Characterization of N-cadherin association to lipid rafts in melanoma

RESUME La peau est un organe complex composé de deux parties distinctes: l'épiderme et le derme, séparé par une membrane basale. Dans la couche basale de l'épiderme, les melanocytes synthétisent la mélanine dans des mélanosomes. Les mélanosomes sont ensuite transportés des mélanocytes vers les kératinocytes, protégeant ainsi la peau des dégâts dus aux radiations U.V. La E-cadhérine assure l'adhésion entre les mélanocytes et les kératinocytes. Au cours de la transformation du mélanocyte en cellule malignes, les mélanocytes perdent l'expression de la E-cadhérine et, simultanément, se mettent à exprimer la N-cadhérine, ce phénomène est nommé « cadherin switch ». La perte de l'expression de la E-cadhérine permet au mélanocytes d'échapper au contrôle des kératinocytes, tandis que l'expression de la N-cadhérine promeut l'invasion métastasique des cellules de mélanome. Préalablement, nous avons trouvé qu'une fraction de la N-cadhérine était localisée les microdomaines membranaires spécialisés, enrichi en cholestérol et en glycosphingolipides, appelés « lipid rafts ». Une des particularité des « lipid rafts » est qu'ils sont riches en molécules permettant la transmission de signaux d'activation. De plus, des travaux récents rapportent qu'un sous-type de « lipid rafts » appelé caveolae pourrai contribuer à la progression tumorale. S'appuyant sur le rôle prépondérant de la N-cadhérine dans la progression du mélanome ainsi que sur sa présence dans les « lipid rafts », nous avons émis l'hypothèse que l'association de la N-cadhérine avec les « lipid rafts » pourrai contribuer à la progression du mélanome. Le but de ce projet à été de caractériser l'association de la Ncadhérine avec les « lipid rafts » au cours de la progression du mélanome. Au moyen de lignées cellulaires humaines, dérivées de mélanomes à différents stades de progression, nous avons trouvé que (1) la N-cadhérine est partiellement associée aux «lipid rafts » dans six lignées dérivées de mélanome en phase avancée de progression et dans des tumeurs expérimentales, mais pas dans deux lignées dérivées de mélanome à un stade plus précoce ; (2) l'association de la N-cadhérine dans les « lipid rafts » ne dépent pas de son niveau d'expression ; (3) la E-cadhérine n'est pas présente dans les « lipid rafts »d'une lignée de cellule de mélanome ayant conservé l'expression de la E-cadhérine ; (4) la localisation de la N-cadhérine dans les « lipid rafts »n'est pas modulée par les facteurs de croissance bFGF, IGF-I, et HRG1-β1, ni par des voies de signalisation impliquant MEK, PKA, les kinases de la famille Src, et PI3K ; (5) l'association de la N-cadhérine avec les « lipid rafts » n'est pas requise pour la stabilisation des jonctions adhérentes et n'est pas perturbée par la destruction de ces dernières ; (6) la N-cadhérine dans les « lipid rafts » forme un complexe avec β-caténine, p 120ctn et α-caténine. En conclusion, cette étude originale montre pour la première fois que dans des cellules de mélanome agressifs, une fraction de la N-cadhérine est localisée dans les « lipid rafts » en association avec β-caténine, p 120ctn et α-caténine. Comme la présence de la N-cadhérine dans les « lipid rafts » ne contribue pas à la formation de jonction adhérentes, cette étude suggère une nouvelle fonction pour la N-cadhérine dans les « lipid rafts ». SUMMARY Human skin is a complex organ composed of two layers separated by a basement membrane: the epidermis and the dermis. In the basal layer of the epidermis, the melanin-producing cells of the skin, the melanocytes deliver melanin-containing melanosomes to keratinocytes, thereby protecting the epidermis and the dermis from the deleterious effects of ultraviolet light. Melanocytes physically interact with keratinocytes through E-cadherin-mediated adhesion. During malignant transformation into melanoma cells, melanocytes lose E-cadherin expression and concomitantly gain expression of N-cadherin, a phenomenon referred to as "cadherin switch". Loss of E-cadherin allows melanocytes to escape the regulatory effects of neighbouring keratinocytes, while gain of N-cadherin expression promotes migration, invasion and metastatic abilities of melanoma cells. In preliminary experiments, we found that a fraction of N-cadherin localized to specialized membrane microdomains enriched in cholesterol- and glycosphingolipid, called lipid rafts. One particular feature of lipid rafts is that they are rich in signalling molecules and they possibly modulate transmembrane signalling events. Moreover, recent reports suggested that a specialized type of rafts called caveolae might contribute to tumor progression. Based on the documented role of N-cadherin in melanoma progression and its presence in lipid rafts of melanoma cells, we raised the hypothesis that the association of N-cadherin with lipid rafts might be relevant to melanoma progression. The aim of this project was to characterize N-cadherin associated to lipid rafts during melanoma progression. Using human melanoma cell lines derived from melanoma at different stages of progression, we found that (1) N-cadherin is partly associated to lipid rafts in six cell lines derived from melanomas at late stages of progression and in experimental tumors, but not in two melanoma cell lines derived from early stages; (2) N-cadherin targeting to lipid rafts does not depend on its expression level; (3) E-cadherin is not localized in lipid rafts of a melanoma cell line that retained E-cadherin expression; (4) N-cadherin localization to lipid rafts is not modulated by the growth factors bFGF, IGF-I, and HRG1-β1, nor by MEK-, PKA-, Src family kinases-, and PI3K-mediated signalling events; (5) the association of N-cadherin with lipid rafts is not required for adherens junctions stability nor it is perturbed by adherens junctions disruption; (6) N-cadherin in lipid rafts is in complex with β-catenin, p 120ctm and α-catenin. In conclusion, this study provides original evidence that in aggressive melanoma cells a pool of N-cadherin is localized in lipid rafts in association with β-catenin, p 120 and α-catenin. The presence of N-cadherin in lipid rafts independently of its involvement in adherens junctions formation, suggests a possible new role for N-cadherin recruited to lipid rafts. Further studies investigating the biological meaning of this localization promise to uncover new properties of this molecule.

