Galectin-3 Binding Research Articles

Disruption of galectin-3 and galectin-3 binding protein (G3BP) interaction by dietary pectic polysaccharides (DPP) - Arrest of metastasis, inhibition of proliferation and induction of apoptosis

Galectin-3 and galectin-3 binding proteins (G3BP) are implicated as key players in metastasis. In the current study, we evaluated the effect of pectic polysaccharides on galectin-3 and G3BP mediated metastasis in vitro (cells) and in vivo (tissues). In vitro study (double immunostaining) confirms the presence of galectin-3 on the cell surface and G3BP in the interlinking region of the cells confirming the role of G3BP in bridging galectin-3 molecules. Dietary carrot (Daucus carota) pectic polysaccharide (CRPP) blocked the expression of galectin-3 and G3BP more effectively (80%), whereas the expressions were reduced to 60% upon treatment with swallow root (Decalepis hamiltonii) pectic polysaccharide (SRPP), β‑carotene and deferoxamine (antiproliferative drug). Ginger (Gingiber officinale) pectic polysaccharide (GRPP) showed only 20% reduction. CRPP reduced 80% of tumor incidence followed by cyclophosphamide - a chemotherapeutic drug (77%), SRPP (67%) and GRPP (45%). Further 3–5 folds reduction in galectin-3/G3BP expression followed by infiltration of macrophages into the deeper layer of the skin by CRPP and SRPP suggested the anticancer property via immunomodulation. Surface Plasmon Resonance (SPR) studies confirm galectin-3 and G3BP interaction, which are disrupted during the treatment with dietary pectic polysaccharides (DPP) (Supplementary Scheme-1). Overall data demonstrate the role of DPPs as potential anticancer alternatives.

Abstract 581: Determining the influence of galectin-3 binding properties on its suppression of tumor antigen-specific CD8+ T cells

Abstract Improving the efficacy of immunotherapeutic cancer treatments requires increased understanding of the factors that contribute to a suppressive tumor microenvironment (TME). Recently, galectin-3 has emerged as an important player in the development and progression of cancer, favoring immune suppression and dysfunction at disease sites. While this protein is consistently identified as a tumor-promoting factor, its unique properties make the mechanisms of its functions difficult to study, and impede the development of therapies against it. Galectin-3 binds β-galactoside carbohydrate residues through a highly-conserved C-terminal domain (CRD) characteristic of mammalian galectins. It is made unique by its ability to self-associate into homo-oligomers through a disordered N-terminal domain (NTD). We have previously shown that galectin-3 knockout in a mouse model of tumor tolerance improves effector function of antitumor CD8+ T cells and increases duration of tumor-free survival, suggesting a suppressive role for galectin-3 in antitumor immunity. Our current work aims to elucidate how the distinct biochemical properties of galectin-3 contribute to this suppression. To this end, we are developing a new assay to model the suppression of CD8+ T cells by galectin-3 in vitro. Our assay utilizes TCR transgenic CD8+ T cells specific for rodent Erbb2, the Her-2/neu (Neu) oncogene. These cells are isolated from either normal transgenic mice or those deficient in galectin-3. The T cells are stimulated in culture, then co-cultured with NT2.5 Neu-expressing tumor cells. We use flow cytometry to analyze the levels of CD8+ effector molecules interferon gamma (IFNγ) and Granzyme B, as well as cell surface binding of galectin-3 after co-culture. To further elucidate the roles of carbohydrate binding and self-association in this process, we have developed recombinant mutant galectin-3 proteins deficient in carbohydrate binding through CRD point mutation, lacking the ability to self-associate through NTD truncation, or both. By introducing these recombinant proteins into the co-culture with normal or galectin-3 deficient transgenic T cells, we expect to be able to learn whether galectin-3-mediated suppression is more dependent on carbohydrate binding, self-association, or both properties in synergy. Current therapies targeting galectin-3 focus only on its carbohydrate binding properties through use of small molecules and glycomimetics, but these have been of limited efficacy. We hypothesize that self-association of galectin-3 through the NTD is an equally important factor in its unique ability among galectins to contribute to an immunosuppressive TME. Learning how these properties each contribute to the mechanism of galectin-3-mediated immunosuppression will allow us to refine strategies for breaking tumor tolerance, contributing to improved outcomes for cancer immunotherapies. Citation Format: Alexandra B. Pucsek, Todd D. Armstrong, Elizabeth M. Jaffee. Determining the influence of galectin-3 binding properties on its suppression of tumor antigen-specific CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 581.

