Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhhibitor GX15-070 as a Novel Therapeutic Strategy in AML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2584-2584
Author(s):  
Julie C. Watt ◽  
Marina Konopleva ◽  
Rooha Contractor ◽  
Ismael Samudio ◽  
David Harris ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Small Molecule ◽  
Chemotherapeutic Agents ◽  
Leukemic Cells ◽  
Phase I Clinical Trials ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Mitochondrial Inner Membrane ◽  
Induced Apoptosis

Abstract GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in leukemias. In this study, we investigated the activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 cell lines at IC50’s of 0.1, 0.5, 0.5 and 2.5μM, respectively, at 72 hours. Neither overexpression of Bcl-2 or Bcl-XL nor loss of expression of Bax conferred resistance to GX15-070. GX15-070 inhibited Bim/Bcl-2 heterodimerization and induced association of activated Bak with Bax in OCI-AML3 cells, as demonstrated by co-immunoprecipitation studies using CHAPS buffer. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity, caspase activation and a decrease in mitochondrial inner membrane potential. Notably, GX15-070 induced cytochrome c release from isolated mitochondria of leukemic cells. GX15-070 synergized with both AraC and the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells, a notoriously chemoresistant cell line (GX15-070 and ABT-737 average CI value 0.3; GX15-070 and AraC average CI value 0.36). In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. GX15-070 potently inhibited clonogenic ability of AML blasts at sub-micromolar doses (58.5±10.6% CFU-Blast at 0.1μM and 38.1±10.5% at 0.25μM, n=7). In summary, BH3 inhibitor GX15-070 induces apoptosis in AML cells via inhibition of association of pro-survival Bcl-2 family proteins and BH3-only proteins, followed by Bax/Bak activation and initiation of the intrinsic apoptotic pathway. Hence, GX15-070 alone or in combination with chemotherapeutic agents may have utility in AML therapy.

Resistance of leukemic cells to 2-chlorodeoxyadenosine is due to a lack of calcium-dependent cytochrome c release

Blood ◽  
2002 ◽  
Vol 99 (2) ◽  
pp. 655-663 ◽  
Author(s):  
Joya Chandra ◽  
Emma Mansson ◽  
Vladimir Gogvadze ◽  
Scott H. Kaufmann ◽  
Freidoun Albertioni ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Dna Fragmentation ◽  
Cell Lines ◽  
Resistant Cell ◽  
Leukemic Cells ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Deoxyguanosine Kinase ◽  
Induced Apoptosis ◽  
Resistant Cells

Abstract The purine nucleoside 2-chlorodeoxyadenosine (CdA) is often used in leukemia therapy. Its efficacy, however, is compromised by the emergence of resistant cells. In the present study, 3 CdA-resistant cell lines were generated and characterized. Their ability to accumulate 2-chloroadenosine triphosphate (CdATP) varied, reflecting differences in activities of deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Nonetheless, the selected lines were uniformly resistant to CdA-induced apoptosis, as assessed by caspase activation and DNA fragmentation. In contrast, cytosols from resistant cells were capable of robust caspase activation when incubated in the presence of cytochrome c and dATP. Moreover, replacement of dATP with CdATP also resulted in caspase activation in the parental and some of the resistant cell lines. Strikingly, CdA-induced decreases in mitochondrial transmembrane potential and release of cytochrome c from mitochondria were observed in the parental cells but not in any resistant lines. The lack of cytochrome c release correlated with an increased ability of mitochondria from resistant cells to sequester free Ca2+. Consistent with this enhanced Ca2+buffering capacity, an early increase in cytosolic Ca2+after CdA treatment of parental cells but not resistant cells was detected. Furthermore, CdA-resistant cells were selectively cross-resistant to thapsigargin but not to staurosporine- or Fas-induced apoptosis. In addition, CdA-induced caspase-3 activation and DNA fragmentation were inhibited by the Ca2+ chelator BAPTA-AM in sensitive cells. Taken together, the data indicate that the mechanism of resistance to CdA may be dictated by changes in Ca2+-sensitive mitochondrial events.

