Effect of glycosaminoglycan structure on all-trans-retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells.

Glycosaminoglycan polysaccharides are components of animal extracellular matrices and regulate cell functions based on their various sulfation and epimerization pattern structures. The present study aimed to find glycosaminoglycan structures to promote neural differentiation. We investigated the effect of exogenous glycosaminoglycans with well-defined structures on the all-trans-retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells, which is an ideal model culture system for studying neural differentiation. We found that chondroitin sulfate E and heparin, but not any other glycosaminoglycans, upregulated the expressions of neural specific markers but not a grail specific marker. Chondroitin sulfate E was suggested to function during spheroid formation, however, equimolar concentration of its oligosaccharide did not show promotive effect on the neural differentiation. Another finding was that hyaluronan oligosaccharide mixture markedly downregulated the expressions of a myelin specific marker. These findings suggested that the specific sulfation pattern and/or chain length of exogenous added glycosaminoglycan is important to regulate neural differentiation and myelination.

Enzymatic synthesis of hyaluronan hybrid urinary trypsin inhibitor.

Human urinary trypsin inhibitor is a proteoglycan that has a single low-sulfated chondroitin 4-sulfate chain at the seryl residue in position 10 of the core protein as a glycosaminoglycan moiety, and is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, the functions of the glycosaminoglycan moiety have not yet been elucidated in detail. In the present study, the glycosaminoglycan chains of a native urinary trypsin inhibitor were remodeled to hyaluronan chains, with no changes to the core protein, using transglycosylation as a reverse reaction of the hydrolysis of bovine testicular hyaluronidase, and the properties of the hybrid urinary trypsin inhibitor were then analyzed. The trypsin inhibitory activitiy of the hyaluronan hybrid urinary trypsin inhibitor was similar to that of the native type; however, its inhibitory effect on the hydrolysis of hyaluronidase were not as strong as that of the native type. This result demonstrated that the native urinary trypsin inhibitor possessed hyaluronidase inhibitory activity on its chondroitin sulfate chain. The hyaluronan hybrid urinary trypsin inhibitors obtained affinity to a hyaluronan-binding protein not exhibited by the native type. The interactions between the hyaluronan hybrid urinary trypsin inhibitors and phosphatidylcholine (abundant in the outer layer of plasma membrane) were stronger than that of the native type. Hyaluronan hybrid urinary trypsin inhibitors may be useful for investigating the functions of the glycosaminoglycan chains of urinary trypsin inhibitors and hyaluronan, and our hybrid synthesizing method may be used widely in research for future medical applications.

Parathyroid hormone-related protein expression, in combination with nodal status, predicts bone metastasis and prognosis of breast cancer patients.

Parathyroid hormone-related protein (PTHrP) has been known to play an important role in the formation of meta-static lesions in the bone. However, there remains controversy over its practical role in predicting the occurrence of bone metastasis and the prognosis of breast cancer patients. In this study, we attempted to investigate the clinical value of PTHrP expression status in the primary lesions of breast cancer patients. We immunohistochemically investigated PTHrP expression in surgically resected specimens from 125 primary breast cancer patients whose clinicopathological background and long-term prognosis were available. Positive PTHrP staining was demonstrated in 79 (63.2%) tumors. PTHrP was expressed significantly more frequently in the tumors of premenopausal patients. Bone metastases were significantly more common in patients with T4 tumors, with a positive node, with distant metastasis and with PTHrP-positive tumors. Multivariate logistic analysis revealed positive PTHrP expression as an independent risk factor for predicting bone metastasis. PTHrP expression was significantly related to a shorter overall survival. Bone metastasis was found significantly more frequently (28.3%) in PTHrP- and node-positive cases than in double-negative cases, and the rate was more pronounced in postmenopausal cases (32.1%). Expression of PTHrP in primary lesions, in combination with positive nodal status, is indicative of an increased risk of bone metastasis in breast cancer patients.

[A case of diffuse infiltrating carcinoma of large intestine with liver metastases effectively treated with systemic chemotherapy].

We report a case of diffuse infiltrating carcinoma of the large intestine effectively treated by operation and chemotherapy. A 79-year-old woman with bone and liver metastases due to descending colon carcinoma underwent left hemicolectomy and colostomy. Pathological resected specimen findings showed a diffuse infiltrating carcinoma(lymphangiosis type). She re- ceived chemotherapy with 7 courses of mFOLFOX6, 8 courses of mFOLFOX6/bevacizumab(BV), and 5 courses of FOLFIRI/BV after surgical resection. The liver metastases reduced markedly as observed by abdominal CT scan. Twelve months later, DIC caused the death of the patient. Resection with lymphadenectomy and systemic chemotherapy may be effective for treatment of diffuse infiltrating carcinoma of the large intestine.

