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Maxime Durand,1 Alicia Chateau,1,* Justine Jubréaux,1,* Jérôme Devy,2 Héna Paquot,1 Gautier Laurent,3 Rana Bazzi,3 Stéphane Roux,3 Nicolas Richet,4 Aurélie Reinhard-Ruch,1 Pascal Chastagner,1,5 Sophie Pinel1 1Université de Lorraine, CNRS, CRAN, Nancy, F-54000, France; 2Université de Reims-Champagne-Ardennes, UMR CNRS/URCA 7369, MEDyC, Reims, F-51100, France; 3Université Bourgogne Franche-Comté, UMR 6213 CNRS-UBFC, UTINAM, Besançon, F-25000, France; 4Université de Reims-Champagne-Ardennes, Plateau Technique Mobile de Cytométrie Environnementale MOBICYTE Mobicyte, Reims, F-51100, France; 5CHRU de Nancy, Hôpital d’enfants - Brabois, Vandoeuvre-lès-Nancy, F-54500, France*These authors contributed equally to this workCorrespondence: Sophie Pinel, Email sophie.pinel@univ-lorraine.frPurpose: This study aimed to evaluate the radiosensitizing potential of Au@DTDTPA(Gd) nanoparticles when combined with conventional external X-ray irradiation (RT) to treat GBM.Methods: Complementary biological models based on U87 spheroids including conventional 3D invasion assay, organotypic brain slice cultures, chronic cranial window model were implemented to investigate the impact of RT treatments (10 Gy single dose; 5× 2 Gy or 2× 5 Gy) combined with Au@DTDTPA(Gd) nanoparticles on tumor progression. The main tumor mass and its infiltrative area were analyzed. This work focused on the invading cancer cells after irradiation and their viability, aggressiveness, and recurrence potential were assessed using mitotic catastrophe quantification, MMP secretion analysis and neurosphere assays, respectively.Results: In vitro clonogenic assays showed that Au@DTDTPA(Gd) nanoparticles exerted a radiosensitizing effect on U87 cells, and in vivo experiments suggested a benefit of the combined treatment “RT 2× 5 Gy + Au@DTDTPA(Gd)” compared to RT alone. Invasion assays revealed that invasion distance tended to increase after irradiation alone, while the combined treatments were able to significantly reduce tumor invasion. Monitoring of U87-GFP tumor progression using organotypic cultures or intracerebral grafts confirmed the anti-invasive effect of Au@DTDTPA(Gd) on irradiated spheroids. Most importantly, the combination of Au@DTDTPA(Gd) with irradiation drastically reduced the number, the viability and the aggressiveness of tumor cells able to escape from U87 spheroids. Notably, the combined treatments significantly reduced the proportion of escaped cells with stem-like features that could cause recurrence.Conclusion: Combining Au@DTDTPA(Gd) nanoparticles and X-ray radiotherapy appears as an attractive therapeutic strategy to decrease number, viability and aggressiveness of tumor cells that escape and can invade the surrounding brain parenchyma. Hence, Au@DTDTPA(Gd)-enhanced radiotherapy opens up interesting perspectives for glioblastoma treatment.Graphical Abstract: Keywords: gold nanoparticles, radiotherapy, brain tumor, cancer cell invasion, tumor recurrence

Atomic force microscopy (AFM) enables the characterization of a wide range of samples including live cells. It is generally admitted that cancer cells are significantly softer than their normal counterparts, but imaging live cells by AFM using traditional modes can be at the cost of time or resolution. We describe how this tool can be used to estimate the motility of cancer versus normal cells, based on topographical and mechanical approaches, and coupled to optical imaging.

