l-685458 and Adenocarcinoma

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-3beta (GSK-3beta). 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 G(0)G(1) 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 gamma-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-3beta. Conversely, inhibiting either PTEN or GSK-3beta increased Hes-1 expression and partially counteracted the inhibition of proliferation promoted by exosomal nanoparticles, highlighting reciprocal regulations between Notch signaling and PTEN/GSK-3beta. 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.

Topics: Adenocarcinoma; Amyloid Precursor Protein Secretases; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Carbamates; Caspase 3; Cell Differentiation; Cell Proliferation; Dipeptides; Exosomes; Flow Cytometry; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Homeodomain Proteins; Humans; Mitochondria; Nanoparticles; Pancreatic Neoplasms; PTEN Phosphohydrolase; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factor HES-1; Tumor Cells, Cultured

Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by gamma-secretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting gamma-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that gamma-secretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells.. Three estrogen receptor (ER) positive cell lines, MCF-7, BT474, and T47D, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. Cytotoxicity was measured by using an MTS cell viability/proliferation assay. Inhibition of gamma-secretase activity was measured by both immunoblotting and immunofluorescent microscopy in order to detect active Notch1 intracellular domain. Proteasome inhibition was determined by using a cell-based proteasome activity assay kit, by immunoblotting to detect accumulation of polyubiquitylated protein, and by immunofluorescent microscopy to detect redistribution of cellular ubiquitin.. We found that blocking gamma-secretase activity by DAPT and L-685,458 had no effect on the survival and proliferation of a panel of six breast cancer cell lines while Z-LLNle-CHO could cause cell death even at concentrations that inhibited gamma-secretase activity less efficiently. Furthermore, we observed that Z-LLNle-CHO could inhibit proteasome activity and the relative cellular sensitivity of these six breast cancer cell lines to Z-LLNle-CHO was the same as observed for three proteasome inhibitors. Finally, we found that the cell killing effect of Z-LLNle-CHO could be reversed by a chemical that restored the proteasome activity.. We conclude that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.

Topics: Adenocarcinoma; Amyloid Precursor Protein Secretases; Breast Neoplasms; Carbamates; Cell Line, Tumor; Dipeptides; Drug Delivery Systems; Estrogens; Female; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptor, Notch1; Receptors, Estrogen