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates.

Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products.

Novel proteoglycan glycotechnology: chemoenzymatic synthesis of chondroitin sulfate-containing molecules and its application.

Proteoglycans consist of a protein core, with one or more glycosaminoglycan chains (i.e., chondroitin sulfate, dermatan sulfate and heparin sulfate) bound covalently to it. The glycosaminoglycan chains account for many of the functions and properties of proteoglycans. The development of proteoglycan glycotechnology to exploit the functionality of glycosaminoglycan chains is an extremely important aspect of glycobiology. Here we describe an efficient and widely applicable method for chemoenzymatic synthesis of conjugate compounds comprising intact long chondroitin sulfate (ChS) chains. An alkyne containing ChS was prepared by an enzymatic transfer reaction and linked with a chemically synthesized core compound containing an azido group using click chemistry. This method enabled highly efficient introduction of ChS into target materials. Furthermore, the ChS-introduced compounds had marked stability against proteolysis, and the chemically linked ChS chain contributed to the stability of these core compounds. We believe the present method will contribute to the development of proteoglycan glycobiology and technology.

Novel products in hyaluronan digested by bovine testicular hyaluronidase.

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.

Structural interactions in chondroitin 4-sulfate mediated adherence of Plasmodium falciparum infected erythrocytes in human placenta during pregnancy-associated malaria.

Infection with Plasmodium falciparum during pregnancy results in the adherence of infected red blood cells (IRBCs) in placenta, causing pregnancy-associated malaria with severe health complications in mothers and fetuses. The chondroitin 4-sulfate (C4S) chains of very low sulfated chondroitin sulfate proteoglycans (CSPGs) in placenta mediate the IRBC adherence. While it is known that partially sulfated but not fully sulfated C4S effectively binds IRBCs, structural interactions involved remain unclear and are incompletely understood. In this study, structurally defined C4S oligosaccharides of varying sulfate contents and sizes were evaluated for their ability to inhibit the binding of IRBCs from different P. falciparum strains to CSPG purified from placenta. The results clearly show that, with all parasite strains studied, dodecasaccharide is the minimal chain length required for the efficient adherence of IRBCs to CSPG and two 4-sulfated disaccharides within this minimal structural motif are sufficient for maximal binding. Together, these data demonstrate for the first time that the C4S structural requirement for IRBC adherence is parasite strain-independent. We also show that the carboxyl group on nonreducing end glucuronic acid in dodecasaccharide motif is important for IRBC binding. Thus, in oligosaccharides containing terminal 4,5-unsaturated glucuronic acid, the nonreducing end disaccharide moiety does not interact with IRBCs due to the altered spatial orientation of carboxyl group. In such C4S oligosaccharides, 14-mer but not 12-mer constitutes the minimal motif for inhibition of IRBC binding to placental CSPG. These data have important implications for the development and evaluation of therapeutics and vaccine for placental malaria.

Effects of proteoglycan on dextran sulfate sodium-induced experimental colitis in rats.

Proteoglycans (PG) are macromolecules composed of glycosaminoglycan chains covalently attached to a protein core. In this study, we examined the effects of PG on dextran sulfate sodium (DSS)-induced experimental colitis in rats. First, to examine whether PG may ameliorate acute established DSS colitis, PG was administered orally for 5 days to the model animals. We evaluated the effects of PG on the basis of clinical symptoms, hematological analysis, macroscopic observation, and microscopic examination. We then examined whether PG administered orally to rats was detectable in their colonic lumen. After administration of PG, the colonic contents were collected, and the molecular weight of PG in the sample was analyzed by gel filtration high-performance liquid chromatography. Furthermore, we examined whether orally administered PG affected the concentrations of short-chain fatty acids (SCFAs) in the colonic feces. Orally administered PG ameliorated the clinical symptoms of bloody stools and diarrhea, and attenuated the increase in the white blood cell count in rats with established DSS colitis. Histologically, orally administered PG reduced the degree of mucosal erosion and inflammatory cell infiltration into the erosive area induced by DSS. Orally administered PG was detected in rat colon, although its molecular weight was slightly decreased. Orally administered PG significantly increased the concentration of total SCFAs and n-butyrate in rat colonic feces. This is the first study to indicate that exogenous PG ameliorates experimental colitis, suggesting the potential usefulness of PG for clinical treatment of colitis.

