrobial activity wherein they can be used in treatment of infectious diseases and as diagnostic biomarkers in the above-mentioned diseased conditions. KEY POINTS: •Focus on biochemical features, enzymatic regulation, and biomedical applications of LAAOs. •Mechanisms of antimicrobial activity, inflammatory regulation, and immune responses of LAAOs. •Potential biomedical application as an antimicrobial and anti-infection agent, and disease biomarker.

Phosphorylation of Thr328 in hyaluronan synthase 2 is essential for hyaluronan synthesis.

Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked ß-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr328 position of full-length HAS2, which was detected in the cells regardless of the presence of PMA or 4-MU. We next prepared T328A site-directed mutagenesis construct-transfected cells and investigated HA synthesis. The amount of HA was increased in cells expressing full-length HAS2 compared to in mock cells, whereas the amount of HA synthesized by cells transfected with the T328A site-directed mutagenesis construct was the same as that in mock cells. This phosphorylation site corresponded with the casein kinase 1 substrate motif. These results suggest that Thr328 phosphorylation is an essential factor for HA synthesis by HAS2 and the role of HAS2-loop may be useful in analyzing the regulation of HAS2 synthesis in physiological and pathological conditions.

Metal coordination by L-amino acid oxidase derived from flounder Platichthys stellatus is structurally essential and regulates antibacterial activity.

L-amino acid oxidases (LAAOs) have antibacterial activity and play important roles in innate immunity. We have previously identified a LAAO of ~52 kDa in size from the mucus layer of the flounder Platichthys stellate (psLAAO1) and have successfully produced psLAAO1 as a secreted bioactive recombinant protein by using Pichia pastoris (P. pastoris). The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in the mucus layer. In this study, homology modeling of psLAAO1 predicted metal coordination by residues Y241, H348, and D406. We show that the Michaelis constant (Km) of psLAAO1 decreased and the catalytic constant (Kcat/Km) value increased following pre-treatment of the protein with a chelating agent. In contrast to the non-chelated protein sample, enzymatic activity of EDTA-treated psLAAO1 gradually decreased or was absent after one or two freeze-thaw cycles. The H348A psLAAO1 mutant generated by site-directed mutagenesis and recombinantly produced by P. pastoris did not display antibacterial activity. The results of the metal detection assay revealed that for the non-metal coordinating histidine mutant (H209A, control), the levels of iron, zinc, and magnesium were similar to those of wild-type psLAAO1, whereas magnesium was not detected in the H348A mutant sample. A wild-type psLAAO1 sample treated with chelating agent did not contain zinc and magnesium ions. In conclusion, metal coordination by psLAAO1 affects enzymatic activity, and H348 is involved in the coordination of magnesium, and metal coordination by psLAAO1 provides essential structural stability. KEY POINTS: • Homology modeling of psLAAO1 predicted metal coordination by residue H348 • The H348A psLAAO1 mutant showed no antibacterial activity or magnesium coordination • Metal coordination by H348 affects enzyme activity and structural stability.

Proteoglycan-substrate gel zymography for the detection of chondroitin sulfate-degrading enzymes.

Chondroitin sulfate (CS), a linear polysaccharide, is a major component of the cartilage matrix. Although CS plays various roles in several biological and pathological processes, most details regarding its metabolism are still poorly understood. Some CS-degrading enzymes have been identified in mammals, but their expression patterns and localizations remain unclear. Here we present a simple zymography procedure to detect CS-degrading enzymes using salmon nasal cartilage proteoglycans as substrates. This method should be useful to explore CS-degrading enzymes.

UDP/P2Y6 receptor signaling regulates IgE-dependent degranulation in human basophils.

BACKGROUND: P2Y purinergic receptors (P2YR) are G protein-coupled receptors that are stimulated by extracellular nucleotides. They mediate cellular effects by regulating cAMP production, protein kinase C activation, inositol trisphosphate generation, and Ca2+ release from intracellular stores. The P2Y6 receptor of this family is selectively stimulated by UDP, and selectively inhibited by MRS2578. In the present study, we examined the effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in human basophils. METHODS: Basophils were purified from human peripheral blood. The mRNA expression of genes encoding P2YR and ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) was measured by RT-PCR. Intracellular Ca2+ influx via UDP/P2Y6 receptor signaling in basophils was detected using a calcium probe. The effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in basophils was confirmed by measuring CD63 expression by flow cytometry. Autocrine secretion of nucleotides was detected by HPLC analysis. RESULTS: We showed that purified basophils express P2Y6 mRNA and that UDP increased intracellular Ca2+, which was reduced by MRS2578 treatment. UDP promoted IgE-dependent degranulation. Furthermore, MRS2578 inhibited IgE-dependent degranulation in basophils. HPLC analysis indicated that basophils spontaneously secrete UTP. In addition, basophils expressed the extracellular nucleotide hydrolases ENTPDase2, ENTPDase3, and ENTPDase8. CONCLUSIONS: This study showed that UDP/P2Y6 receptor signaling is involved in the regulation of IgE-dependent degranulation in basophils, which might stimulate the P2Y6 receptor via the autocrine secretion of UTP. Thus, this receptor represents a potential target to regulate IgE-dependent degranulation in basophils during allergic diseases.