Induction of Trop-2 expression through the binding of galectin-3 to MUC1

Both mucin 1 (MUC1) and trophoblast cell surface antigen 2 (Trop-2) are overexpressed in various epithelial tumor cells, and their high expression is correlated with a poor prognosis. Both proteins were expressed in a human breast cancer cell line, MCF-7 cells, but neither was in a human colon cancer cell line, HCT116 cells. When MUC1 cDNA was introduced into HCT116 cells (HCT116/MUC1), expression of Trop-2 was induced. Reciprocally, treatment of MCF-7 cells with MUC1 siRNA reduced the level of Trop-2. Mithramycin A, an inhibitor of specificity protein 1 (Sp1) transcription factor, effectively inhibited the expression of Trop-2. Consistently, treatment with Sp1 siRNA reduced the expression of Trop-2. To reveal the relationship between MUC1 and Sp1, coimmunoprecipitation assays were performed. Sp1 was coimmunoprecipitated with MUC1 and the level of coimmunoprecipitated Sp1 increased in relation to the level of induced Trop-2. It is known that galectin-3 is an endogenous ligand of MUC1. Binding of galectin-3 to MUC1 elevated the recruitment of Sp1 to MUC1, and knockdown of galectin-3 reduced the level of Trop-2. These results suggest that the binding of galectin-3 to MUC1 enhances the recruitment of Sp1, leading to promotion of the transcription of Trop-2.

NMR-based insight into galectin-3 binding to endothelial cell adhesion molecule CD146: Evidence for noncanonical interactions with the lectin's CRD β-sandwich F-face.

Galectin-3 (Gal-3) binds to cell adhesion glycoprotein CD146 to promote cytokine secretion and mediate endothelial cell migration. Here, we used Nuclear Magnetic Resonance (NMR) 15N-Heteronuclear Single Quantum Coherence (HSQC) spectroscopy to investigate binding between 15N-labeled Gal-3 and the extracellular domain (eFL) of purified CD146 (five Ig-like ectodomains D1-D5) and a shorter, D5-deleted version of CD146 (D1-D4). Binding of Gal-3 and its carbohydrate recognition domain (CRD) to CD146 D1-D4 is greatly reduced vis-à-vis CD146 eFL, supporting the proposal of a larger number of glycosylation sites on D5. Even though the canonical sugar-binding β-sheet S-face (β-strands 1, 10, 3, 4, 5, 6) of the Gal-3 β-sandwich is involved in interactions with CD146 (e.g. N-linked glycosylation sites), equivalent HSQC spectral perturbations at residues on the opposing Gal-3 F-face β-sheet (β-strands 11, 2, 7, 8, 9) indicate involvement of the Gal-3 F-face in binding CD146. This is supported by the observation that addition of lactose, while significantly attenuating Gal-3 binding (primarily with the S-face) to CD146 eFL, does not abolish it. Bio-Layer Interferometry studies with Gal-3 F-face mutants yield KD values to demonstrate a significant decrease (L203A) or increase (V204A, L218A, T243A) in net binding to CD146 eFL compared to wild type Gal-3. However, HSQC lactose titrations show no highly significant effects on sugar binding to the Gal-3 CRD S-face. Overall, our findings indicate that Gal-3 binding to CD146 is more involved than simple interactions with β-galactoside epitopes on the cell receptor, and that there is a direct role for the lectin's CRD F-face in the CD146 binding process.

Distinct Effects of Carrageenan and High-Fat Consumption on the Mechanisms of Insulin Resistance in Nonobese and Obese Models of Type 2 Diabetes.

Exposure to low concentration of the common food additive carrageenan (10 mg/L) for only six days led to glucose intolerance and insulin resistance in the C57BL/6J mouse. Longer exposure produced fasting hyperglycemia but with no increase in weight, in contrast to the HFD. Glucose intolerance was attributable to carrageenan-induced inflammation and to increased expression of GRB10. Both HFD and carrageenan increased p(Ser32)-IκBα and p(Ser307)-IRS1, and the increases were greater following the combined exposure. The effects of carrageenan were inhibited by the combination of the free radical inhibitor Tempol and BCL10 siRNA, which had no impact on the HFD-mediated increase. In contrast, the PKC inhibitor sotrastaurin blocked the HFD-induced increases, without an effect on the carrageenan-mediated effects. HFD had no impact on the expression of GRB10. Both carrageenan and high fat increased hepatic infiltration by F4/80-positive macrophages. Serum galectin-3 and galectin-3 binding to the insulin receptor increased by carrageenan and by HFD. Tyrosine phosphorylation of the insulin receptor declined following either exposure and was further reduced by their combination. Carrageenan reduced the activity of the enzyme N-acetylgalactosamine-4-sulfatase (ARSB; arylsulfatase B), which was unchanged following HFD. Dietary exposure to both high fat and carrageenan can impair insulin signaling through both similar and distinct mechanisms.

