Estrogen receptors regulate galectin­3 in androgen­independent DU­145 prostate cancer cells.

The aim of the present study was to investigate the role of estrogen receptor (ER)α and ERß, and galectin­3 (GAL­3) in migration and invasion of androgen­independent DU­145 prostate cancer cells, and to examine the regulation of the expression of GAL­3 by the activation of these receptors. Wound healing and cell invasion assays were performed using the control (basal level of cellular function) and treated DU­145 cells. At 24 h of treatment, 17ß­estradiol (E2), the ERα­selective agonist, 4,4',4"­(4­propyl­(1H)­pyrazole­1,3,5­triyl)trisphenol (PPT), or the ERß­selective agonist, 2,3­bis(4­hydroxyphenyl)­propionitrile (diarylprepionitrile; DPN), increased the migration and invasion of the DU­145 cells. Pre­treatment with the ERα­ and ERß­selective antagonists blocked these effects, indicating that ERα and ERß are upstream receptors regulating these processes. Western blot analysis and immunofluorescence staining for the detection of the GAL­3 were performed using the control and treated DU­145 cells. Treatment of the DU­145 cells with E2, PPT or DPN for 24 h increased the expression of the GAL­3 compared to the control. Furthermore, a specific inhibitor of GAL­3 (VA03) inhibited the migration and invasion of DU­145 cells, indicating the involvement of the complex ERα/GAL­3 and ERß/GAL­3 in the regulation of these processes. On the whole, the present study demonstrates that the activation of both ERs increases the expression and signaling of GAL­3, and promotes the migration and invasion of DU­145 cells. The findings of the present study provide novel insight into the signatures and molecular mechanisms of ERα and ERß in DU­145 cells.

New in vivo model to assess macroscopic, histological, and molecular changes in Peyronie's disease.

BACKGROUND: Peyronie's Disease (PD) is a connective tissue disorder that affects the tunica albuginea (TA) of the penis causing curvature and erectile dysfunction. The pathophysiology is not well understood and, for this reason, treatment options are limited. OBJECTIVE: The aim of the present study is to analyze and compare whether single or multiple instillations of plasma in the TA of rats is capable of triggering macroscopic, histopathological, and molecular changes consistent with PD. MATERIAL/METHODS: Fifty male Wistar rats were divided into four groups: Group 1: a single instillation of plasma in the TA; Group 2: a single instillation of distilled water in the TA; Group 3: four instillations of plasma in the TA (1x per week); and Group 4: four instillations of distilled water in the TA (1× per week). Forty-five days after the last instillation a manual inspection of the corpus cavernosum, a penile erection test and a penectomy were performed to obtain material for histopathological and molecular analysis. RESULTS: It was observed that 31.25% of the rats that received repeated instillations of plasma presented penile curvature according to the erection test, while none of the rats from the control group or group with one instillation of plasma presented curvature. In the animals that received four instillations of plasma, the following differences were observed in relation to the control group: increase in fibrosis and the deposition of collagen I. The protein expression of heparanase (HPSE) and TGF-ß increased in the groups that received a single or four instillations of plasma, and the protein expression of heparanase-2 (HPSE-2), metalloproteinases (MMP-2, MMP-9) and metalloproteinase inhibitor (TIMP-2) showed an increase in the group that received four instillations of plasma. There was a significant increase in the gene expression of HPSE, MMP-9, and TGF-ß in the group that received four instillations of plasma. In the analysis of the glycosaminoglycans, an increase was observed in the secretion of galactosaminoglycans chondroitin sulfate and dermatan sulfate (CS/DS) in the group that received four instillations of plasma. DISCUSSION: Previous studies have demonstrated increased protein expression. of HPSE, MMP-9 and TGF-ß with instillation of blood in the TA; however, there was no increase in gene expression. In the present study, the increase in the expression of TGF-ß with plasma instillations, proved to be more reliable. The two models with plasma (one or four instillations) demonstrated significant histopathological and molecular changes when compared to the control group. However, only in the group with four plasma instillations there was a macroscopic change. The idea is that repeatedly extravasation of TGF-ß present in plasma of predisposed individuals acts as a trigger for the development and maintenance of changes in the extracellular matrix that perpetuate an anomalous inflammatory process present in PD. CONCLUSION: The present study shows that the repeated instillation of plasma is a low cost in vivo model for the study of PD.

