Assessment of mitochondrial membrane potential and release of mitochondrial matrix proteins in drug-induced apoptosis in a leucemia cell line

CAVALIERE V. PAPADEMETRIO D, BLANCO G.,AULICINO J., HAJOS S., AND ALVAREZ É

Río de Janeiro, Brasil

Congreso; 13th Internacional Congreso of Immunology; 2007

Permeabilisation of mitochondrial outer membrane, disruption of mitochondrial membrane potential (ÄYm) and release of matrix inner proteins are relevant in apoptotic pathways. We have previously determined that MG-132, CAPE (Caffeic Acid Phenethyl Ester), Doxorubicine and Vincristine induced apoptosis in a B lymphoid tumor cell line, PL104, established in our laboratory. The aim of this work was to determine whether apoptosis induced by these chemotherapeutic agents was associated with disruption of mitochondrial membrane potential and release of mitochondrial inner proteins. MG-132 (2uM) showed no decrease in ÄYm as evaluated by flow cytometry using TMRE (0.05uM). Cytochrome c cytosolic protein was increased by this chemoterapeutic drug at 2, 15 and 24 hs while Smac expression was increased at 6 hs post treatment as evaluated by western blot. CAPE (180uM) decreased ÄYm after 15 hs ((17.65±0.43)% depolarized cells (p<0.05)). High Cytochrome c cytosolic protein level was observed after 24 hs with no change on Smac levels. Doxorubicine (1.5uM) increased Cytochrome c after 6 hs, whereas no difference was observed in ÄYm and Smac expression. Vincristine (1uM) induced ÄYm disruption after 15 hs ((19.05±1.82)% depolarized cells (p<0.01)), with maximum increase of Cytochrome c at 6 hs. We conclude that these four chemoterapeutic drugs can release mitochondrial proapoptotic proteins. However only CAPE and Vincristine showed collapse of ÄYm either before or after release of mitochondrial proteins at the times assessed.