Dominique Lombardo

Essential role of Notch signaling in apoptosis of human pancreatic tumoral cells mediated by exosomal nanoparticles.

We previously reported that exosomal nanoparticles secreted by human pancreatic tumoral cell lines decrease tumoral cell proliferation through the mitochondria‐dependent apoptotic pathway, because of activation of pro‐apoptotic phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and of glucose synthase kinase‐3β (GSK‐3β). Interactions between exosomal nanoparticles and cells are thought to involve membrane lipid rafts. However, the underlying mechanism is unknown. Here, we report that the interaction of exosomal nanoparticles with pancreatic cancer cells led to decreased expression of hairy and enhancer‐of‐split homolog‐1 (Hes‐1), the intranuclear target of Notch‐1 signaling pathway, and to activation of the apoptotic pathway after a cell cycle arrest in G0G1 phase. Strikingly, the expression level of Notch‐1 pathway components was critical, because exosomal nanoparticles decreased the proliferation of cells in which these partners are either weakly represented, in differentiated adenocarcinoma cells, or inhibited, in poorly differentiated carcinoma cells, by blocking presenilin in the γ‐secretase complex that regulates the Notch‐1 pathway. Overexpression of Notch‐1 intracellular domain resulted in the reversion of the cell proliferation inhibition promoted by exosomal nanoparticles. Blocking presenilin unexpectedly resulted in activation of PTEN and GSK‐3β. Conversely, inhibiting either PTEN or GSK‐3β increased Hes‐1 expression and partially counteracted the inhibition of proliferation promoted by exosomal nanoparticles, highlighting reciprocal regulations between Notch signaling and PTEN/GSK‐3β. We concluded that interactions of exosomal nanoparticles with target cells, at lipid rafts where Notch‐1 pathway partners are localized, hampered the functioning of the Notch‐1 survival pathway and activated the apoptotic pathway, which determines tumoral cell fate.

Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.

Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2+/-0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 microM, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 microM), with increasing cholate concentration there was an increase in the hydrolysis of LDL- and HDL3-CE; at 100 microM cholate, the present hydrolysis per hour was 32+/-2 and 1.6+/-0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL3-TG hydrolysis was also observed, but was only approximately 5-10% of that for HDL3-CE at either 10 or 100 microM cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content.

O-Glycosylation of C-terminal tandem-repeated sequences regulates the secretion of rat pancreatic bile salt-dependent lipase.

Amino acid sequences rich in Pro, Glu, Ser, and Thr (PEST) are common to rapidly degraded proteins (Rogers, S., Wells, R. & Rechsteiner, M. (1986)Science 234, 364–368). On pancreatic bile salt-dependent lipase (BSDL), PEST sequences are present in the C-terminal region of the enzyme to which is associated theO-glycosylation. We have postulated that theO-glycosylation of BSDL may contribute to mask PEST sequences and to trigger the secretion of this enzyme instead of its delivery into a degradative pathway (Bruneau, N., and Lombardo, D. (1995) J. Biol. Chem. 270, 13524–13523). To further examine the role of the O-linked glycosylation on BSDL metabolism, rat pancreatic BSDL cDNA was stably transfected into two Chinese hamster ovary (CHO) cell lines, the CHO K1 wild-type line and the O-glycosylation defective CHO ldlD line. In these latter cells, O-glycosylation can be reversibly modulated by culture conditions. Results indicate that the rate of BSDL synthesis by transfected CHO K1 or CHO ldlD cells reflects, independently of culture conditions, the amount of mRNA specific for BSDL present in these transfected cells. Nevertheless, the rate of secretion of the enzyme depends upon cell culture conditions and increases with the cell capability to O-glycosylate C-terminal tandem-repeated sequences. Immunoprecipitation experiments performed on cell lysates suggested that a rapid degradation of BSDL occurred particularly when transfected CHO ldlD cells were cultured under non-permissive conditions. We further showed that BSDL secreted by CHO ldlD cells grown under non-permissive conditions that normally prevent O-glycosylation incorporated galactose and was reactive with peanut agglutinin, which recognizes the core structure ofO-linked glycans. We concluded that the BSDL expressed by CHO ldlD cells grown under non-permissive conditions was rapidly degraded but a fraction of the enzyme was allowed toO-glycosylate and consequently was secreted.