Ex vivo expansions of megakaryocytopoiesis from placental and umbilical cord blood CD34(+) cells in serum-free culture supplemented with proteoglycans extracted from the nasal cartilage of salmon heads and the nasal septum cartilage of whale.

As a possible approach to the treatment of thrombopocytopenia, the ex vivo expansion of megakaryocytic progenitor cells may be a useful tool to accelerate platelet recovery in vivo. Our objective was to assess the promoting effect of proteoglycans in a serum-free culture condition using human cord blood CD34(+) cells. Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads and the nasal septum cartilage of a whale were applied to the ex vivo expansion of megakaryocytopoiesis and thrombopoiesis from placental and umbilical cord blood CD34(+) cells in serum-free cultures stimulated with a combination of thrombopoietin (TPO) and interleukin-3 (IL-3). Each PG (0.5 and 5 mug) was applied to the culture with three different concentrations of TPO (50, 5 and 0.5 ng/ml) and IL-3 (100, 10 and 1 ng/ml). Both of the PGs showed no promoting effects on the mononuclear cell proliferation rate in any of the cultures. However, the whale-PG promoted the generation of megakaryocytic progenitor cells and megakaryocytes in the culture with a lower dose of cytokines, respectively. In addition, whale-PG led to a significant increase in CD42a(+) particles which seemed to be platelets. While the salmon-PG failed to promote such production in almost all of the cultures. Although whale-PG is an attractive molecule for the ex vivo expansion of human megakaryocytopoiesis, its action may depend on the glycosaminoglycans sulfation pattern and the ability of the binding affinity and the kinetics to interact with the cytokines and hematopoietic stem/progenitor cells.

Up-regulation of hyaluronan synthase genes in cultured human epidermal keratinocytes by UVB irradiation.

Hyaluronan controls keratinocyte proliferation and regeneration. We examined effect of UV on the expression of hyaluronan synthases (HASs) and hyaluronidases in cultured normal human newborn foreskin epidermal keratinocytes, NHEK(F). HAS3 mRNA was expressed predominantly and HAS2 mRNA expressed in lesser amounts and both were up-regulated after a single irradiation with moderate UVB but hyaluronidases was unchanged. Increased accumulation of hyaluronan in the culture medium mirrored the UVB-induced increase in the mRNA levels of HAS3 and HAS2. Unexpectedly, hyaluronan derived from UVB-irradiated and non-irradiated cells had identical size distribution. Increased expression of KGF and IL-1beta was detected just prior to the increase of HAS3 and HAS2 mRNAs after UVB irradiation. Antibody-neutralization study revealed that KGF and/or IL-1beta were at least involved in the up-regulation of HAS3 and HAS2 expressions. UVB-irradiated cells may enhance hyaluronan production to maintain homeostasis through up-regulation of HAS3 and HAS2 genes via cytokine response mechanism.

4-Methylumbelliferone inhibits hyaluronate synthesis in human uterine cervical fibroblasts.

AIM: Hyaluronate plays an important role in the regulation of cervical function during parturition. In our previous study we showed that 4-methylumbelliferone (MU) suppresses hyaluronate synthesis by cultured human skin fibroblasts. The present study investigated the effects of MU on fibroblasts obtained from the human uterine cervix and assessed the possibility of controlling cervical ripening with MU. METHODS: Human uterine cervical fibroblasts were collected from uterine cervices obtained from the uteri of three patients who had a total hysterectomy for uterine myoma at Hirosaki University Hospital. The fibroblasts were cultured in Dulbecco's modified Eagle's medium until confluence. They were then cultured in medium containing [3H]glucosamine (0.074 MBq/mL) with various MU doses. Hyaluronate synthesis was evaluated by assessing the incorporation of [3H]glucosamine into the soluble fraction of hyaluronate. Three independent studies were carried out on each specimen to clarify whether MU causes compositional changes or promotes hyaluronate degradation, whether the inhibitory effects of MU on hyaluronate synthesis are dose-dependent, and whether the effects of MU are reversible. RESULTS: MU added to the medium of the cultured cells reduced the synthesis of hyaluronate in a dose-dependent manner. After MU was removed from the medium, hyaluronate synthesis recommenced, and the amount of [3H]hyaluronate synthesized was similar to the control level. CONCLUSIONS: MU inhibits the synthesis of hyaluronate in human uterine cervical fibroblasts.

[A case of liver metastasis of gastric cancer undergoing non-curative operation responding completely to combination therapy of S-1 and CDDP].

