The Role of CD36/GPIV in Platelet Biology.

CD36 (also known as platelet glycoprotein IV) is expressed by a variety of different cell entities, where it possesses functions as a signaling receptor, but additionally acts as a transporter for long-chain fatty acids. This dual function of CD36 has been investigated for its relevance in immune and nonimmune cells. Although CD36 was first identified on platelets, the understanding of the role of CD36 in platelet biology remained scarce for decades. In the past few years, several discoveries have shed a new light on the CD36 signaling activity in platelets. Notably, CD36 has been recognized as a sensor for oxidized low-density lipoproteins in the circulation that mitigates the threshold for platelet activation under conditions of dyslipidemia. Thus, platelet CD36 transduces atherogenic lipid stress into an increased risk for thrombosis, myocardial infarction, and stroke. The underlying pathways that are affected by CD36 are the inhibition of cyclic nucleotide signaling pathways and simultaneously the induction of activatory signaling events. Furthermore, thrombospondin-1 secreted by activated platelets binds to CD36 and furthers paracrine platelet activation. CD36 also serves as a binding hub for different coagulation factors and, thus, contributes to the plasmatic coagulation cascade. This review provides a comprehensive overview of the recent findings on platelet CD36 and presents CD36 as a relevant target for the prevention of thrombotic events for dyslipidemic individuals with an elevated risk for thrombosis.

Exostosin 1 Knockdown Induces Chemoresistance in MV3 Melanoma Cells by Upregulating JNK and MEK/ERK Signaling.

Heparan sulfate proteoglycans (HSPGs) possess various functions driving malignancy of tumors. However, their impact on tumor cell sensitivity to cytotoxic treatment is far less understood. Aiming to investigate this, we depleted HSPGs by downregulating Exostosin 1 (EXT1), a key enzyme in HS formation, or upregulating heparanase in human MV3 human melanoma cells, and investigated their response to cytotoxic drugs. Cytotoxicity of trametinib, doxorubicin, and mitoxantrone was detected by MTT assay. Insights into intracellular signaling was provided by kinome protein profiler array, and selected kinases were inhibited to investigate their impact on cell sensitization and migratory dynamics. EXT1 knockdown (EXT1kd) in MV3 cells affected the activity of doxorubicin and mitoxantrone, significantly increasing EC50 values two- or fourfold, respectively. Resistance formation was scarcely related to HSPG deficiency, suggested by enzymatic cleavage of HSPG in control cells. Notably, EXT1kd induced an upregulation of EGFR signaling via JNK and MEK/ERK, and hence blocking these kinases returned resistance to a sensitive level. JNK appeared as a key signal component, also inducing higher migratory activity of EXT1kd cells. Furthermore, EXT1kd upregulated thrombotic properties of MV3 cells, indicated by tissue factor and PAR-1 expression, functionally reflected by a stronger activation of platelet aggregation. EXT1 was confirmed to act as a tumor suppressor, shown here for the first time to affect chemosensitivity of melanoma cells.

Tumour cell-activated platelets modulate the immunological activity of CD4+, CD8+, and NK cells, which is efficiently antagonized by heparin.

Platelets, key players in haemostasis, are progressively investigated with respect to their role in immunity and inflammation. Although the platelet support to haematogenous cancer cell metastasis has been the subject of multiple studies, their impact on anti-cancer immunity remains unaddressed. Here, we investigated the immunomodulatory potential of platelets upon their activation by MDA-MB-231 breast cancer cells in various in vitro approaches. We provide evidence that platelets as well as their tumour cell-induced releasates increased the ratio of regulatory T cells, shaping an immunosuppressive phenotype in isolated CD4+ cultures. The influence on CD8+ T cells was assessed by detecting the expression of activation markers CD25/CD69 and release of cytolytic and pro-inflammatory proteins. Notably, the platelet preparations differentially influenced CD8+ T cell activation, while platelets were found to inhibit the activation of CD8+ T cells, platelet releasates, in contrast, supported their activation. Furthermore, the NK cell cytolytic activity was attenuated by platelet releasates. Low molecular weight heparin (LMWH), the guideline-based anticoagulant for cancer-associated thrombotic events, is known to interfere with tumour cell-induced platelet activation. Thus, we aimed to investigate whether, unfractionated heparin, LMWH or novel synthetic heparin mimetics can also reverse the immunosuppressive platelet effects. The releasate-mediated alteration in immune cell activity was efficiently abrogated by heparin, while the synthetic heparin mimetics partly outperformed the commercial heparin derivatives. This is the first report on the effects of heparin on rebalancing immunosuppression in an oncological context emerging as a novel aspect in heparin anti-tumour activities.