Exosomal Lipids Impact Notch Signaling and Induce Death of Human Pancreatic Tumoral SOJ-6 Cells

Exosomes are of increasing interest as alternative mode of cell-to-cell communication. We previously reported that exosomes secreted by human SOJ-6 pancreatic tumor cells induce (glyco)protein ligand-independent cell death and inhibit Notch-1 pathway, this latter being particularly active during carcinogenesis and in cancer stem cells. Therefore, we asked whether exosomal lipids were key-elements for cell death and hypothesized that cholesterol-rich membrane microdomains were privileged sites of exosome interactions with tumor cells. To address these questions and based on the lipid composition of exosomes from SOJ-6 cells (Ristorcelli et al. (2008) FASEB J. 22; 3358–3369) enriched in cholesterol and sphingomyelin (lipids forming liquid-ordered phase, Lo) and depleted in phospholipids (lipids forming liquid-disordered phase, Ld), we designed Synthetic Exosome-Like Nanoparticles (SELN) with ratios Lo/Ld from 3.0 to 6.0 framing that of SOJ-6 cell exosomes. SELN decreased tumor cell survival, the higher the Lo/Ld ratio, the lower the cell survival. This decreased survival was due to activation of cell death with inhibition of Notch pathway. FRET analyses indicated fusions/exchanges of SELN with cell membranes. Fluorescent SELN co-localized with the ganglioside GM1 then with Rab5A, markers of lipid microdomains and of early endosomes, respectively. These interactions occurred at lipid microdomains of plasma and/or endosome membranes where the Notch-1 pathway matures. We thus demonstrated a major role for lipids in interactions between SELN and tumor cells, and in the ensued cell death. To our knowledge this is the first report on such effects of lipidic nanoparticles on tumor cell behavior. This may have implications in tumor progression.

Histone deacetylase (HDAC) encoding gene expression in pancreatic cancer cell lines and cell sensitivity to HDAC inhibitors.

Purpose: Multiple biochemical and molecular alterations occur in pancreatic cancer cells. In the present study, attempts were made for the first time, to explore the level of expression of members of histone deacetylase encoding genes (HDACs) in four pancreatic tumor cell lines: Panc-1, BxPC-3, SOJ-6 and MiaPaCa-2; and two non-related tumor cells: Jurkat and HeLa. Furthermore, we examined the possible relationship between the levels of HDACs expression and the sensitivity/resistance to HDAC inhibitors (TSA, Nicotinamide and Sirtinol). Methods: We have used four human pancreatic tumor cell lines and two-non related tumor cells, to evaluate the expression of HDAC encoding genes by RT-PCR and Western blot analysis. We also measured the effect of certain HDAC inhibitors (HDIs) on cell growth, cell cycle alteration, membrane phosphatidyl-serine exposure, DNA fragmentation, and mitochondrial membrane potential loss.Results: We have found that although a slight variation in the profiles of gene expression among cell lines could be evidenced, HDACs protein synthesis seem to be similar. Furthermore, the cells were equally sensitive to inhibition by Sirtinol whereas some variation in the IC50 could be seen in the case of TSA. We also demonstrate that the drugs had the capacity to induce the death of cells by apoptosis. Conclusions: Taken together, our data support the notion that the level of cell sensitivity to the HDIs might be related to the level of expression of genes such as those encoding proteins playing a role in cell cycle checkpoints control but not HDAC per se.

Protective effect of resveratrol against LPS-induced extracellular lipoperoxidation in AR42J cells partly via a Myd88-dependent signaling pathway

Lipopolysaccharides (LPS) are major components of the cell wall of Gram negative bacteria implicated in the pathogenesis of bacterial infection. Resveratrol is a polyphenolic phytoalexin exhibiting antioxidant and anti-inflammatory properties. We investigated the protective effects of this natural compound on LPS-induced proinflammatory effect using non-myeloid AR42J pancreatic cells. We found that LPS dose-dependently increased extracellular malondialdehyde (MDA) and nitric oxide without affecting their intracellular level whereas resveratrol abolished all these deleterious effects. LPS increased CD14 expression; IRAK1 and a phosphorylated form of p38 MAPK protein. Resveratrol counteracted LPS effect by decreasing CD14 and IRAK1 expression but unexpectedly increased the p38 MAPK protein phosphorylation. Altogether, our data highlighted the functionality of the TLR4-Myd88 signaling pathway in LPS pro-oxidant effect using non-myeloid cells. They further suggested that resveratrol exerted antioxidant properties either by a Myd88-dependent way not involving IRAK1 or by a TRIF dependent pathway.

