SMAC-Mimetic BV-6 Sensitizes Therapeutic Agents-Induced Apoptosis In AML Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2177-2177
Author(s):  
Duncan H Mak ◽  
Christa Manton ◽  
Michael Andreeff ◽  
Bing Z Carter
Keyword(s):  
Cell Death ◽  
Vector Control ◽  
Caspase 3 ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Smac Mimetic ◽  
P53 Activation ◽  
Induced Apoptosis

Abstract Abstract 2177 The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs) is antagonized by mitochondria-released SMAC protein. The IAP-member XIAP suppresses apoptosis by directly binding and inhibiting caspase-9 and caspase-3, while cIAP1, a component of the cytoplasmic signaling complex containing TNF receptor associated factors, suppresses apoptosis via the caspase-8-mediated pathway. BV-6 (Genentech) is a bivalent SMAC-mimetic and has been shown to promote cell death by inducing cIAP autoubiquitination, NF-κB activation, and TNFα-dependent apoptosis. We examined its effect on leukemic cells and found that BV-6 only moderately induced apoptosis. The EC50 was found to be 15.3±5.1 μM at 48 hours in OCI-AML3 cells which are relatively sensitive. We then determined whether BV-6 sensitizes leukemic cells to the HDM2-inhibitor nutlin-3a and to Ara-C. p53 modulates the expression and activity of Bcl-2 family proteins and promotes the mitochondrial-mediated apoptosis. We showed previously that activation of p53 by nutlin-3a sensitizes AML cells to XIAP inhibition induced-death in part by promoting the release of SMAC from mitochondrion (Carter BZ et al., Blood 2010). We treated OCI-AML3 cells with BV-6, nutlin-3a or Ara-C, and BV-6+nutlin-3a or BV-6+Ara-C and found that the combination of BV-6 and nutlin-3a or BV-6 and Ara-C synergistically induced cell death in OCI-AML3 cells with a combination index (CI) of 0.27±0.11 and 0.22±0.05 (48 hours), respectively. To demonstrate that p53 activation is essential for the synergism of BV-6+nutlin-3a combination, we treated OCI-AML3 vector control and p53 knockdown cells with these two agents and found that the combination synergistically promoted cell death in the vector control (CI=0.47±0.15) but not in the p53 knockdown cells, as expected, while BV6+Ara-C was synergistic in both vector control and p53 knockdown cells (CI=0.15±0.03 and 0.08±0.03, respectively, 48 hours). BV-6 induced activation of caspase-8, caspase-9, and caspase-3 and decreased XIAP levels, but did not cause rapid cIAP1 degradation, as reported by others. To assess the contribution of death receptor-mediated apoptosis in BV-6-induced cell death, we treated Jurkat and caspase-8 mutated Jurkat cells (JurkatI9.2) with BV-6 and found that BV-6 induced cell death and significantly potentiated TRAIL-induced apoptosis in Jurkat cells (CI=0.14±0.08, 48 hours). Caspase-8 mutated JurkatI9.2 cells were significantly less sensitive to BV-6 than Jurkat cells and as expected, JurkatI9.2 was completely resistant to TRAIL. Collectively, we showed that the bivalent SMAC-mimetic BV-6 potentiates p53 activation-, chemotherapy-, and TRAIL-induced cell death, but has only minimal activity by itself in leukemic cells. SMAC-mimetics could be useful in enhancing the efficacy of different classes of therapeutic agents used in AML therapy. Disclosures: No relevant conflicts of interest to declare.

Triptolide-Induced Apoptosis Is Dependent on Caspase Activation and NFκB Signaling and Mediated by XIAP and Survivin Downregulation through the Mitochondrial Pathway in Leukemic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3394-3394
Author(s):  
Bing Z. Carter ◽  
Wendy D. Schober ◽  
Teresa McQueen ◽  
Randall L. Evans ◽  
Michael Andreeff
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Chinese Herb ◽  
Mitochondrial Pathway ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Parp Cleavage ◽  
Caspase 8 ◽  
Imatinib Resistance ◽  
Induced Apoptosis

