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.

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.

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

Blood ◽  
2006 ◽  
Vol 108 (2) ◽  
pp. 630-637 ◽  
Author(s):  
Bing Z. Carter ◽  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Teresa McQueen ◽  
David Harris ◽  
...  
Keyword(s):  
Cell Death ◽  
Anticancer Agents ◽  
Chinese Herb ◽  
Mitochondrial Pathway ◽  
Leukemic Cells ◽  
Cytochrome C Release ◽  
Knock Out ◽  
Dependent Cell ◽  
Induced Apoptosis

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-XL 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.

Survivin-Driven Therapy for Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2749-2749
Author(s):  
Hugo Caldas ◽  
Rachel A. Altura
Keyword(s):  
Gene Therapy ◽  
Cell Death ◽  
Cell Growth ◽  
Cancer Cells ◽  
Suicide Gene ◽  
Post Treatment ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Mitochondrial Potential ◽  
Transformed Cell Lines

Abstract Survivin, a member of the inhibitor of apoptosis (IAP) family of proteins, is widely expressed in transformed cell lines and in many different primary cancer cells, including both hematopoietic and non-hematopoietic malignancies. It is not expressed in many non-malignant adult tissues, but is essential for fetal development, as demonstrated by conventional gene-targeting experiments in mice that show embryonic lethality at day 4–6 of development. In adult cancers, including lymphoma and many epithelial carcinomas (colon, breast, gastric) the expression level of survivin, as assayed by immunohistochemical analysis and RT-PCR, correlates with overall survival. We have designed a novel gene therapy approach that takes advantage of the high expression levels of survivin in malignant cells, in which the survivin promoter is used to drive the expression of a suicide gene to kill cancer cells by programmed cell death. Our system is based on perforin-independent granzyme B cytotoxicity and therefore does not require pro-drug activation. We designated it SAGA, for survivin and granzyme apoptosis. We used Jurkat cells as an in vitro model for T-cell leukemia, and 697Bcl2 cells as a model for pre-B Bcl2-expressing leukemia, to show this approach is more efficient in killing leukemic cells than conventional chemotherapy. Jurkat cells responded to both vincristine therapy and SAGA whereas 697Bcl2 were unaffected by vincristine, but responded to SAGA. Cell growth curves of Jurkat cells and 697Bcl2 cells are shown in Figure 1. Our approach not only inhibits cell growth, but also induces apoptosis. We detected apoptotic events by Annexin V staining and by changes in mitochondrial potential, as early as 12 hours post-treatment. Rates of early apoptotic events are shown in Table 1. In addition to these events, we also documented DNA fragmentation and caspase-3 activation in treated cells. Cytotoxicity was clearly visible by microscopic analysis 24 hours post-treatment (Figure 2). Our results strongly suggest that survivin-driven suicide gene therapy effectively enhances cell death of leukemic blasts derived from two common sub-types of ALL, one of which expresses the potent anti-apoptotic inhibitor, Bcl-2, known to be clinically more resistant to standard therapy. Experiments evaluating the in vivo effects of SAGA in mouse leukemia models are currently underway. Cell death at 24 hours Early Apoptosis Necrosis control vincristine SAGA control vincristine SAGA T-ALL 2% 46% 41% 2% 18% 19% B-ALL 1% 2% 29% 0.4% 1% 30% Figure Figure Figure Figure

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.

scholarly journals Antileukemic Natural Product Induced Both Apoptotic and Pyroptotic Programmed Cell Death and Differentiation Effect

2021 ◽  
Vol 22 (20) ◽  
pp. 11239
Author(s):  
Wohn-Jenn Leu ◽  
Hsun-Shuo Chang ◽  
Ih-Sheng Chen ◽  
Jih-Hwa Guh ◽  
She-Hung Chan
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Electron Microscopic Examination ◽  
Leukemic Cells ◽  
Caspase 8 ◽  
Electron Microscopic ◽  
Medical Needs ◽  
Response To Chemotherapy ◽  
Apoptosis Protein ◽  
Induced Apoptosis