Catalytic and anticancer activities of sawhorse-type diruthenium tetracarbonyl complexes derived from fluorinated fatty acids

The reaction of fluorinated fatty acids, perfluorobutyric acid (C3F7CO2H), and perfluorododecanoic acid (C11F23CO2H), with dodecacarbonyltriruthenium (Ru-3(CO)(12)) under reflux in tetrahydrofuran, followed by addition of two-electron donors (L) such as pyridine, 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane, or triphenylphosphine, gives stable diruthenium complexes Ru-2(CO)(4)((2)-(2)-O2CC3F7)(2)(L)(2) (1a, L=C5H5N; 1b, L=PTA; 1c, L=PPh3) and Ru-2(CO)(4)((2)-(2)-O2CC11F23)(2)(L)(2) (2a, L=C5H5N; 2b, L=PTA; 2c, L=PPh3). The catalytic activity of the complexes for hydrogenation of styrene under supercritical carbon dioxide has been assessed and compared to the analogous triphenylphosphine complexes with non-fluorinated carboxylato groups Ru-2(CO)(4)((2)-(2)-O2CC3H7)(2)(PPh3)(2) (3) and Ru-2(CO)(4)((2)-(2)-O2CC11H23)(2)(PPh3)(2) (4). In addition, the cytotoxicities of the fluorinated complexes 1 were also evaluated on several human cancer cell lines (A2780, A549, Me300, HeLa). The complexes appear to be moderately cytotoxic, showing greater activity on the Me300 melanoma cells. Single-crystal X-ray structure analyses of 1a and 3 show the typical sawhorse-type arrangement of the diruthenium tetracarbonyl backbone with two bridging carboxylates and two terminal ligands occupying the axial positions.

The Bcl-2/Bcl-XL inhibitor ABT-737 promotes death of retinoblastoma cancer cells.

Purpose: Retinoblastoma is a malignant tumor that usually develops in early childhood. During retinoblastoma spreading, RB1 gene inactivation is followed by additional genomic modifications which progressively lead to resistance of tumor cells to death. Drugs that act at downstream levels of death signaling pathways should therefore be interesting in killing retinoblastoma cells. ABT-737, a BH3 mimetic molecule effective at the mitochondrial level, has been shown to induce apoptosis in different human tumoral cell lines as well as in primary patient-derived cells, and in a mouse xenograph model. Methods: In this report, we analyzed the pro-death effect of ABT-737 on two human retinoblastoma cell lines, Y79 and WERI-Rb, as well as on the mouse photoreceptor cell line 661W. Results: We observed that ABT-737 was very effective as a single agent in inducing human WERI-Rb cells apoptosis without affecting the mouse 661W photoreceptor cells. However human Y79 cells were resistant to ABT-737, as a probable consequence of the absence of Bax. The high sensitivity of WERI-Rb to ABT-737 can be increased by downregulating Mcl-1 using the proteasome inhibitor MG-132. Preliminary analysis in primary mouse retinoblastoma tumoral cell lines predicts high sensitivity to ABT-737. Conclusion: Our data suggest that ABT-737 or related compounds could be a highly effective drug in the treatment of some retinoblastomas.

p21 as a transcriptional co-repressor of S-phase and mitotic control genes.