Abstract Triptolide, an immunosuppressor isolated from the Chinese herb, Tripterygium wilfordii Hook. F, has recently shown anti-tumor activities in a broad range of solid tumors. We examined its effects on leukemic cells and investigated mechanisms of apoptosis. Triptolide, at less than 100 nM, arrested cell growth and potently induced cell death in myeloid and lymphoid leukemic cells tested, including OCI-AML3, U937, Jurkat, KBM5, and K562 cells. In OCI-AML3 cells, triptolide induced caspase 3 activation, PARP cleavage and annexin V positivity with an IC50 of about 30 nM, at 24 hrs, all of which were inhibited by a general caspase inhibitor suggesting caspase dependent cell death. However, Triptolide-induced cell growth arrest was not affected by caspase inhibition. Treatment of OCI-AML3 cells with triptolide decreased XIAP and survivin expression, but did not affect Bcl2 and BclXL levels. Forced overexpression of XIAP attenuated Triptolide-induced cell death. Triptolide induced Bid cleavage, but Jurkat cells deficient in caspase 8 were only slightly less sensitive to triptolide than the wild-type counterpart indicating that Triptolide-induced cell death is caspase 8 independent. Jurkat cells deficient in receptor interacting protein (RIP) and therefore deficient in NFκB activation were resistant to Triptolide demonstrating that NFκB signaling is essential for Triptolide-induced cell death. Triptolide treatment induced cytosolic release of cytochrome C and loss of mitochondrial membrane potential, overexpression of Bcl2 effectively suppressed apoptosis induced by Triptolide, and caspase 9 knockout MEF cells were resistant to Triptolide suggesting criticality of the mitochondrial pathway. The antioxidants GSH (5 mM) and vitamin C (150 μM) did not protect from apoptotic cell death induced by Triptolide. In addition, Triptolide-induced apoptosis of blast crisis CML KBM5 cells was independent of their sensitivity or resistance to Imatinib: Triptolide killed Imatinib resistant KBMSTI cells as effectively as Imatinib sensitive KBM5 cells. Ex vivo studies showed that Triptolide also induced cell death in primary AML blasts. Collectively, our studies demonstrate that Triptolide potently induces caspase-dependent apoptosis and arrests cell growth in leukemic cells. Triptolide-induced cell death is dependent on NFκB signaling, and mediated by downregulation of XIAP and survivin through the mitochondrial pathway. The potent anti-leukemic activity of Triptolide in vitro warrants further investigation of this compound for the treatment of leukemia and other malignancies. This drug may also be potentially useful in overcoming Imatinib resistance in CML and Philadelphia chromosome positive ALL.

Tumor-induced apoptosis of T lymphocytes: elucidation of intracellular apoptotic events

Blood ◽  
2000 ◽  
Vol 95 (6) ◽  
pp. 2015-2023 ◽  
Author(s):  
Brian R. Gastman ◽  
Daniel E. Johnson ◽  
Theresa L. Whiteside ◽  
Hannah Rabinowich
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
T Lymphocytes ◽  
Tumor Cells ◽  
Caspase 3 ◽  
Jurkat Cells ◽  
Caspase 8 ◽  
Induced Apoptosis ◽  
Fluoromethyl Ketone

Abstract Our recent studies suggest that human squamous cell carcinoma of the head and neck (SCCHN) is capable of activating an intrinsic mechanism of programmed-cell death in interacting lymphocytes in situ and in vitro. The current study used Jurkat T-cell line as a model to investigate intracellular apoptotic events in T cells interacting with SCCHN. Apoptosis induced in T lymphocytes by tumor cells was in part Fas-mediated, since it was partially, but significantly, inhibited in the presence of anti-Fas ligand Ab or in Fas-resistant Jurkat cells. The synthetic caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and N-benzyloxycarbonyl-Asp-glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK), effectively blocked apoptosis of Jurkat cells co-incubated with SCCHN cell lines, suggesting the involvement of caspases in tumor-induced apoptosis of lymphocytes. Overexpression of CrmA, an inhibitor of caspase-1 and caspase-8, partially inhibited tumor-induced T-cell death. Caspase-8 and caspase-3 were identified as effector molecules in the execution of tumor-induced T-cell death, since the proform enzymes were processed into active subunits during co-incubation of T cells with tumor cells. Furthermore, co-incubation with tumor cells resulted in cleavage of poly(ADP-ribose) polymerase (PARP), a common caspase-3 substrate, and in cleavage of TcR-ζ chain, shown by us to be a T-cell specific caspase-3 substrate. Overexpression of Bcl-2 did not provide protection of T cells from SCCHN-induced DNA degradation. Instead, the Bcl-2 protein was cleaved in the target T cells during their co-incubation with tumor cells. These findings demonstrate that tumor cells can trigger in T lymphocytes caspase-dependent apoptotic cascades, which are not effectively protected by Bcl-2.

