Sulfated Glycopeptide Research Articles

Differential effects of Gekko sulfated glycopeptide on breast and liver cancer cell lines

Differential effects of Gekko sulfated glycopeptide on breast and liver cancer cell lines

EXTH-18. PEPTIDE NANO-STRUCTURES ENHANCE PEDIATRIC BRAIN TUMOR CHEMOTHERAPEUTIC EFFICACY

Abstract Pediatric gliomas, particularly high-grade gliomas, which include diffuse intrinsic pontine gliomas (DIPGs), are among the most formidable and devastating cancers in children. These tumors remain incurable, despite many treatment approaches. We recently identified a small population of glioma cells with stem-like features in pediatric gliomas (glioma stem cells: GSCs), that may be responsible, for therapeutic resistance. Bone morphogenetic protein 4 (BMP4), essential for CNS development, increases GSC therapeutic sensitivity and is a promising adjuvant for glioma treatment. Mechanisms through which BMP4 increases therapeutic sensitivity need to be elucidated, as this can lead to identification of additional treatment targets and delivery systems for BMP4 administration in a clinical setting. Additionally, extension of BMP4 short half-life would enhance its’ clinical application. Here we show that BMP4 increases chemosensitivity by decreasing H3K4me3 at the promoter of multidrug resistant gene 1 (MDR1), resulting in decreased MDR1 expression. BMP4 appears to bring about this effect by decreasing hSETD1A, an H3K4me3 methyltransferase. Our work also demonstrates the first use of a novel sulfated glycopeptide (glyco-PA) nanostructure as a vector for BMP4 delivery. Glyco-PA markedly extended and enhanced BMP4 function, and increased chemotherapeutic anti-tumor activity against pediatric malignant glioma cells in culture. Overall, this work illuminates BMP4 effects on pediatric glioma therapeutic sensitivity through epigenetic mechanisms, and demonstrates the potential of bioactive glyco-PA nanostructures as a delivery mechanism for treating pediatric malignant gliomas and other tumors.

The effects of Gekko sulfated glycopeptide and basic fibroblast growth factor on human fibrosarcoma HT1080 cells

The effects of Gekko sulfated glycopeptide and basic fibroblast growth factor on human fibrosarcoma HT1080 cells

Abstract 3175: Sulfated glycopeptide nanostructures activate and extend bone morphogenetic protein 4 ability to increase pediatric malignant glioma chemotherapeutic efficacy

Abstract Pediatric gliomas, particularly high-grade gliomas, are among the most formidable and devastating cancers in children. These tumors have remained incurable, regardless of the many treatment approaches attempted. We recently identified a small population of glioma cells with stem-like features in pediatric gliomas (glioma stem cells: GSCs), that may be responsible, for therapeutic resistance in pediatric gliomas. Signaling by bone morphogenetic protein 4 (BMP4), an essential molecule for central nervous system (CNS) development, increases GSC therapeutic sensitivity, and whose activation is a promising adjuvant for glioma treatment. Mechanisms through which BMP4 increases therapeutic sensitivity need to be elucidated as this can lead to the identification of additional targets for treating malignant gliomas in children. Delivery systems for administering BMP4 in a clinical setting also need to be developed. Here, we show that BMP4 increases chemosensitivity by decreasing H3 lysine 4 trimethylation (H3K4me3) at the promoter of multidrug resistant gene 1 (MDR1) that, in turn, results in increased BMP4 expression. This appears to be the result of BMP4 decreasing levels of hSETD1A, a critical methyltransferase for H3K4me3. Our work also demonstrates the first use of a novel sulfated glycopeptide (glycol-PA) nanostructure as a vector for delivery of BMP4. Glycol-PA markedly extended and enhanced BMP4 function, and increased chemotherapeutic anti-tumor activity against pediatric malignant glioma cells in culture. Overall, our results expand understanding of how BMP4 brings about glioma therapeutic sensitization through epigenetic mechanisms, and show that highly bioactive glyco-PA nanostructures have potential as a novel delivery mechanism for treating pediatric malignant glioma, as well as other tumors. Citation Format: Guifa Xi, Benjamin Best, Sonali Nayak, Mark McClendon, Barbara Mania-Farnell, John Kessler, Charles David James, Samuel Stupp, Tadanori Tomita. Sulfated glycopeptide nanostructures activate and extend bone morphogenetic protein 4 ability to increase pediatric malignant glioma chemotherapeutic efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3175.

