BH3I-2' Induces Both Oxidation Of Cardiolipin And Cytochrome C Release From Mitochondria

Bcl-2 family proteins are major regulators of cell death and cell survival. BH3-containing peptides induce apoptosis by binding to the hydrophobic pocket of the anti-apoptotic proteins, such as Bcl-2 or Bcl-XL. A small cell-permeable compound, BH3I-2' (3-iodo-5-chloro-N-[2-chloro-5-((4-chlorophenyl)sulphonyl) phenyl]-2-hydroxybenzamide), has been recently reported to have a function similar to Bak BH3 peptide. BH3I-2' induces apoptosis by disrupting interactions, mediated by the BH3 domain, between pro-apoptotic and anti-apoptotic members of the Bcl-2 family. In this study, we report that BH3I-2' induced cytochrome c release from the mitochondria! outer membrane in a Bax-dependent manner and this is correlated with the sensitivity of leukemic cells to apoptosis. Moreover, it also induced rapid damage to the inner mitochondria! membrane, with loss of cardiolipin content and collapse of mitochondrial membrane potential ( m), prior to the cytochrome c release. This occurs both in whole cells and isolated mitochondria and is not associated with the sensitivity of cells to BH3I-2'-induced apoptosis. An inhibitor of reactive oxygen species (ROS), N-acetylcysteine (NAC), partly inhibits BH3I-2'-induced activation of caspase-3 and apoptosis, indicating ROS play a role, at least in part, in BH3I-2'-mediated apoptosis. Exogenous Bcl-2 or Bcl-XL neutralizes BH3I-2' in vitro and diminishes its effect on the inner mitochondria! membrane.Taking these results together, we have shown that BH3I-2'-induced rapid damage to the inner mitochondrial membrane is an early event prior to cytochrome c release from mitochondria That is I an important event. This might be mediated by the generation of ROS, because BH3I-2'-induced apoptosis can be partly inhibited by NAC at the whole cell level, especially, at the mitochondrial level. Exogenous Bcl-2 or Bcl-XL neutralized BH3I-2' in vitro and diminished its effect on the inner mitochondrial membrane. This study reveals that through binding to anti-Bcl-2 family proteins, BH31-2' exerts it pro-apoptotic effect by acting at two different steps in the mechanism of cytochrome c release That is, BH3I-2' not only induces cytochrome c release from the outer mitochondrial membrane but also mediates cardiolipin oxidation which may facilitate cytochrome c release. Therefore, development of BH3 mimetic compounds may have great beneficial effect in the treatment of human leukaemia.