Elevated Platelet Galectin-3 and Rho-Associated Protein Kinase Activity Are Associated with Hemodialysis Arteriovenous Shunt Dysfunction among Subjects with Diabetes Mellitus.

Introduction Hyperglycemia is a major factor in influencing the patency rate of arteriovenous shunts, potentially associated with the RhoA/Rho-associated protein kinase (ROCK) pathway. Besides, galectin-3 mediates thrombotic mechanisms in venous thrombosis and peripheral artery disease. We hypothesized that high ROCK activity and galectin-3 levels are associated with arteriovenous shunt dysfunction. Methods We prospectively enrolled 38 patients diagnosed with arteriovenous shunt dysfunction. 29 patients received a complete follow-up and each provided two blood samples, which were collected at the first visit for occluded status of arteriovenous shunts and 1 month later for patent status. A Western blot assay for a myosin phosphatase target subunit (MYPT) was performed to examine Rho-kinase activity. A Western blot assay for platelet galectin-3 and enzyme-linked immunosorbent assay (ELISA) for circulating galectin-3 were completed. Results Higher platelet MYPT ratios and galectin-3 levels were identified at occluded arteriovenous shunts (MYPT ratio: 0.5 [0.3–1.4] vs. 0.4 [0.3–0.6], p = 0.01; galectin-3: 1.2 [0.4–1.6] vs. 0.7 [0.1–1.2], p = 0.0004). The plasma galectin-3 binding protein ELISA was also higher at occluded arteriovenous shunts (8.4 [6.0–9.7] μg/mL vs. 7.1 [4.5–9.1] μg/mL, p = 0.009). Biomarker ratios (occluded/patent status) trended high in patients with poorly controlled diabetes (MYPT ratio: 1.7 [1.0–3.0] vs. 1.1 [0.7–1.3], p = 0.06; galectin-3: 1.6 [1.3–3.4] vs. 1.1 [0.8–1.9], p = 0.05). Conclusion High platelet ROCK activity and galectin-3 levels are associated with increased risk in arteriovenous shunt dysfunction, especially in patients with poorly controlled diabetes.

E-105 Mesenchymal stromal cells: Mediators of inflammation in cancer

Cancer-associated fibroblasts (CAF) have been identified in the tumor microenvironment (TME) of many cancers. CAF represent a heterogeneous population of cells that can have pro-tumorigenic and anti-tumorigenic functions. We have isolated from primary human neuroblastoma tumors, a population of CAF that share phenotypic and functional characteristics of bone marrow-derived mesenchymal stromal cells (MSC). These cells are CD73+, CD90+, CD105+, CD34−, CD45−, CD14−, CD19−, CD31−, and VEGFR2−, and retain the ability to differentiate into osteoblasts, chondrocytes and adipocytes, a feature of MSC. However, we show that these cells also express fibroblast activation protein alpha (FAP/DPPIV), fibroblast specific protein 1 (FSP1/S1004A) and alpha smooth muscle actin (SMA), markers of cancer-associated fibroblasts. When in the presence of tumor cells, these MSC/CAF stimulate the production of multiple pro-tumorigenic cytokines and chemokines including interleukin (IL)-6, IL-8, CCL2/monocyte chemoattractant protein 1, CXCL-12/stromal-derived factor 1 (SDF1), CXCL1, vascular endothelial growth factor (VEGF-A), plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor (TGF) β. The production of these cytokines/chemokines by MSC/CAF is stimulated by the production of soluble factors by tumor cells including galectin-3 binding protein and extracellular vesicles. In addition to stimulating angiogenesis, the recruitment and M2 polarization of macrophages, and to suppress immune cells, MSC/CAF have a direct effect on tumor cells by stimulating tumor cell growth, survival and resistance to chemotherapy in an ERK1/2 and STAT3-dependent manner. We then show that targeting ERK1/2 and STAT3 or targeting MSC in combination with chemotherapy or immunotherapy in mice orthotopically implanted with human neuroblastoma tumors, inhibits tumor growth and extends survival. In contrast, targeting a single cytokine like IL-6 has no significant effect. These data thus identify a new type of CAF sharing characteristics of bone marrow-derived MSCs that contribute to a pro-tumorigenic inflammatory reaction in the TME and provide preclinical evidence that targeting these cells or the pathways they activated in tumor cells is a valuable therapeutic approach.

Urinary Proteomics Profiles Are Useful for Detection of Cancer Biomarkers and Changes Induced by Therapeutic Procedures.

