Release of mitochondrial cytochrome C in both apoptosis and necrosis induced by beta-lapachone in human carcinoma cells.
BACKGROUND
There are two fundamental forms of cell death: apoptosis and necrosis. Molecular studies of cell death thus far favor a model in which apoptosis and necrosis share very few molecular regulators. It appears that apoptotic processes triggered by a variety of stimuli converge on the activation of a member of the caspase family, such as caspase 3, which leads to the execution of apoptosis. It has been suggested that blocking of caspase activation in an apoptotic process may divert cell death to a necrotic demise, suggesting that apoptosis and necrosis may share some upstream events. Activation of caspase is preceded by the release of mitochondrial cytochrome C.


METHODS
We first studied cell death induced by beta-lapachone by MTT and colony-formation assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the PI staining procedure to determine the sub-G1 fraction and the Annexin-V staining for externalization of phophatidylserine. We next compared the release of mitochondrial cytochrome C in apoptosis and necrosis. Mitochondrial cytochrome C was determined by Western blot analysis. To investigate changes in mitochondria that resulted in cytochrome C release, the mitochondrial membrane potential (delta psi) was analyzed by the accumulation of rhodamine 123, a membrane-permeant cationic fluorescent dye. The activation of caspase in apoptosis and necrosis were measured by using a profluorescent substrate for caspase-like proteases, PhiPhiLuxG6D2.


RESULTS
beta-lapachone induced cell death in a spectrum of human carcinoma cells, including nonproliferating cells. It induced apoptosis in human ovary, colon, and lung cancer cells, and necrotic cell death in four human breast cancer cell lines. Mitochondrial cytochrome C release was found in both apoptosis and necrosis. This cytochrome C release occurred shortly after beta-lapachone treatment when cells were fully viable by trypan blue exclusion and MTT assay, suggesting that cytochrome C release is an early event in beta-lapachone induced apoptosis as well as necrosis. The mitochondrial cytochrome C release induced by beta-lapachone is associated with a decrease in mitochondrial transmembrane potential (delta psi). There was activation of caspase 3 in apoptotic cell death, but not in necrotic cell death. This lack of activation of CPP 32 in human breast cancer cells is consistent with the necrotic cell death induced by beta-lapachone as determined by absence of sub-G1 fraction, externalization of phosphatidylserine.


CONCLUSIONS
beta-lapachone induces either apoptotic or necrotic cell death in a variety of human carcinoma cells including ovary, colon, lung, prostate, and breast, suggesting a wide spectrum of anti-cancer activity in vitro. Both apoptotic and necrotic cell death induced by beta-lapachone are preceded by a rapid release of cytochrome C, followed by the activation of caspase 3 in apoptotic cell death but not in necrotic cell death. Our results suggest that beta-lapachone is a potential anti-cancer drug acting on the mitochondrial cytochrome C-caspase pathway, and that cytochrome C is involved in the early phase of necrosis.
Li
Y Z
YZ
Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
Li
C J
CJ
Pinto
A V
AV
Pardee
A B
AB
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D002277:Carcinoma / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000502:physiology*; D003574:Cytochrome c Group / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009285:Naphthoquinones / Q000494:pharmacology*; D009336:Necrosis; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; D003574:Cytochrome c Group; D009285:Naphthoquinones; C014638:beta-lapachone; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases