Molecular ordering of apoptosis induced by anticancer drugs in neuroblastoma cells.
Apoptosis mediated by anticancer drugs may involve activation of death-inducing ligand/receptor systems such as CD95 (APO-1/Fas), cleavage of caspases, and perturbance of mitochondrial functions. We investigated the sequence of these events in SHEP neuroblastoma cells transfected with Bcl-2 or Bcl-X(L) using two different drugs, namely, doxorubicin (Doxo), which activates the CD95/CD95 ligand (CD95-L) system, and betulinic acid (Bet A), which does not enhance the expression of CD95 or CD95-L and which, as shown here, directly targets mitochondria. Apoptosis induced by both drugs was inhibited by Bcl-2 or Bcl-X(L) overexpression or by bongkrekic acid, an agent that stabilizes mitochondrial membrane barrier function, suggesting a critical role for mitochondria. After Doxo treatment, enhanced CD95/CD95-L expression and caspase-8 activation were not blocked by Bcl-2 or Bcl-X(L) and were found in cells with a mitochondrial transmembrane potential (delta psi(m)) that was still normal (delta psi(m)high cells). In marked contrast, after Bet A treatment, caspase-8 activation occurred in a Bcl-2- or Bcl-X(L)-inhibitable fashion and was confined to cells that had lost their delta psi(m) (delta psi(m)low cells). Mitochondria from cells treated with either Doxo or Bet A induced cleavage of both caspase-8 and caspase-3 in cytosolic extracts. Thus, caspase-8 activation may occur upstream or downstream of mitochondria, depending on the apoptosis-initiating stimulus. In contrast to caspase-8, cleavage of caspase-3 or poly(ADP-ribose)polymerase was always restricted to delta psi(m)low cells, downstream of the Bcl-2- or Bcl-X(L)-controlled checkpoint of apoptosis. Cytochrome c, released from mitochondria undergoing permeability transition, activated caspase-3 but not caspase-8 in a cell-free system. However, both caspases were activated by apoptosis-inducing factor, indicating that the mechanism of caspase-8 activation differed from that of caspase-3 activation. Taken together, our findings demonstrate that perturbance of mitochondrial function constitutes a central coordinating event in drug-induced cell death.
Fulda
S
S
University Children's Hospital, Ulm, Germany.
Susin
S A
SA
Kroemer
G
G
Debatin
K M
KM
D000970:Antineoplastic Agents / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects; D004273:DNA, Neoplasm / Q000378:metabolism; D004317:Doxorubicin / Q000633:toxicity*; D053222:Fas Ligand Protein; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D009447:Neuroblastoma / Q000473:pathology* / Q000503:physiopathology; D053978:Pentacyclic Triterpenes; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D014315:Triterpenes / Q000633:toxicity*; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein; D019014:fas Receptor / Q000235:genetics; D000094062:Betulinic Acid
D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; D004273:DNA, Neoplasm; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D053978:Pentacyclic Triterpenes; D019253:Proto-Oncogene Proteins c-bcl-2; D014315:Triterpenes; D051020:bcl-X Protein; D019014:fas Receptor; D004317:Doxorubicin; D000094062:Betulinic Acid