Melanoma is the most aggressive form of skin cancer and the most rapidly expanding cancer in terms of worldwide incidence. If primary cutaneous melanoma is mostly treated with a curative wide local excision, malignant melanoma has a poor prognosis and needs other therapeutic approaches. Angiogenesis is a normal physiological process essential in growth and development, but it also plays a crucial role in crossing from benign to advanced state in cancer. In melanoma progression, angiogenesis is widely involved during the vertical growth phase. Currently, no anti-angiogenic agents are efficient on their own, and combination of treatments will probably be the key to success. In the past, phenacetin was used as an analgesic to relieve pain, causing side effects at large dose and tumor-inducing in humans and animals. By contrast, Phenacetinum low-dilution is often used in skin febrile exanthema, patches profusely scattered on limbs, headache, or flushed face without side effects. Herein are described the in vitro, in vivo, and ex vivo anti-angiogenic and anti-tumoral potentials of Phenacetinum low-dilution in a B16F1 tumor model and endothelial cells. We demonstrate that low-diluted Phenacetinum inhibits in vivo tumor growth and tumor vascularization and thus increases the survival time of B16F1 melanoma induced-C57BL/6 mice. Moreover, Phenacetinum modulates the lung metastasis in a B16F10 induced model. Ex vivo and in vitro, we evidence that low-diluted Phenacetinum inhibits the migration and the recruitment of endothelial cells and leads to an imbalance in the pro-tumoral macrophages and to a structural malformation of the vascular network. All together these results demonstrate highly hopeful anti-tumoral, anti-metastatic, and anti-angiogenic effects of Phenacetinum low-dilution on melanoma. Continued studies are needed to preclinically validate Phenacetinum low-dilution as a complementary or therapeutic strategy for melanoma treatment.

International audience; Ultrasmall polyaminocarboxylate-coated gold nanoparticles (NPs), Au@DTDTPA and Au@TADOTAGA, that have been recently developed exhibit a promising potential for image-guided radiotherapy. In order to render the radiosensitizing effect of these gold nanoparticles even more efficient, the study of their localization in cells is required to better understand the relation between the radiosensitizing properties of the agents and their localization in cells and in tumors. To achieve this goal, post-functionalization of Au@DTDTPA nanoparticles by near-infrared (NIF) organic dyes (aminated derivative of cyanine 5, Cy5-NH2) was performed. The immobilization of organic Cy5-NH2 dyes onto the gold nanoparticles confers to these radiosensitizers fluorescence properties which can be exploited for monitoring their internalization in cancerous cells, for determining their localization in cells by fluorescence microscopy (a common and powerful imaging tool in biology), and for following up on their accumulation in tumors after intravenous injection.

LRP-1 (low-density lipoprotein receptor-related protein-1) receptor is a multifunctional endocytosis receptor that is part of the LDL receptor family. Due to its capacity to control the pericellular level of various growth factors and proteases, LRP-1 plays a crucial role in controlling the dynamics of the membrane proteome. [1] LRP-1 overexpression in breast cancer and in particular in triple negative breast cancer (TNBC), prompted us to take an interest in its involvement in tumor progression and more specifically to the role it could play in controlling angiogenesis. For that purpose, an RNA interference strategy in a TNBC model (MDA-MB-231 line) in vivo was used, based on shRNA stable expression. 42 days following an orthotopic injection of shLRP-1 MDA-MB-231 cells (in which LRP-1 expression is repressed by 70%) and shCtrl MDA-MB-231 cells (comparable to wild type MDA-MB-231 cells) in BALB/c nude mice, a delay of 40% in tumor growth was observed (257 mm3 vs 432 mm3, p Citation Format: Océane Campion, Jessica Thevenard-Devy, Nicolas Etique, Anne-Aurélie Raymond, Jean-William Dupuy, Stéphane Dedieu, Jérôme Devy. The matricellular receptor LRP-1 acts as a pro-tumorigenic receptor by supporting tumor angiogenesis in a triple negative breast cancer model [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO020.

The aim of the study was to get more insight into the role of LRP-1 in the mechanism of tumor progression in triple negative breast cancer. Atomic force microscopy, videomicroscopy, confocal microscopy and Rho-GTPAse activity assay were used on MDA-MB-231 and LRP-1-silenced cells. Silencing of LRP-1 in MDA-MB-231 cells was shown to led to a dramatic increase in the Young's modulus in parallel to a spectacular drop in membrane extension dynamics as well as a decrease in the cells migration abilities on both collagen I and fibronectin substrates. These results were perfectly correlated to a corresponding change in cell morphology and spreading capacity as well as in Rho-GTPases activity. By a multi-technique approach, it was demonstrated that LRP-1 played a crucial role in the migration of MDA-MB-231 cells by modulating the membrane extension dynamic. The originality of this AFM investigation lies in the non-invasive aspect of the measurements.