A novel strategy for evasion of NK cell immunity by tumours expressing core2 O-glycans.

The O-glycan branching enzyme, core2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT), forms O-glycans containing an N-acetylglucosamine branch connected to N-acetylgalactosamine (core2 O-glycans) on cell-surface glycoproteins. Here, we report that upregulation of C2GnT is closely correlated with progression of bladder tumours and that C2GnT-expressing bladder tumours use a novel strategy to increase their metastatic potential. Our results showed that C2GnT-expressing bladder tumour cells are highly metastatic due to their high ability to evade NK cell immunity and revealed the molecular mechanism of the immune evasion by C2GnT expression. Engagement of an NK-activating receptor, NKG2D, by its tumour-associated ligand, Major histocompatibility complex class I-related chain A (MICA), is critical to tumour rejection by NK cells. In C2GnT-expressing bladder tumour cells, poly-N-acetyllactosamine was present on core2 O-glycans on MICA, and galectin-3 bound the NKG2D-binding site of MICA through this poly-N-acetyllactosamine. Galectin-3 reduced the affinity of MICA for NKG2D, thereby severely impairing NK cell activation and silencing the NK cells. This new mode of NK cell silencing promotes immune evasion of C2GnT-expressing bladder tumour cells, resulting in tumour metastasis.

Antibacterial properties of L-amino acid oxidase: mechanisms of action and perspectives for therapeutic applications.

Venom, the mucus layer covering the body surface, ink glands, mammary glands, milk, and various animal secretory functions as both a physical and chemical defense barrier against bacteria and virus infections. Previously, several studies reported that L-amino acid oxidases (LAAOs) present in animal secretary fluids have strong antimicrobial activities and selective cytotoxic activities against Gram-positive and Gram-negative bacteria, various pathogenic bacteria, viruses, and parasite species. These LAAOs catalyze oxidative deamination of an L-amino acid substrate with the generation of hydrogen peroxide. The antibacterial activity of LAAOs is completely inhibited by catalase; thus, LAAOs kill bacteria by the hydrogen peroxide generated from the oxidation of L-amino acid substrates. This review focuses on the selective, specific, and local antibacterial actions of various LAAOs that may be used as novel therapeutic agents against infectious diseases. LAAOs that are suitable leads for combating multidrug-resistant bacterial infections are also studied.

Recombinant production and evaluation of an antibacterial L-amino acid oxidase derived from flounder Platichthys stellatus.

Fish produce mucus substances as a defensive outer barrier against several bacterial infections. We have recently identified an antibacterial L-amino acid oxidase (psLAAO1) in the mucus layer of the flounder Platichthys stellate. In this study, the antibacterial protein psLAAO1 was expressed as a secretory bioactive recombinant protein in the methylotrophic yeast Pichia pastoris. The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in mucus. In particular, Staphylococci and Yersinia were strongly suppressed, showing the highest growth retardation of the 21 species and strains tested. Moreover, Staphylococcus epidermidis was most sensitive to psLAAO1 with a minimum inhibitory concentration (MIC) of 0.078 µg/mL, whereas Escherichia coli was essentially resistant to psLAAO1 with a MIC of >10 µg/mL. Interestingly, psLAAO1-treated E. coli were found to upregulate the expression of the btuE gene, which encodes glutathione peroxidase (GPx). The biochemical function of GPx is to reduce free hydrogen peroxide and is induced under response to reactive oxygen species (ROS). Thus, E. coli confers resistance to the reduced free hydrogen peroxide produced by psLAAO1 by increasing GPx levels. Furthermore, the growth of Staphylococcus aureus was completely inhibited in the presence of recombinant psLAAO1. The morphology of psLAAO1-treated S. aureus showed cell surface damage, the formation of large aggregates and the cells showed severe deformations. Western blot analysis showed that psLAAO1 binds to the surface of S. aureus. Therefore, psLAAO1 binds to the surface of LAAO-sensitive S. aureus and directs peroxidative activity at the surface of the bacterial membrane.