Boron neutron capture therapy (BNCT) is a binary cancer treatment modality where two different agents (10B and thermal neutrons) have to be present to produce an effect. A dedicated trial design is necessary for early clinical trials. The concentration of 10B in tissues is an accepted surrogate to predict BNCT effects on tissues. Tissue, blood, and urines were sampled after infusion of two different boron carriers, namely BSH and BPA in the frame of the European Organisation for Research and Treatment of Cancer (EORTC) trial 11001. In this study, urine samples were used to identify protein profiles prior and after drug infusion during surgery. Here, an approach that is based on the mass spectrometry (MS)-based proteomic analysis of urine samples from head and neck squamous cell carcinoma (HNSCC) and thyroid cancer patients is presented. This method allowed the identification of several inflammation- and cancer-related proteins, which could serve as tumor biomarkers. In addition, changes in the urinary proteome during and after therapeutic interventions were detected. In particular, a reduction of three proteins that were involved in inflammation has been observed: Galectin-3 Binding Protein, CD44, and osteopontin. The present work represents a proof of principle to follow proteasome changes during complex treatments based on urine samples.

Extracellular and intracellular small-molecule galectin-3 inhibitors

Galectin-3 is a carbohydrate binding protein which has important roles in cancer and immunity. Potent galectin-3 inhibitors have been synthesized, for experimental purposes and potential clinical use. As galectin-3 is implicated in both intra- and extracellular activities, permeability of galectin-3 inhibitors is an important parameter determining biological effects. We compared the cellular uptake of galectin-3 inhibitors and their potency in the intracellular or extracellular space. The inhibitors differed in their polar surface area (PSA), but had similar affinities for galectin-3. Using a well-established permeability assay, we confirmed that the uptake was significantly higher for the inhibitor with the lowest PSA, as expected. To analyze intracellular activity of the inhibitors, we developed a novel assay based on galectin-3 accumulation around damaged intracellular vesicles. The results show striking differences between the inhibitors intracellular potency, correlating with their PSAs. To test extracellular activity of the inhibitors, we analyzed their potency to block binding of galectin-3 to cell surfaces. All inhibitors were equally able to block galectin-3 binding to cells and this was proportional to their affinity for galectin-3. These inhibitors may serve as useful tools in exploring biological roles of galectin-3 and may further our understanding of intracellular versus extracellular roles of galectin-3.

Neuroprotective effect of placenta-derived mesenchymal stromal cells: role of exosomes.

We have established early-gestation chorionic villus-derived placenta mesenchymal stromal cells (PMSCs) as a potential treatment for spina bifida (SB), a neural tube defect. Our preclinical studies demonstrated that PMSCs have the potential to cure hind limb paralysis in the fetal lamb model of SB via a paracrine mechanism. PMSCs exhibit neuroprotective function by increasing cell number and neurites, as shown by indirect coculture and direct addition of PMSC-conditioned medium to the staurosporine-induced apoptotic human neuroblastoma cell line, SH-SY5Y. PMSC-conditioned medium suppressed caspase activity in apoptotic SH-SY5Y cells, suggesting that PMSC secretome contributes to neuronal survival after injury. As a part of PMSC secretome, PMSC exosomes were isolated and extensively characterized; their addition to apoptotic SH-SY5Y cells mediated an increase in neurites, suggesting that they exhibit neuroprotective function. Proteomic and RNA sequencing analysis revealed that PMSC exosomes contain several proteins and RNAs involved in neuronal survival and development. Galectin 1 was highly expressed on the surface of PMSCs and PMSC exosomes. Preincubation of exosomes with anti-galectin 1 antibody decreased their neuroprotective effect, suggesting that PMSC exosomes likely impart their effect via binding of galectin 1 to cells. Future studies will include in-depth analyses of the role of PMSC exosomes on neuroprotection and their clinical applications.-Kumar, P., Becker, J. C., Gao, K., Carney, R. P., Lankford, L., Keller, B. A., Herout, K., Lam, K. S., Farmer, D. L., Wang, A. Neuroprotective effect of placenta-derived mesenchymal stromal cells: role of exosomes.

P022 Galectin-3, galectin-9, and galectin-3 binding protein in patients with inflammatory bowel diseases

P022 Galectin-3, galectin-9, and galectin-3 binding protein in patients with inflammatory bowel diseases

Hybrid ligands with calixarene and thiodigalactoside groups: galectin binding and cytotoxicity.

Galectins have diverse functions and are involved in many biological processes because of their complex intra- and extracellular activities. Selective and potent inhibitors for galectins will be valuable tools to investigate the biological functions of these proteins. Therefore, we describe here the synthesis of galectin inhibitors with a potential “chelate effect”. These compounds are designed to bind to two different binding sites on galectins simultaneously. In this paper a series of asymmetric “hybrid” compounds are prepared, which combine two galectin ligands (1) a substituted thiodigalactoside derivative and (2) an antagonist calixarene-based therapeutic agent. NMR spectroscopy was used to evaluate the interactions of these compounds with Galectin-1 and -3. In addition, cellular experiments were conducted to compare the cytotoxic effects of the hybrids with those of a calixarene derivative. While only the thiodigalactoside part of the hybrids showed strong binding, the calixarene part was responsible for observed cytoxoxicity effects, suggesting that the calixarene moiety may also be addressing a non-galectin target.

Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context

Galectins compose a protein family defined by a conserved sequence motif conferring affinity for β-galactose-containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their β-galactoside-binding site, as revealed by extensive testing against panels of purified glycans. However, in cells, galectins bind glycans on glycoproteins and glycolipids in the context of other cellular components, such as at the cell surface. Because of difficulties in characterizing natural cellular environments, we currently lack a detailed understanding of galectin-binding specificities in the cellular context. To address this challenge, we used a panel of genetically stable glycosylation mutated CHO cells that express defined glycans to evaluate the binding affinities of 10 different carbohydrate-recognition domains in galectins to N-glycans and mucin-type O-glycans. Using flow cytometry, we measured the cell-surface binding of the galectins. Moreover, we used fluorescence anisotropy to determine the galectin affinities to recombinant erythropoietin used as a reporter glycoprotein produced by the glycoengineered cells and to synthetic N-glycans with defined branch structures. We found that all galectins, apart from galectin-8N, require complex N-glycans for high-affinity binding. Galectin-8N targeted both N- and O-linked glycans with high affinity, preferring 2,3-sialylated N-acetyllactosamine (LacNAc) structures. Furthermore, we found that 2,3-sialylation suppresses high-affinity binding of select galectins, including galectin-2, -3, -4N, and -7. Structural modeling provided a basis for interpreting the observed binding preferences. These results underscore the power of a glycoengineered platform to dissect the glycan-binding specificities of carbohydrate-binding proteins.

A Novel Glycomimetic Compound (GMI-1757) with Dual Functional Antagonism to E-Selectin and Galectin-3 Demonstrates Inhibition of Thrombus Formation in an Inferior Vena Cava Model

▪E-selectin functions in venous thrombosis by binding and activating host cells to initiate the coagulation cascade. The E-selectin antagonist, Uproleselan (GMI-1271), has been shown to attenuate thrombus formation following electrical stimulation in a preclinical inferior vena cava (IVC) model without significantly affecting hemostasis (Culmer DL et al. Thromb Haemost. 117:1171-1181, 2017). A recent study has reported that galectin-3 (gal-3) and gal-3 binding protein are associated with murine thrombogenesis where they interact at the thrombus-vein wall interface, and that gal-3 may be contributing to thrombosis through proinflammatory mechanisms (Elise P et al. Blood 125:1813-1821, 2015). Collectively these studies suggest that pharmacologic targeting of both E-selectin and gal-3 function may afford a new class of therapeutics for treatment of venous thrombosis. In the present studies, we report on the activity of a novel glycomimetic compound with dual functional antagonism of E-selectin and gal-3, and demonstrate its anti-thrombotic activity in an IVC model.Binding affinities of GMI-1757 in solution or to immobilized E-selectin or gal-3 were determined by surface plasmon resonance (SPR) or microscale thermophoresis (MST), respectively. By SPR the mean (+/- SD) KD of GMI-1757 was 61 +/- 4 nM and 276 +/- 98 nM for E-selectin and gal-3, respectively (N=2 determinations). By MST the mean KD of GMI-1757 was 563 +/- 158 nM and 83 +/- 9 nM for E-selectin and gal-3, respectively (N=2-3 determinations). GMI-1757 was subsequently evaluated for its ability to inhibit binding of (a) sialyl Lea to immobilized human E-selectin and (b) gal-3 to an immobilized Galb1-3GlcNAc carbohydrate structure. The median IC50 of GMI-1757 for ligand binding of E-selectin or gal-3 was 800 nM (n=9 independent assays) and 110 nM (n=7 independent assays), respectively.The pharmacokinetics of GMI-1757 was evaluated following intravenous (IV) administration in male CD-1 mice at 5 mg/kg. Blood samples were collected up to 24 hours post dose, and GMI-1757 concentrations were determined with a qualified LC-MS/MS method. Following IV dosing of GMI 1757 at 5 mg/kg, the average half-life was 3.5 hours. Its average clearance rate was 1.39 +/- 0.180 L/hr/kg and the average volume of distribution was 1.35 +/- 0.651 L/kg.The in vivo activity of GMI-1757 following intraperitoneal (IP) administration was assessed in an acute model of IVC thrombosis.Immediately following the induction of a non-occlusive thrombosis via electrical stimulation (250 mAmp) of the IVC, cohorts of male C57BL/6J mice were given a single IP injection of saline (0.1 mL, n=9); Uproleselan (40 mg/kg, n=7); or GMI-1757 (40 mg/kg, n=6), and twenty-four hrs post thrombus induction the IVC was harvested from all mice and thrombus weights were determined. All treatments were well tolerated. The median thrombus weight from mice treated with Uproleselan was 0.0138 g and was significantly reduced from mice receiving saline (0.0208 g, P=0.004). Treatment with GMI-1757 resulted in a further reduction in median thrombus weight (0.0098 g) which was statistically differentiated from both saline and Uproleselan treatment (P= 0.002 and 0.049, respectively). Mechanism-of-action studies continue to be pursued to fully understand the impact of E-selectin and gal-3 inhibition in this model and other models where disease progression is dependent on both inflammation and fibrosis.In summary, an innovative dual antagonist of E-selectin and gal-3, GMI-1757, has been produced that demonstrates marked inhibition of thrombus formation in a murine IVC model following electrical stimulation. [Display omitted] DisclosuresPeterson:GlycoMimetics: Employment, Equity Ownership. Vohra:GlycoMimetics: Employment, Equity Ownership. Locatelli-Hoops:GlycoMimetics: Employment, Equity Ownership. Lee:GlycoMimetics: Employment, Equity Ownership. Deng:GlycoMimetics: Employment, Equity Ownership. Smith:GlycoMimetics: Employment, Equity Ownership. Fogler:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Abstract 291: Galectin-3 as a Risk Predictor of Short-Term Mortality in the Resuscitated Cohorts of Out-of-Hospital Cardiac Arrest