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor that interacts with advanced glycation end products, but also with C3a, CpG DNA oligonucleotides, and alarmin molecules such as HMGB1 to initiate a proinflammatory reaction. Systemic lupus erythematosus is an autoimmune disorder associated with the accumulation of RAGE ligands. We generated mice invalidated for RAGE in the lupus-prone B6-MRL Fas lpr/j background to determine the role of RAGE in the pathogenesis of systemic lupus erythematosus. We compared the phenotype of these mice with that of their wild-type and B6-MRL Fas lpr/j littermates. Lymphoproliferative syndrome, production of anti-dsDNA Abs, lupus nephritis, and accumulation of CD3+B220+CD4−CD8− autoreactive T cells (in the peripheral blood and the spleen) were significantly increased in B6-MRL Fas lpr/j RAGE−/− mice compared with B6-MRL Fas lpr/j mice (respectively p < 0.005, p < 0.05, p < 0.001, and p < 0.001). A large proportion of autoreactive T cells from B6-MRL Fas lpr/j mice expressed RAGE at their surface. Time course studies of annexin V expression revealed that autoreactive T cells in the spleen of B6-MRL Fas lpr/j-RAGE−/− mice exhibited a delay in apoptosis and expressed significantly less activated caspase 3 (39.5 ± 4.3%) than T cells in B6-MRL Fas lpr/j mice (65.5 ± 5.2%) or wild-type mice (75.3 ± 2.64%) (p = 0.02). We conclude that the deletion of RAGE in B6-MRL Fas lpr/j mice promotes the accumulation of autoreactive CD3+B220+CD4−CD8− T cells, therefore exacerbating lymphoproliferative syndrome, autoimmunity, and organ injury. This suggests that RAGE rescues the apoptosis of T lymphocytes when the death receptor Fas/CD95 is dysfunctional.

Further improvements in Photodynamic therapy (PDT) necessitate that the dye targets more selectively tumour tissues or neovascularization than healthy cells. Different enzymes such as matrix metalloproteinases (MMPs) are overexpressed in tumour areas. Among these MMPs, gelatinases (MMP-2 and MMP-9) and its activator MMP-14 are known to play a key role in tumour angiogenesis and the growth of many cancers such as glioblastoma multiforme (GBM), an aggressive malignant tumour of the brain. These last years, the concept of photodynamic molecular beacons (PMB) became interesting for controlling the photosensitizer’s ability to generate singlet oxygen (1O2) close to target biomolecules as MMPs. We report herein novel PMBs triggered by MMP-2 and/or MMP-9 and/or MMP-14, comprising a photosensitizer and a singlet oxygen quencher linked by MMP cleavable peptide linker (H-GRIGFLRTAKGG-OH). First of all, we focused on the synthesis and the photophysical study of different derivatives photosensitizer-peptide. This preliminary work concluded on an influence of the nature and the distance from the peptide, but not of the position of the photosensitizer in these derivatives on the proteolytic enzymatic action. The nature of the quencher used (a blackberry quencher (BBQ-650) or a black hole quencher (BHQ3)) does not influence the enzymatic action. We also studied the influence of an additional PEG spacer. Finally, the synthesis, the singlet oxygen quenching efficiency and the enzymatic activation of these new MMP- cleavable-PMBs were compared.

International audience; Angiogenesis plays a pivotal role in tumorigenesis and also contributes to the pathogenesis of hematologic malignancies. A number of plant compounds have shown efficacy in preclinical and clinical studies and some of them possess an anti-angiogenic activity. Our present findings report anti-angiogenic activities of ethoxyfagaronine (etxfag), a synthetic derivative of fagaronine. Once determined the non-cytotoxic concentration of etxfag, we showed that the drug inhibits VEGF-induced angiogenesis in a Matrigel™ plug assay and suppresses ex vivo sprouting from VEGF-treated aortic rings. Each feature leading to neovascularization was then investigated and results demonstrate that etxfag prevents VEGF-induced migration and tube formation in human umbilical vein endothelial cells (HUVEC). Moreover, etxfag also suppresses VEGF-induced VEGFR-2 phosphorylation and inhibits FAK phosphorylation at Y-861 as well as focal adhesion complex turnover. Beside these effects, etxfag modifies MT1-MMP localization at the endothelial cell membrane. Finally, immunoprecipitation assay revealed that etxfag decreases VEGF binding to VEGFR-2. As we previously reported that etxfag is able to prevent leukemic cell invasiveness and adhesion to fibronectin, all together our data collectively support the anti-angiogenic activities of etxfag which could represent an additional approach to current anti-cancer therapies.

