benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Lymphoma--Large-B-Cell--Diffuse

We have previously shown that actinonin causes inhibition of cellular proliferation in U937 leukemia cells. In this report we demonstrate that the inhibition of cell growth by actinonin occurs through the induction of apoptosis. Signs of apoptosis at high actinonin concentration included DNA fragmentation, exposure of phosphatidylserine and condensation of cell nuclei. Apoptosis caused by actinonin was inhibited by Z-VAD-FMK, a broad specificity inhibitor of caspases, implicating the caspase pathway of apoptosis. Further, apoptosis was associated with a large increase in intracellular caspase-3 and -7 activities.

Topics: Amino Acid Chloromethyl Ketones; Anti-Bacterial Agents; Apoptosis; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; DNA Damage; Humans; Hydroxamic Acids; Leukemia; Lymphoma, Large B-Cell, Diffuse; Tumor Cells, Cultured

Motexafin gadolinium (MGd, Xcytrin) is a tumor-localizing redox mediator that catalyzes the oxidation of intracellular reducing molecules including NADPH, ascorbate, protein and non-protein thiols, generating reactive oxygen species (ROS). MGd localizes to tumors and cooperates with radiation and chemotherapy to kill tumor cells in tissue culture and animal models. In this report, we demonstrate that MGd triggers the mitochondrial apoptotic pathway in the HF-1 lymphoma cell line as determined by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspase-9 prior to caspase-8, cleavage of PARP and annexin V binding. There was minimal effect on MGd-induced apoptosis by the caspase inhibitor z-VAD-fmk, even though caspase-3 activity (as measured by DEVD-cleavage) was completely inhibited. However, MGd-induced apoptosis was reduced to baseline levels by the more potent caspase inhibitor Q-VD-OPh, demonstrating that MGd-induced apoptosis is indeed caspase-dependent. Apoptosis induced by dexamethasone, doxorubicin and etoposide (mediated through the mitochondrial pathway) was also more sensitive to inhibition by Q-VD-OPh than z-VAD-fmk. Our results demonstrating differential sensitivity of drug-induced apoptosis to caspase inhibitors suggest that the term "caspase-independent apoptosis" cannot be solely defined as apoptosis that is not inhibited by z-VAD-fmk as has been utilized in some published studies.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Lymphoma, Follicular; Lymphoma, Large B-Cell, Diffuse; Metalloporphyrins; Mitochondria; Poly(ADP-ribose) Polymerases; Quinolines