Analysis of the neutral polysaccharide fraction of MCP and its inhibitory activity on galectin-3

The pH-modified citrus pectin (MCP) has been demonstrated to inhibit galectin-3 in cancer progression. The components and structures of MCP related to this inhibition remained unknown. In this paper, we fractionated MCP on DEAE-cellulose column into a homogenous neutral fraction MCP-N (about 20?kDa) and a pectin mixture fraction MCP-A (wide molecular distribution on Sepharose CL-6B chromatography). Both MCP-N and MCP-A inhibited hemagglutination mediated by galectin-3 with minimum inhibition concentration (MIC) 625 and 0.5?μg/ml, respectively. MCP-N was identified to be a type I arabinogalactan (AG-I) with a main chain of β-1→4-galactan. MCP-N was digested by α-L-arabinofuranosidase to give its main chain structure fraction (M-galactan, around 18?kDa), which was more active than the original molecule, MIC 50?μg/ml. The acidic degradation of M-galactan increased the inhibitory activity, MIC about 5 times lower than M-galactan. These results above showed that the functional motif of the β-1→4-galactan fragment might lie in the terminal residues rather than in the internal region of the chain. Therefore, MCP-N and its degraded products might be developed to new potential galectin-3 inhibitors. This is the first report concerning the fractionation of MCP and its components on galectin-3 inhibition. The information provided in this paper is valuable for screening more active galectin-3 inhibitors from natural polysaccharides.     

Production of extracellular pectinase enzymes by a mutant (Pol6) of Penicillium occitanis

Summary Penicillium occitanis strain Pol6, a mutant developed for hyperproduction of cellulase and pectinase enzymes was used for the study of extracellular pectinase production when pectins from different sources (apple and citrus) and with varying degree of esterification (DE) were used as inducers. Highly esterified citrus pectins were found to be suitable substrates for polygalacturonase, pectinase and pectin methyl esterase production, while low esterified citrus pectin favoured pectin lyase (PL) production. Apple pectins induced other hydrolytic enzymes (e.g., -1,3-glucanase, -glucosidase, -galactosidase), in addition to pectolytic enzymes. Moreover, the combination of high and low esterified citrus pectins induced the production of a complete pectinase complex. The extent of degradation of the substrate and the affinity for PL decreased with decreasing DE irrespective of the source. There was no evidence of PL activity in this strain. No significant effect of cations (Ca⁺⁺, Mn⁺⁺, Na⁺) on PL activity was observed. However, EDTA (100 mm) inhibited 50% of the activity, when tested on highly esterified (rapid set citrus) pectin. Offprint requests to: S. Jain     

In vivo UVA irradiation of mouse is more efficient in promoting pulmonary melanoma metastasis than in vitro

Background

We have previously shown in vitro that UVA increases the adhesiveness of mouse B16-F1 melanoma cells to endothelium. We have also shown in vivo that UVA exposure of C57BL/6 mice, i.v. injected with B16-F1 cells, increases formation of pulmonary colonies of melanoma. The aim of the present animal study was to confirm the previously observed in vivo UVA effect and to determine whether in vitro UVA-exposure of melanoma cells, prior the i.v. injection, will have an enhancing effect on the pulmonary colonization capacity of melanoma cells. As a second aim, UVA-derived immunosuppression was determined.

Methods

Mice were i.v. injected with B16-F1 cells into the tail vein and then immediately exposed to UVA. Alternatively, to study the effect of UVA-induced adhesiveness on the colonization capacity of B16-F1 melanoma, cells were in vitro exposed prior to i.v. injection. Fourteen days after injection, lungs were collected and the number of pulmonary nodules was determined under dissecting microscope. The UVA-derived immunosuppression was measured by standard contact hypersensitivity assay.