International audience; Thrombospondin-1 (TSP-1) is a matricellular glycoprotein known for being highly expressed within a tumor microenvironment, where it promotes an aggressive phenotype particularly by interacting with the CD47 cell-surface receptor. While it originates from the stromal compartment in many malignancies, melanoma is an exception as invasive and metastatic melanoma cells overexpress TSP-1. We recently demonstrated that a new molecular agent that selectively prevents TSP-1 binding to CD47, called TAX2, exhibits anti-cancer properties when administered systemically by decreasing viable tumor tissue within subcutaneous B16 melanoma allografts. At the same time, emerging evidence was published suggesting a contribution of TSP-1 in melanoma metastatic dissemination and resistance to treatment. Through a comprehensive systems biology approach based on multiple genomics and proteomics databases analyses, we first identified a TSP-1-centered interaction network that is overexpressed in metastatic melanoma. Then, we investigated the effects of disrupting TSP-1:CD47 interaction in A375 human malignant melanoma xenografts. In this model, TAX2 systemic administrations induce tumor necrosis by decreasing intra-tumoral blood flow, while concomitantly making tumors less infiltrative. Besides, TAX2 treatment also drastically inhibits B16F10 murine melanoma cells metastatic dissemination and growth in a syngeneic experimental model of lung metastasis, as demonstrated by histopathological analyses as well as longitudinal and quantitative µCT follow-up of metastatic progression. Altogether, the results obtained by combining bioinformatics and preclinical studies strongly suggest that targeting TSP-1/CD47 axis may represent a valuable therapeutic alternative for hampering melanoma spreading.

A method was developed to characterize the adhesion properties of single cells by using protein-functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin-functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM-based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.

Background: Elastin peptides possess several biological activities and in vitro data suggest they could be involved in the early phase of melanoma growth. Methods: Using diverse in vitro and in vivo techniques (cell proliferation, invasion and migration assays, zymography, western blots, collagen degradation assay, reverse transcription PCR, melanoma allographs and immunohistochemistry), we analysed the effect of elastin-derived peptides (EDPs) on B16F1 melanoma growth and invasion, as well as on the proteolytic systems involved. Results: We found that EDPs dramatically promote in vivo tumour development of B16F1 melanoma, as well as their in vitro migration and invasion. The inhibition of serine proteases and matrix metalloproteinases (MMPs) activities, by aprotinin and galardin, respectively, demonstrated that these enzymes were involved in these processes. However, we found that EDPs did not increase urokinase-type plasminogen activator, tissue-type plasminogen activator or MMP-2 expression and/or activation, neither in vitro nor in vivo. Nevertheless, we observed a strong increase of pro-MMP-9 secretion in EDPs-treated tumours and, more importantly, an increase in the expression and activation of the murine counterpart of MMP-1, named murine collagenase-A (Mcol-A). Moreover, we show that plasminogen system inhibition decreases collagen degradation by this enzyme. Finally, the use of a specific blocking antibody against Mcol-A abolished EDP-induced B16F1 invasion in vitro, showing that this MMP was directly involved in this process. Conclusion: Our data show that in vivo, EDPs are involved in melanoma growth and invasion and reinforced the concept of elastin fragmentation as a predictive factor.

Besides its ability to inhibit MMP activity, TIMP-1 exhibits other biological functions. We earlier reported that TIMP-1 induced UT-7 erythroid cell survival through activation of the JAK2/PI 3-kinase/Akt pathway and we now aim to determine whether the TIMP-1 anti-apoptotic effect requires MMP involvement. We first show that proMMP-9 was expressed in UT-7 cells and associated with the cell plasma membrane. Such proMMP-9 localization was crucial for TIMP-1 intracellular signalling since (i) TIMP-1 specifically bound to proMMP-9 and (ii) proMMP-9 silencing abrogated the TIMP-1 effect. We also demonstrated that TIMP-1 anti-apoptotic effect was independent on MMP inhibition since MMP-9 function blocking antibodies as well as a synthetic MMP inhibitor were unable to reproduce TIMP-1 effect. Nevertheless, these compounds prevented TIMP-1 binding to proMMP-9 and subsequently abolished TIMP-1-induced cell survival. We finally demonstrated that CD44 anchored proMMP-9 to the plasma membrane and enabled TIMP-1-mediated signal transduction. Therefore, our results indicate that the anti-apoptotic signalling of TIMP-1 depends on the formation of a ternary complex between TIMP-1, proMMP-9 and CD44 at the UT-7 erythroid cell surface.