Estrogen Receptor Signaling Pathways Involved in Invasion and Colony Formation of Androgen-Independent Prostate Cancer Cells PC-3.

Castration-resistant prostate cancer (CRPC) is an advanced and androgen-independent form of prostate cancer. Recent studies of rapid actions mediated by estrogen in the prostate and its relationship with CRPC are emerging. We have previously shown that estrogen receptor (ER) promotes migration and invasion of the androgen-independent prostate cancer cells PC-3, but the signaling pathways involved in these events remain to be elucidated. Therefore, this study aimed to analyze the role of ERα and ERß in the activation of SRC, and the involvement of SRC and PI3K/AKT on invasion and colony formation of the PC-3 cells. Our results showed that the activation of ERα (using ERα-selective agonist PPT) and ERß (using ERß-selective agonist DPN) increased phosphorylation of SRC in PC-3 cells. In the presence of the selective inhibitor for SRC-family kinases PP2, the effects of DPN and PPT on transmigration and soft agar colony formation assays were decreased. Furthermore, SRC is involved in the expression of the non-phosphorylated ß-catenin. Finally, using PI3K specific inhibitor Wortmannin and AKT inhibitor MK2206, we showed that PI3K/AKT are also required for invasion and colony formation of PC-3 cells simulated by ER. This study provides novel insights into molecular mechanisms of ER in PC-3 cells by demonstrating that ER, located outside the cell nucleus, activates rapid responses molecules, including SRC and PI3K/AKT, which enhance the tumorigenic potential of prostate cancer cells, increasing cell proliferation, migration, invasion, and tumor formation.

ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the delineation of HS biosynthesis.

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.

Anti-IIa activity and antitumor properties of a hybrid heparin/heparan sulfate-like compound from Litopenaeus vannamei shrimp.

In this present study, the anti-IIa activity and the antitumor properties of a hybrid heparin/heparan sulfate-like compound (sH/HS) from Litopenaeus vannamei shrimp heads are related. In addition to inhibiting 90.7% of thrombin activity at the lowest tested concentration (0.5 µg/mL), sH/HS compound stimulated the synthesis of antithrombotic heparan sulfate by endothelial cells in a dose-dependent manner. In vitro experiments demonstrated that the molecule from shrimp displayed a potent anti-angiogenic effect, reducing over 80% of the tubular structures formation at 50 and 100 µg/mL. In addition, sH/HS compound was able to inhibit the migration of B16F10 cells at all tested concentrations without affecting the cell viability. Although the studied compound had no effect on the proliferation of such cells during a period of 24 h, it had a significant long-term anti-proliferative effect, reducing about 80% of colony formation and anchorage-independent growth at 50 and 100 µg/mL concentrations. When its effectiveness was tested in vivo, it was demonstrated that sH/HS promoted a reduction of more than 90% of tumor growth. In the context of thromboembolic disorders associated with cancer, such findings make the sH/HS compound an excellent target for studies on inhibiting of development and tumor progression, and the prevention of coagulopathies.

2,3-Di-O-sulfo glucuronic acid: An unmodified and unusual residue in a highly sulfated chondroitin sulfate from Litopenaeus vannamei.

The occurrence of a natural and unmodified highly sulfated chondroitin sulfate from Litopenaeus vannamei heads (sCS) is herein reported. Its partial digestion by Chondroitinases AC and ABC together with its electrophoretic migration profile revealed it as a highly sulfated chondroitin sulfate despite its average molecular weight being similar to CSA. Using orthogonal 1D/2D NMR experiments, the anomeric signals (δ 4.62/106.0) corresponding to unusual 2,3-di-O-Sulfo-GlcA (∼36%), U33S (δ 4.42/84.1, ∼63%) and U22S (4.12/80.1, ∼50%) substitutions were confirmed. In addition, non-sulfated GlcA (δ 4.5/106.3) linked to 4-O- (A14S, 36%) or 6-O-Sulfo (A16S, 28%) GalNAc (δ 4.64/103.5) was observed. Although the biological role of sCS in shrimp is unknown, its influence on hemostasis was also demonstrated. The sCS identification brings to light new questions about the hierarchical model of GAGs biosynthesis and contributes to the better understanding of the subtle relationship between GAGs structure and function.

Extracellular matrix alterations after blood instillation in tunica albuginea of rats.

The cause of Peyronie's disease (PD) is still not completely understood. The objective of this study, therefore, was to analyze the histological and biochemical alterations that occur after the instillation of blood in the tunica albuginea (TA) of rats with an emphasis on the remodeling process of ECM. Thirty male Wistar rats were divided into 4 groups: two control groups with instillation of distilled water in TA followed by penectomy after 15 days or 45 days, respectively and two experimental groups with instillation of blood in TA followed by penectomy after 15 days or 45 days, respectively. Histological, immunofluorescent and immunohistochemical analyses were performed. The higher presence of fibrotic tissue in rats injected with blood demonstrated alterations in TA similar to inflammation found in PD. The increased expression of TGF-ß, MMP9, HPSE, and biglycan associated with the decreased expression of syndecan-1 and aggrecan in the experimental groups suggested an enhancement in the remodeling of ECM. The results contribute to show that blood instillation on TA appears to trigger alterations in the ECM similar to the ones found in inflammatory diseases such as PD.

19F labelled glycosaminoglycan probes for solution NMR and non-linear (CARS) microscopy.

Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of 19F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing 19F NMR spectroscopy is followed. Furthermore, the ability of 19F labelled GAGs to be imaged using CARS microscopy is demonstrated. 19F labelled GAGs enable both 19F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals.

Glycosaminoglycans affect heparanase location in CHO cell lines.

Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-ß-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [(35)S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.

The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth.

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

Activation of the low molecular weight protein tyrosine phosphatase in keratinocytes exposed to hyperosmotic stress.

Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.

Ultra-low-molecular-weight heparins: precise structural features impacting specific anticoagulant activities.

Ultra-low-molecular-weight heparins (ULMWHs) with better efficacy and safety ratios are under development; however, there are few structural data available. The main structural features and molecular weight of ULMWHs were studied and compared to enoxaparin. Their monosaccharide composition and average molecular weights were determined and preparations studied by nuclear magnetic resonance spectroscopy, scanning ultraviolet spectroscopy, circular dichroism and gel permeation chromatography. In general, ULMWHs presented higher 3-O-sulphated glucosamine and unsaturated uronic acid residues, the latter being comparable with their higher degree of depolymerisation. The analysis showed that ULMWHs are structurally related to LMWHs; however, their monosaccharide/oligosaccharide compositions and average molecular weights differed considerably explaining their different anticoagulant activities. The results relate structural features to activity, assisting the development of new and improved therapeutic agents, based on depolymerised heparin, for the prophylaxis and treatment of thrombotic disorders.

Glycosaminoglycan backbone is not required for the modulation of hemostasis: effect of different heparin derivatives and non-glycosaminoglycan analogs.

Heparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. In the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide, C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. In addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. The data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is not always crucial for certain activities. The results also suggest that non-GAG structures may provide an alternative route for the pharmaceutical control of hemostasis.

Structural and pharmacological profile of generic enoxaparins used in Brazil.

Generic active pharmaceutical ingredients (APIs) have been commonly used in Brazil, since 1999, but most of them are synthetic and small molecules. Recently, a large number of generic enoxaparins were introduced into the market raising concerns related to product-to-product interchangeability, efficiency, and drug counterfeiting. These drugs are produced from biological sources and their production involves complex procedures and purification processes. The present article evaluates several generic enoxaparins, structurally and pharmacologically, and compares them with the branded products. Structural analysis showed that the generic products are, indeed, quite similar to the branded products, however, this similarity cannot be extended to their pharmacological activities. The results showed that generic products must go through extensive structural, pharmacological, and clinical evaluation in order to assess their quality, efficacy and, ultimately, avoid drug counterfeiting before clinical use. Variation was also observed between the branded products, showing that such drugs must be at constant surveillance.

A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model.

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.

Heparan sulfate proteoglycans: structure, protein interactions and cell signaling.

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.

Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.

Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam sulfatos possuem papel na sinalização celular como receptores ou coreceptores para diferentes ligantes. Esta ligação dispara vias de sinalização celular levam à fosforilação de diversas proteínas citosólicas ou com ou sem interações diretas com o citoesqueleto, culminando na regulação gênica. O papel dos proteoglicanos de heparam sulfato na sinalização celular e vias de captação endocítica também são discutidas nesta revisão.