Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium (II)

Rozenberg, Gabriela I.; Espada, Jesús; Cidre, Lilia Lauria de; Eiján, Ana María; Calvo, Juan; Bertolesi, Gabriel E.

doi:10.1002/1522-2683(200101)22:1<3::aid-elps3>3.0.co;2-g

Cited by 18 publications

(10 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Heparinase I recognizes highly sulfated regions and is specific for heparin. Heparinase II digests both heparin and HS, while heparinase III degrades less‐sulfated regions and is more active on HS . We found that HS20 cell binding decreased 80% following heparinase I treatment, and both heparinase II and heparinase III treatments led to a 96% decrease in HS20 binding.…”

Section: Resultsmentioning

confidence: 73%

Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy

et al. 2014

View full text Add to dashboard Cite

Wnt signaling is important for cancer pathogenesis and is often upregulated in hepatocellular carcinoma (HCC). Heparan sulfate proteoglycans (HSPGs) function as co-receptors or modulators of Wnt activation. Glypican-3 (GPC3) is a HSPG that is highly expressed in HCC, where it can attract Wnt proteins to the cell surface and promote cell proliferation. Thus, GPC3 has emerged as a candidate therapeutic target in liver cancer. While monoclonal antibodies to GPC3 are currently being evaluated in preclinical and clinical studies, none have shown an effect on Wnt signaling. Here, we first document the expression of Wnt3a, multiple Wnt receptors and GPC3 in several HCC cell lines, and demonstrate that GPC3 enhanced the activity of Wnt3a/β-catenin signaling in these cells. Then, we report the identification of HS20, a human monoclonal antibody against GPC3, which preferentially recognized the heparan sulfate chains of GPC3, both the sulfated and non-sulfated portions. HS20 disrupted the interaction of Wnt3a and GPC3 and blocked Wnt3a/β-catenin signaling. Moreover, HS20 inhibited Wnt3a-dependent cell proliferation in vitro and HCC xenograft growth in nude mice. In addition, HS20 had no detectable undesired toxicity in mice. Taken together, our results show that a monoclonal antibody primarily targeting the heparin sulfate chains of GPC3 inhibited Wnt/β-catenin signaling in HCC cells and had potent anti-tumor activity in vivo. Conclusion Here, we provide one of the first examples of an antibody directed against the heparan sulfate of a proteoglycan that showed efficacy in blocking Wnt signaling and HCC growth, suggesting a novel strategy for liver cancer therapy.

show abstract

Section: Resultsmentioning

confidence: 73%

Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Thus, we examined whether Pep-1 might also recognize heparan sulfates through the above HA-like epitope. Pretreatment of XS106 DC with heparinase III, which is known to selectively degrade heparan sulfates (38), reduced the extent of Pep-1 binding, albeit modestly even at the highest concentration (330 U/ml; Fig. 3A).…”

Section: Molecular Targets Of Pep-1 On DCmentioning

confidence: 99%

Synthesis and Surface Expression of Hyaluronan by Dendritic Cells and Its Potential Role in Antigen Presentation