Low-density lipoprotein receptor-related protein 1 (LRP-1) can internalize proteases involved in cancer progression and is thus considered a promising therapeutic target. However, it has been demonstrated that LRP-1 is also able to regulate the endocytosis of membrane-anchored proteins. Thus, strategies that target LRP-1 to modulate proteolysis could also affect adhesion and cytoskeleton dynamics. Here, we investigated the effect of LRP-1 silencing on parameters reflecting cancer cells' invasiveness by atomic force microscopy (AFM). The results show that LRP-1 silencing induces changes in the cells' adhesion behavior, particularly the dynamics of cell attachment. Clear alterations in morphology, such as more pronounced stress fibers and increased spreading, leading to increased area and circularity, were also observed. The determination of the cells' mechanical properties by AFM showed that these differences are correlated with an increase in Young's modulus. Moreover, the measurements show an overall decrease in cell motility and modifications of directional persistence. An overall increase in the adhesion force between the LRP-1-silenced cells and a gelatin-coated bead was also observed. Ultimately, our AFM-based force spectroscopy data, recorded using an antibody directed against the β1 integrin subunit, provide evidence that LRP-1 silencing modifies the rupture force distribution. Together, our results show that techniques traditionally used for the investigation of cancer cells can be coupled with AFM to gain access to complementary phenotypic parameters that can help discriminate between specific phenotypes associated with different degrees of invasiveness.

Curcumin, the active ingredient from the spice turmeric (Curcuma longa Linn), is known to be an anti-oxidant and an anti-inflammatory agent. It has been demonstrated recently to possess anti-angiogenic effects and pro-apoptotic activities against Ehrlich ascites tumor cells. In the current study, curcumin was found to be cytotoxic in vitro for B16-R melanoma cells resistant to doxorubicin either cultivated as monolayers or grown in three-dimensional (3-D) cultures (spheroids). We have demonstrated that the cytotoxic effect observed in the 2 culture types can be related to the induction of programmed cell death. In our in vivo studies, we examined the effectiveness of a prophylactic immune preparation of soluble proteins from B16-R cells, or a treatment with curcumin as soon as tumoral appearance, alone or in combination, on the murine melanoma B16-R. The combination treatment resulted in substantial inhibition of growth of B16-R melanoma, whereas each treatment by itself showed little effect. Moreover, animals receiving the combination therapy exhibited an enhancement of their humoral anti-soluble B16-R protein immune response and a significant increase in their median survival time (>82.8% vs. 48.6% and 45.7% respectively for the immunized group and the curcumin-treated group). Our study shows that curcumin may provide a valuable tool for the development of a therapeutic combination against the melanoma. © 2004 Wiley-Liss, Inc.

