pepstatin and Cell-Transformation--Neoplastic

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

We have established several glioma-relevant oncogene-engineered cancer cells to reevaluate the oncogene-selective cytotoxicity of previously well-characterized anticancer drugs, such as etoposide, doxorubicin, staurosporine, and carmustine. Among several glioma-relevant oncogenes (activated epidermal growth factor receptor, Ras, and Akt, as well as Bcl-2 and p53DD used in the present study), the activated epidermal growth factor receptor, Ras, and Akt exerted oncogenic transformation of Ink4a/Arf(-/-) murine astrocyte cells. We identified that etoposide, a topoisomerase II inhibitor, caused selective killing of myristylated Akt (Akt-myr)-transduced Ink4a/Arf(-/-) astrocytes and U87MG cells in a dose- and time-dependent manner. Etoposide-selective cytotoxicity in the Akt-myr-transduced cells was shown to be caused by nonapoptotic cell death and occurred in a p53-independent manner. Etoposide caused severe reactive oxygen species (ROS) accumulation preferentially in the Akt-myr-transduced cells, and elevated ROS rendered these cells highly sensitive to cell death. The etoposide-selective cell death of Akt-myr-transduced cells was attenuated by pepstatin A, a lysosomal protease inhibitor. In the present study, we show that etoposide might possess a novel therapeutic activity for oncogenic Akt-transduced cancer cells to kill preferentially through ROS-mediated damage.

Topics: Animals; Antineoplastic Agents; Astrocytes; Brain Neoplasms; Cell Death; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p16; Dose-Response Relationship, Drug; Etoposide; Mice; Mice, Nude; Oncogene Protein v-akt; Pepstatins; Reactive Oxygen Species; Time Factors; Transduction, Genetic; Tumor Suppressor Protein p53

Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway.

Topics: Aged; Alkaloids; Amino Acid Chloromethyl Ketones; Aprotinin; Benzophenanthridines; Benzyl Compounds; Caspase 3; Caspases; Cell Division; Cell Transformation, Neoplastic; Colonic Neoplasms; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; DNA; DNA Fragmentation; Down-Regulation; Humans; Hydrocarbons, Fluorinated; Leucine; Leupeptins; Male; Pepstatins; Phenanthridines; Protein Kinase C; Pyridines; Tumor Cells, Cultured