Results and Discussion

Obtained results have confirmed that mice, i.v. injected with B16-F1 cells and thereafter exposed to UVA, developed 4-times more of melanoma colonies in lungs as compared with the UVA non-exposed group (p < 0.01). The in vitro exposure of melanoma cells prior to their injection into mice, led only to induction of 1.5-times more of pulmonary tumor nodules, being however a statistically non-significant change. The obtained results postulate that the UVA-induced changes in the adhesive properties of melanoma cells do not alone account for the 4-fold increase in the pulmonary tumor formation. Instead, it suggests that some systemic effect in a mouse might be responsible for the increased metastasis formation. Indeed, UVA was found to induce moderate systemic immunosuppression, which effect might contribute to the UVA-induced melanoma metastasis in mice lungs.     

Effect of <Emphasis Type="Italic">Cordyceps sinensis</Emphasis> on TIMP-1 secretion from mouse melanoma cell

Cordyceps sinensis is a Chinese medicinal fungus traditionally used in cancer treatments. In a previous study, we investigated the antimetastatic activity of Cordyceps sinensis (WECS) extract using liver metastatic model mice injected with B16-F0 mouse melanoma cells into the spleen. WECS reduced the number of metastatic nodules of B16-F0 cells in the liver of C57BL/6 mice, and significantly prolonged survival of the mice. Furthermore, we examined the effects of WECS on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. WECS was shown to significantly reduce HGF-accelerated B16-F0 cell invasion. In the present study, we investigated the effect of WECS on Tissue Inhibitor of Metalloproteinase (TIMP)-1 secretion from B16-F0 cells in order to identify clues to the mechanism underlying the anti-invasive action of WECS. As a result, WECS significantly increased the secretion of TIMP-1 from B16-F0 cells. Moreover, we investigated the effect of cordycepin (3′-deoxyadenosine), a component of WECS, on TIMP-1 secretion from B16-F0 cells to potentially identify the pharmacologically active ingredient in WECS extract. Cordycepin was shown to significantly accelerate the release of TIMP-1 from cells. These findings suggest that WECS exerts anti-invasive activity, in part by increasing TIMP-1 secretion from melanoma cells, and that cordycepin potentially functions as the effective component.     

Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier

Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, β-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins.Mucosal surfaces comprise more than 400 m² of the total surface area in humans (compared with 1.8 m² for skin) and are, thus, by far the largest area of contact with the environment (1). Epithelial cells in mucosal surfaces are continuously faced with the critical function of forming a protective apical barrier that prevents cellular damage and infection while allowing the exchange of molecules with the extracellular milieu. Loss of barrier function is ascribed to numerous mucosal pathologies, such as dry eye (a disease affecting more than 5 million people in the United States), severe asthma, and inflammatory bowel disease (2–4). Integral to the apical surface of mucosal epithelia are cell surface-associated mucins, a group of high molecular weight glycoproteins defined by the presence of long amino-terminal, extracellular domains containing extensive sites for O-glycan attachment. O-Glycosylation is the most abundant post-translational modification of mucins and constitutes up to 80% of mucin''s mass. It is thought that specific cell surface mucins and their O-glycans provide protection to the mucosal surface (5). Data from knock-out mice deficient in cell surface mucin MUC1 and core 3 β-1,3-N-acetylglucosaminyltransferase, an enzyme involved in the synthesis of mucin-type O-glycans in human colon, indicate the requirement for mucins and their O-glycans in maintaining barrier integrity in the gastrointestinal tract and the eye (6–9). However, the mechanism by which cell surface-associated mucins and their O-glycans contribute to forming the mucosal barrier on the epithelial glycocalyx remains poorly characterized.Galectins are a family of animal β-galactoside-binding lectins, defined by their evolutionarily conserved carbohydrate recognition domain (10, 11). As many as 15 galectins have been identified in mammals, and they are widely distributed among different types of cells and tissues (12). Galectins have been implicated in numerous biological processes, including tumor cell adhesion and progression, immunity, inflammation, wound healing, and development (11, 13, 14). Galectin-3 is a 35-kDa protein originally identified as Mac-2, a cell surface antigen expressed on murine thioglycollate-elicited peritoneal macrophages (15). It is now established that galectin-3, like other galectins, can interact in a multivalent fashion and cross-link glycan ligands on cell surface receptors, such as with epidermal growth factor receptors and α5β1 integrin, to generate molecular lattices (16, 17). In this study we investigate whether galectin-3 participates in mucosal barrier function through its interaction with cell surface-associated mucins. We demonstrate here that two distinct cell surface mucins, MUC1 and MUC16, interact with galectin-3 on the apical surface of epithelial cells and that carbohydrate-mediated mucin-galectin-3 interactions play an important role in maintaining mucosal barrier function.     