Reactive oxygen species and caspase activation mediate silica-induced apoptosis in alveolar macrophages

2001 ◽  
Vol 280 (1) ◽  
pp. L10-L17 ◽  
Author(s):  
Han-Ming Shen ◽  
Zhuo Zhang ◽  
Qi-Feng Zhang ◽  
Choon-Nam Ong
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Alveolar Macrophages ◽  
Caspase 3 ◽  
Reactive Oxygen ◽  
Parp Cleavage ◽  
Oxygen Species ◽  
Caspase Activation ◽  
Caspase 9 ◽  
Induced Apoptosis

Alveolar macrophages (AMs) are the principal target cells of silica and occupy a key position in the pathogenesis of silica-related diseases. Silica has been found to induce apoptosis in AMs, whereas its underlying mechanisms involving the initiation and execution of apoptosis are largely unknown. The main objective of the present study was to examine the form of cell death caused by silica and the mechanisms involved. Silica-induced apoptosis in AMs was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and cell cycle/DNA content analysis. The elevated level of reactive oxygen species (ROS), caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage in silica-treated AMs were also determined. The results showed that there was a temporal pattern of apoptotic events in silica-treated AMs, starting with ROS formation and followed by caspase-9 and caspase-3 activation, PARP cleavage, and DNA fragmentation. Silica-induced apoptosis was significantly attenuated by a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp aldehyde, and ebselen, a potent antioxidant. These findings suggest that apoptosis is an important form of cell death caused by silica exposure in which the elevated ROS level that results from silica exposure may act as an initiator, leading to caspase activation and PARP cleavage to execute the apoptotic process.

A Novel Ring-Substituted Diindolylmethane 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) Methane Abrogates ERK Activation and Induces Apoptosis in Acute Myeloid Leukemia (AML).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3399-3399
Author(s):  
Rooha Contractor ◽  
Ismael J. Samudio ◽  
Zeev Estrov ◽  
David Harris ◽  
James A. McCubrey ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Caspase 9 ◽  
Apoptotic Pathway ◽  
Erk Activation ◽  
New Class ◽  
Induced Apoptosis ◽  
Extracellular Regulated Kinase

Abstract We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane (DIM) derivative, named, 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in myeloid leukemic cells. DIM #34 inhibited leukemic cell growth via induction of apoptosis. DIM #34 inhibited clonogenic growth and induced apoptosis of AML CD34+ progenitor cells but spared normal progenitors. DIM #34 induced loss of mitochondrial membrane potential, which was accompanied by the release of cytochrome c into the cytosol and early cleavage of caspase-9 followed by the cleavage of caspases -8, and -3. Bcl-2 overexpression and caspase-9-deficient cells were partially protected against DIM #34-induced apoptosis, suggesting activation of the intrinsic apoptotic pathway. DIM #34 induced Bax cleavage, and Bax knockout cells were partially resistant to cell death. Furthermore, DIM #34 transiently inhibited the phosphorylation and the activity of the extracellular-regulated kinase (ERK) and abrogated Bcl-2 phosphorylation. Because other methylene substituted DIM analogs transactivate the nuclear receptor PPARγ, we studied the role of PPARγ in apoptosis induction. Although the co-treatment of cells with a selective PPARγ antagonist T007, and a low dose of DIM #34 partially diminished apoptosis, apoptosis was not inhibited at higher concentrations of DIM #34, suggesting the involvement of both, receptor-dependent and independent mechanisms. Co-treatment with RXR- and RAR-ligands enhanced DIM #34-induced cell death. Together, these findings showed that substituted DIMs represent a new class of compounds that selectively induce apoptosis in AML cells through interference with ERK and activation of PPARγ signaling pathways.

