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 ne crosis. Molecular studies of cell death thus far favor a model in which apo ptosis and necrosis share very few molecular regulators. It appears that ap optotic processes triggered by a variety of stimuli converge on the activat ion of a member of the caspase family, such as caspase 3, which leads to th e execution of apoptosis. It has been suggested that blocking of caspase ac tivation in an apoptotic process may divert cell death to a necrotic demise , suggesting that apoptosis and necrosis may share some upstream events. Ac tivation of caspase is preceded by the release of mitochondrial cytochrome C. Materials and Methods: We first studied cell death induced by beta-lapachon e by MTT and colony-formation assay. To determine whether the cell death in duced by beta-lapachone occurs through necrosis or apoptosis, we used the P I staining procedure to determine the sub-G1 fraction and the Annexin-V sta ining for externalization of phophatidylserine. We next compared the releas e of mitochondrial cytochrome C in apoptosis and necrosis. Mitochondrial cy tochrome C was determined by Western blot analysis. To investigate changes in mitochondria that resulted in cytochrome C release, the mitochondrial me mbrane potential (delta psi) was analyzed by the accumulation of rhodamine 123, a membrane-permeant cationic fluorescent dye. The activation of caspas e in apoptosis and necrosis were measured by using a profluorescent substra te 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 ova ry, colon, and lung cancer cells, and necrotic cell death in four human bre ast cancer cell lines. Mitochondrial cytochrome C release was found in both apoptosis and necrosis. This cytochrome C release occurred shortly after b eta-lapachone treatment when cells were fully viable by trypan blue exclusi on and MTT assay, suggesting that cytochrome C release is an early event in beta-lapachone induced apoptosis as well as necrosis. The mitochondrial cy tochrome 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 wit h the necrotic cell death induced by beta-lapachone as determined by absenc e 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, prosta te, and breast, suggesting a wide spectrum of anti-cancer activity in vitro . Both apoptotic and necrotic cell death induced by beta-lapachone are prec eded by a rapid release of cytochrome C, followed by the activation of casp ase 3 in apoptotic cell death but not in necrotic cell death. Our results s uggest that beta-lapachone is a potential anti-cancer drug acting on the mi tochondrial cytochrome C-caspase pathway, and that cytochrome C is involved in the early phase of necrosis.