Optimized extraction and purification technology of Gekko sulfated glycopeptide based on its anticancer activities

Optimized extraction and purification technology of Gekko sulfated glycopeptide based on its anticancer activities

Sulfated glycopeptide nanostructures for multipotent protein activation.

Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. We report here on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with very different polysaccharide binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signaling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than expected. These super-bioactive nanostructures may enable many therapies in the horizon involving proteins.

Optimized extraction and purification technology of Gekko sulfated glycopeptide based on its anticancer activities

Optimized extraction and purification technology of Gekko sulfated glycopeptide based on its anticancer activities

The Antitumor Effect of Gekko Sulfated Glycopeptide by Inhibiting bFGF-Induced Lymphangiogenesis.

Objective. To study the antilymphangiogenesis effect of Gekko Sulfated Glycopeptide (GSPP) on human lymphatic endothelial cells (hLECs). Methods. MTS was conducted to confirm the antiproliferation effect of GSPP on hLECs; flow cytometry was employed to detect hLECs cycle distribution; the antimigration effect of GSPP on hLECs was investigated by wound healing experiment and transwell experiment; tube formation assay was used to examine its inhibitory effect on the lymphangiogenesis; western blotting was conducted to detect the expression of extracellular signal-regulated kinase1/2 (Erk1/2) and p-Erk1/2 after GSPP and basic fibroblast growth factor (bFGF) treatment. Nude mice models were established to investigate the antitumor effect of GSPP in vivo. Decreased lymphangiogenesis caused by GSPP in vivo was verified by immunohistochemical staining. Results. In vitro, GSPP (10 μg/mL, 100 μg/mL) significantly inhibited bFGF-induced hLECs proliferation, migration, and tube-like structure formation (P < 0.05) and antagonized the phosphorylation activation of Erk1/2 induced by bFGF. In vivo, GSPP treatment (200 mg/kg/d) not only inhibited the growth of colon carcinoma, but also inhibited the tumor lymphangiogenesis. Conclusion. GSPP possesses the antitumor ability by inhibiting bFGF-inducing lymphangiogenesis in vitro and in vivo, which may further inhibit tumor lymphatic metastasis.

Interplay between Tumor Microenvironment and Cancer Cells.