Design, synthesis, and characterization of PROTACs targeting the androgen receptor in prostate and lung cancer models.

Although the androgen receptor (AR) is a validated target for the treatment of prostate cancer, resistance to antiandrogens necessitates the development of new therapeutic modalities. Exploiting the ubiquitin-proteasome system with proteolysis-targeting chimeras (PROTACs) has become a practical approach to degrade specific proteins and thus to extend the portfolio of small molecules used for the treatment of a broader spectrum of diseases. Herein, we present three subgroups of enzalutamide-based PROTACs in which only the exit vector was modified. By recruiting cereblon, we were able to demonstrate the potent degradation of AR in lung cancer cells. Furthermore, the initial evaluation enabled the design of an optimized PROTAC with a rigid linker that degraded AR with a DC50 value in the nanomolar range. These results provide novel AR-directed PROTACs and a clear rationale for further investigating AR involvement in lung cancer models.

The Antibiotic Negamycin Crosses the Bacterial Cytoplasmic Membrane by Multiple Routes.

Negamycin is a natural pseudodipeptide antibiotic with promising activity against Gram-negative and Gram-positive bacteria, including Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus, and good efficacy in infection models. It binds to ribosomes with a novel binding mode, stimulating miscoding and inhibiting ribosome translocation. We were particularly interested in studying how the small, positively charged natural product reaches its cytoplasmic target in Escherichia coli Negamycin crosses the cytoplasmic membrane by multiple routes depending on environmental conditions. In a peptide-free medium, negamycin uses endogenous peptide transporters for active translocation, preferentially the dipeptide permease Dpp. However, in the absence of functional Dpp or in the presence of outcompeting nutrient peptides, negamycin can still enter the cytoplasm. We observed a contribution of the DppA homologs SapA and OppA, as well as of the proton-dependent oligopeptide transporter DtpD. Calcium strongly improves the activity of negamycin against both Gram-negative and Gram-positive bacteria, especially at concentrations around 2.5 mM, reflecting human blood levels. Calcium forms a complex with negamycin and facilitates its interaction with negatively charged phospholipids in bacterial membranes. Moreover, decreased activity at acidic pH and under anaerobic conditions points to a role of the membrane potential in negamycin uptake. Accordingly, improved activity at alkaline pH could be linked to increased uptake of [3H]negamycin. The diversity of options for membrane translocation is reflected by low resistance rates. The example of negamycin demonstrates that membrane passage of antibiotics can be multifaceted and that for cytoplasmic anti-Gram-negative drugs, understanding of permeation and target interaction are equally important.

Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39.

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

Another structural correction for 1-oxo-1H-phenalene-2,3-dicarbonitriles: Synthesis of a potent BCL-2 inhibiting 7-phenoxy derivative.

Aromatic scaffolds are an important part of biologically active compounds and molecular probes used to study biochemical pathways and the involved targeted proteins of interest. 1-Oxo-1H-phenalene-2,3-dicarbonitrile-based compounds have been described as inhibitors of the BCL-2 family of proteins, and this core structure represents numerous possibilities for modifications that could lead to improved inhibitory potencies. Many studies demonstrated intriguing characteristics of these compounds in terms of reactivity and, interestingly, some contradictory literature reports appeared about reaction outcomes to synthesize them. Here, we initially provide a condensed overview of transformations performed on the phenalene scaffold, followed by the resynthesis of a 6-phenoxy-substituted derivative. We show that the initial determination of this particular structure was wrong and provide two-dimensional nuclear magnetic resonance (NMR) evidence to assign the structure properly. When preparing new derivatives using the same synthetic route, we observed 6- and 7-substituted regioisomers. After confirming their structures by NMR experiments, the ability of these compounds to inhibit BCL-2 was evaluated. The most potent 1-oxo-1H-phenalene-2,3-dicarbonitrile derivatives inhibited BCL-2 in the nanomolar range and showed double-digit micromolar cytotoxicity against four different cancer cell lines.

Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells.

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.

A Combined Activity of Thrombin and P-Selectin Is Essential for Platelet Activation by Pancreatic Cancer Cells.

Pancreatic cancer patients have an elevated risk of suffering from venous thrombosis. Among several risk factors that contribute to hypercoagulability of this malignancy, platelets possess a key role in the initiation of clot formation. Although single mechanisms of platelet activation are well-known in principle, combinations thereof and their potential synergy to mediate platelet activation is, in the case of pancreatic cancer, far from being clear. Applying an inhibitor screening approach using light transmission aggregometry, dense granule release, and thrombin formation assays, we provide evidence that a combination of tissue factor-induced thrombin formation by cancer cells and their platelet P-selectin binding is responsible for AsPC-1 and Capan-2 pancreatic cancer cell-mediated platelet activation. While the blockade of one of these pathways leads to a pronounced inhibition of platelet aggregation and dense granule release, the simultaneous blockade of both pathways is inevitable to prevent platelet aggregation completely and minimize ATP release. In contrast, MIA PaCa-2 pancreatic cancer cells express reduced levels of tissue factor and P-selectin ligands and thus turn out to be poor platelet activators. Consequently, a simultaneous blockade of thrombin and P-selectin binding seems to be a powerful approach, as mediated by heparin to crucially reduce the hypercoagulable state of pancreatic cancer patients.

Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor.

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane-bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram-positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid-encoded and secreted virulence-associated protein A (VapA) participates in exclusion of the proton-pumping vacuolar-ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH-neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid-less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH-neutral and hence growth-promoting intracellular niche. VapA represents a new type of Gram-positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton-pumping ATPase, and consequently disarming host defences.

Targeting Discoidin Domain Receptor 1 (DDR1) Signaling and Its Crosstalk with ß1-integrin Emerges as a Key Factor for Breast Cancer Chemosensitization upon Collagen Type 1 Binding.

Collagen type 1 (COL1) is a ubiquitously existing extracellular matrix protein whose high density in breast tissue favors metastasis and chemoresistance. COL1-binding of MDA-MB-231 and MCF-7 breast cancer cells is mainly dependent on ß1-integrins (ITGB1). Here, we elucidate the signaling of chemoresistance in both cell lines and their ITGB1-knockdown mutants and elucidated MAPK pathway to be strongly upregulated upon COL1 binding. Notably, Discoidin Domain Receptor 1 (DDR1) was identified as another important COL1-sensor, which is permanently active but takes over the role of COL1-receptor maintaining MAPK activation in ITGB1-knockdown cells. Consequently, inhibition of DDR1 and ERK1/2 act synergistically, and sensitize the cells for cytostatic treatments using mitoxantrone, or doxorubicin, which was associated with an impaired ABCG2 drug efflux transporter activity. These data favor DDR1 as a promising target for cancer cell sensitization, most likely in combination with MAPK pathway inhibitors to circumvent COL1 induced transporter resistance axis. Since ITGB1-knockdown also induces upregulation of pEGFR in MDA-MB-231 cells, inhibitory approaches including EGFR inhibitors, such as gefitinib appear promising for pharmacological interference. These findings provide evidence for the highly dynamic adaptation of breast cancer cells in maintaining matrix binding to circumvent cytotoxicity and highlight DDR1 signaling as a target for sensitization approaches.

Insight into Cisplatin-Resistance Signaling of W1 Ovarian Cancer Cells Emerges mTOR and HSP27 as Targets for Sensitization Strategies.