Introduction: Out of Hospital Cardiac Arrest (OHCA) is a common and catastrophic manifestation of multiple cardiac and non-cardiac illnesses. Recent studies showed increase in survival rate, reaching to about one third of those who suffer a bystander witnessed OHCA with a shockable rhythm; yet biomarkers to predict outcomes in this group is still missing. Hypothesis: We tested the hypothesis that serum galectin-3, alone or together with galectin-3 binding protein (G3BP),brain natriuretic peptide (BNP) and troponin, can predict the risk of early (30-day) mortality in ResCOHS. Methods: ResCOHS prospective multicenter pilot study enrolled 117 patients with aborted out-of-hospital cardiac arrest, and 10 healthy volunteers. Transthoracic Echocardiogram was performed for cardiac morphology and function. Serum levels of galectin-3, G3BP, BNP and peak troponin were analyzed. Patients were prospectively followed up for 30-days to determine all-cause in-hospital mortality. Results: Among the ResCOHS, patients who died within 30-days had higher BNP and galectin-3 levels, with no differences in the G3BP, peak troponin, CK and CK-MB levels. Receiver operating characteristic analysis for mortality prediction showed that, for 30-day prognosis, galectin-3 had the greatest area under the curve (AUC) at 0.764 (p = 0.001), whereas G3BP, BNP and troponin had an AUC of 0.518 (p = 0.735), 0.759 (p = 0.001)and 0.540 (p=0.460) respectively. In a multivariate logistic regression analysis, an elevated level of galectin-3 was the strongest independent predictor of 30-day mortality (odds ratio 1.04, p = 0.002). The Kaplan-Meier analyses showed that the combination of an elevated galectin-3 with NT-proBNP was a better predictor of mortality than either of the 2 markers alone. Conclusions: In the ResCOHS, higher serum galectin-3 levels are associated with increased chances of death within 30-days of the first episode of cardiac arrest. These findings have implications to target specific therapies to those at the greatest risk of mortality after OHCA.

Chemoenzymatic Synthesis of Galectin Binding Glycopolymers.

Chemoenzymatic synthesis is an important strategy for the formation of glycopolymers. The use of a smaller number of traditional chemical steps and enzyme catalyzed reactions increases the yield of glycopolymer that can be produced by reducing the overall number of synthetic steps. In addition, chemoenzymatic routes are likely to be more accessible to those without a background in carbohydrate synthesis, making glycopolymers more available for studies across a broader range of scientists. Here, the enzymatic addition of galactose to N-acetylglucosamine functionalized glycodendrimers reduced the requisite number of synthetic steps for the full chemical synthesis of N-acetyl lactosamine (Lac NAc) functionalized dendrimers to four steps. Unpurified cell lysate was used in the enzyme catalyzed glycosylation, and product glycodendrimers were readily purified by dialysis after enzymatic degradation of all protein components of the lysate in the crude reaction mixture. Lac NAc functionalized dendrimers were used very effectively in homotypic cancer cellular aggregation assays and were found to either inhibit or enhance galectin-3 mediated cancer cellular aggregation, with differences in outcomes dependent on the generation of Lac NAc functionalized dendrimers that were used.