Intracellular NAD+ Depletion Enhances Bortezomib-Induced Myeloma Cytotoxicity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 330-330
Author(s):  
Antonia Cagnetta ◽  
Michele Cea ◽  
Chirag Acharya ◽  
Teresa Calimeri ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Cell Death ◽  
Synergistic Effect ◽  
Cell Lines ◽  
Caspase 3 ◽  
Statistical Significance ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Nicotinamide Phosphoribosyltransferase

Abstract Abstract 330 Background: Our previous study demonstrated that inhibition of nicotinamide phosphoribosyltransferase (Nampt) acts by severely depleting intracellular NAD+ content and thus eliciting mitochondrial dysfunction and autophagic MM cell death. The proteasome inhibitor Bortezomib induces anti-MM activity by affecting a variety of signaling pathways. However, as with other agents, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we demonstrate that combining Nampt inhibitor and bortezomb induces synergistic anti-MM cell death both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. Material and Methods: We utilized MM.1S, MM.1R, RPMI-8226, and U266 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies. Cell viability and apoptosis assays were performed using Annexin V/PI staining. Intracellular NAD+ level and proteasome activity were quantified after 12, 24, and 48h exposure to single/combination drugs by specific assays. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, and tubulin. CB-17 SCID male mice (n = 28; 7 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (3 weeks after MM cell injection), mice were treated for three weeks with vehicle alone, FK866 (30mg/kg 4 days weekly), Bortezomib (0.5 mg/kg twice weekly), or FK866 (30 mg/kg) plus Bortezomib (0.5 mg/kg). Statistical significance of differences observed in FK866, Bortezomib or combination-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index < 1.0 indicates synergism. Results/Discussion: Combining FK866 and Bortezomib induces synergistic anti-MM activity in vitro against MM cell lines (P<0.005, CI < 1) or patient CD138-positive MM cells (P< 0.004). FK866 plus Bortezomib-induced synergistic effect is associated with: 1)activation of caspase-8, caspase-9, caspase-3, and PARP; 2) improved intracellular NAD+ dissipation; 3) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteolytic activities; 4) inhibition of NF-kappa B signaling; and 5) inhibition of angiogenesis. Importantly, the ectopic overexpression of Nampt rescues this observed synergistic effect; conversely, Nampt knockdown by RNAi significantly enhances the anti-MM effect of bortezomib. In the murine xenograft MM model, low dose combination FK866 (30 mg/kg) and Bortezomib (0.5 mg/kg) is well tolerated, significantly inhibits tumor growth (P < 0.001), and prolongs host survival (2–2.5 months in mice receiving combined drugs, P = 0.001). These findings demonstrate that intracellular NAD+ levels represent a major determinant in the ability of bortezomib to induce apoptosis of MM cells, providing the rationale for clinical protocols evaluating FK866 together with Bortezomib to improve patient outcome in MM. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

scholarly journals Caspase 3 is Activated through Caspase 8 instead of Caspase 9 during H2O2-induced Apoptosis in HeLa Cells

2011 ◽  
Vol 27 (5) ◽  
pp. 539-546 ◽  
Author(s):  
Yinyuan Wu ◽  
Dianjun Wang ◽  
Xiaodong Wang ◽  
Yinyin Wang ◽  
Fangli Ren ◽  
...  
Keyword(s):  
Hela Cells ◽  
Caspase 3 ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Induced Apoptosis

scholarly journals NeosedumosideIII induced Apoptosis of Human Hepatocellular Carcinoma HepG2 cells and SMMC-7721 Cells and Related Mechanism

2021 ◽  
Author(s):  
Xin-Yu Li ◽  
Xin Zhou ◽  
Yu- Liu ◽  
Feng Qiu ◽  
Qing-Qing Zhao
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Hepg2 Cells ◽  
Caspase 3 ◽  
Human Hepatocellular Carcinoma ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Induced Apoptosis