Modified citrus pectin anti-metastatic properties: one bullet, multiple targets

In this minireview, we examine the ability of modified citrus pectin (MCP), a complex water soluble indigestible polysaccharide obtained from the peel and pulp of citrus fruits and modified by means of high pH and temperature treatment, to affect numerous rate-limiting steps in cancer metastasis. The anti-adhesive properties of MCP as well as its potential for increasing apoptotic responses of tumor cells to chemotherapy by inhibiting galectin-3 anti-apoptotic function are discussed in the light of a potential use of this carbohydrate-based substance in the treatment of multiple human malignancies.     

Fluorescence probes in metastatic B16 melanoma membranes

Fluorescence probe molecules, trans-parinaric acid and 1,6-diphenylhexatriene, were utilized to characterize the structure of plasma membranes, microsomes and mitochondria from B16 melanoma cells. High metastatic B16-F10 and low metastatic B16-F1 melanoma cell lines had markedly different membrane structures. The fluorescence polarization, fluorescence lifetime and limiting anisotropy of trans-parinaric acid were significantly lower ($\text{P < 0.05}$) in all three membrane fractions of the B16-F1 cell line than in the corresponding membranes of the B16-F10 cell line. These data indicated less restriction to rotational motion in the solid lipid domains of B16-F1 cell membranes preferentially sensed by trans-parinaric acid. The limiting anisotropy of both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene was significantly lower in the outer monolayer than the inner monolayer of the plasma membrane of B16-F1 cells but not in B16-F10 cells. A breakpoint in Arrhenius plots of fluorescence near 30–34°C indicated the presence of a phase separation that was assigned to the inner monolayer of the plasma membrane. However, no differences in this breakpoint temperature were noted between the B16-F1 and B16-F10 melanoma membranes. Thus, more fluid solid membrane domains and a distinct transbilayer fluidity difference were characteristic of plasma membranes from low metastatic B16-F1 melanoma cells in contrast to high metastatic B16-F10 melanoma cells.     

Modified citrus pectin reduces galectin-3 expression and disease severity in experimental acute kidney injury

Galectin-3 is a β-galactoside binding lectin with roles in diverse processes including proliferation, apoptosis, inflammation and fibrosis which are dependent on different domains of the molecule and subcellular distribution. Although galectin-3 is known to be upregulated in acute kidney injury, the relative importance of its different domains and functions are poorly understood in the underlying pathogenesis. Therefore we experimentally modulated galectin-3 in folic acid (FA)-induced acute kidney injury utilising modified citrus pectin (MCP), a derivative of pectin which can bind to the galectin-3 carbohydrate recognition domain thereby predominantly antagonising functions linked to this role. Mice were pre-treated with normal or 1% MCP-supplemented drinking water one week before FA injection. During the initial injury phase, all FA-treated mice lost weight whilst their kidneys enlarged secondary to the renal insult; these gross changes were significantly lessened in the MCP group but this was not associated with significant changes in galectin-3 expression. At a histological level, MCP clearly reduced renal cell proliferation but did not affect apoptosis. Later, during the recovery phase at two weeks, MCP-treated mice demonstrated reduced galectin-3 in association with decreased renal fibrosis, macrophages, pro-inflammatory cytokine expression and apoptosis. Other renal galectins, galectin-1 and -9, were unchanged. Our data indicates that MCP is protective in experimental nephropathy with modulation of early proliferation and later galectin-3 expression, apoptosis and fibrosis. This raises the possibility that MCP may be a novel strategy to reduce renal injury in the long term, perhaps via carbohydrate binding-related functions of galectin-3.     