One set system for the synthesis and purification of glycosaminoglycan oligosaccharides reconstructed using a hyaluronidase-immobilized column.

Using the transglycosylation reaction as a reverse reaction for the hydrolysis of hyaluronidase, new artificial oligosaccharides may be synthesized by reconstructing natural glycosaminoglycans (GAGs) according to preliminary planned arrangements. However, as some problems have been associated with the method, including the low yields of reaction products and complicated processes of separation and purification, improvements in this method were investigated. Transglycosylation reactions were carried out using bovine testicular hyaluronidase-immobilized resin packed in a column. For the transglycosylation reaction, pyridylaminated (PA) GAG hexasaccharides, which were the minimum size for hydrolysis sensitivity and the transglycosylation reaction, were used as acceptors, whereas large size GAGs were used as donors. The reaction mixture was pooled after incubation in the hyaluronidase-immobilized resin column and was then introduced into continuously joined HPLC columns constructed from three steps: the first step of ion-exchange HPLC for concentrating newly synthesized GAG oligosaccharides as reaction products, the second step of reverse phase HPLC for separating PA oligosaccharides from non-PA oligosaccharides, and the third step of size fractionation HPLC for fractionating newly synthesized oligosaccharides. Newly synthesized oligosaccharides were obtained by one complete cycle of the transglycosylation reaction and separation.

Hyaluronan-chondroitin hybrid oligosaccharides as new life science research tools.

Hyaluronan and chondroitin are glycosaminoglycans well-known as components of pharmaceutical agents and health foods. From these attractive molecules, using transglycosylation reaction of testicular hyaluronidase, we synthesized hybrid neo-oligosaccharides not found in nature. We also found a new site between the chondroitin disaccharide unit and hyaluronan disaccharide unit recognized by a hyaluronan lyase specific to hyaluronan using these hybrid oligosaccharides as substrates. We hope that these hybrid oligosaccharides will help to elucidate the involvement of hyaluronan, chondroitin, and chondroitin sulfates in the mechanisms of cell functions and diseases, based on the structures of these glycosaminoglycans.

Requirement for FBP17 in invadopodia formation by invasive bladder tumor cells.

PURPOSE: Invadopodia (protrusions of the plasma membrane formed by invasive tumor cells) have an essential role in bladder tumor invasion. To understand the process of bladder tumor invasion it is crucial to investigate the molecular mechanisms of invadopodia formation. We found that invasive bladder tumor cells express FBP17. In this study we examined the role of FBP17 in bladder tumor cell invadopodia formation and invasion. MATERIALS AND METHODS: We used the 3 bladder tumor cell lines YTS-1, T24 and RT4 (ATCC®), and primary culture of bladder tumors from patients. Cells were stained with phalloidin for invadopodia formation. FBP17 knockdown cells were tested for invadopodia formation and subjected to invasion assay using a Transwell® cell culture chamber. We also examined the role of the extended FER-CIP4 homology and Src homology 3 domains of FBP17 in invadopodia formation in FBP17 mutant constructs. RESULTS: Invadopodia formation was observed in invasive bladder tumor cells and FBP17 was localized to invadopodia in invasive cells. FBP17 knockdown decreased invadopodia formation in invasive cells to 13% to 14% (p <0.0005) and decreased their invasive capacity to 14% to 16% (p <0.001). The extended FER-CIP4 homology and Src homology 3 domains of FBP17 were necessary for invadopodia formation and invasion. CONCLUSIONS: Invadopodia formation requires membrane deformation activity and recruitment of dynamin-2 mediated by FBP17. FBP17 has a critical role in the process of bladder tumor cell invasion by mediating invadopodia formation.

Evidence for the heparin-binding ability of the ascidian Xlink domain and insight into the evolution of the Xlink domain in chordates.

The vertebrate Xlink domain is found in two types of genes: lecticans and their associated hyaluronan-and-proteoglycan-binding-link-proteins (HAPLNs), which are components of the extracellular matrix, and those represented by CD44 and stabilins, which are expressed on the surface of lymphocytes. In both types of genes, Xlink functions as a hyaluronan binding domain. We have already reported that protochordate ascidians possess only the latter type of gene. The present analysis of the expression of ascidian Xlink domain genes revealed that these genes function in blood cell migration and apoptosis. While the Xlink domain is found in various metazoans, including ascidians and nematodes, hyaluronan is believed to be specific for vertebrates. In comprehensive genome surveys for hyaluronan synthase (HAS), we found no HAS gene in ascidians. We also established that hyaluronan is absent from the ascidian body biochemically. Therefore, ascidians possess the Xlink domain, but they lack HA. We recovered one ascidian Xlink domain gene that encoded a heparin-binding protein, although it shows no affinity for hyaluronan. Based on these findings, we conclude that in invertebrates, the Xlink domain serves as heparin-binding protein domain and functions in blood cell migration and apoptosis. Its binding affinity for HA might have been acquired in the vertebrate lineage.