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.

Covalent binding of C3b to monoclonal antibodies selectively up-regulates heavy chain epitope recognition by T cells.

Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigen-presenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,kappa and IgG2a,kappa murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and purified in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of kappa light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigen-presenting cells.

MHC class I-related chain A conjugated to antitumor antibodies can sensitize tumor cells to specific lysis by natural killer cells.

PURPOSE: As a first step for the development of a new cancer immunotherapy strategy, we evaluated whether antibody-mediated coating by MHC class I-related chain A (MICA) could sensitize tumor cells to lysis by natural killer (NK) cells. EXPERIMENTAL DESIGN: Recombinant MICA (rMICA) was chemically conjugated to Fab' fragments from monoclonal antibodies specific for tumor-associated antigens, such as carcinoembryonic antigen, HER2, or CD20. RESULTS: Flow cytometry analysis showed an efficient coating of MICA-negative human cancer cell lines with the Fab-rMICA conjugates. This was strictly dependent on the expression of the appropriate tumor-associated antigens in the target cells. Importantly, preincubation of the tumor cells with the appropriate Fab-rMICA conjugate resulted in NK cell-mediated tumor cell lysis. Antibody blocking of the NKG2D receptor in NK cells prevented conjugate-mediated tumor cell lysis. CONCLUSIONS: These results open the way to the development of immunotherapy strategies based on antibody-mediated targeting of MICA.

The role of the δ-opioid receptor in skin homeostasis and wound healing

Au regard des agressions environnementales constantes que la peau doit endurer, l'équilibre fragile entre l'expression et la répression des gènes épidermiques, nécessaire à la différentiation et la prolifération des kératinocytes, pourrait facilement être perturbé en l'absence des mécanismes de stabilisation robustes. La présence d'un système neuroendocrinien local est donc importante afin de coordonner une réponse aux éventuelles irritations. En effet, l'expression de plusieurs neurohormones, des neurotransmetteurs et des neuropeptides, y compris des dérivés pro-opiomélanocortine comme la ß-endorphine et [Met5]-enképhaline, ainsi que l'expression du récepteur 8-opioïde (DOR) a été démontré dans la peau. Cependant, les mécanismes moléculaires par lesquels ils modulent la fonction des kératinocytes sont mal connus. Le présent travail démontre que la voie de signalisation DOR active spécifiquement la voie ERK 1/2 MAPK dans les lignées cellulaires de kératinocytes humains, inhibant la prolifération des cellules et entraîne une diminution de l'épaisseur épidermique dans un modèle organotypique de peau. De plus, l'expression de DOR retarde nettement l'induction de la kératine 10 (KRT 10) et la kératine 1 (KRT 1) dans une modèle 2D de différentiation in vitro, et supprime l'induction de KRT 10 dans un modèle organotypique de peau. Ceci est accompagné de la dérégulation de l'involucrine (IVL), la loricrine (LOR) et la fïlaggrin (FLG), résultant en une induction nettement réduite de leur expression lors de l'initiation de la différentiation in vitro. De plus, POU2F3 a été identifié comme un facteur de transcription régulant les gènes de différentiation des kératinocytes modulés par DOR. Il a été démontré que la régulation négative de POU2F3 via la voie DOR-ERK affecte les principaux aspects de la fonction des kératinocytes. Toutefois, il est évident que des facteurs supplémentaires influencent la fonctionnalité de la voie DOR elle-même. Le calcium et le contact cellule-cellule augmentent la quantité des récepteurs à la surface cellulaire des kératinocytes. Les kératinocytes dont les récepteurs sont internalisés ne répondent pas de la même manière que ceux possédant des récepteurs fonctionnels localisée à la membrane. Ce travail suggère que lors de signaux intrinsèques ou extrinsèques spécifiques, les kératinocytes sont capable de répondre via le système opioïdergique neuro-epidermique. Cette réponse doit être spatialement et temporairement contrôlée afin d'éviter un déséquilibre de l'homéostasie épidermique et un retard de cicatrisation. La compréhension de ce processus très complexe pourrait permettre à terme le développement de meilleurs traitements des affections cutanées pathologiques. En complément des études précédentes sur des souris DOR-défïcientes, ces données suggèrent que l'activation de DOR dans les kératinocytes humains influence la morphogenèse et l'homéostasie de l'épiderme, et pourrait jouer un rôle lors du processus de cicatrisation. - In view of the constant environmental assaults that the skin must endure, the delicate balance of an eloquent sequence of epidermal gene expression and repression, that is required for appropriate differentiation and proliferation of keratinocytes, might easily become derailed in the absence of robust stabilizing mechanisms. The presence of a local neuroendocrine system is thereby important to coordinate a response towards irritations. In fact, the expression of several neurohormones, neurotransmitters, and neuropeptides, including proopiomelanocortin derivatives, such as ß- endorphin and [Met5]-enkephalin has been shown in skin, as well as expression of the 6-opioid receptor (DOR). However, there is currently a lack of understanding of the molecular mechanisms by which their signalling modulates keratinocyte function. The present work demonstrates that DOR signalling specifically activates the ERK 1/2 MAPK pathway in human keratinocyte cell lines. This activation inhibits cell proliferation, resulting in decreased epidermal thickness in an organotypic skin model. Furthermore, DOR expression markedly delays induction of keratin intermediate filament Keratin 10 (KRT 10) and KRT 1 during in vitro differentiation, and abolishes the induction of KRT 10 in the organotypic skin model. This is accompanied by deregulation of involucrin (IVL), loricrin (LOR), and filaggrin (FLG), illustrated by a markedly reduced induction of their expression upon initiation of differentiation in vitro. Additionally, POU2F3 was identified as a transcription factor mediating the DOR induced regulation of keratinocyte differentiation related genes. It was revealed that DOR-mediated ERK-dependent downregulation of this factor affects key aspects of keratinocyte function. However, it is evident that additional triggers influence the functionality of the DOR itself. Calcium at concentrations above 0.1 mM and cell-cell contact both enhance the presence of receptor molecules on the keratinocytes cell surface. Keratinocytes with internalized receptor do not respond to DOR ligands in the same way as keratinocytes with a functional membrane localized receptor.

Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms.

PURPOSE: In this study, we investigated the mechanisms by which temozolomide enhances radiation response in glioblastoma cells. EXPERIMENTAL DESIGN: Using a panel of four primary human glioblastoma cell lines with heterogeneous O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression, normal human astrocytes, and U87 xenografts, we investigated (a) the relationship of MGMT status with efficacy of temozolomide-based chemoradiation using a panel of in vitro and in vivo assays; (b) underlying mechanisms by which temozolomide enhances radiation effect in glioblastoma cells; and (c) strategies to overcome resistance to radiation + temozolomide. RESULTS: Temozolomide enhances radiation response most effectively in glioblastomas without detectable MGMT expression. On concurrent radiation + temozolomide administration in MGMT-negative glioblastomas, there seems to be decreased double-strand DNA (dsDNA) repair capacity and enhanced dsDNA damage compared either with radiation alone or with sequentially administered temozolomide. Our data suggest that O(6)-benzylguanine can enhance the antitumor effects of concurrent radiation + temozolomide in MGMT-positive cells by enhancing apoptosis and the degree of dsDNA damage. O(6)-Benzylguanine was most effective when administered concurrently with radiation + temozolomide and had less of an effect when administered with temozolomide in the absence of radiation or when administered sequentially with radiation. Our in vivo data using U87 xenografts confirmed our in vitro findings. CONCLUSIONS: The present study shows that temozolomide enhances radiation response most effectively in MGMT-negative glioblastomas by increasing the degree of radiation-induced double-strand DNA damage. In MGMT-positive glioblastomas, depletion of MGMT by the addition of O(6)-benzylguanine significantly enhances the antitumor effect of concurrent radiation + temozolomide. These are among the first data showing mechanisms of synergy between radiation and temozolomide and the effect of MGMT.

Cellular delivery of pyrenyl-arene ruthenium complexes by a water-soluble arene ruthenium metalla-cage.