Acute myeloid leukemia (AML) is one of the most common forms of leukemia. Despite advances in the management of such malignancies and the progress of novel therapies, unmet medical needs still exist in AML because of several factors, including poor response to chemotherapy and high relapse rates. Ardisianone, a plant-derived natural component with an alkyl benzoquinone structure, induced apoptosis in leukemic HL-60 cells. The determination of dozens of apoptosis-related proteins showed that ardisianone upregulated death receptors and downregulated the inhibitor of apoptosis protein (IAPs). Western blotting showed that ardisianone induced a dramatic increase in tumor necrosis factor receptor 2 (TNFR2) protein expression. Ardisianone also induced downstream signaling by activating caspase-8 and -3 and degradation in Bid, a caspase-8 substrate. Furthermore, ardisianone induced degradation in DNA fragmentation factor 45 kDa (DFF45), a subunit of inhibitors of caspase-activated DNase (ICAD). Q-VD-OPh (a broad-spectrum caspase inhibitor) significantly diminished ardisianone-induced apoptosis, confirming the involvement of caspase-dependent apoptosis. Moreover, ardisianone induced pyroptosis. Using transmission electron microscopic examination and Western blot analysis, key markers including gasdermin D, high mobility group box1 (HMGB1), and caspase-1 and -5 were detected. Notably, ardisianone induced the differentiation of the remaining survival cells, which were characterized by an increase in the expression of CD11b and CD68, two markers of macrophages and monocytes. Wright–Giemsa staining also showed the differentiation of cells into monocyte and macrophage morphology. In conclusion, the data suggested that ardisianone induced the apoptosis and pyroptosis of leukemic cells through downregulation of IAPs and activation of caspase pathways that caused gasdermin D cleavage and DNA double-stranded breaks and ultimately led to programmed cell death. Ardisianone also induced the differentiation of leukemic cells into monocyte-like and macrophage-like cells. The data suggested the potential of ardisianone for further antileukemic development.

scholarly journals G1 Cell Cycle Arrest and Extrinsic Apoptotic Mechanisms Underlying the Anti-Leukemic Activity of CDK7 Inhibitor BS-181

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3845
Author(s):  
Shin Young Park ◽  
Ki Yun Kim ◽  
Do Youn Jun ◽  
Su-Kyeong Hwang ◽  
Young Ho Kim
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Jurkat Cells ◽  
Parp Cleavage ◽  
Caspase 8 ◽  
Down Regulation ◽  
Blocking Antibody ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest ◽  
Induced Apoptosis

In vitro antitumor activity of the CDK7 inhibitor BS-181 against human T-ALL Jurkat cells was determined. Treatment of Jurkat clones (JT/Neo) with BS-181 caused cytotoxicity and several apoptotic events, including TRAIL/DR4/DR5 upregulation, c-FLIP down-regulation, BID cleavage, BAK activation, ΔΨm loss, caspase-8/9/3 activation, and PARP cleavage. However, the BCL-2-overexpressing Jurkat clone (JT/BCL-2) abrogated these apoptotic responses. CDK7 catalyzed the activating phosphorylation of CDK1 (Thr161) and CDK2 (Thr160), and CDK-directed retinoblastoma phosphorylation was attenuated in both BS-181-treated Jurkat clones, whereas only JT/BCL-2 cells exhibited G1 cell cycle arrest. The G1-blocker hydroxyurea augmented BS-181-induced apoptosis by enhancing TRAIL/DR4/DR5 upregulation and c-FLIP down-regulation. BS-181-induced FITC–annexin V-positive apoptotic cells were mostly in the sub-G1 and G1 phases. BS-181-induced cytotoxicity and mitochondrial apoptotic events (BAK activation/ΔΨm loss/caspase-9 activation) in Jurkat clones I2.1 (FADD-deficient) and I9.2 (caspase-8-deficient) were significantly lower than in A3 (wild-type). Exogenously added recombinant TRAIL (rTRAIL) markedly synergized BS-181-induced apoptosis in A3 cells but not in normal peripheral T cells. The cotreatment cytotoxicity was significantly reduced by the DR5-blocking antibody but not by the DR4-blocking antibody. These results demonstrated that the BS-181 anti-leukemic activity is attributed to extrinsic TRAIL/DR5-dependent apoptosis preferentially induced in G1-arrested cells, and that BS-181 and rTRAIL in combination may hold promise for T-ALL treatment.