We experienced a case with liver metastasis of gastric cancer that disappeared by S-1 administration following non-curative operation. A distal gastrectomy for advanced gastric cancer with liver metastasis was performed on a 71-year-old male. S-1 was administered at 100 mg/body/day for 4 weeks followed by withdrawal for 2 weeks, and CDDP was prescribed at 5 mg/body/day div, for 2 days per a week as 1 course. After one course of treatment, the liver metastatic lesion decreased in size (reduction ratio was 87.4%). For side effect, S-1 100 mg alone was administered beginning with the second course. This lesion became CR after four courses. The adverse events of grade 3 observed during S-1 administration were neutropenia and diarrhea. We changed S-1 to UFT after nine courses, and the patient has now survived 1 year without recurrence after the disappearance of liver metastasis.

Prognostic significance of lymphovascular invasion diagnosed by lymphatic endothelium immunostaining in breast cancer patients.

Prognostic factors for breast cancer include axillary lymph node status, tumor size, histology, nuclear grade, presence of estrogen and progesterone receptors, HER2/neu status, and mean microvessel density (MVD). In this study, we evaluated the usefulness of a new marker, D2-40, by investigating lymph vascular invasion of the tumor immunohistochemically in 132 patients with breast cancer and compared it with those of well-known prognostic indicators. Positive immunostaining of lymphatic endothelium with D2-40 outlining tumor emboli in the lumen of lymphatics was defined as D2-LVI, and lymphatic invasion following conventional hematoxylin and eosin staining was defined as HE-LVI. Significant correlation was observed between HE-LVI and D2-LVI (p<0.001), between lymph node status and HE-LVI (p=0.005), and between recurrent status and D2-LVI (p=0.008) by univariate analysis. Based on multivariate analysis, lymph node status (p<0.001, OR=6.993), tumor size (p=0.005, OR=5.504), D2-LVI (p=0.006, OR=4.740), and MVD (p=0.002, OR=4.484) were independent prognostic factors of disease recurrence. A significant difference in disease-free survival was also found between patients with and without D2-LVI (p=0.0067), but not with or without HE-LVI. Even in node-positive cases, D2-LVI had prognostic meaning. D2-LVI may play a crucial role for predicting recurrence of breast cancers much more than expected. Our data identifying D2-LVI expression in tumors of patients with a poor disease-free survival prognosis provides an easier and more accurate prognostic method than identifying HE-LVI.

Application of proteoglycan extracted from the nasal cartilage of salmon heads for ex vivo expansion of hematopoietic progenitor cells derived from human umbilical cord blood.

Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads was applied to the ex vivo expansion of hematopoietic progenitor cells prepared from human umbilical cord blood in serum-free cultures supplemented with the combination of early-acting cytokines, thrombopoietin (TPO), interleukin-3 (IL-3) and stem cell factor (SCF). PG showed no promoting effects on the cell proliferation rate; however, they promoted the generation of progenitor cells for granulocyte-macrophages, erythrocytes and/or megakaryocytes in culture with TPO alone or SCF plus TPO. However, no promoting effect was observed in a combination of IL-3 plus SCF, which showed the highest cell proliferation rate. PG failed to promote the generation of mixed colony-forming units (i.e. the relatively immature cells in hematopoiesis). These results suggest that PG acts on the relatively mature stem/progenitor cells, and may function as a regulatory factor in the differentiation pathway of hematopoiesis.

Diversity in the degree of sulfation and chain length of the glycosaminoglycan moiety of urinary trypsin inhibitor isomers.

Five isomers with different electric charge were fractionated from human urinary trypsin inhibitor (UTI) by anion exchange HPLC. Intact low-sulfated chondroitin 4-sulfate chains from the isomers were analyzed by HPLC and mass spectrometry. Unsaturated disaccharide composition analysis of the chondroitin sulfate chain revealed that the five isomers differ in the numbers of 4-sulfated disaccharide units. Intriguingly, we detected the presence of multiple novel isomers with different numbers of non-sulfated disaccharide units even in the same charge isomer fraction. Our results demonstrate that UTI can vary in terms of both the degree of sulfation and the length of the low-sulfated chondroitin 4-sulfate chain.

Salmon cartilage proteoglycan modulates cytokine responses to Escherichia coli in mouse macrophages.

Proteoglycans (PGs) are complex glycohydrates, which are composed of core proteins and glycosaminoglycans and widely distributed in connective tissues and on the cell surface of mammalian tissues. We investigated the effect of PG extracted from salmon cartilage on cytokine responses to stimulation with heat-killed Escherichia coli (HKEC) in a mouse macrophage cell line, RAW264.7. PG exhibited the suppression of tumor necrosis factor-alpha production compared with chondroitin 4 sulfate (C4S) and chondroitin 6 sulfate (C6S). PG also revealed the up-regulation of interleukin-10 production. HKEC-induced Toll-like receptor 4 (TLR4) and inducible nitric oxide synthase expression was dose-dependently suppressed by treatment with PG, C4S or C6S, and the PG showed the strongest suppressive effect among 3 compounds. Only PG dramatically up-regulated the expression of signal transducer and activator of transcription 3 (STAT3), and the phosphorylation of STAT3 in mouse macrophages. Our results suggested that the novel interaction might exist between the extracellular matrix and immune system.