// Albin Jeanne 1, 2, 3 , Emilie Sick 1, 4 , Jerome Devy 1, 2 , Nicolas Floquet 5 , Nicolas Belloy 2, 6 , Louis Theret 1, 2 , Camille Boulagnon-Rombi 2, 7 , Marie-Daniele Diebold 2, 7 , Manuel Dauchez 2, 6 , Laurent Martiny 1, 2 , Christophe Schneider 1, 2 , Stephane Dedieu 1, 2 1 Universite de Reims Champagne-Ardenne, Laboratoire SiRMa, UFR Sciences Exactes et Naturelles, Reims, France 2 CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire, MEDyC, Reims, France 3 SATT Nord, Lille, France 4 Universite de Strasbourg, CNRS UMR 7213, Illkirch, France 5 Institut des Biomolecules Max Mousseron (IBMM), CNRS UMR 5247, Universite de Montpellier, Ecole Normale Superieure de Chimie de Montpellier, Faculte de Pharmacie, Montpellier, France 6 Plateforme de Modelisation Moleculaire Multi-echelle (P3M), Universite de Reims Champagne-Ardenne, Reims, France 7 CHU de Reims, Laboratoire Central d’Anatomie et de Cytologie Pathologiques, Reims, France Correspondence to: Stephane Dedieu, e-mail: stephane.dedieu@univ-reims.fr Keywords: TSP-1, CD47, CD36, cancer, angiogenesis Received: March 24, 2015 Accepted: May 09, 2015 Published: May 22, 2015 ABSTRACT The multi-modular glycoprotein thrombospondin-1 (TSP-1) is considered as a key actor within the tumor microenvironment. Besides, TSP-1 binding to CD47 is widely reported to regulate cardiovascular function as it promotes vasoconstriction and angiogenesis limitation. Therefore, many studies focused on targeting TSP-1:CD47 interaction, aiming for up-regulation of physiological angiogenesis to enhance post-ischemia recovery or to facilitate engraftment. Thus, we sought to identify an innovative selective antagonist for TSP-1:CD47 interaction. Protein-protein docking and molecular dynamics simulations were conducted to design a novel CD47-derived peptide, called TAX2. TAX2 binds TSP-1 to prevent TSP-1:CD47 interaction, as revealed by ELISA and co-immunoprecipitation experiments. Unexpectedly, TAX2 inhibits in vitro and ex vivo angiogenesis features in a TSP-1-dependent manner. Consistently, our data highlighted that TAX2 promotes TSP-1 binding to CD36-containing complexes, leading to disruption of VEGFR2 activation and downstream NO signaling. Such unpredicted results prompted us to investigate TAX2 potential in tumor pathology. A multimodal imaging approach was conducted combining histopathological staining, MVD, MRI analysis and μCT monitoring for tumor angiography longitudinal follow-up and 3D quantification. TAX2 in vivo administrations highly disturb syngeneic melanoma tumor vascularization inducing extensive tumor necrosis and strongly inhibit growth rate and vascularization of human pancreatic carcinoma xenografts in nude mice.

DNA aggregation by polyamines has acquired importance as a prerequisite for the cellular uptake of DNA for gene therapy. Intracellular polyamines are constitutive components of mammalian cells and their availability is critical for cell proliferation. Interference of polyamine biosynthesis by synthetic polyamines leads to cytotoxicity. Optimization of the polyamine structural parameters is necessary to control their DNA aggregation, cytotoxic or enzyme inhibitory activities. We designed two series of tetra- and hexamines and compared their human DNA topoisomerase I (top1) inhibitory effects with the DNA aggregation properties. We show that hexamines are more efficient inhibitors of DNA relaxation by top1 than tetramines and that they suppress the top1-mediated DNA cleavage while tetramines do not. The DNA aggregation abilities within two series of polyamines correlate with the length of their central methylene chain. By contrast, the top1 inhibition within two series does not show the same correlation but demonstrates a threshold inhibitory effect on going from the (CH(2))(12) to the (CH(2))(14) central chain. We show further that the structures of DNA aggregates formed by polyamines with the (CH(2))(10-12) or with the (CH(2))(14-16) chains are very different. The first are a fluid cholesteric-type phases, whereas the second are well-structured aggregates similar to columnar liquid crystals with high packing density of DNA duplexes. The structures of polyamines-induced DNA aggregates are proposed to be crucial for top1 catalysis. The structure-function correlation described here may serve as a guide for rational design of polyamines with desired DNA-aggregation or anti-top1 activities.

DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016–50 μm concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(−1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH2 group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects.

International audience; Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the extracellular matrix turnover by inhibiting the proteolytic activity of matrix metalloproteinases (MMPs). TIMP-1 also displays MMP-independent activities that influence the behavior of various cell types including neuronal plasticity, but the underlying molecular mechanisms remain mostly unknown. The trans-membrane receptor low-density lipoprotein receptor-related protein-1 (LRP-1) consists of a large extracellular chain with distinct ligand-binding domains that interact with numerous ligands including TIMP-2 and TIMP-3 and a short transmembrane chain with intracellular motifs that allow endocytosis and confer signaling properties to LRP-1. We addressed TIMP-1 interaction with recombinant ligand-binding domains of LRP-1 expressed by CHO cells for endocytosis study, or linked onto sensor chips for surface plasmon resonance analysis. Primary cortical neurons bound and internalized endogenous TIMP-1 through a mechanism mediated by LRP-1. This resulted in inhibition of neurite outgrowth and increased growth cone volume. Using a mutated inactive TIMP-1 variant we showed that TIMP-1 effect on neurone morphology was independent of its MMP inhibitory activity. We conclude that TIMP-1 is a new ligand of LRP-1 and we highlight a new example of its MMP-independent, cytokine-like functions.