Proteomic Profile Associated With Loss of Spontaneous Human Immunodeficiency Virus Type 1 Elite Control.

Elite controllers (ECs) spontaneously control plasma human immunodeficiency virus type 1 (HIV-1) RNA without antiretroviral therapy. However, 25% lose virological control over time. The aim of this work was to study the proteomic profile that preceded this loss of virological control to identify potential biomarkers. Plasma samples from ECs who spontaneously lost virological control (transient controllers [TCs]), at 2 years and 1 year before the loss of control, were compared with a control group of ECs who persistently maintained virological control during the same follow-up period (persistent controllers [PCs]). Comparative plasma shotgun proteomics was performed with tandem mass tag (TMT) isobaric tag labeling and nanoflow liquid chromatography coupled to Orbitrap mass spectrometry. Eighteen proteins exhibited differences comparing PC and preloss TC timepoints. These proteins were involved in proinflammatory mechanisms, and some of them play a role in HIV-1 replication and pathogenesis and interact with structural viral proteins. Coagulation factor XI, α-1-antichymotrypsin, ficolin-2, 14-3-3 protein, and galectin-3-binding protein were considered potential biomarkers. The proteomic signature associated with the spontaneous loss of virological control was characterized by higher levels of inflammation, transendothelial migration, and coagulation. Galectin-3 binding protein could be considered as potential biomarker for the prediction of virological progression and as therapeutic target in ECs.

Bergenin: a computationally proven promising scaffold for novel galectin-3 inhibitors

Bergenin is a C-glycoside of 4-O-methylgallic acid that is isolated from medicinal plants such as Flueggea leucopyrus, Bergenia crassifolia, Mallotus philippensis, Corylopsis spicata, Caesalpinia digyna, Mallotus japonicus, and Sacoglottis gabonensis. Even though there appears to be ample evidence from South Asian traditional medicine that bergenin possesses strong anticancer activity, no comprehensive scientific study has been carried out to test its anticancer potency. Therefore, in this study, the potential mechanisms of action for bergenin's postulated anticancer activity were examined using computational techniques. Firstly, bergenin was tested for its toxicity as a drug candidate using in silico toxicity analysis. It was found that bergenin is nontoxic according to modern toxicity measures. The optimized structure of bergenin was obtained at the DFT-B3LYP/6-31G(d) level of theory. Potential biological targets of bergenin were identified using reverse docking calculations. Reverse docking results suggested that galectin-3 is a potential target of bergenin. Gelectin-3 is an enzyme that plays a major role in cell-cell adhesion, cell-matrix interactions, macrophage activation, angiogenesis, metastasis, and apoptosis in cancer, making it a popular target in anticancer drug design. Among the many potential biological targets predicted by reverse docking calculations, galectin-3 was selected as it complies with the primary objective of this study. The binding of bergenin to galectin-3 was studied by conventional forward docking calculations. Classical molecular dynamics (MD) simulations were used to study the stability of the galectin-3:bergenin complex. Docking calculations indicated that bergenin has the potential to effectively bind to the carbohydrate recognition domain (CRD) of galectin-3. As well as electrostatic and van der Waals interactions, a few strong hydrogen bonds were found to be involved in the binding of bergenin to galectin-3. There is also a plausible π-stacking interaction between the aromatic moiety of bergenin and the His158 residue at the binding site. A 50-ns MD simulation was carried out for the bergenin:galectin-3 complex in a cubic water box with periodic boundary conditions. The MD results showed that the bergenin:galectin-3 complex is highly stable and confirmed the veracity of the docking results, which suggested that bergenin potentially exerts an inhibitory effect on galectin-3. This study therefore sheds new light on the anticancer activity of bergenin and demonstrates that bergenin could potentially be used to develop more potent galectin-3 inhibitors. The study also provides scientific evidence supporting the use of bergenin-containing plants in cancer treatments in Eastern traditional medicine. Graphical abstract Bergenin in the galectin-3 binding site.

Abstract 1776: High-affinity anti-galectin-3 antibodies targeting oncogenic properties in serous ovarian cancer