Three pyrenyl-arene ruthenium complexes (M(1)-M(3)) of the general formula [Ru(η(6)-arene-pyrenyl)Cl(2)(pta)] (pta = 1,3,5-triaza-7-phosphaadamantane) have been synthesised and characterised. Prior to the coordination to ruthenium, pyrene was connected to the arene ligand via an alkane chain containing different functional groups: ester (L(1)), ether (L(2)) and amide (L(3)), respectively. Furthermore, the pyrenyl moieties of the M(n) complexes were encapsulated within the hydrophobic cavity of the water soluble metalla-cage, [Ru(6)(η(6)-p-cymene)(6)(tpt)(2)(donq)(3)](6+) (tpt = 2,4,6-tri-(pyridin-4-yl)-1,3,5-triazine; donq = 5,8-dioxydo-1,4-naphthoquinonato), while the arene ruthenium end was pointing out of the cage, thus giving rise to the corresponding host-guest systems [M(n)⊂Ru(6)(η(6)-p-cymene)(6)(tpt)(2)(donq)(3)](6+) ([M(n)⊂cage](6+)). The antitumor activity of the pyrenyl-arene ruthenium complexes (M(n)) and the corresponding host-guest systems [M(n)⊂cage][CF(3)SO(3)](6) were evaluated in vitro in different types of human cancer cell lines (A549, A2780, A2780cisR, Me300 and HeLa). Complex M(2), which contains an ether group within the alkane chain, demonstrated at least a 10 times higher cytotoxicity than the reference compound [Ru(η(6)-p-cymene)Cl(2)(pta)] (RAPTA-C). All host-guest systems [M(n)⊂cage](6+) showed good anticancer activity with IC(50) values ranging from 2 to 8 μM after 72 h exposure. The fluorescence of the pyrenyl moiety allowed the monitoring of the cellular uptake and revealed an increase of uptake by a factor two of the M(2) complex when encapsulated in the metalla-cage [Ru(6)(η(6)-p-cymene)(6)(tpt)(2)(donq)(3)](6+).

The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment.

The fasting-induced adipose factor (FIAF, ANGPTL4, PGAR, HFARP) was previously identified as a novel adipocytokine that was up-regulated by fasting, by peroxisome proliferator-activated receptor agonists, and by hypoxia. To further characterize FIAF, we studied regulation of FIAF mRNA and protein in liver and adipose cell lines as well as in human and mouse plasma. Expression of FIAF mRNA was up-regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARbeta/delta agonists in rat and human hepatoma cell lines and by PPARgamma and PPARbeta/delta agonists in mouse and human adipocytes. Transactivation, chromatin immunoprecipitation, and gel shift experiments identified a functional PPAR response element within intron 3 of the FIAF gene. At the protein level, in human and mouse blood plasma, FIAF was found to be present both as the native protein and in a truncated form. Differentiation of mouse 3T3-L1 adipocytes was associated with the production of truncated FIAF, whereas in human white adipose tissue and SGBS adipocytes, only native FIAF could be detected. Interestingly, truncated FIAF was produced by human liver. Treatment with fenofibrate, a potent PPARalpha agonist, markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. Levels of both truncated and native FIAF showed marked interindividual variation but were not associated with body mass index and were not influenced by prolonged semistarvation. Together, these data suggest that FIAF, similar to other adipocytokines such as adiponectin, may partially exert its function via a truncated form.

CD1d-antibody fusion proteins target iNKT cells to the tumor and trigger long-term therapeutic responses.

Despite the well-established antitumor activity of CD1d-restricted invariant natural killer T lymphocytes (iNKT), their use for cancer therapy has remained challenging. This appears to be due to their strong but short-lived activation followed by long-term anergy after a single administration of the CD1d agonist ligand alpha-galactosylceramide (αGC). As a promising alternative, we obtained sustained mouse iNKT cell responses associated with prolonged antitumor effects through repeated administrations of tumor-targeted recombinant sCD1d-antitumor scFv fusion proteins loaded with αGC. Here, we demonstrate that CD1d fusion proteins bound to tumor cells via the antibody fragment specific for a tumor-associated antigen, efficiently activate human iNKT cell lines leading to potent tumor cell lysis. The importance of CD1d tumor targeting was confirmed in tumor-bearing mice in which only the specific tumor-targeted CD1d fusion protein resulted in tumor inhibition of well-established aggressive tumor grafts. The therapeutic efficacy correlated with the repeated activation of iNKT and natural killer cells marked by their release of TH1 cytokines, despite the up-regulation of the co-inhibitory receptor PD-1. Our results demonstrate the superiority of providing the superagonist αGC loaded on recombinant CD1d proteins and support the use of αGC/sCD1d-antitumor fusion proteins to secure a sustained human and mouse iNKT cell activation, while targeting their cytotoxic activity and cytokine release to the tumor site.

Novel Bioconjugates of Aminolevulinic Acid with Nucleosides

Esters and amino acid derivatives of 5-aminolevulinic acid (ALA) are efficient prodrugs for the production of protoporphyrin IX (PpIX), which has been used in photodynamic cancer therapy (PDT). The synthesis of novel bioconjugates combining ALA with adenosine and thymidine is reported. The novel bioconjugates have been made using a robust methodology. The new class of prodrugs contains one, two, or three ALA per molecule. Preliminary cell tests in human cancer cell lines indicate that the thymidine conjugate of ALA is an efficient prodrug for PDT.

Immunotherapies for uro-genital cancers

Les cancers du col utérin et de la vessie prennent tous deux leur origine dans les sites muqueux et peuvent évoluer lentement de lésions superficielles (lésions squameuses intra-épithéliales de bas à haut grade (HSIL) et carcinomes in situ du col utérin (CIS); ou tumeurs non musculo-invasives de la vessie (NMIBC)) à des cancers invasifs plus avancés. L'éthiologie de ces deux cancers est néanmoins très différente. Le cancer du col utérin est, à l'échelle mondiale, le deuxième cancer le plus mortel chez la femme. Ce cancer résulte de l'infection des cellules basales de l'épithélium stratifié du col utérin par le papillomavirus humain à haut risque (HPV). Les vaccins prophylactiques récemment développés contre le HPV (Gardasil® et Cervarix®) sont des moyens de prévention efficaces lorsqu'ils sont administrés chez les jeunes filles qui ne sont pas encore sexuellement actives; cependant ces vaccins ne permettent pas la régression des lésions déjà existantes. Malgré un développement actif, les vaccins thérapeutiques ciblant les oncogènes viraux E6/E7 n'ont montré qu'une faible efficacité clinique jusqu'à présent. Nous avons récemment démontré qu'une immunisation sous-cutanée (s.c.) était capable de faire régresser les petites tumeurs génitales chez 90% des souris, mais chez seulement 20% des souris présentant de plus grandes tumeurs. Dans cette étude, nous avons développé une nouvelle stratégie où la vaccination est associée à une application locale (intra-vaginale (IVAG)) d'agonistes de TLR. Celle-ci induit une augmentation des cellules T CD8 totales ainsi que T CD8 spécifiques au vaccin, mais pas des cellules T CD4. L'attraction sélective des cellules T CD8 est permise par leur expression des récepteurs de chemokines CCR5 et CXCR3 ainsi que par les ligants E-selectin. La vaccination, suivie de l'application IVAG de CpG, a conduit, chez 75% des souris, à la régression de grandes tumeurs établies. Le cancer de la vessie est le deuxième cancer urologique le plus fréquente. La plupart des tumeurs sont diagnostiquées comme NMIBC et sont restreintes à la muqueuse de la vessie, avec une forte propension à la récurrence et/ou progression après une résection locale. Afin de développer des vaccins contre les antigènes associés à la tumeur (TAA), il est nécessaire de trouver un moyen d'induire une réponse immunitaire CD8 spécifique dans la vessie. Pour ce faire, nous avons comparé différentes voies d'immunisation, en utilisant un vaccin composé d'adjuvants et de l'oncogène de HPV (E7) comme modèle. Les vaccinations s.c. et IVAG ont toutes deux induit un nombre similaire de cellules T CD8 spécifiques du vaccin dans la vessie, alors que l'immunisation intra-nasale fut inefficace. Les voies s.c. et IVAG ont induit des cellules T CD8 spécifiques du vaccin exprimant principalement aL-, a4- et le ligand d'E-selectin, suggérant que ces intégrines/sélectines sont responsables de la relocalisation des cellules T dans la vessie. Une unique immunisation avec E7 a permis une protection tumorale complète lors d'une étude prophylactique, indépendemment de la voie d'immunisation. Dans une étude thérapeutique, seules les vaccinations s.c. et IVAG ont efficacement conduit, chez environ 50% des souris, à la régression de tumeurs de la vessie établies, alors que l'immunisation intra-nasale n'a eu aucun effet. La régression de la tumeur est correlée avec l'infiltration dans la tumeur des cellules T CD8 spécifiques au vaccin et la diminution des cellules T régulatrices (Tregs). Afin d'augmenter l'efficacité de l'immunisation avec le TAA, nous avons testé une vaccination suivie de l'instillation d'agonistes de TLR3 et TLR9, ou d'un vaccin Salmonella Typhi (Ty21a). Cette stratégie a entraîné une augmentation des cellules T CD8 effectrices spécifiques du vaccin dans la vessie, bien qu'à différentes échelles. Ty21a étant l'immunostimulant le plus efficace, il mérite d'être étudié de manière plus approfondie dans le contexte du NMIBC. - Both cervical and bladder cancer originates in mucosal sites and can slowly progress from superficial lesions (low to high-grade squamous intra-epithelial lesions (HSIL) and carcinoma in