Triptolide Sensitizes Leukemic Cells to TRAIL Induced Apoptosis Via Decrease of XIAP and p53-Mediated Increase of DR5.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 528-528
Author(s):  
Bing Z. Carter ◽  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Martin Dietrich ◽  
Clemencia Pinilla ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Chinese Herb ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
U937 Cells ◽  
Apoptosis Resistance ◽  
Protein Levels ◽  
P53 Signaling ◽  
Induced Apoptosis

Abstract Triptolide, a recently identified anticancer agent from a Chinese herb, has been shown to synergistically enhance TRAIL-induced cell death in various solid tumor cell lines. We have found that triptolide potently induces apoptosis in leukemic cell lines and blasts from AML patients at least in part by decreasing XIAP levels. XIAP is known to be a resistance factor in TRAIL-induced cell death. XIAP is highly expressed in AML blasts and primary AML cells generally are insensitive to TRAIL. We therefore hypothesize that triptolide will sensitize AML cells to TRAIL-induced apoptosis. We treated OCI-AML3, U937, and Jurkat cells with sub-optimal concentrations of triptolide, TRAIL, and their combination. At concentrations showing no or minimal effects by each agent alone, their combinations significantly promoted cell death with combination indices (CI) <1.0 for all three leukemia cell lines. To ensure that XIAP contributes to TRAIL resistance, we treated U937 cells overexpressing XIAP (U937XIAP) and the control cells (U937neo) with TRAIL. U937XIAP cells were 6-fold more resistant to TRAIL (IC50=455.8 ng/ml) than U937neo cells (IC50=74.3 ng/ml). We then treated these cells with TRAIL and 1396–11, a small molecule XIAP antagonist that binds to BIR2 of XIAP and promotes caspase-dependent apoptosis. At 3μM, 1396–11 had no significant effect on survival of either U937neo or U939XIAP cells. When combined with TRAIL, 1396–11 augmented TRAIL-induced cell death of both U937neo (IC50=45.0 ng/ml with 1396-11 vs. IC50=74.3 ng/ml without) and U939XIAP cells (IC50=318.9 ng/ml with 1396–11 vs. IC50=455.8 ng/ml without). Furthermore, we observed that triptolide decreased MDM2 and increased p53 protein levels in p53 wild type OCI-AML3 but not in p53 null U937 cells. TRAIL receptor DR5 has been shown to be regulated by p53. To elucidate the role of p53 in TRAIL-induced cell death sensitized by triptolide, we treated OCI-AML3 and U937 cells with triptolide and examined DR5 expression. We found that triptolide induced DR5 protein levels in OCI-AML3, but much less so in U937 cells. Treatment of OCI-AML3 cells with Nutlin3a, a MDM2 inhibitor that binds to MDM2 and stabilizes p53, increased DR5 protein levels and sensitized to TRAIL-induced cell death. Knockdown of p53 with retrovirus expressing p53siRNA in OCI-AML3 cells abolished cellular responses to nutlin3a and significantly decreased the sensitization to TRAIL by nutlin3a. Finally, we treated OCI-AML3 cells with 1396–11, nutlin3a, and TRAIL and our results showed that the triple combination (CI=0.045 at 24 hours, average of ED50, ED75, and ED90) was more effective in inducing cell death than either 1396–11 and TRAIL (CI=0.066) or nutlin3a and TRAIL (CI=0.190) combinations supporting our notion that triptolide sensitizes to TRAIL-induced cell death by modulating both XIAP expression and p53 signaling. Collectively, our studies suggest that inhibition of XIAP and induction of DR5 mediated by p53 activation both independently sensitize leukemic cells to TRAIL-induced apoptosis. Triptolide not only inhibits XIAP, which is overexpressed in AML, but also activates p53 signaling, which is intact in the majority of AMLs. Thus, combinations of triptolide and TRAIL may provide a novel strategy for treating AML patients by overcoming critical mechanisms of apoptosis resistance.

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.

scholarly journals Leukotoxin (LtxA/Leukothera) induces ATP expulsion via pannexin-1 channels and subsequent cell death in malignant lymphocytes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Derek J. Prince ◽  
Deendayal Patel ◽  
Scott C. Kachlany
Keyword(s):  
Cell Death ◽  
Combined Treatment ◽  
White Blood Cells ◽  
Atp Release ◽  
Death Receptor ◽  
Jurkat Cells ◽  
Parp Cleavage ◽  
Lymphocyte Function ◽  
Pannexin 1 ◽  
Induced Apoptosis