scholarly journals d,l-Methadone causes leukemic cell apoptosis via an OPRM1-triggered increase in IP3R-mediated ER Ca2+ release and decrease in Ca2+ efflux, elevating [Ca2+]i

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
JungKwon Lee ◽  
Jesusa L. Rosales ◽  
Hee-Guk Byun ◽  
Ki-Young Lee
Keyword(s):  
Cytochrome C ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Specific Target ◽  
Induced Apoptosis

AbstractThe search continues for improved therapy for acute lymphoblastic leukemia (aLL), the most common malignancy in children. Recently, d,l-methadone was put forth as sensitizer for aLL chemotherapy. However, the specific target of d,l-methadone in leukemic cells and the mechanism by which it induces leukemic cell apoptosis remain to be defined. Here, we demonstrate that d,l-methadone induces leukemic cell apoptosis through activation of the mu1 subtype of opioid receptors (OPRM1). d,l-Methadone evokes IP3R-mediated ER Ca2+ release that is inhibited by OPRM1 loss. In addition, the rate of Ca2+ extrusion following d,l-methadone treatment is reduced, but is accelerated by loss of OPRM1. These d,l-methadone effects cause a lethal rise in [Ca2+]i that is again inhibited by OPRM1 loss, which then prevents d,l-methadone-induced apoptosis that is associated with activation of calpain-1, truncation of Bid, cytochrome C release, and proteolysis of caspase-3/12. Chelating intracellular Ca2+ with BAPTA-AM reverses d,l-methadone-induced apoptosis, establishing a link between the rise in [Ca2+]i and d,l-methadone-induced apoptosis. Altogether, our findings point to OPRM1 as a specific target of d,l-methadone in leukemic cells, and that OPRM1 activation by d,l-methadone disrupts IP3R-mediated ER Ca2+ release and rate of Ca2+ efflux, causing a rise in [Ca2+]i that upregulates the calpain-1-Bid-cytochrome C-caspase-3/12 apoptotic pathway.

Nitric Oxide–Induced Apoptosis in Human Leukemic Lines Requires Mitochondrial Lipid Degradation and Cytochrome C Release

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2342-2352 ◽  
Author(s):  
Alexey Ushmorov ◽  
Frank Ratter ◽  
Volker Lehmann ◽  
Wulf Dröge ◽  
Volker Schirrmacher ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cytochrome C ◽  
Mitochondrial Protein ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Cytochrome C Release ◽  
Mitochondrial Functions ◽  
Mitochondrial Lipid ◽  
Induced Apoptosis

Abstract We have previously shown that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through activation of caspases not only via CD95/CD95L interaction, but also independently of such death receptors. Here we investigated mitochondria-dependent mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor glycerol trinitrate (at the concentration, which induces apoptotic cell death) caused (1) a significant decrease in the concentration of cardiolipin, a major mitochondrial lipid; (2) a downregulation in respiratory chain complex activities; (3) a release of the mitochondrial protein cytochrome c into the cytosol; and (4) an activation of caspase-9 and caspase-3. These changes were accompanied by an increase in the number of cells with low mitochondrial transmembrane potential and with a high level of reactive oxygen species production. Higher resistance of the CD95-resistant Jurkat subclone (APO-R) cells to NO-mediated apoptosis correlated with the absence of cytochrome c release and with less alterations in other mitochondrial parameters. An inhibitor of lipid peroxidation, trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat cells, whereas bongkrekic acid (BA), which blocks mitochondrial permeability transition, provided only a moderate antiapoptotic effect. Transfection of Jurkat cells with bcl-2 led to a complete block of apoptosis due to the prevention of changes in mitochondrial functions. We suggest that the mitochondrial damage (in particular, cardiolipin degradation and cytochrome c release) induced by NO in human leukemia cells plays a crucial role in the subsequent activation of caspase and apoptosis.