The microenvironment possesses a strong impact on the tumor chemoresistance when cells bind to components of the extracellular matrix. Here we elucidate the signaling pathways of cisplatin resistance in W1 ovarian cancer cells binding to collagen type 1 (COL1) and signaling interference with constitutive cisplatin resistance in W1CR cells to discover the targets for sensitization. Proteome kinase arrays and Western blots were used to identify the signaling components, their impact on cisplatin resistance was evaluated by inhibitory or knockdown approaches. W1 cell binding to COL1 upregulates integrin-associated signals via FAK/PRAS40/mTOR, confirmed by ß1-integrin (ITGB1) knockdown. mTOR appears as key for resistance, its blockade reversed COL1 effects on W1 cell resistance completely. W1CR cells compensate ITGB1-knockdown by upregulation of discoidin domain receptor 1 (DDR1) as alternative COL1 sensor. COL1 binding via DDR1 activates the MAPK pathway, of which JNK1/2 appears critical for COL1-mediated resistance. JNK1/2 inhibition inverts COL1 effects in W1CR cells, whereas intrinsic cisplatin resistance remained unaffected. Remarkably, knockdown of HSP27, another downstream MAPK pathway component overcomes intrinsic resistance completely sensitizing W1CR cells to the level of W1 cells for cisplatin cytotoxicity. Our data confirm the independent regulation of matrix-induced and intrinsic chemoresistance in W1 ovarian cancer cells and offer novel targets for sensitization.

Glycosaminoglycans as Tools to Decipher the Platelet Tumor Cell Interaction: A Focus on P-Selectin.

Tumor cell-platelet interactions are regarded as an initial crucial step in hematogenous metastasis. Platelets protect tumor cells from immune surveillance in the blood, mediate vascular arrest, facilitate tumor extravasation, growth, and finally angiogenesis in the metastatic foci. Tumor cells aggregate platelets in the bloodstream by activation of the plasmatic coagulation cascade and by direct contact formation. Antimetastatic activities of unfractionated or low molecular weight heparin (UFH/LMWH) can undoubtedly be related to attenuated platelet activation, but molecular mechanisms and contribution of contact formation vs. coagulation remain to be elucidated. Using a set of non-anticoagulant heparin derivatives varying in size or degree of sulfation as compared with UFH, we provide insight into the relevance of contact formation for platelet activation. Light transmission aggregometry and ATP release assays confirmed that only those heparin derivatives with P-selectin blocking capacities were able to attenuate breast cancer cell-induced platelet activation, while pentasaccharide fondaparinux was without effects. Furthermore, a role of P-selectin in platelet activation and signaling could be confirmed by proteome profiler arrays detecting platelet kinases. In this study, we demonstrate that heparin blocks tumor cell-induced coagulation. Moreover, we identify platelet P-selectin, which obviously acts as molecular switch and controls aggregation and secretion of procoagulant platelets.

The Low Molecular Weight Heparin Tinzaparin Attenuates Platelet Activation in Terms of Metastatic Niche Formation by Coagulation-Dependent and Independent Pathways.

An intimate interplay with platelets is an initial key issue for tumor cells in terms of hematogenous metastasis. Tumor cells activate platelets by different pathways and receive, upon forming a platelet cloak, protection from immune surveillance and support in metastatic niche creation. Therapeutic intervention with this early interaction is promising to antagonize the whole metastatic cascade. Here we aimed to investigate the capability of low molecular weight heparin (LMWH), unfractionated heparin (UFH), and a non-anticoagulant heparin derivative or FXa inhibitor fondaparinux to interfere with platelet activation by tumor cells. Coagulation-dependent and independent pathways of platelet activation by three tumor cell lines, and interference therewith were analyzed by fluorigenic thrombin formation assay, platelet aggregometry, ATP and VEGF release and endothelial tube formation assay. LMWH and UFH were found to repress various routes of platelet activation, reflected by attenuated endothelial tube formation. This confirms the duality of anti-coagulative and anti-adhesive properties of heparin. While non-anticoagulative heparin (RO-heparin) depressed platelets' ATP and VEGF release by contact inhibition sufficiently, fondaparinux just attenuated tissue factor mediated thrombin generation. Concluding, these data suggest that LMWH as a guideline-based drug for anticoagulative strategies in oncology is promising to provide additional benefit for interference with metastatic activities.

The Impact of the Low Molecular Weight Heparin Tinzaparin on the Sensitization of Cisplatin-Resistant Ovarian Cancers-Preclinical In Vivo Evaluation in Xenograft Tumor Models.