AbstractLeukotoxin (LtxA) (Trade name, Leukothera) is a protein that is secreted from the oral bacterium Aggregatibacter actinomycetemcomitans, which targets and kills activated white blood cells (WBCs) by binding to lymphocyte function associated antigen-1 (LFA-1). Interaction between LtxA and Jurkat T-cells results in cell death and is characterized by increased intracellular Ca2+, activation of caspases, clustering of LtxA and LFA-1 within lipid rafts, and involvement of the Fas death receptor. Here, we show that LtxA can kill malignant lymphocytes via apoptotic and necrotic forms of cell death. We show that LtxA causes activation of caspases and PARP, cleavage of pannexin-1 (Panx1) channels, and expulsion of ATP, ultimately leading to cell death via apoptosis and necrosis. CRISPR-Cas9 mediated knockout (K/O) of Panx1 in Jurkat cells prevented ATP expulsion and resulted in resistance to LtxA for both apoptotic and necrotic forms of death. Resistance to necrosis could only be overcome when supplementing LtxA with endogenous ATP (bzATP). The combination of LtxA and bzATP promoted only necrosis, as no Panx1 K/O cells stained positive for phosphatidylserine (PS) exposure following the combined treatment. Inhibition of LtxA/bzATP-induced necrosis was possible when pretreating Jurkat cells with oATP, a P2X7R antagonist. Similarly, blockage of P2X7Rs with oATP prevented the intracellular mobilization of Ca2+, an important early step in LtxA induced cell death. We show that LtxA is able to kill malignant lymphocytes through an apoptotic death pathway which is potentially linked to a Panx1/P2X7R mediated necrotic form of death. Thus, inhibition of ATP release appears to significantly delay the onset of LtxA induced apoptosis while completely disabling the necrotic death pathway in T-lymphocytes, demonstrating the crucial role of ATP release in LtxA-mediated cell death.

scholarly journals Inhibition of KSP by ARRY-520 Induces Cell Cycle Block and Cell Death Via the Mitochondrial Pathway in AML Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2782-2782 ◽  
Author(s):  
Bing Z. Carter ◽  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Richard Woessner ◽  
Stefan Gross ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Cycle Progression ◽  
Mitotic Arrest ◽  
Mitochondrial Pathway ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Cycle Progression ◽  
Bipolar Spindle ◽  
Cell Cycle Block

Abstract Kinesin spindle protein (KSP or Eg5), a microtubule-associated motor protein, plays an important role in establishing a bipolar spindle during mitosis and is essential for cell cycle progression. It has been demonstrated that inhibition of KSP prevents bipolar spindle formation leading to mitotic arrest and cell death. As such, a KSP inhibitor may have anti-tumor potential without toxicities associated with anti-microtubule agents such as taxanes. We found that KSP is highly expressed in various acute leukemic cells. Inhibition of KSP by a specific inhibitor, ARRY-520, at low nanomolar concentrations, blocked cell cycle progression and led to subsequent cell death in OCI-AML3, Molm13, HL-60, U937, and Jurkat cells. Knocking down p53 by p53shRNA in OCI-AML3 cells did not alter the effectiveness of ARRY-520. U937 cells overexpressing XIAP (U937XIAP) and Jurkat cells lacking caspase 8 (JurkatI2.1) showed unchanged sensitivities to ARRY-520 compared with their respective control cells, suggesting that cell cycle block and cell death induced by KSP inhibition is independent of p53 status, XIAP levels, and the activation of the extrinsic apoptotic pathway. However, although ARRY-520 blocked cell growth and induced mitotic arrest in both HL-60 cells and HL-60 cells overexpressing Bcl-2 (HL-60Bcl-2), cell death (determined by annexin V staining and changes in mitochondrial potential) was significantly abolished in HL-60Bcl-2 cells. These results suggest that cell death following cell cycle blockade by KSP inhibition is mediated through the intrinsic mitochondrial pathway. Furthermore, ARRY-520 induced the protein level of Bim, a proapoptotic BH3-only Bcl-2 family protein, prior to the activation of caspases in HL-60 cells. Although KSP inhibition by ARRY-520 had no effect on the survival of non-dividing AML blasts in vitro, ARRY-520 significantly inhibited the colony formation capacities of AML blasts, further supporting the critical role of KSP in cell proliferation. Our studies demonstrated that inhibition of KSP by ARRY-520 potently induces blockage of cell cycle progression which leads to cell death of various leukemic cells via the mitochondrial pathway and has the potential to eradicate AML progenitor cells.

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