Activation of the JNK Pathway Mediates Chemoresistance in T-Cell Acute Lymphoblastic Leukemia by Phosphorylation and Proteosomal Degradation of BimEL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2372-2372
Author(s):  
Kam Tong Leung ◽  
Karen Kwai Har Li ◽  
Samuel Sai Ming Sun ◽  
Paul Kay Sheung Chan ◽  
Yum Shing Wong ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cytochrome C ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
Jnk Pathway ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Induced Apoptosis

Abstract Despite progress in the development of effective treatments against T-cell acute lymphoblastic leukemia (T-ALL), about 20% of patients still exhibit poor response to the current chemotherapeutic regimens and the cause of treatment failure in these patients remains largely unknown. In this study, we aimed at finding mechanisms that drive T-ALL cells resistant to chemotherapeutic agents. By screening etoposide sensitivity of a panel of T-ALL cell lines using DNA content and PARP cleavage as apoptosis markers, we identified an apoptosis-resistant cell line, Sup-T1. Western blot analysis and caspase activity assay showed that Sup-T1 cells were deficient in etoposide-induced activation of caspase-3 and caspase-9. In addition, mitochondrial cytochrome c release was not evident in etoposide-treated Sup-T1 cells. However, addition of exogenous cytochrome c in cell-free apoptosis reactions induced prominent caspase-3 activation, indicating that the chemoresistance observed in Sup-T1 cells was due to its insusceptibility to the drug-induced mitochondrial alterations. Analysis of the basal expression of the Bcl-2 family proteins revealed that the levels of Bcl-2 was higher in Sup-T1 cells, while Bax and BimEL levels were lower, when compared to etoposide-sensitive T-ALL cell lines. Gene silencing using antisense oligonucleotide to Bcl-2 and overexpression of Bax did not resensitize cells to etoposide-induced apoptosis. On the contrary, transient transfection of BimEL into Sup-T1 cells significantly restored etoposide sensitivity. Further experiments revealed that the lack of BimEL expression in Sup-T1 cells was due to the rapid degradation of newly-synthesized BimEL by the proteosomal pathway, as treatment of Sup-T1 cells with a proteosome inhibitor significantly restored the protein level of BimEL. Moreover, treatment with proteosome inhibitor resulted in mobility shift of BimEL, which was sensitive to phosphatase digestion. Furthermore, treatment of Sup-T1 cells with JNK inhibitor resulted in accumulation of BimEL, and pretreatment with JNK inhibitor restored sensitivity of Sup-T1 cells to etoposide-induced apoptosis, indicating that constitutive activation of the JNK pathway in Sup-T1 cells was responsible for promoting BimEL phosphorylation, and this may serve as a signal targeting BimEL to the proteosome for degradation. Altogether, our findings provide the first evidence that JNK activation correlates inversely with BimEL level by promoting its phosphorylation and degradation. This, in turn, reduces the sensitivity of T-ALL cells to chemotherapeutic agents.

Mechanisms of Apoptosis Induction by BH3 Inhibitor ABT-737 in AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 244-244
Author(s):  
Michael Andreeff ◽  
Rooha Contractor ◽  
Peter P. Ruvolo ◽  
Xingming Deng ◽  
Ismael Samudio ◽  
...  
Keyword(s):  
Cell Lines ◽  
Phosphorylation Site ◽  
Leukemia Cell ◽  
Mek Inhibitor ◽  
Chemotherapeutic Agents ◽  
Site Directed Mutagenesis ◽  
Apoptotic Pathway ◽  
Induced Apoptosis ◽  
In Cells