Abstract Objective: Galectin-3 (LGALS3) regulates the interaction of surface proteins with the extracellular membrane domain and mediates a signal cascade leading to invasion, oncogene activation and growth. We targeted LGALS3 cancer promotion by developing high-affinity anti-galectin-3 antibodies directed either at the carbohydrate recognition domain (CRD) of the galectin-3 carboxyl-terminus (to block sugar binding) or at the N-terminus (NT) (to inhibit N-terminal mediated oligomerization of galectin-3) in order to prevent formation of the cell surface galectin lattice and suppress the oncogenic effects of the glycoprotein. Methods: Two distinct types of murine monoclonal antibodies (MAb) were generated by creating hybridomas from mice immunized either with a fusion protein derived from the 128-amino acid LGALS3 carboxyl-terminus fused to a human IgG1-Fc backbone followed by immunization with a recombinant human LGALS3 protein, or the mice were further immunized with a 25-amino acid peptide derived from the LGALS3-N-terminus conjugated to KLH. The resulting antibodies were confirmed to recognize either the LGALS3 CRD or NT by ELISA. Functional assays (Matrigel invasion, inhibition of laminin binding, and oncogene expression) and cell surface expression by FACS analysis were utilized to confirm MAb binding and activity. Mice bearing A2780 and SKOV3 ovarian cancer cell lines were treated with purified MAb to determine the effects of MAb on tumor growth in vivo. Data were analyzed for statistical significance using Student's t-test. Results: Initial screening yielded two candidate antibodies, of which one (named 14D11.2D2) had superior binding to galectin-3 by ELISA and Surface Plasmon Resonance (SPR). The antibody dissociation constant was 14.6 nM by SPR. At a concentration as low as 150 nM, the 14D11.2D2 MAb decreased galectin-3 binding to laminin by 36.6% compared to untreated control. In a Matrigel invasion assay, the anti-CRD MAb significantly inhibited invasion by MUC16 expressing SKOV3 cells and by wild-type MUC16 positive OVCAR3 cells. MUC16 expressing A2780 cells showed a statistically nonsignificant decrease in invasion. In vivo experiments with MUC16 expressing A2780 or SKOV3 tumors showed a significant inhibition of tumor growth in mice treated with anti-CRD MAb. Six candidate antibodies directed against the LGALS3-N-terminus have been identified. Characterization of two NT antibodies (1F5.D4 and 7D4.A1) is under way via in in vitro and in vivo experiments. Conclusion: High-affinity CRD and NT anti-galectin-3 antibodies inhibit MUC16-induced cellular invasion and growth. Anti-LGALS3 MAbs hold therapeutic potential for inhibition of glycosylation-dependent invasion. Citation Format: Dharmarao Thapi, Marina Stasenko, Thomas White, Sven J. Walderich, Noah Feit, Frances Weis-Garcia, David R. Spriggs. High-affinity anti-galectin-3 antibodies targeting oncogenic properties in serous ovarian cancer [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 1776.

Abstract 5713: Promising predictive biomarkers for immunotherapy in metastatic melanoma

Abstract Aim: to explore biomarkers in order to predict outcome in patients with metastatic melanoma who have had immunotherapy with ipilimumab in a real-world setting. Metastatic melanoma is a very aggressive, incurable cancer with historically few therapeutic options and poor survival. Immunotherapy represents a revolution for metastatic melanoma treatment but there is a lack of biomarkers to predict treatment response. Material and methods: In the Norwegian National Phase 4 Multicenter Study, IPI4, 150 patients were included to receive ipilimumab (anti-CTLA3). A subgroup of 73 patients (4 screening failure) was included at Oslo University Hospital -The Norwegian Radium Hospital. Serum was available from 56 patients of this subgroup and were examined before and during ipilimumab treatment concerning possible predictive biomarkers. Expression of a panel of 17 inflammatory markers reflecting different inflammatory pathways including extra cellular matrix remodeling and fibrosis, vascular inflammation, notch signaling, inflammation in general and monocyte/macrophage activation were measured at baseline and at the 2nd and/or 3rd treatment with ipilimumab. Results: During an average 33.7 months follow-up, 33 (59%) patients died. Six promising candidates (endostatin, osteoprotegerin, C-reactive protein, pulmonary and activation-regulated chemokine and galectin-3 binding-protein) were higher in non-survivors. In particular, high endostatin and galectin-3 binding protein levels were independently associated with poor long time survival also in adjusted analysis (age, gender, lactate dehydrogenase). A 1 standard deviation (SD) increase in Gal3BP gave a 1.8 x times higher risk of death (95% CI 1.10-2.95, p=0.019) while a 1 SD increase in endostatin was associated with a 2x higher risk of death (95% CI 1.12-3.64, p=0.020) in the final model. Conclusion: Endostatin and galectin-3 binding protein may represent biomarkers for prognosis during immunotherapy with ipilimumab and should be further evaluated. Citation Format: Marta Nyakas, Elin Aamdal, Tormod Guren, Steinar Aamdal, Kari Dolven Jacobsen, Paal Brunsvig, Kristin Austlid Tasken, Gunhild Mælandsmo, Arne Yndestad, Bente Halvorsen, Paal Aukrust, Thor Ueland. Promising predictive biomarkers for immunotherapy in metastatic melanoma [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 5713.