Abstract Purpose: NeosedumosideIII (Neo) is a megastigmanes and belongs to monocyclic sesquiterpenoids compound with antioxidant, anti-inflammatory and other pharmacological activities. In order to explore the anti-cancer effect and possible mechanism of Neo, the study examined the anti-proliferation and apoptosis effect of Neo against human hepatocellular carcinoma HepG2 cells and SMMC-772 cells and related mechanism in vitro. Methods ：The anti-proliferation effect of Neo was detected on HepG2 cells and SMMC-772 cells by MTT assay and IC50 with increasing dose and time. Cell cycle and apoptosis were detected by flow cytometer. The changes of Bcl-2, Bax, Caspase-3, Caspase-8 and Caspase-9 proteins were detected by western blotting.Results ：The results indicated that Neo could inhibited proliferation of HepG2 cells and SMMC-772 cells in vitro and promoted apoptosis, it significantly induced apoptosis of HepG2 cells and SMMC-772 cells arrested cell cycle at G0/G1 phase in a dose-dependent manner, reduce the expression of Bcl-2 protein, and increase the expression of Bax and Caspase-3, Caspase-8 and Caspase-9 proteins. Conclusion：Neo could inhibit proliferation and induce apoptosis of HepG2 cells and SMMC-7721 cells in vivo which suggested that it might be served as a promising candidate for the treatment of liver cancer.

Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L–induced apoptosis of human acute leukemia cells

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3900-3906 ◽  
Author(s):  
Jinghai Wen ◽  
Nimmanapalli Ramadevi ◽  
Diep Nguyen ◽  
Charles Perkins ◽  
Elizabeth Worthington ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Fas Ligand ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Leukemia Cells ◽  
Maximum Effect ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Induced Apoptosis

Abstract In present studies, treatment with tumor necrosis factor (TNF)–related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand [Apo-2L]) is shown to induce apoptosis of the human acute leukemia HL-60, U937, and Jurkat cells in a dose-dependent manner, with the maximum effect seen following treatment of Jurkat cells with 0.25 μg/mL of Apo-2L (95.0% ± 3.5% of apoptotic cells). Susceptibility of these acute leukemia cell types, which are known to lack p53wt function, did not appear to correlate with the levels of the apoptosis-signaling death receptors (DRs) of Apo-2L, ie, DR4 and DR5; decoy receptors (DcR1 and 2); FLAME-1 (cFLIP); or proteins in the inhibitors of apoptosis proteins (IAP) family. Apo-2L–induced apoptosis was associated with the processing of caspase-8, Bid, and the cytosolic accumulation of cytochrome c as well as the processing of caspase-9 and caspase-3. Apo-2L–induced apoptosis was significantly inhibited in HL-60 cells that overexpressed Bcl-2 or Bcl-xL. Cotreatment with either a caspase-8 or a caspase-9 inhibitor suppressed Apo-2L–induced apoptosis. Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 but not DR4, Fas, DcR1, DcR2, Fas ligand, or Apo-2L levels. Importantly, sequential treatment of HL-60 cells with etoposide, Ara-C, or doxorubicin followed by Apo-2L induced significantly more apoptosis than treatment with Apo-2L, etoposide, doxorubicin, or Ara-C alone, or cotreatment with Apo-2L and the antileukemic drugs, or treatment with the reverse sequence of Apo-2L followed by one of the antileukemic drugs. These findings indicate that treatment with etoposide, Ara-C, or doxorubicin up-regulates DR5 levels in a p53-independent manner and sensitizes human acute leukemia cells to Apo-2L–induced apoptosis.

The Novel, Orally Active Proteasome Inhibitor, NPI-0052, Induces Apoptosis in Leukemia Lymphoma Cell Lines and Patient Specimens.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 241-241
Author(s):  
Claudia P. Miller ◽  
Kechen Ban ◽  
Stacey L. Ruiz ◽  
Saskia Neuteboom ◽  
Michael Palladino ◽  
...  
Keyword(s):  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Caspase 8 ◽  
Proteolytic Activities ◽  
Induced Apoptosis ◽  
Orally Active