Resistance formation of tumors against chemotherapeutics is the major obstacle in clinical cancer therapy. Although low molecular weight heparin (LMWH) is an important component in oncology referring to guideline-based antithrombotic prophylaxis of tumor patients, a potential interference of LMWH with chemoresistance is unknown. We have recently shown that LMWH reverses the cisplatin resistance of A2780cis human ovarian cancer cells in vitro. Here we address the question whether this LMWH effect is also valid under in vivo conditions. Therefore, we established tumor xenografts of A2780 and cisplatin resistant A2780cis cells in nude mice and investigated the impact of daily tinzaparin applications (10 mg/kg BW) on anti-tumor activity of cisplatin (6 mg/kg BW, weekly) considering the tumor growth kinetics. Intratumoral platinum accumulation was detected by GF-AAS. Xenografts of A2780 and A2780cis cells strongly differed in cisplatin sensitivity. As an overall consideration, tinzaparin co-treatment affected the response to cisplatin of A2780cis, but not A2780 tumors in the later experimental time range. A subgroup analysis confirmed that initially smaller A2780cis tumors benefit from tinzaparin, but also small A2780 xenografts. Tinzaparin did not affect cisplatin accumulation in A2780cis xenografts, but strongly increased the platinum content in A2780, obviously related to morphological differences in both xenografts. Although we cannot directly confirm a return of A2780cis cisplatin resistance by tinzaparin, as shown in vitro, the present findings give reason to discuss heparin effects on cytostatic drug efficiency for small tumors and warrants further investigation.

Contribution of very late antigen-4 (VLA-4) integrin to cancer progression and metastasis.

The integrin "very late antigen-4" (VLA-4) is expressed by numerous cells of hematopoietic origin and possesses a key function in the cellular immune response, e.g., by mediating leukocyte tethering, rolling, binding, and finally transmigration of the vascular wall at inflammatory sites. Thus, VLA-4 is a valuable target in medical sciences to interfere with pathological inflammations. In addition, leukemic cells and different solid tumors, which express VLA-4, make use of these adhesive functions and confer VLA-4 a progressive role in the metastatic spread. With a growing insight into the molecular mechanisms for creating a tumor-friendly microenvironment at metastatic sites and various tumor host interactions, the multiple functions of VLA-4 became evident recently, e.g., in leukocyte recruitment to micrometastases, the protection of tumors from immune surveillance, or contribution to a chemoresistance. Nevertheless, despite accumulating evidence for several functions of VLA-4 in tumorigenicity, a therapeutic interference with VLA-4 in cancer sciences has not been developed yet to the clinical level, undoubtedly by a marked impact on the physiological immune response. This review gives an up to date insight into the multiple functional role of VLA-4 in cancer and introduces this integrin as a promising target worthwhile to attract attention in biomedical cancer research.

A liposomal formulation of the synthetic curcumin analog EF24 (Lipo-EF24) inhibits pancreatic cancer progression: towards future combination therapies.

BACKGROUND: Pancreatic cancer is one of the most lethal of human malignancies known to date and shows relative insensitivity towards most of the clinically available therapy regimens. 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), a novel synthetic curcumin analog, has shown promising in vitro therapeutic efficacy in various human cancer cells, but insufficient water solubility and systemic bioavailability limit its clinical application. Here, we describe nano-encapsulation of EF24 into pegylated liposomes (Lipo-EF24) and evaluation of these particles in preclinical in vitro and in vivo model systems of pancreatic cancer. RESULTS: Transmission electron microscopy and size distribution studies by dynamic light scattering confirmed intact spherical morphology of the formed liposomes with an average diameter of less than 150 nm. In vitro, treatment with Lipo-EF24 induced growth inhibition and apoptosis in MIAPaCa and Pa03C pancreatic cancer cells as assessed by using cell viability and proliferation assays, replating and soft agar clonogenicity assays as well as western blot analyses. Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha. In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine. In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues. CONCLUSION: Due to its promising therapeutic efficacy and favorable toxicity profile Lipo-EF24 might be a promising starting point for development of future combinatorial therapeutic regimens against pancreatic cancer.

Vascular cell adhesion molecule-1 (VCAM-1)--an increasing insight into its role in tumorigenicity and metastasis.