Cancer cell interaction with microenvironment plays a very important role in cancer cell survival, proliferation, and/or metastasis. The cancer cell-microenvironment interaction is a highly complex process and has been one of the leading areas of research in cancer biology. Over the last decade, several components in tumor microenvironment have been identified to have a role in cancer cell survival, proliferation, or metastasis. The underlying mechanisms of tumor cell-microenvironment interactions have been investigated and exploited for cancer therapy. The present issue covers several aspects of cancer cell and tumor microenvironment. This issue contains interesting articles, which were accepted after a rigorous peer review process. The monocarboxylate transporters (MCTs) are proton-linked plasma membrane transporters that mediate transport of monocarboxylates such as pyruvate and lactate across cellular membranes. Tumor cells depend heavily on anaerobic glycolysis, which generates enormous amount of lactic acid as a byproduct. Lactic acid is toxic to cells and has to be excreted out of the cells through MCTs. An interesting meta-analysis paper by C. D. Bovenzi et al. showed the high expression of MCT4 and CD147 in multiple cancer cells. The elevated MCT4 and CD147 levels were strongly associated with poor overall survival of cancer patients. This work highlighted the importance of MCT4 and CD147 expression in several different cancers and establishes rationale for targeting these molecules for cancer therapy. As discussed above, as well as by several other reports, mostly the overabundance of ion channels in biological membranes has been implicated in pathogenesis of cancers. Only recently did few studies identify antitumor function of ion channels in some type of cancers. Interestingly, the paper by Z. Xia et al. showed the overexpression of potassium ions in liver cancer cells by the treatment with potassium ions that trigger cancer cell apoptosis, providing convincing evidence in favor of tumor suppression function of potassium ion channels in liver cancer. Given this, the levels of potassium ions in tumor microenvironment can influence the survival of liver cancer cells. Further investigation in this regard is warranted. Lymphatic vessels are integral part of tumor microenvironment. Although lymphatic vessels were mostly viewed as passive transporters of cancer cells, recent studies demonstrated the active role of these vessels in cancer metastasis. Indeed, these studies collectively established the rationale for targeting lymphangiogenesis for cancer treatment. The paper by X.-L. Ding et al. describes the antilymphangiogenesis effects of Gekko Sulfated Glycopeptide (GSPP) and discusses the potential therapeutic benefits of GSPP in colon carcinoma. The future studies could explore the translational potential of GSPP or related molecules in colon carcinoma or related cancers. The hormones play a key role in several types of cancers. Gender differences in hormone can influence several aspects of cancer. The paper by S. Caceres et al. developed a male mice xenograft model for mammary tumor and studied the impact of gender differences in hormones on mammary tumor cell proliferation and metastasis. Their preliminary analysis showed the less aggressive nature of mammary tumor cells in male mice compared to female. This work elaborates on the vast literature that describes the importance of extracellular hormone in tumor cell behavior. The traditional Chinese medicines are being used for cancer treatment for centuries. The review article by J. Xu et al. provides a very nice overview of the selected traditional Chinese medicines that suppress cancer cells by interfering with tumor microenvironment. This article also discusses the synergistic benefits of combining few selected traditional Chinese medicines. Collectively, this issue will advance our researchers and readers knowledge on various aspects of tumor cell-microenvironment interactions and will provide insight into new research focused in this area and potential therapeutic interventions. Kallesh D. Jayappa Ramesh C. Kovi Sumanta Chatterjee

Harnessing the synthetic capabilities of glycopeptide antibiotic tailoring enzymes: characterization of the UK-68,597 biosynthetic cluster.

In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81-kb biosynthetic cluster for the unusual sulfated glycopeptide UK-68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram-positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK-68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes.

Gekko-sulfated Glycopeptide Inhibits Tumor Angiogenesis by Targeting Basic Fibroblast Growth Factor

Basic fibroblast growth factor (bFGF) is a therapeutic target of anti-angiogenesis. Here, we report that a novel sulfated glycopeptide derived from Gekko swinhonis Guenther (GSPP), an anticancer drug in traditional Chinese medicine, inhibits tumor angiogenesis by targeting bFGF. GSPP significantly decreased the production of bFGF in hepatoma cells by suppressing early growth response-1. GSPP inhibited the release of bFGF from extracellular matrix by blocking heparanase enzymatic activity. Moreover, GSPP competitively inhibited bFGF binding to heparin/heparan sulfate via direct binding to bFGF. Importantly, GSPP abrogated the bFGF-stimulated proliferation and migration of endothelial cells, whereas it had no inhibitory effect on endothelial cells in the absence of bFGF. Further study revealed that GSPP prevented bFGF-induced neovascularization and inhibited tumor angiogenesis and tumor growth in a xenograft mouse model. These results demonstrate that GSPP inhibits tumor angiogenesis by blocking bFGF production, release from the extracellular matrix, and binding to its low affinity receptor, heparin/heparan sulfate.

