Weekes CD, Song D, Arcaroli J, Wilson LA, Rubio-Viqueira B, Cusatis G, Garrett-Mayer E, Messersmith WA, Winn RA, Hidalgo M. proteins. Furthermore SELN6.0 decreased the amount of NICD, which consecutively decreased the expression of Hes-1, its nuclear target. Although SELN affected the survival of human pancreatic cancer SOJ-6 cells the Notch pathway inhibition, the MiaPaCa-2 cells were particularly resistant to exosomal particles and to SELN hypothetically due to the fact that this cell line poorly expresses Notch pathway partners [10, 12]. MiaPaCa-2 cells are also resistant to gemcitabine the gold-standard drug for pancreatic cancer therapies. This intrinsic resistance of MiaPaCa-2 cells to curative drugs has been attributed to their cancer stem-like cells or initiating cells characteristics, notably the aldehyde dehydrogenase (ALDH) overexpression [13, 14]. In pancreatic cancer this ALDH-expressing cell population is particularly sensitive to cyclopamine, an inhibitor of the Hedgehog self-renewal embryonic pathway , one of the numerous misregulated signaling pathways in pancreatic cancer . We wondered whether the resistance of MiaPaCa-2 cells to SELN6.0 could be either due to a time-delayed answer to SELN6.0 or to an antagonistic effect of these lipid particles on the inhibition of the Notch-1 survival pathway. The CXCR4-SDF-1 signaling axis has been implicated in pancreatic cancer drug resistance . Therefore we hypothesized that the CXCR4-SDF-1 signaling axis could be involved in the resistance of MiaPaCa-2 cells. Here we showed that in MiaPaCa-2 SELN-resistant cells  SELN6.0 impacted on the Notch-1 pathway as already observed with SELN-sensitive SOJ-6 cells but do not affect MiaPaCa-2 cells survival. We observed that SELN6.0 induced the activation of NF-kinase (IKK/) phosphorylation at residues Ser176/Ser180 increased after 12h incubation of MiaPaCa-2 cells with SELN6.0 to reach a significant difference after 24h incubation. The phosphorylation then decreased to the basal level after 96h incubation. Meanwhile the expression of the NF-activated, ) on Ser536 (Figure ?(Figure2C)2C) and translocated to the nucleus (Figure ?(Figure3).3). These data suggested that SELN6.0 induced the activation of the NF-p65 phosphorylation with a significant activation observed after 12h incubation time. Open in a separate window Figure 2 Effects of SELN6.0 on the NF-kB signalingMiaPaCa-2 cells were grown until 60-70% confluence and starved 24h prior incubation with SELN6.0 or PBS (control). At each time supernatants were removed, cells were lysed, centrifuged 30 min at 12 000g Norfluoxetine to obtain proteins. 80 g of proteins were loaded for electrophoresis and transferred onto a nitrocellulose membrane. After saturation, the membrane has been incubated overnight with the primary antibody to Ser176/180-phosphorylated p-IKK (A) and to Ser32-phosphorylated ICXCR7 (central panel). Going further we showed that the invalidation of CXCR4 expression does not allow the reversion of the SELN6.0-conditioned medium Norfluoxetine effects on cell survival inhibition in the presence of CPA (right panel). This result demonstrates that CXCR4 is the target of SDF-1. Taken as a whole those data demonstrated that 1/the CXCR4-SDF-1 axis seems inefficient in MiaPaCa-2 cells in normal conditions (in the absence of SELN6.0), and 2/this axis is activated in the presence of SELN6.0 to reverse the CPA effects on MiaPaCa-2 cells survival. Open in a separate window Figure 7 Expression of CXCR4 and CXCR7 by MiaPaCa-2 cellsA : MiaPaCa-2 cells were seeded on 1.2 cm-diameter cover slips in Norfluoxetine 12-wells plate, once adherent cells were seeded in appropriate medium on cover-slips in 12 well-plates. Cells were fixed (2 % paraformaldehydein PBS, 37 C, 15 min) and saturated (4% BSA in PBS, 30 min). The cells were then incubated successively with the primary antibodies to CXCR4 or to CXCR7 for 90 min and then with secondary antibody to IgG coupled to AlexaFluor 488 for 45 min. The cell nuclei were labelled 30 min with 1 M Draq5, a Norfluoxetine far-red fluorescent DNA dye. All the later stages were carried out at 4C. (Scale bar = 500 m). B: Subconfluent monolayers of MiaPaCa-2 cells were harvested and suspended in DMEM containing 10 %10 % FCS during 30 min ETV4 at 37 C. The single cell suspension (106 cells/ml) was incubated for 90 min at 4C in the presence of antibodies to CXCR4 or to CXCR7. Cells were rinsed three times with ice-cold PBS and then incubated for 45 min at 4C with the appropriate secondary conjugated antibody. Cell-bound fluorescence was quantified (Flowjo program). Each value represents.