Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway in leukemic cells

Abstract

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.

Figure 1.

Triptolide induced significant cell growth arrest and cell death in leukemic cells. Cells at a density of 0.2 × 10⁶/mL were treated with various concentrations of triptolide. (A) Structure of triptolide. (B) Viable leukemic cells treated with triptolide, as determined by trypan blue exclusion after 24 and 48 hours. (C) Cell death induced by triptolide in various leukemic cells, as determined by annexin V staining with PI after 24 or 48 hours of treatment.

Figure 2.

Triptolide induced significant cell death in AML samples. (A) Comparison of triptolide-induced cell death in AML samples with that in normal bone marrow samples. Cells from 11 AML blasts and 3 normal bone marrow samples at a density of 1 × 10⁶/mL were treated with various concentrations of triptolide. Cell death was determined by annexin V staining with PI after 24 hours of treatment. Normal bone marrows were stained with CD34-APC after triptolide treatment, and PS/annexin V was determined in the CD34⁺ population. (B) Results of colony-formation assays in blasts from 5 AML samples and cells from 3 normal bone marrow samples treated with the indicated concentrations of triptolide.

Figure 3.

Triptolide reduced the XIAP level and induced caspase-dependent cell death in leukemic cells. OCI-AML3 cells at a density of 0.2 × 10⁶/mL were treated for 24 hours with various concentrations of triptolide in the absence or presence of the general caspase inhibitor IDN-1965 (20 μM). (A) Caspase-3 activation and PARP cleavage were analyzed by Western blot. (B) Cell viability was determined by annexin V staining with PI. (C) XIAP levels were analyzed by Western blot. (D) U937 and HL60 cells at a density of 0.2 × 10⁶/mL and (E) AML blasts (n = 6) at a density of 1 × 10⁶/mL were treated for 24 hours with various concentrations of triptolide. XIAP and caspase-3 protein levels were determined by Western blot.

Figure 4.

Triptolide-induced cell death is mediated, at least in part, through XIAP down-regulation. (A) OCI-AML3 cells at a density of 0.2 × 10⁶/mL were treated with various concentrations of triptolide for 24 hours in the presence or absence of the caspase inhibitor IDN-1965 (20 μM) or the proteasome inhibitor MG132 (0.2 μM). The XIAP protein level was determined by Western blot. (B) OCI-AML3 cells at a density of 0.2 × 10⁶/mL were treated for 24 hours with various concentrations of triptolide. RNA was isolated, and the XIAP RNA level was determined by TaqMan RT-PCR. (C) U937neo and U937XIAP cells at a density of 0.2 × 10⁶/mL were treated for 24 hours with various concentrations of triptolide. Cell death was determined by annexin V staining with PI.

Figure 5.

Mitochondria- not death receptor–mediated caspase activation is essential for triptolide-induced cell death. (A) MEFs, MEFs deficient in caspase-9 (MEF-caspase-9^–/–), Jurkat cells, and Jurkat cells deficient in caspase-8 (JurkatI2.1) were treated with the indicated concentrations of triptolide for 24 hours. Cell death was determined by annexin V staining with PI. (B) Measurement of cytosolic cytochrome C by Western blot of OCI-AML3 cells treated with triptolide for 24 hours. (C) Determination of MMP by CMXRos-MitoTracker Green staining and flow cytometry analysis of OCI-AML3 cells treated with triptolide for 24 hours. Area I represents the cells with intact mitochondria; area II; cells with partial loss of MMP; and area III, complete loss of MMP. (D) OCI-AML3vec and OCI-AML3Bcl-2 cells at a density of 0.2 × 10⁶/mL were treated for 24 hours with various concentrations of triptolide. Cell death was determined by annexin V staining with PI. (E) Mcl-1 levels in OCI-AML3, U937, and HL-60 cells and cells from patients with AML (n = 6) treated with triptolide for 24 hours determined by Western blot.

Figure 6.

Effect of triptolide in combination with Dox, gemtuzumab ozogamicin, or Ara-C on cell death in AML cells. OCI-AML3 cells (0.2 × 10⁶/mL) were treated with low concentrations of triptolide, Dox, gemtuzumab ozogamicin, Ara-C, or their combinations, as indicated, for 48 hours. (A) XIAP and Mcl-1 protein levels in OCI-AML3 cells treated with low concentrations of triptolide at 48 hours. (B) Cell viability determined by annexin V staining in OCI-AML3 cells treated with triptolide, Dox, gemtuzumab ozogamicin, Ara-C, or the combinations for 48 hours. (C) Cell cycle distribution of OCI-AML3 cells treated with triptolide, Dox, gemtuzumab ozogamicin, Ara-C, or their combinations at 48 hours, as shown by PI staining. (D) Blasts (1 × 10⁶/mL) from patients with AML (n = 4) were treated with low concentrations of triptolide, Dox, gemtuzumab ozogamicin, or their combinations for 48 hours. Cell viability was determined by annexin V staining.