Nanoparticules et apoptose (cas des cellules tumorales pancréatiques)

Nous avons caractérisé les nanoparticules exprimées par les cellules tumorales pancréatiques tant en ce qui concerne leur composition protéique que leur composition lipidique, avec une richesse particulière en sphingomyéline et cholestérol. Les nanoparticules interagissent avec la membrane des cellules cibles, pour affecter le fonctionnement de la voie de survie Notch-1. L interaction des nanoparticules avec la cellule provoque l activation de PTEN qui constitue un complexe avec la GSK-3b inactive et l actine. Dans ce complexe la b-caténine n est pas transférée vers le noyau et ne peut induire la transcription des gènes impliqués dans la survie cellulaire. PTEN joue un rôle essentiel en activant la GSK-3b qui inhibe l activité de la PDH. L augmentation du rapport Bax/Bcl2, l activation des caspases -9 et -3, le clivage de PARP, l expression diminuée de Hes-1 et de la cycline D1 sont un ensemble d évènements conduisant la cellule à l apoptose via la voie mitochondriale.AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocSudocFranceF

Lectin-like Ox-LDL Receptor Is Expressed in Human INT-407 Intestinal Cells: Involvement in the Transcytosis of Pancreatic Bile Salt–dependent Lipase

We have recently shown that the pancreatic bile salt–dependent lipase (BSDL) can be taken up by intestinal cells and transported to the blood circulation. This mechanism likely involves (specific) receptor(s) able to bind BSDL and located at the apical intestinal cell membrane. In this study, using Int407 human intestinal cells cultured to form a tight epithelium, we attempted to characterize (the) BSDL receptor(s). We found that an apical 50-kDa protein was able to bind BSDL. Further, we have demonstrated that Int407 cells expressed the lectin-like oxidized-LDL receptor (LOX-1), the upregulation of which by oxidized-LDL potentiates the transcytosis of BSDL, whereas carrageenan and to a lesser extent polyinosinic acid and fucoidan decrease the enzyme transcytosis. The mAb JTX92, which blocks the LOX-1 receptor function, also impaired the BSDL transcytosis. To confirm these results, the cDNA encoding the human intestinal receptor LOX-1 has been cloned, inserted into vectors, and transfected into Int407 cells. Overexpression of LOX-1 by these cells leads to a substantial increase in the BSDL transcytosis. Globally, these data support the view that LOX-1 could be an intestinal receptor for BSDL, which is implicated in the transcytosis of this enzyme throughout Int407 cells

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

International audienceExosomes 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

Synthetic exosome-like nanoparticles (SELN).

<p>(A) Synthetic exosome-like nanoparticles (SELN) were examined by electron microscopy. SELN3.0, SELN4.5 and SELN6.0 preparations were incubated at room temperature, then 3 µl were removed at 0, 24 h and 48 h. SELN were then disposed on top of Formvar-coated 300-mesh carbon grids and treated with 2% phosphotungstic acid. Several fields were photographed and used to determine the diameter of SELN. Scale bars on microphotographs represent 50 nm. (B) The range of observed diameters for lipid structures as represented in A was statistically represented by the box plot. The dashed line inside the box represents the median diameter of SELN (n = 20). The box represents the interquartile range (50% of values). Tails extend to values within 1.5 times the interquartile range.</p

Lipid composition of synthetic exosome-like nanoparticles (SELN).

*<p>Data from ref. 11, given for comparison.</p>**<p>Ratio in starting lipid mix to prepare SELN of various raft lipids over non-raft lipids ratios.</p>***<p>Ratio in SELN after isolation, data are average of two independent assays.</p

Proliferation of MiaPaCa-2 and SOJ-6 cells in the presence of drugs.

<p>MiaPaCa-2 and SOJ-6 cells were incubated 1h with drugs affecting lipid metabolism at the indicated concentration then SELN6.0 (16 nmoles cholesterol/ml) were added for 24h in the presence or absence (control) of drugs at indicated concentration. At the end of incubation, proliferation was measured by MTT assay and expressed as % of control. Results are means (± SD) of independent experiments, (n = 24, Student’s <i>t</i>-test).</p

Effect of SELN on apoptosis.

<p>(A) SOJ-6 and MiaPaCa-2 cells were starved then treated for 24h with SELN (16 nmoles cholesterol/ml) and lysed. Cell lysate proteins were separated on SDS-PAGE (80 µg of proteins/lane) and electrotransferred onto nitrocellulose membrane. The levels of Bax, Bcl-2 and β-actin were determined by probing membranes with specific antibodies as indicated. Western blots are representative of three independent experiments. Lane 1; control performed in the absence of SELN, Lane 2; in the presence of SELN 3.0, Lane 3; in the presence of SELN 6.0. The right panels in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047480#pone-0047480-g004" target="_blank">figure 4A</a> represents the quantification of western blots and the ratio Bax/Bcl-2. Data are means (± SD) of three independent experiments using the NIH Image program (Mann-Whitney test). (B) Apoptosis was determined using the Terminal Transferase dUTP Nick End Labeling (TUNEL). SOJ-6 cells were starved then treated with SELN6.0 (16 nmoles cholesterol/ml) for 24h then fragmented DNA was stained according to the protocol given with the ApopTag® Red In Situ Apoptosis Detection Kit (right pictures). Nuclei were counterstained with DAPI (left pictures). Apoptotic cells were visualized under a Zeiss fluorescence microscope equipped with a digital camera. The ratio of apoptotic cells to total cells was counted in 5-to-10 random area, in both control and SELN6.0 treated SOJ-6 cells (right histogram, (means (± SD) of three independent experiments, Mann-Whitney test), scale bar = 100 µm. (C) SOJ-6 cells were incubated 4 h with medium (control), with SELN6.0 (16 nmoles of cholesterol/ml, column 2), with SELN6.0 and Z-IETD-fmk (a caspase-8 inhibitor, 10 µM, column 3) or Z-LEHD-fmk (a caspase-9 inhibitor, 10 µM, column 4). After adding freshly prepared SELN6.0, cells were incubated for another 24h. After washing and fixation, cleaved caspase-positive cells were counted under fluorescent microscopy. Results are means (± SD) of fluorescent cell amounts collected in 10 fields per assay (at least 3 independent assays). Results are expressed as percentage of cleaved caspase-positive cells relatively to controls (Mann-Whitney test).</p