pepstatin and Lung-Neoplasms

Topics: alpha 1-Antitrypsin; Animals; Aprotinin; Breast Neoplasms; Cathepsin B; Cathepsins; Cell Transformation, Neoplastic; Female; Humans; Laryngeal Neoplasms; Lung Neoplasms; Mice; Neoplasms; Ovarian Neoplasms; Pancreatic Neoplasms; Pepstatins; Peptide Hydrolases; Protease Inhibitors; Rats; Stomach Neoplasms

Napsin A is an intracellular aspartic protease and biomarker of various malignancies like lung adenocarcinoma and ovarian clear cell carcinoma, but its detection is usually limited to immunohistochemical techniques gaining excellent information on its distribution but missing information about posttranslational modifications (e.g. maturation state) of the protein. We present a protocol for specific enrichment of napsin A from clinical or biological specimens, that facilitates detailed analysis of the protein. By using the exceptionally broad pH range under which napsin A binds to its inhibitor pepstatin A we achieve highly selective binding of napsin A while other aspartic proteases have negligible affinity. Using this method we demonstrate that lung napsin A in many mammals is a heterogeneous enzyme with a characteristic ladder-like appearance in SDS-PAGE that might be caused by proteolytically processed N- and/or C-termini, in contrast to the more homogeneous form found in kidneys and primary lung adenocarcinoma.

Topics: Adenocarcinoma; Animals; Aspartic Acid Endopeptidases; Blotting, Western; Cattle; Guinea Pigs; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Lung; Lung Neoplasms; Mice; Pepstatins; Protein Binding; Rabbits; Rats; Sheep; Species Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Pemetrexed, a multitarget antifolate used to treat malignant mesothelioma and non-small cell lung cancer (NSCLC), has been shown to stimulate autophagy. In this study, we determined whether autophagy could be induced by pemetrexed and simvastatin cotreatment in malignant mesothelioma and NSCLC cells. Furthermore, we determined whether inhibition of autophagy drives apoptosis in malignant mesothelioma and NSCLC cells. Malignant mesothelioma MSTO-211H and A549 NSCLC cells were treated with pemetrexed and simvastatin alone and in combination to evaluate their effect on autophagy and apoptosis. Cotreatment with pemetrexed and simvastatin induced greater caspase-dependent apoptosis and autophagy than either drug alone in malignant mesothelioma and NSCLC cells. 3-Methyladenine (3-MA), ATG5 siRNA, bafilomycin A, and E64D/pepstatin A enhanced the apoptotic potential of pemetrexed and simvastatin, whereas rapamycin and LY294002 attenuated their induction of caspase-dependent apoptosis. Our data indicate that pemetrexed and simvastatin cotreatment augmented apoptosis and autophagy in malignant mesothelioma and NSCLC cells. Inhibition of pemetrexed and simvastatin-induced autophagy was shown to enhance apoptosis, suggesting that this could be a novel therapeutic strategy against malignant mesothelioma and NSCLC.

Topics: Adenine; AMP-Activated Protein Kinases; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Autophagy-Related Protein 5; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Humans; Lung Neoplasms; Macrolides; Mesothelioma; Mesothelioma, Malignant; Mice, Nude; Microtubule-Associated Proteins; Pemetrexed; Pepstatins; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Simvastatin; Time Factors; TOR Serine-Threonine Kinases; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion--exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Apoptosis; Caspase 3; Cathepsin D; Cell Line, Tumor; Cell Membrane Permeability; Glutathione; Humans; Intracellular Membranes; Lung Neoplasms; Lysosomes; Membrane Potential, Mitochondrial; Mitochondria; Pepstatins; Reactive Oxygen Species; Sesquiterpenes