Abstract Bcl2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus, strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). In this report, we investigate mechanisms of action of the novel small molecule Bcl2 inhibitor ABT-737 in AML. ABT-737 effectively killed AML patient blast cells and colony-forming cell lines at nanomolar concentrations with no effect on normal hematopoietic cells. Notably, CD34+38−123+ AML stem cells are highly sensitive to the compound. ABT-737-induced apoptosis is initiated by disruption of Bcl2:Bax dimers and activation of the intrinsic apoptotic pathway. ABT-737 works synergistically with chemotherapeutic agents such as ara-C and doxorubicin. To investigate the role of Bcl-2 phosphorylation in the sensitivity to BH3 inhibitor, we used IL-3 dependent NSF.N1/H7 mouse myeloid cells modified by site-directed mutagenesis to produce various Bcl-2 phospho-mutants. NSF.N1/H7 cells stably transfected with phosphomimetic T69E/S70E/S87E (EEE) Bcl-2 mutants were resistant to ABT-737 (IC50>500 nM) as compared to cells expressing wt-Bcl-2 or the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutants (IC50s of 50 and 25 nM). Consistent with a mechanism whereby increased Bcl2 phosphorylation impedes ABT-737 suppression of Bcl2 dimerization with Bax, ABT-737 potently blocked Bcl2:Bax association in cells expressing exogenous WT Bcl2 and AAA mutant Bcl2 but not in cells expressing exogenous phosphomimetic EEE mutant Bcl2. Since the S70E phosphorylation site of Bcl-2 is a known ERK substrate, we examined combined effects of ABT-737 and MEK inhibitor PD98059 in OCI-AML3 cells resistant to ABT-737 alone. The combined activity of PD98059 and ABT-737, evaluated by isobologram analysis, revealed a striking synergistic interaction between the MEK and BH3 inhibitors, with combination indices (CI) of 0.08±0.003. OCI-AML3 cells exhibit the highest expression of Mcl-1 among the acute leukemia cell lines tested. We propose that loss of Mcl-1 expression as a result of suppression of ERK may also be involved in the ability of PD98059 to enhance ABT-737-induced apoptosis. siRNA to Mcl-1 strikingly sensitized OCI-AML3 cells to ABT-induced apoptosis (14% apoptosis in parental cells at 2.5μM ABT-737, 64% apoptosis in siRNA-transfected cells at 10-fold lower concentration of 0.25μM). We have further demonstrated that ABT-737 reduced leukemia burden and significantly (p=0.0018) prolonged survival of mice in an in vivo mouse model. These findings suggest that: 1) ABT-737 reduces apoptosis through disruption of Bcl2:Bax heterodimers; 2) its activity is limited by Bcl2 phosphorylation and Mcl-1 overexpression; 3) combination with MEK inhibition results in inhibition of Bcl2 phosphorylation, downregulation of Mcl-1 and dramatic enhancement of ABT-737-induced apoptosis in AML.