Anti-Migration Effects of Gekko Sulfated Glycopeptide on Human Hepatoma SMMC-7721 Cells

Gekko swinhonis Guenther has been used as an anti-cancer drug in traditional Chinese medicine for hundreds of years. Previous studies showed that the Gekko sulfated polysaccharide-protein complex suppressed the proliferation and migration of hepatoma cells. Gekko sulfated glycopeptide α was obtained from Gekko sulfated polysaccharide-protein complex using papain hydrolysis. Gekko sulfated glycopeptide α inhibited the proliferation and migration of SMMC-7721 cells. The secretion of IL-8 and the concentration of intracellular calcium were decreased after Gekko sulfated glycopeptide α exposure. SMMC-7721 cells in the control group showed abnormal features, with a polygonal shape, whereas this changed to a spindle shape after the treatment with Gekko sulfated glycopeptide α. Actin filaments were distributed diffusely along the cell membrane in control cells, whereas those were polymerized and preferentially accumulated in the cytoplasm of treated cells. Microtubules distributed in the cytoplasm of untreated cells were located diffusely whereas those in treated cells were polymerized. Therefore, Gekko sulfated glycopeptide α inhibit the migration of hepatoma cells via reducing the secretion of IL-8 and the concentration of intracellular calcium, as well as regulating the reorganization of cytoskeleton.

Tailoring Enzyme-Rich Environmental DNA Clones: A Source of Enzymes for Generating Libraries of Unnatural Natural Products

A detailed bioinformatics analysis of six glycopeptide biosynthetic gene clusters isolated from soil environmental DNA (eDNA) megalibraries indicates that a subset of these gene clusters contains collections of tailoring enzymes that are predicted to result in the production of new glycopeptide congeners. In particular, sulfotransferases appear in eDNA-derived gene clusters at a much higher frequency than would be predicted from the characterization of glycopeptides from cultured Actinomycetes . Enzymes found on tailoring-enzyme-rich eDNA clones associated with these six gene clusters were used to produce a series of new sulfated glycopeptide derivatives in both in vitro and in vivo derivatization studies. The derivatization of known natural products with eDNA-derived tailoring enzymes is likely to be a broadly applicable strategy for generating libraries of new natural product variants.

Preparation of a novel sulfated glycopeptide complex and inhibiting L1210 cell lines property in vitro

A native glycopeptide, LZ-D-4, was purified from the fruiting bodies of Ganoderma lucidum, and its derivative, LZ-D-9, was prepared using sulfated agent (CSA: Pyr = 1:6). HPLC experiment showed that both of them were homogeneity and had molecular weights of 1.56 × 10 4 and 1.30 × 10 4 Da, respectively. Some properties including amino acid and monosaccharide compositions were investigated for the two compounds, along with anti-tumour bioactivity in vitro. The results indicated that two monosaccharides (Xyl and Ara) were completely destroyed and most of amino acids in LZ-D-4 were broken during sulfated process. Anti-tumour test in vitro showed that both complexes have properties to inhibit growth of L1210 cell lines and have similar bioactivities, but might have different mechanism.

Biosynthesis of Sulfated Glycopeptide Antibiotics by Using the Sulfotransferase StaL

The unique glycopeptide antibiotic A47934, produced by Streptomyces toyocaensis, possesses a nonglycosylated heptapeptide core that is sulfated on the phenolic hydroxyl of the N-terminal 4-hydroxy-L-phenylglycine residue. Genetic and biochemical experiments confirmed that StaL is a sulfotransferase capable of sulfating the predicted crosslinked heptapeptide substrate to produce A47934 both in vivo and in vitro. Incubation of purified His(6)-StaL with various substrates in vitro revealed substrate specificity and yielded two sulfo-glycopeptide antibiotics: sulfo-teicoplanin aglycone and sulfo-teicoplanin. Quantification of the antibacterial activity of desulfo-A47934, A47934, teicoplanin, and sulfo-teicoplanin demonstrated that sulfation slightly increased the minimum inhibitory concentration. This unique modification by sulfation expands glycopeptide diversity with potential application for the development of new antibiotics.

Activation of macrophages by sulfated glycopeptides in ovomucin, yolk membrane, and chalazae in chicken eggs.