Abstract NPI-0052 is the second proteasome inhibitor with potential for clinical use since the FDA approval of bortezomib. NPI-0052 is a novel, orally active, non-peptide small molecule inhibitor discovered by Nereus Pharmaceuticals during the fermentation of a new marine Gram-positive actinomycete, Salinospora sp. In human erythrocyte derived 20S proteasomes, with EC50 values in the picomolar and nanomolar range, NPI-0052 inhibits all three proteolytic activities: the chymotrypsin-like, the trypsin-like and caspase-like activities. In the present study, exposure of 1uM NPI-0052 for 1 h inhibited the chymotryptic and caspase-like activities by greater than 90% in Jurkat and ML-1 cells. The trypsin-like activity was also inhibited to a lesser extent. NPI-0052 demonstrated varying degrees of apoptosis in cell lines representative of AML (ML-1), ALL (Jurkat), and Burkitt lymphoma (BL-41) and in mononuclear cells isolated from CLL and ALL patients, as measured by propidium iodide staining and subsequent FACS analysis. In addition, treatment of Jurkat cells with NPI-0052 resulted in activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP). Further experiments revealed that caspase activity might be initiated differently in myeloid versus lymphoid leukemia cell lines. NPI-0052 caused cleavage of caspase-8 as demonstrated by SDS-PAGE analysis and when combined with an inhibitor specific for caspase-8 (IETD-fmk), Jurkat cells (of lymphoid origin) were protected against NPI-0052 induced apoptosis whereas ML-1 (of myeloid origin) were not. The cleaved product of Bid was detected by immunoblotting in NPI-0052 treated Jurkat cells, suggesting amplification of caspase-8 activity through mitochondria. NPI-0052 induced loss of mitochondrial membrane potential and release of cytochrome c in Jurkat cells. Cell lines of lymphocytic origin exposed to 4h of NPI-0052 resulted in increased levels of peroxide and superoxide prior to cell death. Furthermore, the antioxidant, N-acetyl cysteine (NAC), conferred protection in Jurkat cells against NPI-0052 induced apoptosis. CLL and Ph+ ALL patient material confirmed that lymphocytes from these patients are protected from NPI-0052 induced apoptosis by antioxidants. In summary, NPI-0052 inhibits all three major proteolytic activities of 20S proteasome in leukemia cells and induces apoptosis in leukemic cells and patient samples. The cytotoxic effects of NPI-0052 in leukemia and lymphoma cells warrant further testing to determine if this compound is clinically effective.

Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L–induced apoptosis of human acute leukemia cells

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3900-3906 ◽  
Author(s):  
Jinghai Wen ◽  
Nimmanapalli Ramadevi ◽  
Diep Nguyen ◽  
Charles Perkins ◽  
Elizabeth Worthington ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Fas Ligand ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Leukemia Cells ◽  
Maximum Effect ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Induced Apoptosis

In present studies, treatment with tumor necrosis factor (TNF)–related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand [Apo-2L]) is shown to induce apoptosis of the human acute leukemia HL-60, U937, and Jurkat cells in a dose-dependent manner, with the maximum effect seen following treatment of Jurkat cells with 0.25 μg/mL of Apo-2L (95.0% ± 3.5% of apoptotic cells). Susceptibility of these acute leukemia cell types, which are known to lack p53wt function, did not appear to correlate with the levels of the apoptosis-signaling death receptors (DRs) of Apo-2L, ie, DR4 and DR5; decoy receptors (DcR1 and 2); FLAME-1 (cFLIP); or proteins in the inhibitors of apoptosis proteins (IAP) family. Apo-2L–induced apoptosis was associated with the processing of caspase-8, Bid, and the cytosolic accumulation of cytochrome c as well as the processing of caspase-9 and caspase-3. Apo-2L–induced apoptosis was significantly inhibited in HL-60 cells that overexpressed Bcl-2 or Bcl-xL. Cotreatment with either a caspase-8 or a caspase-9 inhibitor suppressed Apo-2L–induced apoptosis. Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 but not DR4, Fas, DcR1, DcR2, Fas ligand, or Apo-2L levels. Importantly, sequential treatment of HL-60 cells with etoposide, Ara-C, or doxorubicin followed by Apo-2L induced significantly more apoptosis than treatment with Apo-2L, etoposide, doxorubicin, or Ara-C alone, or cotreatment with Apo-2L and the antileukemic drugs, or treatment with the reverse sequence of Apo-2L followed by one of the antileukemic drugs. These findings indicate that treatment with etoposide, Ara-C, or doxorubicin up-regulates DR5 levels in a p53-independent manner and sensitizes human acute leukemia cells to Apo-2L–induced apoptosis.

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