Vascular cell adhesion molecule-1 (VCAM-1) first attracted attention more than two decades ago as endothelial adhesion receptor with key function for leukocyte recruitment in term of cellular immune response. The early finding of VCAM-1 binding to melanoma cells, and thus a suggested mechanistic contribution to metastatic spread, was the first and for a long time the only link of VCAM-1 to cancer sciences. In the last few years, hallmarked by a growing insight into the molecular understanding of tumorigenicity and metastasis, an impressive variety of VCAM-1 functionalities in cancer have been elucidated. The present review aims to provide a current overview of VCAM-1 relevance for tumor growth, metastasis, angiogenesis, and related processes. By illustrating the intriguing role of VCAM-1 in cancer disease, VCAM-1 is suggested as a new and up to now underestimated target in cancer treatment and in clinical diagnosis of malignancies.

Structural variations of the cell wall precursor lipid II and their influence on binding and activity of the lipoglycopeptide antibiotic oritavancin.

Oritavancin is a semisynthetic derivative of the glycopeptide antibiotic chloroeremomycin with activity against Gram-positive pathogens, including vancomycin-resistant staphylococci and enterococci. Compared to vancomycin, oritavancin is characterized by the presence of two additional residues, a hydrophobic 4'-chlorobiphenyl methyl moiety and a 4-epi-vancosamine substituent, which is also present in chloroeremomycin. Here, we show that oritavancin and its des-N-methylleucyl variant (des-oritavancin) effectively inhibit lipid I- and lipid II-consuming peptidoglycan biosynthesis reactions in vitro. In contrast to that for vancomycin, the binding affinity of oritavancin to the cell wall precursor lipid II appears to involve, in addition to the D-Ala-D-Ala terminus, other species-specific binding sites of the lipid II molecule, i.e., the crossbridge and D-isoglutamine in position 2 of the lipid II stem peptide, both characteristic for a number of Gram-positive pathogens, including staphylococci and enterococci. Using purified lipid II and modified lipid II variants, we studied the impact of these modifications on the binding of oritavancin and compared it to those of vancomycin, chloroeremomycin, and des-oritavancin. Analysis of the binding parameters revealed that additional intramolecular interactions of oritavancin with the peptidoglycan precursor appear to compensate for the loss of a crucial hydrogen bond in vancomycin-resistant strains, resulting in enhanced binding affinity. Augmenting previous findings, we show that amidation of the lipid II stem peptide predominantly accounts for the increased binding of oritavancin to the modified intermediates ending in D-Ala-D-Lac. Corroborating our conclusions, we further provide biochemical evidence for the phenomenon of the antagonistic effects of mecA and vanA resistance determinants in Staphylococcus aureus, thus partially explaining the low frequency of methicillin-resistant S. aureus (MRSA) acquiring high-level vancomycin resistance.

Latent heparanase facilitates VLA-4-mediated melanoma cell binding and emerges as a relevant target of heparin in the interference with metastatic progression.

Heparanase is an endo-ß-glucuronidase that enzymatically cleaves heparan sulfates (HS) and heparan sulfate proteoglycan (HSPG) structures. Heparanase expression levels by tumors were correlated with cell invasion, angiogenic activity, and poor prognosis. Heparanase can also possess pro-tumorigenic effects independent of its enzymatic activity. Using human melanoma MV3 cells, we demonstrate that latent heparanase activates in a tightly temporary-regulated manner the binding function of the integrin very late antigen-4 (VLA-4), an important component in the metastatic spread of melanoma cells. shRNA-mediated knockdown of syndecan-4 (SDC-4) indicated that this proteoglycan is the key element to convey heparanase binding via focal adhesion complex formation, detected by vinculin staining, to an upregulated VLA-4 binding function. This inside-out signaling pathway of VLA-4 involved activated FAK and Akt, but apparently not PKCα/δ. VLA-4, however, appears representative of other integrins which together impact the heparanase/integrin activation axis in tumorigenicity. Biosensor measurements provided an insight as to how heparin can interfere with this activation process. While low-molecular-weight heparin (LMWH) cannot replace heparanase bound to SDC-4, LMWH can compete with SDC-4 binding of heparanase. Since blockade of heparanase by LMWH has functional consequences for reduced VLA-4 binding, latent heparanase appears as a novel, so far unnoticed target of heparin, underlying its antimetastatic activity.