Invadopodia formation by bladder tumor cells.

A major cause of death in patients with bladder tumors is recurrence with metastasis. Bladder tumor metastasis is largely dependent upon the invasive capacity of tumor cells. Tumor cell invasion is mainly mediated by actin-rich protrusive membrane structures called invadopodia. The formation of invadopodia was observed in various types of invasive tumors such as breast cancer and melanomas. However, invadopodia formation so far has not been described in bladder tumor cells. We here report that human bladder tumor cells form functionally active invadopodia. By using a confocal laser scanning microscope, we demonstrated that invasive bladder tumor cell lines, YTS-1 and T24, with high Matrigel degradation activity form invadopodia but that noninvasive bladder tumor cell lines, RT4 and KK-47, form no detectable invadopodia. Invadopodia formed by YTS-1 cells had the ability to secrete matrix metalloproteases and degrade extracellular matrix to invade surrounding areas. Moreover, we observed that primary tumor cells obtained from patients with invasive bladder tumors also form invadopodia, validating the results from bladder tumor cell lines. Our results provide evidence that invasive human bladder tumor cells form invadopodia for tumor invasion.

Structural change of retinoic-acid receptor-related orphan receptor induced by binding of inverse-agonist: Molecular dynamics and ab initio molecular orbital simulations.

To elucidate structural changes in the retinoic acid receptor-related orphan receptor gamma (RORγt) induced by the binding of an agonist or an inverse agonist, we conducted molecular dynamics (MD) simulations in explicit water. In addition, ab initio fragment molecular orbital calculations were carried out for certain characteristic structures obtained from the MD simulations to reveal important interactions between the amino acid residues of RORγt, and to distinguish the different effects in the binding of an agonist and an inverse agonist on the structure of RORγt. The results elucidate that the hydrogen bond between His479 of helix11 (H11) and Tyr502 of helix12 (H12) is important to keep the H12 conformation in the agonist-bound RORγt. In contrast, in the inverse-agonist-bound RORγt, the side chain of His479 rotates, significantly weakening the interaction between His479 and Tyr502, leading to a conformational change in H12. Therefore, the present molecular simulations clearly indicate that the conformational change in the side chain of His479 in the inverse-agonist-bound RORγt is the main reason for the H12 destabilization induced by the binding of the inverse agonist. Such a conformational change does not occur on the binding of the agonist in RORγt, owing to the strong hydrogen bond between His479 and Tyr502.

Stimulation of small proteoglycan synthesis by the hyaluronan synthesis inhibitor 4-methylumbelliferone in human skin fibroblasts.

Human skin fibroblasts cultured with 4-methylumbelliferone (MU), a hyaluronan synthesis inhibitor, produce a hyaluronan-deficient extracellular matrix (See [9]). Our present study investigated the effects of MU on proteoglycan, which is the other main component of the extracellular matrix, and interacts with hyaluronan. Proteoglycans isolated from culture medium in the presence or absence of MU were characterized by gel-filtration chromatography, ion-exchange HPLC, electrophoresis, and immunoblotting. We found that MU had only a negligible effect on the synthesis of large proteoglycan but increased the production of small proteoglycan in comparison with cultures lacking MU. This small proteoglycan was identified by immunoblotting as decorin. The structures of decorin synthesized in the presence and absence of MU were compared by gel-filtration chromatography, and the data indicated that cells incubated with MU produced a larger decorin molecule than cells incubated without MU. Furthermore, the two decorins had galactosaminoglycan chains of different sizes. These results suggest that MU inhibits the synthesis of hyaluronan and accelerates production of the larger decorin in the extracellular matrix.

Dermatan sulfate oligosaccharides having reducing end 2, 5-anhydro-d-talose inhibit bovine testicular hyaluronidase activity.

Dermatan sulfate oligosaccharides having reducing end 2,5-anhydro-d-talose were prepared by partial N-deacetylation of dermatan sulfate polysaccharide with hydrazine followed by deamination with nitrous acid, and the effect of these oligosaccharides on the activity of bovine testicular hyaluronidase was investigated. Hydrolysis activity and transglycosylation activity of this enzyme were assessed in an independent reaction system by analyzing the products by HPLC. Dermatan sulfate oligosaccharides inhibited hyaluronan hydrolysis by bovine testicular hyaluronidase. Kinetic analysis of the hydrolysis reaction revealed that the inhibition mode by dermatan sulfate oligosaccharides was as competitive as that previously shown by chondroitin sulfate oligosaccharides. Transglycosylation of hyaluronan by bovine testicular hyaluronidase, as a reverse reaction of hydrolysis of hyaluronan, was also inhibited. These inhibitory effects were dependent on the dose and sulfation degree of dermatan sulfate.

Change in binding states between catabolite activating protein and DNA induced by ligand-binding: molecular dynamics and ab initio fragment molecular orbital calculations.

The transcription mechanism of genetic information from DNA to RNA is efficiently controlled by regulatory proteins, such as catabolite activator protein (CAP), and their ligands. When cyclic AMP (cAMP) binds to CAP, the complex forms a dimer and binds specifically to DNA to activate the transcription mechanism. On the other hand, when cyclic GMP (cGMP) binds to CAP, the complex has no marked effect on the mechanism. In our previous study, based on molecular dynamics (MD) and ab initio fragment molecular orbital (FMO) methods, we elucidated which residues of CAP are important for the specific interactions between CAP and DNA in the CAP-monomer+DNA + cAMP complex. However, this monomer model for CAP cannot describe real interactions between the CAP-dimer and DNA because CAPs form a dimer before binding to DNA. Accordingly, here, we investigated stable structures and their electronic states for the CAP-dimer+DNA complex with cAMP or cGMP ligand, to clarify the influence of ligand-binding on the interactions between CAP-dimer and DNA. The MD simulations elucidated that the DNA-binding domains of CAP-dimer behave differently depending on the ligand bound to the CAP-dimer. In addition, FMO calculations revealed that the binding energy between CAP-dimer and DNA for the CAP-dimer+DNA + cAMP complex is larger than that for the CAP-dimer+DNA + cGMP complex, being consistent with experiments. It was also highlighted that the Arg185 and Lys188 residues of CAP-dimer are important for the binding between CAP-dimer and DNA. These results provide useful information for proposing new compounds that efficiently control the transcription mechanism.

Increase in muscle endurance in mice by dietary Yamabushitake mushroom (Hericium erinaceus) possibly via activation of PPARδ.

Skeletal muscle fiber is largely classified into two types: type 1 (slow-twitch) and type 2 (fast-twitch) fibers. Meat quality and composition of fiber types are thought to be closely related. Previous research showed that overexpression of constitutively active peroxisome proliferator-activated receptor (PPAR)δ, a nuclear receptor present in skeletal muscle, increased type 1 fibers in mice. In this study, we found that hexane extracts of Yamabushitake mushroom (Hericium erinaceus) showed PPARδ agonistic activity in vitro. Eight-week-old C57BL/6J mice were fed a diet supplemented with 5% (w/w) freeze-dried Yamabushitake mushroom for 24 hr. After the treatment period, the extensor digitorum longus (EDL) muscles were excised. The Yamabushitake-supplemented diet up-regulated the PPARδ target genes Pdk4 and Ucp3 in mouse skeletal muscles in vivo. Furthermore, feeding the Yamabushitake-supplemented diet to mice for 8 weeks resulted in a significant increase in muscle endurance. These results indicate that Yamabushitake mushroom contains PPARδ agonistic ligands and that dietary intake of Yamabushitake mushroom could activate PPARδ in skeletal muscle of mice. Unexpectedly, we observed no significant alterations in composition of muscle fiber types between the mice fed control and Yamabushitake-supplemented diets.

Regulation of radiosensitivity by 4-methylumbelliferone via the suppression of interleukin-1 in fibrosarcoma cells.

Tumor recurrence and distant metastasis following radiotherapy, which can lead to poor prognosis, are caused by residual cancer cells that acquire radioresistance. Chemotherapy or a combination of targeted inhibitors can potentially enhance radiation sensitivity and prevent metastasis. It was previously reported that co-administration of the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) enhanced the lethality of X-ray irradiation in HT1080 human fibrosarcoma cells and decreased their invasiveness to a greater extent than either treatment alone. To clarify the molecular basis of these effects, the present study conducted mRNA expression profiling by cDNA microarray to identify the signaling pathways that are altered under this combination treatment. The activation state of the signaling pathways was classified by z-scores in the Ingenuity Pathway Analysis. The results revealed that the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 were activated by 2 Gy X-ray irradiation, an effect that was abolished by co-administration of 4-MU. Similar trends were observed for the upstream signaling component IL-1. These results indicate that the radiosensitivity of fibrosarcoma cells is improved by suppressing inflammation through the administration of 4-MU.