Inhibition of the Pan-Bcl-2 Family by the Small Molecule GX15-070 Induces Apoptosis in Mantle Cell Lymphoma (MCL) Cells and Enhances the Activity of Two Proteasome Inhibitors (NPI-0052 and Bortezomib), and Doxorubicin Chemotherapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2532-2532 ◽  
Author(s):  
Victor Y. Yazbeck ◽  
Georgios V. Georgakis ◽  
Yang Li ◽  
David McConkey ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Small Molecule ◽  
Cell Lymphoma ◽  
Proteasome Inhibitors ◽  
Dependent Manner ◽  
Intrinsic Apoptotic Pathway ◽  
Phase I Clinical Trials ◽  
Apoptotic Pathway ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) accounts for 6–8% of all non-Hodgkin lymphomas (NHLs). It is an aggressive lymphoma with a poor prognosis--it is generally considered incurable with conventional treatments, and median survival is 3–4 years with a 10-year survival of only 10–15%. There is no accepted standard of care and effective treatments are greatly needed. Bcl-2 family proteins are important regulators of the intrinsic apoptotic pathway and are involved in oncogenesis and chemoresistance of a variety of tumor types, including lymphoma. Antiapoptotic proteins of the Bcl-2 family are overexpressed in mantle cell lymphoma (MCL) cells and may be responsible, in part, for drug resistance. GX15-070 is a small-molecule antagonist of the BH3-binding groove of the Bcl-2 family of proteins, and is currently in Phase I clinical trials. Consequently, we determined the activity of GX15-070 in 3 MCL cell lines (Jeko-1, Mino, and SP53). Cell viability was determined by MTS assay, apoptosis by Annexin-V binding and FACS analysis, and molecular changes by western blot. GX15-070 induced apoptosis in all three MCL cell lines in a dose and time-dependent manner. In the SP53 cell line, GX15-070 decreased MCL-1 and Bak levels, increased Bax and cleaved caspase 3. Furthermore, GX15-070 activated both the extrinsic and intrinsic apoptotic pathway as evident by cleavage of caspase 8, 9, and Bid. Both bortezomib and the novel proteasome inhibitor NPI-0052 induced single agent antiproliferative activity in MCL. GX15-070 enhanced the effect of both proteasome inhibitors. Additionally, GX15-070 showed an additive effect with doxorubicin. These studies suggest that GX15-070 may have a therapeutic value in MCL either alone or in combination with proteasome inhibitors or chemotherapy.

Inhibition of hypoxia/reoxygenation-induced apoptosis in metallothionein-overexpressing cardiomyocytes

2001 ◽  
Vol 280 (5) ◽  
pp. H2292-H2299 ◽  
Author(s):  
Guang-Wu Wang ◽  
Zhanxiang Zhou ◽  
Jon B. Klein ◽  
Y. James Kang
Keyword(s):  
Cytochrome C ◽  
Caspase 3 ◽  
Ischemia Reperfusion ◽  
Annexin V ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Fetal Bovine ◽  
Induced Apoptosis ◽  
Hypoxia Reoxygenation

To study possible mechanisms for metallothionein (MT) inhibition of ischemia-reperfusion-induced myocardial injury, cardiomyocytes isolated from MT-overexpressing transgenic neonatal mouse hearts and nontransgenic controls were subjected to 4 h of hypoxia (5% CO2-95% N2, glucose-free modified Tyrode's solution) followed by 1 h of reoxygenation in MEM + 20% fetal bovine serum (FBS) (5% CO2-95% air), and cytochrome c-mediated caspase-3 activation apoptotic pathway was determined. Hypoxia/reoxygenation-induced apoptosis was significantly suppressed in MT-overexpressing cardiomyocytes, as measured by both terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling and annexin V-FITC binding. In association with apoptosis, mitochondrial cytochrome c release, as determined by Western blot, was observed to occur in nontransgenic cardiomyocytes. Correspondingly, caspase-3 was activated as determined by laser confocal microscopic examination with the use of FITC-conjugated antibody against active caspase-3 and by enzymatic assay. The activation of this apoptotic pathway was significantly inhibited in MT-overexpressing cells, as evidenced by both suppression of cytochrome c release and inhibition of caspase-3 activation. The results demonstrate that MT suppresses hypoxia/reoxygenation-induced cardiomyocyte apoptosis through, at least in part, inhibition of cytochrome c-mediated caspase-3 activation.

