pepstatin and Glioma

Cancer cells invade by secreting enzymes that degrade the extracellular matrix and these are sequestered in lysosomal vesicles. In this study, the effects of the selective lysosome lysing drug GPN and the lysosome exocytosis inhibitor vacuolin-1 on lysosome exocytosis were studied to determine their effect on glioma cell migration and invasion. Both GPN and vacuolin-1 evidently inhibited migration and invasion in transwell experiments and scratch experiments. There are more lysosomes located on the cell membrane of glioma cells than of astrocytes. GPN decreased the lysosome number on the cell membrane. We found that rab27A was expressed in glioma cells, and colocalized with cathepsin D in lysosome. RNAi-Rab27A inhibited lysosome cathepsin D exocytosis and glioma cell invasion in an in vitro assay. Inhibition of cathepsin D inhibited glioma cell migration. The data suggest that the inhibition of lysosome exocytosis from glioma cells plays an important modulatory role in their migration and invasion.

Topics: Antineoplastic Agents; Astrocytes; Cathepsin D; Cell Line, Tumor; Cell Movement; Dipeptides; Exocytosis; Gene Knockdown Techniques; Glioma; Heterocyclic Compounds, 4 or More Rings; Humans; Lysosomal Membrane Proteins; Lysosomes; Neoplasm Invasiveness; Pepstatins; Protein Transport; rab GTP-Binding Proteins; rab27 GTP-Binding Proteins; RNA Interference

CD44 is the major adhesion molecule for the extracellular matrix components and is implicated in a wide variety of physiological and pathological processes including the regulation of tumor cell growth and metastasis. Our previous studies have shown that CD44 undergoes sequential proteolytic cleavages in the extracellular and transmembrane domains and the cleavage product derived from CD44 intramembranous cleavage acts as a signal transduction molecule. However, the underlying mechanism of the intramembranous cleavage of CD44 remains to be elucidated. In the present study, we report for the first time that CD44 is a substrate of the presenilin (PS)-dependent gamma-secretase. We demonstrate that the intramembranous cleavage of CD44 induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment or mechanical scraping is blocked by gamma-secretase inhibitors in U251MG cells and that this cleavage is also inhibited in PS-deficient mouse embryonic fibroblasts. Furthermore, we showed that PS1 is redistributed to ruffling areas of the plasma membrane similarly to CD44 after TPA treatment, supporting our biochemical observation that PS1 is involved in the intramembranous cleavage of CD44. Our present findings suggest important implications for understanding CD44-dependent signal transduction and a potential role of PS/gamma-secretase activity in the functional regulation of adhesion molecules.

Topics: Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Carbamates; Cell Membrane; Central Nervous System Neoplasms; Dipeptides; Embryo, Mammalian; Endopeptidases; Fibroblasts; Glioma; Humans; Hyaluronan Receptors; Leupeptins; Membrane Proteins; Mice; Mice, Knockout; Pepstatins; Presenilin-1; Protease Inhibitors; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured