Apoptotic response to homoharringtonine in human wt p53 leukemic cells is independent of reactive oxygen species generation and implicates Bax translocation, mitochondrial cytochrome c release and caspase activation

In the present study, we investigated the in vitro apoptotic response of le ukemic cells to the cellular stress induced by homoharringtonine (HHT), a p lant alkaloid with antileukemic activity which is currently being tested fo r treatment of acute and chronic leukemias. A comparison of leukemic cell l ines with different p53 gene status revealed a considerably higher sensitiv ity to HHT-induced apoptosis in the cells with a wt p53, and apoptotic even ts in wt p53 leukemia cells (MOLT-3 cell line) were studied in more detail. To this end, we examined components of apoptotic cascades including Bax ex pression and its intracellular localization, changes of mitochondrial membr ane potential (MMP), reactive oxygen species (ROS) levels, cytochrome c rel ease from mitochondria and activation of caspases. Bax protein levels did n ot increase despite an up-regulation of bax at mRNA level. However, Bax tra nslocation from cytosol towards mitochondria was observed. In addition, we observed a release of cytochrome c from the mitochondria, and the localizat ion changes of both Bax and cytochrome c were found already at the early, a nnexin V-negative stage of HHT-induced apoptosis, HHT-treated MOLT-3 cells revealed loss of MMP as well as activation of caspases demonstrated by DEVD -, IETD- and LEHD-tetrapeptide cleavage activity in the cell lysates, ROS l evels only slightly increased in HHT-treated cells and antioxidants did not prevent apoptosis and MMP changes. Therefore, wt p53 leukemic cells respon d to HHT-specific cellular stress by induction of ROS-independent apoptotic pathway characterized by translocation of Bax, mitochondrial cytochrome c release and activation of caspases.