Phytochemical potential of Daphne gnidium in inhibiting growth of melanoma cells and enhancing melanogenesis of B16-F0 melanoma

In this study, we have investigated inhibitory capacity of ethyl acetate, total oligomer flavonoid (TOF), aqueous extracts and beta amyrin acetate, a triterpene isolated from ethyl acetate extract obtained from leaves of Daphne gnidium, on mouse melanoma (B16-F0 and B16-F10 cells) proliferation. Influence of these products on percentage cell distribution in cycle phases and melanogenesis was also studied. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and flow cytometry was used to analyse effects of tested compounds on progression through the cell cycle. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Ethyl acetate, TOF and aqueous extracts exhibited significant anti-proliferative activity after incubation with the two types of tumour skin cells B16-F0 and B16-F10. Furthermore, cell cycle analysis revealed that cells treated with ethyl acetate and TOF extracts were arrested predominantly in G2-M phase. Ethyl acetate extract has also the ability to enhance melanogenesis and tyrosinase activity of B16-F0 melanoma cells. Copyright © 2012 John Wiley & Sons, Ltd.     

The role of glycoconjugates in metastatic melanoma blood-borne arrest and cell surface properties

The role of glycoconjugates in cell surface and blood-borne implantation properties of murine metastatic melanoma sublines of low (B16-F1) or high (B 16-F10) potential to colonize lungs was investigated by treating melanoma cells with the antibiotic tunicamycin. This drug prevents glycosylation of glycoproteins by inhibiting the formation of lipid-linked oligosaccharide precursors. The degree of tunicamycin-mediated modifications in glycoproteins was assessed by monitoring the decrease in cell surface sialogalactoproteins by binding of ¹²⁵I-labeled Ricinus communis agglutinin I. Scanning electron microscopy of tunicamycin-treated B16-F1 and B16-F10 cells showed morphologic changes such as cell rounding and formation of numerous surface blebs. Tunicamycin-treated B16-F1 and B16-F10 cells lost their lung colonization abilities when injected intravenously into C57BL/6 mice, concomitant with lowered rates of adhesion to endothelial cell monolayers, endothelial extracellular matrix (basal lamina), and polyvinyl-immobilized fibronectin in vitro, suggesting that this drug inhibits experimental metastasis by modifying the surface glycoproteins involved in determining the adhesive properties of malignant cells.     

Analysis of selected blood and immune cell responses to carbohydrate-dependent surface binding of proto- and chimera-type galectins

Cell surface glycans present docking sites to endogenous lectins. With growing insight into the diversity of lectin families it becomes important to answer the question on the activity profiles of individual family members. Focusing on galectins (-galactoside-binding proteins without Ca²⁺-requirement sharing the jelly-roll-like folding pattern), this study was performed to assess the potency of proto-type galectins (galectins-1 and -7 and CG-16) and the chimera-type galectin-3 to elicit selected cell responses by carbohydrate-dependent surface binding and compare the results. The galectins, except for galectin-1, were found to enhance detergent (SDS)-induced hemolysis of human erythrocytes to different degrees. Their ability to confer increased membrane osmofragility thus differs. Aggregation of neutrophils, thymocytes and platelets was induced by the proto-type galectin-1 but not -7, by CG-16 and also galectin-3. Cell-type-specific quantitative differences and the importance of the fine-specificity of the galectin were clearly apparent. In order to detect cellular responses based on galectin binding and bridging of cells the formation of haptenic-sugar-resistant (HSR) intercellular contacts (an indicator of post-binding signaling) was monitored. It was elicited by CG-16 and galectin-1 but not galectin-3, revealing another level at which activities of individual galectins can differ. Acting as potent elicitor of neutrophil aggregation, CG-16-dependent post-binding effects were further analyzed. Carbohydrate-dependent binding to the neutrophils' surface led to a sustained increase of cytoplasmic Ca²⁺ concentration in a dose-dependent manner. The ability of CG-16 to activate H₂O₂ generation by human peripheral blood neutrophils was primed by the Ca²⁺-ionophor ionomycin and by cytochalasin B. In a general context, these results emphasize that – besides plant lectins as laboratory tools – animal lectins can trigger cell reaction cascades, implying potential in vivo relevance for the measured activities. Within the family of galectins, the activity profiles depend on the target cell type and the individual galectin. Notably, proto-type galectins do not necessarily share a uniform capacity as elicitor.     

Boron-bridged RG-II and calcium are required to maintain the pectin network of the <Emphasis Type="Italic">Arabidopsis</Emphasis> seed mucilage ultrastructure

The structure of a pectin network requires both calcium (Ca²⁺) and boron (B). Ca²⁺ is involved in crosslinking pectic polysaccharides and arbitrarily induces the formation of an “egg-box” structure among pectin molecules, while B crosslinks rhamnogalacturonan II (RG-II) side chain A apiosyl residues in primary cell walls to generate a borate-dimeric-rhamnogalacturonan II (dRG-II-B) complex through a boron-bridge bond, leading to the formation of a pectin network. Based on recent studies of dRG-II-B structures, a hypothesis has been proposed suggesting that Ca²⁺is a common component of the dRG-II-B complex. However, no in vivo evidence has addressed whether B affects the stability of Ca²⁺ crosslinks. Here, we investigated the L-fucose-deficient dwarf mutant mur1, which was previously shown to require exogenous B treatment for phenotypic reversion. Imbibed Arabidopsis thaliana seeds release hydrated polysaccharides to form a halo of seed mucilage covering the seed surface, which consists of a water-soluble outer layer and an adherent inner layer. Our study of mur1 seed mucilage has revealed that the pectin in the outer layer of mucilage was relocated to the inner layer. Nevertheless, the mur1 inner mucilage was more vulnerable to rough shaking or ethylene diamine tetraacetic acid (EDTA) extraction than that of the wild type. Immunolabeling analysis suggested that dRG-II-B was severely decreased in mur1 inner mucilage. Moreover, non-methylesterified homogalacturonan (HG) exhibited obvious reassembly in the mur1 inner layer compared with the wild type, which may imply a possible connection between dRG-II-B deficiency and pectin network transformation in the seed mucilage. As expected, the concentration of B in the mur1 inner mucilage was reduced, whereas the distribution and concentration of Ca²⁺in the inner mucilage increased significantly, which could be the reason why pectin relocates from the outer mucilage to the inner mucilage. Consequently, the disruption of B bridges appears to result in the extreme sensitivity of the mur1 mucilage pectin complex to EDTA extraction, despite the reinforcement of the pectin network by excessive Ca²⁺. Therefore, we propose a hypothesis that B, in the form of dRG-II-B, works together with Ca²⁺to maintain pectin network crosslinks and ultimately the mucilage ultrastructure in seed mucilage. This work may serve to complement our current understanding of mucilage configuration.     

α-MSH Receptor Autoradiography on Mouse and Human Melanoma Tissue Sections and Biopsies

Abstract

MSH receptors and their binding characteristics of [¹²⁵I]-labelled derivatives of α-MSH have been studied extensively on various mouse and human melanoma cell lines in culture. The aim of this study was to determine the binding characteristics of α-MSH radioligands to MSH receptors occurring in experimental mouse and human melanoma tumours as well as in human melanoma biopsies. For this reason, solid tumours were grown on experimental animals by inoculation of murine B16-F1 and human D10 and HBL melanoma cells. After excision and cryosectioning of the tumours, frozen tissue sections were incubated with [(¹²⁵I)Tyr²]-α-MSH or [(¹²⁵I)Tyr²,Nle⁴,D-Phe⁷]-α-MSH and specific α-MSH binding sites were visualized by subsequent autoradiography. The presence of increasing concentrations of unlabelled α-MSH during incubation with tracer led to a dose-dependent displacement of the radioligand. Quantitative analysis of the autoradiograms produced dissociation constants which were comparable with those obtained with cell binding assays: K_D = 1.87 and 1.31 nmol/l for B16 tumours and cells, respectively; 0.32 and 0.33 nmol/l for D10, and 2.24 and 1.36 nmol/l for HBL tumours and cells, respectively. This indicates similar binding properties of α-MSH radioligands to both cultured melanoma cells and tissue sections of melanoma tumours from experimental animals. Similar binding characteristics were also observed with human melanoma tissue sections originating from biopsies of melanoma patients.     

L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway

l-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.Abbreviations NAC N-acetyl-l-cysteine - ROS reactive oxygen species - _m mitochondrial membrane potentialJae Seung Kang and Daeho Cho contributed equally to this work.     

Inhibition of galectin-3 mediated cellular interactions by pectic polysaccharides from dietary sources

Pectic polysaccharides from dietary sources such as Decalepis hamiltonii—swallow root (SRPP), Hemidesmus indicus (HPP), Nigella sativa—black cumin (BCPP), Andrographis serpyllifolia—(APP), Zingiber officinale—ginger (GRPP) and, citrus pectin (CPP) were examined for galectin inhibitory activity. Inhibition of (a) galectin-3 of MDA-MB-231 cells induced hemagglutination of red blood cells; (b) galectin-3 mediated interaction between normal/metastatic human buccal cells (NBC)/(MBC) and; (c) invasion of MDA-MB-231 and MBC in the invasive chamber was assessed. Results indicated that SRPP inhibited hemagglutination at Minimum Inhibitory Concentration (MIC) of 1.86 μg ml⁻¹ equivalent of carbohydrate as apposed to those of BCPP (130 μg ml⁻¹), APP (40 μg ml⁻¹), HPP (40 μg ml⁻¹) and CPP (25 μg ml⁻¹). GRPP even at concentration >1–6 mg ml⁻¹ did not inhibit agglutination. Also SRPP showed ∼15 and 2 fold potent anti hemagglutination activity relative to that of galectin-3 specific sugars—galactose (MIC-27.1 μg ml⁻¹) and lactose (MIC-4.16 μg ml⁻¹) respectively. Further, SRPP at 10 μg ml⁻¹ inhibited agglutination of NBC by galectin-3 of MDA-MB-231 cells. Modified swallow root pectic polysaccharide (MSRPP) of 50 kDa retained anti hemagglutination activity (MIC of 1.03 μg ml⁻¹) and inhibited MDA-MB-231 and MBC invasion by 73 and 50% with an IC₅₀ of 136 and 200 μg ml⁻¹ respectively. Both SRPP and MSRPP induced apoptosis up to 80% at 100 μg ml⁻¹ concentration by activating ∼2 and 8 folds of Caspase-3 activity. Sugar composition analysis and its correlation with the galectin inhibitory property indicated that pectic polysaccharides with higher arabinose and galactose content—arabinogalactan inhibited hemagglutination significantly.     

Pectin induces apoptosis in human prostate cancer cells: correlation of apoptotic function with pectin structure

Treatment options for androgen-independent prostate cancer cells are limited. Therefore, it is critical to identify agents that induce death of both androgen-responsive and androgen-insensitive cells. Here we demonstrate that a product of plant cell walls, pectin, is capable of inducing apoptosis in androgen-responsive (LNCaP) and androgen-independent (LNCaP C4-2) human prostate cancer cells. Commercially available fractionated pectin powder (FPP) induced apoptosis (approximately 40-fold above non-treated cells) in both cell lines as determined by the Apoptosense assay and activation of caspase-3 and its substrate, poly(ADP-ribose) polymerase. Conversely, citrus pectin (CP) and the pH-modified CP, PectaSol, had little or no apoptotic activity. Glycosyl residue composition and linkage analyses revealed no significant differences among the pectins. Mild base treatment to remove ester linkages destroyed FPP's apoptotic activity and yielded homogalacturonan (HG) oligosaccharides. The treatment of FPP with pectinmethylesterase to remove galacturonosyl carboxymethylesters and/or with endopolygalacturonase to cleave nonmethylesterified HG caused no major reduction in apoptotic activity, implicating the requirement for a base-sensitive linkage other than the carboxymethylester. Heat treatment of CP (HTCP) led to the induction of significant levels of apoptosis comparable to FPP, suggesting a means for generating apoptotic pectic structures. These results indicate that specific structural elements within pectin are responsible for the apoptotic activity, and that this structure can be generated, or enriched for, by heat treatment of CP. These findings provide the foundation for mechanistic studies of pectin apoptotic activity and a basis for the development of pectin-based pharmaceuticals, nutraceuticals, or recommended diet changes aimed at combating prostate cancer occurrence and progression.     