Sulfated glycopeptides in ovomucin, chalazae and yolk membrane were found to activate cultured macrophage-like cells, J774.1, and TGC-induced macrophages from the peritoneal cavity of male mice. The macrophage-stimulating activity was estimated by the growth and morphology of the cells, H2O2 generation, and interleukin-1 (IL-1) production from the cells. The in vitro culture assay with macrophages showed that the protease digests of ovomucin, yolk membrane, and chalazae induced morphologic alteration and increased H2O2 generation and IL-1 production in lower concentration (100 micrograms/ml). The isolation of the components having macrophage-stimulating activity was attempted to elucidate the molecular mechanism. The O-linked carbohydrate chains, consisting of N-acetylgalactosamine, galactose, N-acetylneuraminic acid and sulfate, in the sulfated glycopeptide were identified as a component having macrophage-stimulating activity.

Enzymic determination of acidic glycoconjugates in human pancreatic juice.

Acidic glycoconjugates (glycosaminoglycans, sulfated glycopeptide, and sialoglycopeptide) were isolated by precipitation with cetylpyridinium chloride from human pancreatic juice after digestion with pronase. The acidic glycoconjugates were found exclusively in the proteinaceous precipitate that occurred during dialysis against a buffer of low ionic strength. The concentration of the acidic glycoconjugates in normal pancreatic juice was about 2.4 mg/L. The acidic glycoconjugates were characterized by electrophoresis on cellulose acetate membrane and chemical analysis before and after digestion with Streptomyces hyaluronidase, chondroitinase AC, chondroitinase ABC, and heparitinase. It was found that the major acidic glycoconjugates were heparan sulfate (39.3%), sulfated glycopeptide (34.4%), chondroitin sulfate (14.2%), and the minor ones hyaluronic acid (6.4%) and sialoglycopeptide (5.7%).

Effect of sulglycotide treatment on cell kinetics alterations induced by aspirin in humans

The effect on gastric epithelial cell proliferation of small doses of aspirin was evaluated in 9 healthy volunteers, with or without administration of sulglycotide, a sulfated glycopeptide with cytoprotective properties. Cell kinetics study was performed by incubation of gastric biopsies with bromodeoxyuridine (BrdU) and immunohistochemistry. A decrease of BrdU-labeling index and a shortening of the height of gastric columns were observed after treatment with aspirin and placebo. No variations were observed after treatment with aspirin and sulglycotide. A decrease of the epithelial cell renewal could be one of the damaging effects of aspirin on the gastric mucosa. The treatment with sulglycotide seems to be effective to prevent this alteration.

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in the secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, sialogylcopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239–246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridium chloride solution at 100% for 15 min. The extract was then measured by absorbance at 615 nm an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0–25 μg.

Characterization of sulfated forms of the pro-opiomelanocortin amino-terminal glycopeptide in rat intermediate lobe cells

Explants of rat neurointermediate lobes were incubated in the presence of radioactive amino acids, sugars or sulfate and the labeled proteins were separated by two-dimensional gel electrophoresis. A double series of acidic peptides (Mr=16,000–21,500) were identified as variant forms of the amino-terminal glycopeptide of pro-opiomelanocortin (N-POMC). The series of peptides with the higher molecular weights (Mr=18,000–21,500) contain a tryptic fragment (tentatively identified as the tryptic peptide of the “joining peptide”: sequence 77 to 93 of rat POMC) which is absent from the forms of the lower molecular weight series (Mr=16,000 to 18,000). Pulse-chase studies further showed that the high molecular weight forms of N-POMC could be post-translationally cleaved albeit slowly into the species of Mr=16,000–18,000 which constitute, at least in part, the final maturation products of the N-terminal region of the precursor molecule. All the variant forms of the N-POMC glycopeptide could be labeled with [ 35S]sulfate. Our results strongly suggest that most of the sulfate groups are attached to N-linked oligosaccharide side chains of N-POMC. We therefore propose that one of the final maturation products of the N-terminal portion of POMC in rat intermediate lobes is a sulfated glycopeptide (Mr=16,000–18,000) composed of the 1–74 sequence of rat POMC.