Background NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far. Methodology/Principal Findings We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (–38%) and invasive (–52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (–80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (KD = 148±9.54 nM). Conclusion/Significance Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine.

The low-density lipoprotein receptor-related protein 1 (LRP-1) is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. In the field of cancer, LRP-1-mediated endocytosis was first associated with antitumor properties. However, recent results suggested that LRP-1 may coordinate the adhesion-deadhesion balance in malignant cells to support tumor progression. Here, we observed that LRP-1 silencing or RAP (receptor-associated protein) treatment led to accumulation of CD44 at the tumor cell surface. Moreover, we evidenced a tight interaction between CD44 and LRP-1, not exclusively localized in lipid rafts. Overexpression of LRP-1-derived minireceptors indicated that the fourth ligand-binding cluster of LRP-1 is required to bind CD44. Labeling of CD44 with EEA1 and LAMP-1 showed that internalized CD44 is routed through early endosomes toward lysosomes in a LRP-1-dependent pathway. LRP-1-mediated internalization of CD44 was highly reduced under hyperosmotic conditions but poorly affected by membrane cholesterol depletion, revealing that it proceeds mostly via clathrin-coated pits. Finally, we demonstrated that CD44 silencing abolishes RAP-induced tumor cell attachment, revealing that cell surface accumulation of CD44 under LRP-1 blockade is mainly responsible for the stimulation of tumor cell adhesion. Altogether, our data shed light on the LRP-1-mediated internalization of CD44 that appeared critical to define the adhesive properties of tumor cells.

Quaternary benzo[c]phenanthridines such as fagaronine are natural substances which have been reported to exhibit anticancer and anti-leukemic properties. However, the therapeutic use of these molecules is limited due to the high dose required to exhibit anti-tumor activity and subsequent toxicity. In this study, we describe the therapeutic potential of a new derivative of fagaronine, Ethoxyfagaronine (N-methyl-12-ethoxy-2hydroxy-3, 8, 9-trimethoxybenzo[c]-phenanthridiniumchlorhydrate) as an anti-leukemic agent. Cytotoxic activity and cell growth inhibition of Ethoxyfagaronine (Etxfag) was tested on murine L1210 leukemia cells using trypan blue assay and MTT assay. At the concentration of 10(-7) M, Etxfag induced less than 10% of cell death. Etxfag (10(-7) M) was tested on L1210 cell invasiveness using matrigel™ precoated transwell chambers and efficiently reduces the invasive potential of L1210 cells by more than 50% as compared with untreated cells. Western blot and immunofluorescence experiments showed that Etxfag decreased both MT1-MMP expression and activation at the cell surface, decreased plasmin activity by down-regulating u-PAR and uPA expression at the cell surface and increasing PAI-1 secretion in conditioned media. The set of our findings underscore the therapeutic potential of ethoxyfagaronine as a new potential anticancer agent able to prevent leukemic cell dissemination.

International audience; We previously demonstrated that the CNYYSNS peptide derived from tumstatin inhibited in vivo tumor progression. The YSNS motif formed a β-turn crucial for biological activity. More recently, a YSNSG cyclopeptide with a constrained β-turn on the YSNS residues was designed. Intraperitoneal administration of the YSNSG cyclopeptide inhibited in vivo melanoma progression more efficiently than the native linear peptide. In the present article, we showed that the YSNSG cyclopeptide also triggered an inhibition of in vivo tumor neovascularization and we further analyzed its in vitroantiangiogenic effect. The YSNSG cyclopeptide did not alter endothelial cell proliferation but inhibited cell migration by 83% in an in vitro wound healing model. The inhibition was mediated by a decrease in active MT1-MMP at the migration front as well as a decrease in u-PA and u-PAR expression. The cyclopeptide also altered β1-integrin distribution in endothelial cell lamellipodia, induced a strong decrease in the phosphorylated focal adhesion kinase (p125FAK), disorganized F-actin stress fibers and decreased the number of lamellipodia, resulting in a non migratory phenotype. Our results confirm the YSNSG cyclopeptide as a potent antitumor agent, through both the inhibition of invasive properties of tumor cells and the antiangiogenic activity.

In situ hybridization (ISH) associated to immunohistochemistry has become a powerful tool for the examination of gene expression. Application of these techniques in grapevine tissues is limited mainly because of technical difficulties with this plant material. Here we present detailed protocols for ISH and immunohistochemistry, recommended controls and troubleshooting, along with examples of several applications to grapevine tissues.