Saccharomyces boulardii Improves Intestinal Epithelial Cell Restitution by Inhibiting αvβ5 Integrin Activation State

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohns disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. Saccharomyces boulardii (Sb, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2β1 integrin collagen receptors. In the present study, we demonstrated that α2β1 integrin is not the sole cell-extracellular matrix receptor involved during Sb-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that Sb supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased αvβ5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, Sb-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvβ5signaling pathway. We used a monolayer wounding assay that mimics in vivo cell restitution to demonstrate that down-modulation of the αvβ5 integrin-vitronectin interaction is related to Sb-induced cell migration. We therefore postulated that Sb supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvβ5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

Resistance to cisplatin‐induced cell death conferred by the activity of organic anion transporting polypeptides (OATP) in human melanoma cells

Expression of organic anion transporting polypeptides (OATP) transporters can be modified with potential incidence in cancers, yet they have not been considered in melanoma. Here, we demonstrate transcriptional and protein expression of OATP members in human melanoma cell lines with sodium‐independent organic anion uptake activity. Importantly, uptake of different organic anions over 24 h led to a common resistance signal to apoptotic cell death, induced further by cisplatin in 24 h. The mechanism is not dependent on the transport of cisplatin by the OATP, as it is not an OATP substrate. The resistance signal was modulated by PKC, disclosing it as signal mediator. This study suggests that OATP, which can be constantly activated by endobiotics, may contribute to melanoma chemotherapeutic resistance, thereby justifying the development of OATP targeting strategies.

A Novel Tumor-Associated Pancreatic Glycoprotein Is Internalized by Human Dendritic Cells and Induces Their Maturation

Aberrant glycosylation or overexpression of cell-surface glycosylated tumor-associated Ags (TAA) distinguish neoplastic from normal cells. Interactions of TAA MUC1 and HER2/neu with dendritic cells (DC) preclude efficient processing, which impairs immune responses. It is thus important to define the mechanisms of interactions between DC and glycosylated TAA and their trafficking and processing for further T cell activation. In this work, we study interactions between DC and the oncofetal fucose-rich glycovariants of bile salt-dependent lipase (BSDL), expressed in pancreatic cancer tissues and referred to as pathological BSDL carrying the fucosylated J28 glycotope (pBSDL-J28) because it is characterized by the mAb J28. The expression of pBSDL-J28 was assessed by immunohistochemistry and quantified by confocal microscopy. Nontumoral pancreatic tissues and cells do not express pBSDL-J28. Using multidisciplinary approaches and functional studies, we provide the first evidence, to our knowledge, that this tumoral glycoprotein is rapidly internalized by human DC through macropinocytosis and endocytosis via mannose receptors and then transported to late endosomes for processing. Interestingly, pBSDL-J28 per se induced DC maturation with increased expression of costimulatory and CD83 molecules associated with cytokine secretion (IL-8 and IL-6). Surprisingly, DC retained their full ability to internalize Ags, making this maturation atypical. Finally, the allogeneic pBSDL-J28–treated DC stimulated lymphocyte proliferation. Besides, pulsing DC with pBSDL-J28 C-terminal glycopolypeptide and maturation with CD40L triggered CD4+ and CD8+ T cell proliferation. Therefore, interactions of pBSDL-J28, expressed on tumoral pancreatic tissue, with DC may lead to adequate Ag trafficking and processing and result in T cell activation.

Targeting a Novel Onco-glycoprotein Antigen at Tumoral Pancreatic Cell Surface by mAb16D10 Induces Cell Death

The mAb16D10 was raised against a pathological onco-glycoform of bile salt-dependent lipase isolated from the pancreatic juice of a patient suffering from a pancreatic adenocarcinoma. We previously showed that mAb16D10 specifically discriminates human pancreatic tumor tissues from other cancer and nontumor tissues. In this study, we report that mAb16D10 inhibited the proliferation of only human pancreatic tumor cells expressing 16D10 plasma membrane Ag. Interaction of mAb16D10 with its cognate surface Ag on pancreatic cells promoted cell death by activation of the p53- and caspase-dependent apoptotic pathway, and silencing of p53 decreased cell death. The decreased proliferation was also partly due to cell cycle arrest in G1/S phase, mAb16D10 triggering of glycogen synthase kinase-3β (GSK-3β) activation, degradation of β-catenin, and decreased expression of cyclin D1. GSK-3β positively affected p53 expression in pancreatic tumor cells after mAb16D10 binding. Inhibition of GSK-3β activity reversed the effects induced by mAb16D10 in SOJ-6 cells, supporting the pivotal role of GSK-3β signaling in the mechanisms of action induced by mAb16D10. Also, mAb16D10 cell treatment led to membrane overexpression of E-cadherin. Both E-cadherin and tumor Ag were localized in membrane lipid cholesterol-rich microdomains and are thought to belong to signaling platforms involved in the induction of cell cycle arrest and cell death. Overall, this study reveals that mAb16D10 holds great potential to prevent pancreatic tumor proliferation by apoptotic cell death, thus promising therapeutic prospects for treatment of pancreatic adenocarcinoma, a highly lethal disease.