situ (CIS) in the cervix; or non-muscle invasive tumors in the bladder (NMIBC)), to more advanced invasive cancers. The etiology of these two cancers is however very different. Cervical cancer is the second most common cause of cancer death in women worldwide. This cancer results from the infection of the basal cells of the stratified epithelium of the cervix by high-risk human papillomavirus (HPV). The recent availability of prophylactic vaccines (Gardasil® and Cervarix®) against HPV is an effective strategy to prevent this cancer when administered to young girls before sexual activity; however, these vaccines do not induce regression of established lesions. Despite active development, therapeutic vaccines targeting viral oncogenes E6/E7 had limited clinical efficacy to date. We recently reported that subcutaneous (s.c.) immunization was able to regress small genital tumors in 90% of the mice, but only 20% of mice had regression of larger tumors. Here, we developed a new strategy where vaccination is combined with the local (intravaginal (IVAG)) application of TLR agonists. This new strategy induced an increase of both total and vaccine-specific CD8 T cells in cervix-vagina, but not CD4 T cells. The selective attraction of CD8 T cells is mediated by the expression of CCR5 and CXCR3 chemokine receptors and E-selectin ligands in these cells. Vaccination followed by IVAG application of CpG resulted in tumor regression of large established tumors in 75% of the mice. Bladder cancer is the second most common urological malignancy. Most tumors are diagnosed as NMIBC, and are restricted to the mucosal bladder with a high propensity to recur and/or progress after local resection. Aiming to develop vaccines against tumor associated antigens (TAA) it is necessary to investigate how to target vaccine-specific T-cell immune responses to the bladder. Here we thus compared using an adjuvanted HPV oncogene (E7) vaccine, as a model, different routes of immunization. Both s.c. and IVAG vaccination induced similar number of vaccine-specific CD8 T-cells in the bladder, whereas intranasal (i.n.) immunization was ineffective. S.c. and IVAG routes induced predominantly aL-, a4- and E-selectin ligand-expressing vaccine-specific CD8 T-cells suggesting that these integrin/selectin are responsible for T-cell homing to the bladder. A single E7 immunization conferred full tumor protection in a prophylactic setting, irrespective of the immunization route. In a therapeutic setting, only ivag and s.c. vaccination efficiently regressed established bladder-tumors in ca. 50 % of mice, whereas i.n. immunization had no effect. Tumor regression correlated with vaccine- specific CD8 T cell tumor-infiltration and decrease of regulatory T cells (Tregs). To increase efficacy of TAA immunization, we tested vaccination followed by the local instillation of TLR3 or TLR9 agonist or of a Salmonella Typhi vaccine (Ty21a). This strategy resulted in an increase of vaccine-specific effector CD8 T cells in the bladder, although at different magnitudes. Ty21a being the most efficient, it deserves further investigation in the context of NMIBC. We further tested another strategy to improve therapies of NMIBC. In the murine MB49 bladder tumor model, we replaced the intravesical (ives) BCG therapy by another vaccine strain the Salmonella Ty21a. Ives Ty21a induced bladder tumor regression at least as efficiently as BCG. Ty21a bacteria did not infect nor survive neither in healthy nor in tumor-bearing bladders, suggesting its safety. Moreover, Ty21a induced a transient inflammatory response in healthy bladders, mainly through infiltration of neutrophils and macrophages that rapidly returned to basal levels, confirming its potential safety. The tumor regression was associated to a robust infiltration of immune cells, and secretion of cytokines in urines. Infection of murine tumor cell lines by Ty21a resulted in cell apoptosis. The infection of both murine and human urothelial cell lines induced secretion of in vitro inflammatory cytokines. Ty21a may be an attractive alternative for the ives treatment of NMIBC after transurethral resection and thus deserves more investigation.