Polysaccharidmetabolismus in den Zellwänden wachsender Keimlinge von Phaseolus aureus

Summary Quantitative determinations of the cell wall constituents (pectin, hemicellulose and -cellulose) of growing Phaseolus aureus seedlings showed marked changes during early growth. The cell walls of the 2 to 4 days old seedlings were composed of approximately 30% -cellulose, 50% hemicelluloses and 20% pectin. After four weeks the proportion of the different fractions had changed to approximately 60% -cellulose, 30% hemicelluloses and 10% pectin. Quantitative sugar determinations on these polysaccharide fractions have shown that mainly the non-cellulosic fractions (hemicelluloses and pectin) underwent considerable changes in sugar composition during growth. The hemicelluloses contained non-cellulosic polysaccharides with a high glucose content, which were not starch. These were broken down in the cell walls during growth.In a series of experiments in which ¹⁴C-glucose was injected into the hypocotyls of four days old Phaseolus aureus seedlings, the transport of radioactivity to the different plant organs and its incorporation into the cell wall polysaccharides of the bean stem were studied. The major part of the radioactivity was incorporated into the cell wall of the stem tissue. Minor amounts were transported to the roots and leaves. Of the cell wall polysaccharides of the stem, the hemicellulosic fraction showed a higher rate of incorporation of the ¹⁴C-glucose than the -cellulose in the early stages of growth. With increasing age of the plant, radioactivity was transferred from the hemicellulosic fraction to the -cellulose, suggesting turnover of polysaccharides in the growing cell wall.     

New Method for Preparing 3-Ketobutylidene 2-Chloro-4-nitrophenyl β-Maltopentaoside

Polyclonal antibodies against P-l, a pectic polysaccharide fraction extracted with 0.5m NaOH from the kernels of Prunus mume and consisted of arabino-galacturonan, and 1–3, the partial acid (0.1 m trifluoroacetic acid) hydrolysate of P-l, were prepared in Japanese white rabbits. Competitive elisa experiments strongly suggested that anti P-l and anti 1–3 antibodies were different but P–l and 1–3 cross-reacted with each other to recognize a partly similar epitope structure. The reactivities of polysaccharide fractions from the raw flesh of P. mume, and the kernels of apricot and peach extracted with either water or sodium hydroxide were examined using both antisera by the indirect competitive elisa method. The polysaccharide fractions extracted with sodium hydroxide solutions had the reactivities but not those extracted with cold and hot water. These facts suggested that the similar structure of polysaccharides to P-l was present in the flesh of P. mume and the kernels of apricot and peach. However, neither pectin of apple nor citrus had reactivity with each antiserum. P-l would be different in chemical structure from a commercially available pectin, a water-soluble polysaccharide from apple and citrus.     

Poly <Emphasis Type="Italic">N</Emphasis>-acetyllactosamine substitutions on <Emphasis Type="Italic">N</Emphasis>- and not <Emphasis Type="Italic">O</Emphasis>-oligosaccharides or Thomsen–Friedenreich antigen facilitate lung specific metastasis of melanoma cells via galectin-3

Galectin-3 on vascular endothelium has been shown to facilitate lung specific metastasis. Metastatic variants of B16 melanoma were chosen to identify specific ligands that mediate lung colonization via galectin-3. Flow cytometry showed that, galectin-3 binding to cells correlates with surface expression of poly N-acetyllactosamine (polylacNAc) but not with other reported ligands, e.g. Thomsen-Friedenreich (T/Tn) antigen. Immobilized galectin-3 promoted adhesion of melanoma cells in a metastasis dependent manner. Moreover, adhesion and galectin-3 binding to cells were specifically inhibited with lactose. These properties together with lung metastasis were inhibited with N-glycosylation inhibitor Swainsonine (SW), whereas, O-glycosylation inhibitor Benzyl-α-N-acetylgalactosamine (BG) had no effect. BG treatment significantly increased expression of T/Tn antigen on low metastatic cells; however, had no effect on their metastatic potential. The studies very comprehensively demonstrate the importance of polylacNAc substitutions on N-oligosaccharides in galectin-3 mediated lung metastasis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.