TPCK Induces Apoptosis in Human Acute Myeloid Leukemia U-937 Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4478-4478
Author(s):  
Julia Mazar ◽  
Alexandra Lichtenstein ◽  
Leora Katz ◽  
Ofer Shpilberg ◽  
Itai Levi ◽  
...  
Keyword(s):  
Chromatin Condensation ◽  
Annexin V ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Concentration Dependent Manner ◽  
Flip Flop ◽  
Induced Apoptosis

Abstract Many types of antitumor therapy in general and AML in particular exert their effect by activating apoptosis. Apoptosis of AML cells can be induced by cytostatic drugs, corticosteroids, and radiation. Recently, the role of different proteases as possible targets for chemotherapy was described. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a chymotrypsin-like protease (CLP) inhibitor was shown to exert a dual effect on leukemic cells: proapoptotic and antiapoptotic. In the present study the mechanism of its proapoptotic effect was addressed. It was found that the CLP inhibitors, TPCK and 3,4 dichloroisicoumarine induced apoptosis in a time- and concentration-dependent manner. Apoptosis was accompanied by a decrease in mitochondrial membrane potential, cytochrome c release, caspase-3 (but not caspase-8) activation, PS flip-flop (measured by Annexin-V staining followed by flow cytometry analysis) and chromatin condensation, but not fragmentation (detected by acridine orange/ethidium bromide staining). All apoptotic processes induced by TPCK were completely inhibited by cycloheximide. The ability of cycloheximide to inhibit TPCK-induced cell death suggests that protein synthesis plays a role in TPCK-induced apoptosis. Additionaly, the proapoptotic effect of TPCK was abolished by elimination of glucose from the medium. The data supports the role of mitochondria in this process. In the present study the apoptotic pathway driven by inhibition of CLP was demonstrated. Moreover, these findings suggest possible ways of preventing the proapoptotic activity of TPCK and thereby enhancimg its antiapoptotic action.

Small-Molecule XIAP Inhibitors Derepress Downstream Effector Caspases and Induce Apoptosis of Leukemia Cell Lines and Patient Samples.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 759-759
Author(s):  
Aaron D. Schimmer ◽  
Marcela Gronda ◽  
Zhiliang Wang ◽  
Kate Welsh ◽  
Clemencia Pinilla ◽  
...  
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Leukemia Cell ◽  
Chemotherapeutic Agents ◽  
Leukemia Cells ◽  
Hematologic Toxicity ◽  
Phase I Clinical Trials ◽  
Leukemia Cell Lines ◽  
Induced Apoptosis ◽  
Control Compound

Abstract XIAP is a potent inhibitor of caspases 3, 7, and 9 and its over-expression renders malignant cells resistant to chemotherapy. Through an enzymatic derepression assay, we identified chemical XIAP antagonists, including 1396-12, based on a polyphenylurea pharmacophore (Cancer Cell, 1:25–35;2004). This compound binds and inhibits the caspase 3/7 inhibitory BIR2 domain of XIAP. Given the potential therapeutic utility of IAP inhibitors, we tested this XIAP antagonist in leukemia cell lines and primary patient samples. The XIAP antagonist 1396-12, but not the structurally related control compound, directly induced apoptosis in leukemia cell lines at low micromolar concentrations and sensitized leukemia cells to cytarabine. 1396-12 activated downstream caspases 3/7 prior to the activation of upstream caspases 8 and 9, and independent of Bcl-2 or caspase-8, consistent with the inhibition of the BIR2 domain of XIAP. To evaluate this XIAP antagonist as a potential novel therapy for acute myeloid leukemia (AML), primary AML blasts (n= 27), normal bone marrow mononuclear cells (n =1), or normal mobilized peripheral blood stem cells (PBSC) (n =6) were treated with increasing concentrations of 1396-12. Apoptosis was measured 24 hours after treatment by Annexin V staining. Median LD50 among the AML patient samples tested was 6 μM (range: 2μM to >40μM). The XIAP antagonist 1396-12 induced apoptosis of primary AML samples with a LD50 ≥ 10μM in 16 of 27 (60%) samples and with a LD50 >40μM in 7 of 27 (26%) samples. In contrast, 1396-12 was less toxic to the normal PBSC or marrow with a LD50>40μM in all normal samples tested. As a comparison, the inactive control compound was not toxic to any of the AML or normal samples at concentrations up to 40μM. In addition to the short-term cytotoxicity assays, the effects of 1396-12 on AML and normal samples were evaluated in colony formation assays. The XIAP antagonist inhibited clonogenic survival in 4 AML samples tested with a mean LD50 of 4 ± 0.8μM. Treatment with 1396-12 also reduced colony formation by 2 normal PBSC samples with LD50’s of 8.5 ± 0.3μM and 5.6 ± 0.4μM. In the normal PBSC samples, both BFU-E and CFU-GM lineages were equally reduced after treatment with the XIAP antagonist. Treatment with the control compound did not reduce colony growth in the AML or normal samples. Among the primary AML samples, response to the XIAP inhibitors correlated with XIAP protein levels. Low to absent levels of XIAP were associated with a higher probability of resistance to treatment with XIAP inhibitors (p =0.04, by logistic regression analysis). In conclusion, polyphenylurea-based XIAP antagonists directly induce apoptosis in leukemia cells and patient samples at low micromolar concentrations through a mechanism of action distinct from conventional chemotherapeutic agents. These antagonists can be used as biological tools to understand the role of IAPs in normal and malignant hematopoietic cells. They may also serve as lead compounds for the development of useful therapies for the treatment of leukemia and other malignancies, but their potential hematologic toxicity will have to be carefully evaluated in phase I clinical trials.