Molecular characterization of the Carma1-related protein card9

ABSTRACT : Fungal infections have become a major source of diseases in immuncompromised patients, but are quite benign in healthy individuals. As fungi are eukaryotes, and share many biological processes with humans, many antifungal drugs can cause toxicity in the patients. Therefore, the characterization of signaling pathways specific to the anti-fungal immune response is relevant for the better understanding of the disease and the development of new therapeutic approaches. Dectin-1 is the major mammalian pattern recognition receptor for the fungal component zymosan. Dectin-1 is an innate non-Toll-like receptor containing immunoreceptor tyrosine-based activation motifs (ITAMs). Card9, Bc110 and Maltl are proteins that have been shown to play a key role in the Dectin-l-induced signaliñg pathway by controlling Dectin-l-mediated cell activation, cytokine production and innate anti-fungal immunity in mice. Here we investigate the role of the Card9-Bc110-Maltl complex in humans using the monocytic cell line THP-1. We show that Card9 interacts with Bc110 through a CARD-CARD interaction and that interaction of Card9 with Bc110 is required for NF-xB activation. We further demonstrate that Card9 is phosphorylated in its C-terminal part on serine residues. The phosphorylation status of Card9 can influence its ability to active NF-xB, since mutation of the phosphorylation sites increases its ability to activate NF-xB. We find that Card9 is expressed in myeloid derived cells, such as the human monocytic cell lines THP1 and U937, and in human monocyte-enriched PBLs and monocyte-derived DCs. Our findings demonstrate that Card9 is implicated in anti-fungal responses, since silencing of Card9 as well as of Bc110 and Maltl diminishes the capacity of THP1 cells to produce TNF-a in response to zymosan. Interestingly, activation of the NF-xB and MAPK pathway remained normal and levels of TNF-a mRNA produced were also not affected in THP 1 cells silenced for the expression of Card9, Bc110 or Malt1. Using a Malt1 inhibitor, we provide evidence that the proteolytic activity of Malt1 is needed for zymosan-induced TNF-a production in THP 1 cells and bone marrow-derived macrophages of mice, but further experiments are required to confirm these findings and identify the substrate(s) of Malt1. In conclusion, our results reveal an important role for Card9 in the innate immune response of human macrophages to fungi. RÉSUMÉ : Les infections fongiques sont une source majeure de maladie chez les patients immunodéprimés, alors qu'elles sont plutôt bénignes chez les individus sains. Comme les champignons sont des eucaryotes et partagent beaucoup de processus biologiques avec les humains, les médicaments antifongiques peuvent être source de toxicité chez les patients. Il est donc important de mieux caractériser les voies de signalisation intracellulaire des réponses anti-fongiques pour pouvoir développer de nouvelles approches thérapeutiques. La protéine Dectin-1 est le récepteur principal du composé fongique zymosan. Les protéines Card9, Bc110 et Maltl ont été décrites comme jouant un rôle primordial dans les signaux d'activation induits par Dectin-l, en contrôlant l'activité cellulaire, la production de cytokines et la défense anti-fongique dans les souris. Dans cette étude, nous investiguons le rôle du complexe Card9-Bc110-Maltl dans la lignée monocytaire humaine THP1. Nous montrons que Card9 interagit avec Bc110 par une interaction CARD-CARD et que cette interaction est requise pour activer le facteur de transcription NF-xB. Nous observons que Card9 est phosphorylé dans sa partie C-terminale sur des résidus serine et que l'état de phosphorylation de Card9 influence sa capacité à activer NF-xB. En effet, sa capacité à activer NF-xB est augmentée, après mutation des sites de phosphorylation. La génération d'un anticorps spécifique dirigé contre Card9 nous a permis de démontrer que Card9 est exprimé dans des cellules myéloïdes comme les lignées cellulaires monocytiques THP-1 et U-937, ainsi que dans les cellules dendritiques humaines. Nos résultats démontrent que Card9 est impliqué dans la réponse immunitaire antifongique puisque la réduction de l'expression de Card9 ainsi que de Bc110 et de Malt1 diminue la capacité des THP-1 à produire du TNF-a en réponse au zymosan. Par contre, les voies de signalisation NF-xB et MAPK ainsi que les niveaux de mRNA de TNF-a produits en réponse au zymosan ne sont pas affectés dans ces cellules. En utilisant un inhibiteur de Malt1, nous montrons que l'activité protéolytique de Malt1 est nécessaire pour la production de TNF-a induite par le zymosan dans les cellules THP-1 ainsi que dans les macrophages de souris, mais d'autres expériences seront nécessaires pour confirmer cette observation et identifier le(s) substrat(s) de Malt1 responsables de cet effet. En conclusion, nos résultats révèlent un rôle important de la protéine Card9 dans la réponse immunitaire innée antifongique dans les macrophages humains.

Isolation, structural assignment and total synthesis of Barmumycin

Barmumycin was isolated from an extract of the marine actinomycete Streptomyces sp. BOSC-022A and found to be cytotoxic against various human tumor cell lines. Based on preliminary one- and two-dimensional 1H- and 13C-NMR spectra, the natural compound was initially assigned the structure of macrolactone-type compound 1, which was later prepared by two different routes. However, major spectroscopic differences between isolated barmumycin and 1 led to revision of the proposed structure as E-16. Based on synthesis of this new compound, and subsequent spectroscopic comparison of it to an authentic sample of barmumycin, the structure of the natural compound was indeed confirmed as that of E-16.