RESUMO
Triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook.f, has shown antitumor activities in a broad range of solid tumors. Here, we examined its effects on leukemic cells and found that, at 100 nM or less, it potently induced apoptosis in various leukemic cell lines and primary acute myeloid leukemia (AML) blasts. We then attempted to identify its mechanisms of action. Triptolide induced caspase-dependent cell death accompanied by a significant decrease in XIAP levels. Forced XIAP overexpression attenuated triptolide-induced cell death. Triptolide also decreased Mcl-1 but not Bcl-2 and Bcl-X(L) levels. Bcl-2 overexpression suppressed triptolide-induced apoptosis. Further, triptolide induced loss of the mitochondrial membrane potential and cytochrome C release. Caspase-9 knock-out cells were resistant, while caspase-8-deficient cells were sensitive to triptolide, suggesting criticality of the mitochondrial but not the death receptor pathway for triptolide-induced apoptosis. Triptolide also enhanced cell death induced by other anticancer agents. Collectively, our results demonstrate that triptolide decreases XIAP and potently induces caspase-dependent apoptosis in leukemic cells mediated through the mitochondrial pathway at low nanomolar concentrations. The potent antileukemic activity of triptolide in vitro warrants further investigation of this compound for the treatment of leukemias and other malignancies.
Assuntos
Apoptose , Caspases/metabolismo , Diterpenos/farmacologia , Leucemia/patologia , Mitocôndrias/metabolismo , Fenantrenos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Proteínas Reguladoras de Apoptose/análise , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Caspase 8 , Caspase 9 , Linhagem Celular Tumoral , Compostos de Epóxi , Humanos , Leucemia/tratamento farmacológico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/análise , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/farmacologiaRESUMO
The mechanisms utilized to transduce apoptotic signals that originate from within the nucleus, in response to DNA damage for example, are not well understood. Identifying these mechanisms is important for predicting how tumor cells will respond to genotoxic radiation or chemotherapy. The Rb tumor suppressor protein can inhibit apoptosis triggered by DNA damage, but how it does so is unclear. We have previously characterized a death domain protein, p84N5, that specifically associates with an amino-terminal domain of Rb protein. The p84N5 death domain is required for its ability to trigger apoptotic cell death. Association with Rb protein inhibits p84N5-induced apoptosis suggesting that it may be a mediator of Rb's effects on apoptosis. Unlike other death domain-containing apoptotic signaling proteins, however, p84N5 is localized predominantly within the nucleus of interphase cells. Here we test whether p84N5 requires nuclear localization in order to trigger apoptosis. We identify the p84N5 nuclear localization signal and demonstrate that nuclear localization is required for p84N5-induced apoptosis. To our knowledge, this identifies p84N5 as the first death-domain containing apoptotic signaling protein that functions within the nucleus. By analogy to other death domain containing proteins, p84N5 may play some role in apoptotic signaling within the nucleus. Further, p84N5 is a potential mediator of Rb protein's effects on DNA damage induced apoptosis.