Subcellular Distribution and Redistribution of Bcl-2 Family Proteins in Human Leukemia Cells Undergoing Apoptosis

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2353-2359 ◽  
Author(s):  
Li Jia ◽  
Marion G. Macey ◽  
Yuzhi Yin ◽  
Adrian C. Newland ◽  
Stephen M. Kelsey
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Western Blotting ◽  
Leukemic Cells ◽  
Mitochondrial Outer Membrane ◽  
Human Leukemia ◽  
Cell Level ◽  
Cytochrome C Release ◽  
Free System ◽  
Whole Cell

It has been suggested that the ratio of Bcl-2 family proapoptotic proteins to antiapoptotic proteins determines the sensitivity of leukemic cells to apoptosis. However, it is believed that Bcl-2 family proteins exert their function on apoptosis only when they target to the mitochondrial outer membrane. The vinblastine-resistant T-lymphoblastic leukemic cell line CEM/VLB100 has increased sensitivity to tumor necrosis factor- (TNF-)–induced cytochrome crelease, mitochondrial respiratory inhibition, and consequently apoptosis, compared with parental CEM cells. However, there was no difference between the two cell lines in the expression of Bcl-2 family proteins Bcl-2, Bcl-XL, Bcl-XS, Bad, and Bax at the whole cell level, as analyzed by Western blotting. Bcl-2 mainly located to mitochondria and light membrane as a membrane-bound protein, whereas Bcl-XL was located in both mitochondria and cytosol. Similar levels of both Bcl-2 and Bcl-XL were present in the resting mitochondria of the two cell lines. Although the proapoptotic proteins Bcl-XS, Bad, and Bax were mainly located in the cytosol, CEM/VLB100 mitochondria expressed higher levels of these proapoptotic proteins. Subcellular redistribution of the Bcl-2 family proteins was detected in a cell-free system by both Western blotting and flow cytometry after exposure to TNF-. The levels of Bcl-2 family proteins were not altered at the whole cell level by TNF-. However, after exposure to TNF-, Bax, Bad, and Bcl-XS translocated from the cytosol to the mitochondria of both cell lines. An increase in Bcl-2 levels was observed in CEM mitochondria, which showed resistance to TNF-–induced cytochrome c release. By contrast, decreased mitochondrial Bcl-2 was observed in CEM/VLB100 cells, which released cytochrome c from the mitochondria and underwent apoptosis as detected by fluorescence microscopy. We conclude that mitochondrial levels of Bcl-2 family proteins may determine the sensitivity of leukemic cells to apoptosis and that, furthermore, these levels may change rapidly after exposure of cells to toxic stimuli.

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