Study of hyaluronan synthase inhibitor, 4-methylumbelliferone derivatives on human pancreatic cancer cell (KP1-NL).

The structure of 4-methylumbelliferone (MU) consists of coumarin with 4-methyl group and 7-hydroxy group. MU inhibits HA synthesis and pericellular HA matrix formation. In this study, we used 10 MU derivatives which have hydroxy groups and methyl groups at various positions of coumarin to investigate a more effective HA inhibitor than MU. First, human pancreatic cancer cell (KP1-NL) growth assay was analyzed by Alamar Blue to determine the non-toxic concentration of MU derivatives, and the inhibitory effect on HA synthesis in the cell cultures was analyzed by HA measuring kit. Next, cell surfaces of cancer cells were analyzed by particle-exclusion assay. In conclusion, both hydroxy and methyl groups are necessary for HA inhibition by MU, and two hydroxy groups inhibited HA synthesis more strongly than MU.

The effects of glycosaminoglycans on thrombopoietin-induced megakaryocytopoiesis.

BACKGROUND AND OBJECTIVES: The extracellular matrix plays an essential role in normal hematopoiesis. Proteoglycans and glycosaminoglycans (GAG) are major components of the matrix. In this study, the effects of various GAG on the proliferation and differentiation of CD34+ megakaryocytic progenitor cells (CFU-Meg) were evaluated in vitro. DESIGN AND METHODS: CD34+ cells were highly purified from steady-state human peripheral blood. The GAG tested were hyaluronic acid (from humans, pigs and roosters), keratan sulfate, heparan sulfate, chondroitin sulfate (from whale, shark or squid cartilage) and dermatan sulfate (DS). RESULTS: When used alone, none of the GAG supported the clonal growth of CFU-Meg; however, in cultures stimulated by recombinant human thrombopoietin, human hyaluronic acid, whale chondroitin sulfate and DS significantly enhanced such growth. In particular, the addition of DS resulted in increases of about 1.3-fold, 1.6-fold and 2.0-fold in the numbers of total cells, megakaryocytes and CFU-Meg, respectively, compared with the control culture stimulated by thrombopoietin alone after 9-12 days of serum-free liquid culture. Furthermore, DS induced the generation of hyperploid megakaryocytes and promoted pro-platelet formation. Chemical fragmentation and desulfation of DS showed that a chain of at least 12 saccharides is required for colony-promoting activity and that the sulfate groups play an essential role. INTERPRETATION AND CONCLUSIONS: DS acts on an immature population of CD34+ cells, stimulates the proliferation of CFU-Meg, and enhances the terminal maturation of megakaryocytes and thrombopoiesis. These results suggest that DS has a wide spectrum of action in promoting megakaryocytopoiesis and thrombopoiesis.

4-methylumbelliferone, a hyaluronan synthase suppressor, enhances the anticancer activity of gemcitabine in human pancreatic cancer cells.

Hyaluronan (HA) is a ubiquitous, major component of the pericellular matrix and is necessary for various physiological processes. It plays a very important role in biological barriers. We previously reported that 4-methylumbelliferone (MU) inhibits HA synthesis and pericellular HA matrix formation in cultured human skin fibroblasts, Streptococcus equi FM100, and B16F10 melanoma cells. We hypothesized that MU-mediated inhibition of HA synthesis and pericellular HA matrix formation would increase the efficacy of anticancer drugs. We have already demonstrated in vitro, using a sandwich binding protein assay and a particle exclusion assay, that MU inhibits HA synthesis and formation of the pericellular HA matrix, respectively, in human KP1-NL pancreatic cancer cells. AlamarBlue assay revealed that the anticancer effect of gemcitabine in KP1-NL cells was increased by pretreatment with MU. In vivo simultaneous administration of MU and gemcitabine to tumor-bearing mice with severe combined immunodeficiency disease (SCID) decreased the size of the primary and metastatic tumors more than did gemcitabine alone. These data strongly suggest that a combination of MU and gemcitabine is effective against human pancreatic cancer cells. MU may have potential as a chemosensitizer and may provide us with a new anticancer strategy.