The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.

The objective of this work was to prepare microcapsules which would allow protection and slow release of antigens used for melanoma immunotherapy treatment. Hydroxyethylstarch (HES) microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride (TC). They were characterized with respect to morphology (microscopy) and size (in the 4-15 microm range). Bovine serum albumin (BSA) was used as model protein for loading and release studies. Microcapsules were loaded with solutions at different protein concentrations (0.5-5%). The maximum loading efficiency (20%) was observed with the concentration of 2.5%, which allowed a loading capacity near 100%. Confocal laser scanning microscopy (CLSM) visualization showed that BSA was entrapped within the microcapsules and not only associated to their outer surface. BSA-release studies showed a 20% BSA release within 30 min while 80% remained entrapped in the microcapsules for 4 days. Microcapsules were degraded by alpha-amylase and addition of esterase to alpha-amylase enhanced slightly their degradation. In vitro studies on melanoma cells showed that HES microcapsules were non-toxic. Preliminary in vivo studies demonstrated that microcapsules were biodegradable after intraperitoneal injection (i.p.). The observation of peritoneal wash showed a complete degradation within 7 days, indicating a possible application as an in vivo drug delivery system especially to enhance the presentation of antigens.

Multicellular spheroids are known to be the most adapted model to keep the in vitro resistance properties of cells. This in vivo-like tissue-culture representation was applied to investigate the immune reactivity of MCF-7 cells by monocytes.Human blood monocytes, obtained by elutriation, were co-cultured with multicellular tumor spheroids of drug-sensitive (MCF-7S) and doxorubicin-resistant (MCF-7DXR) MCF-7 breast cancer cells.Tumor cells, according to their phenotype, induced differential recruitment and behavior of the immune cells towards the two types of spheroids. The secretion of various cytokines and the expression of several adhesion molecules were analysed. The MCF-7DXR/monocytes co-culture supernatant showed higher levels of IL-6 and IL-8 than the MCF-7S/monocytes co-culture supernatant. Cells from the MCF-7DXR spheroids expressed some adhesion molecules, CD-44 and CD-54, leading to a strong cellular cohesion in comparison with the sensitive spheroids.The two spheroid phenotypes represented an excellent model system for determining the precise tumor microenvironment in which cells move, the crucial molecular requirements and the mechanisms by which immunotherapeutic strategies could be developed to eradicate chemo-resistant tumors.

International audience; A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shellnanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluo-rescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys werestabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs con-jugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We furtherdemonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocalanalysis ofp-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7rbreast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant tophotobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively.The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D imagesof the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained bythe method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, usingimmunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studiesof membrane proteins and cells.

Highly Stable Fluorescent Nanocrystals as a Novel Class of Labels for Immunohistochemical Analysis of Paraffin-Embedded Tissue Sections

New antitumor 12-alkoxy-benzo[c]phenanthridinium derivatives were obtained in high yields through multistep syntheses. Analysis of DNA binding and human DNA topoisomerase I inhibitory activities demonstrates that new compounds, combining 2, 6, and 12 substitutions, interact strongly with DNA and exhibit important topoisomerase I inhibition. The cytotoxicities against solid tumor cell lines are also determined and compared with those for fagaronine and ethoxidine.

Antitumor benzo[c]phenanthiridines (fig. I) fagaronine (fag), isolated from the roots of Fagarara zanthoxyloides Lam. (Rutaceae), and its derivative ethoxidine (ethox) interfere with the human DNA topoisomerase (topo) I cleavage complexes and are potent DNA intercalators [1, 2]. Our recent data show that both fag and ethox inhibit topo I but in two different mechanisms. Fag traps topo I-DNA cleavage complex being the topo I-“poison”. Ethox is a potent suppresser of DNA cleavage by topo I being a modulator of site-specific recognition of DNA by the enzyme. Both drugs are known to be typical intercalators whereas the data on their sequence-specificity are contradictory. DNA recognition and cleavage by topo I is known to be extremely sequence-specific: cleavage is processed in the sequences corresponding to the so-called “topo I-specific” sites which includes obligatory T-base in position (-1) downstream in the sequence. So, the goal of this work was to analyse molecular basis of site-specific interactions between the drugs and DNA, using different DNA sequences.