Mummert

Edelbaum

et al. 2002

The Journal of Immunology

View full text Add to dashboard Cite

Hyaluronan (HA) is a large glycosaminoglycan consisting of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is known to act as a filling material of extracellular matrices and as an adhesive substrate for cellular migration. Here we report that dendritic cells (DC) express mRNAs for HA synthases and hyaluronidases, actively synthesize HA, and display HA on their surfaces. Interestingly, HA expression levels on DC were not significantly altered by their maturation states. With respect to physiological function, three specific HA inhibitors, i.e., bovine proteoglycan, a 12-mer HA-binding peptide (GAHWQFNALTVR) termed Pep-1, and an oligomeric Pep-1 formulation, all interfered with DC-induced activation of CD4+ T cells isolated from DO11.10 TCR transgenic mice. For example, Pep-1 oligomer efficiently inhibited DC-dependent cluster formation, IL-2 and IFN-γ production, and proliferation by DO11.10 T cells in vitro without affecting the viabilities of DC or T cells, DC function to uptake exogenous proteins, or DC-T cell conjugate formation at earlier time points. These observations suggest a paracrine mechanism by which DC-associated HA facilitates some of the late changes in T cell activation. Although T cells constitutively expressed mRNAs for HA synthases and hyaluronidases, their surface HA expression became detectable only after activation. Oligomeric Pep-1 and bovine proteoglycan both inhibited mitogen-triggered T cell activation in the absence of DC, suggesting an autocrine mechanism by which HA expressed by T cells assists their own activation processes. Finally, adoptively transferred DO11.10 T cells showed progressive mitosis when stimulated with Ag-pulsed DC in living animals, and this clonal expansion was inhibited significantly by administration of Pep-1 oligomer. Our findings may introduce a new concept that relatively simple carbohydrate moieties expressed on DC and perhaps T cells play an important immunomodulatory role during Ag presentation.

show abstract

“…in order to evaluate the enzymatic activity of heparanase, heparin degradation was quantified in the nMuMG and lM3 cells incubated with cM from the preA, pDA and MA 3T3-L1 cells (21). Briefly, 15 µl each of the nMuMG or lM3 cells (5x10 6 cells/ml) were incubated with commercial heparin (2 µg/5 µl) at 37˚C for 48 h. After incubation, 6 µl of 4X sample buffer was added, and the samples were loaded on a non-denaturing 20% PaGe gel and stained with rubipy (a cationic dye).…”

Section: Adhesion Of Cells Grown On Stromal Supportmentioning

confidence: 99%

Adipocyte differentiation influences the proliferation and migration of normal and tumoral breast epithelial cells

Creydt

Sacca²,

Tesone³

et al. 2010

Mol Med Rep

View full text Add to dashboard Cite

Abstract. Stromal tissue regulates the development and differentiation of breast epithelial cells, with adipocytes being the main stromal cell type. The aim of the present study was to evaluate the effect of adipocyte differentiation on proliferation and migration, as well as to assess the activity of heparanase and metalloproteinase-9 (MMP-9), in normal (nMuMG) and tumoral (lM3) murine breast epithelial cells. nMuMG and lM3 cells were grown on irradiated 3T3-l1 cells (stromal support, SS) at various degrees of differentiation [preadipocytes (prea), poorly differentiated adipocytes (pda) and mature adipocytes (Ma)] and/or were incubated in the presence of conditioned medium (cM) derived from each of these three types of differentiated cells. cells grown on a plastic support or in fresh medium served as the controls. cell proliferation was measured with a commercial colorimetric kit, and the motility of the epithelial cells was evaluated by means of a wound-healing assay. Heparanase activity was assessed by quantifying heparin degradation, and the expression of MMP-9 was determined using Western blotting. The results indicate that cell proliferation was increased after 24 and 48 h in the nMuMG and lM3 cells grown on prea, pda and Ma SS. in the nMuMG cells cultured on SS in the presence of all three types of cM, proliferation was enhanced. lM3 cell migration was increased in the presence of all three types of cM and in cells grown on prea SS. Heparanase activity was increased in the nMuMG cells incubated with all three types of cM, and in the lM3 cells incubated with the cM from pda and Ma. Both the nMuMG and lM3 cell lines presented basal expression of MMP-9; however, a significant increase in MMP-9 expression was observed in the lM3 cells incubated with each of the three types of cM. in conclusion, adipocyte differentiation influences normal and tumoral breast epithelial cell proliferation and migration. Heparanase and MMP-9 appear to be involved in this regulation. The experimental model presented in this study is in keeping with the characteristics of the physiological environment of breast epithelial cells, in terms of both the soluble and insoluble factors present and the stromal structure per se.

show abstract

Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium (II)

Cited by 18 publications

References 29 publications

Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy

Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy

Synthesis and Surface Expression of Hyaluronan by Dendritic Cells and Its Potential Role in Antigen Presentation

Adipocyte differentiation influences the proliferation and migration of normal and tumoral breast epithelial cells

Contact Info

Product

Resources

About