Thioredoxin inhibitor PX-12 induces mitochondria-mediated apoptosis in acute lymphoblastic leukemia cells

AbstractImbalances in redox homeostasis have been described to be involved in the development, progression and relapse of leukemia. As the thioredoxin (Trx) system, one of the major cellular antioxidant networks, has been implicated in acute lymphoblastic leukemia (ALL), we investigated the therapeutic potential of Trx inhibition in ALL. Here, we show that the Trx inhibitor PX-12 reduced cell viability and induced cell death in a dose- and time-dependent manner in different ALL cell lines. This antileukemic activity was accompanied by an increase in reactive oxygen species (ROS) levels and enhanced PRDX3 dimerization. Pre-treatment with the thiol-containing ROS scavenger N-acetylcysteine (NAC), but not with non-thiol-containing scavengers α-tocopherol (α-Toc) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP), significantly rescued PX-12-induced cell death. Furthermore, PX-12 triggered activation of BAK. Importantly, knockdown of BAK reduced PX-12-stimulated ROS production and cell death. Similarly, silencing of NOXA provided significant protection from PX-12-mediated cell death. The relevance of mitochondria-mediated, caspase-dependent apoptosis was further supported by data showing that PX-12 triggered cleavage of caspase-3 and that addition of the broad-range caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD.fmk) potently blocked cell death upon PX-12 treatment. This study provides novel insights into the mechanisms of PX-12-induced cell death in ALL and further highlights the therapeutic potential of redox-active compounds in ALL.

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Obatoclax (GX15-070) Overcomes Glucocorticoid-Resistance In Acute Lymphoblastic Leukemia through Induction of Apoptosis and Autophagy

Blood ◽

10.1182/blood.v116.21.1809.1809 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1809-1809

Author(s):

Hisashi Harada ◽

Nastaran Heidari ◽

Mark Hicks

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Small Molecule ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Annexin V ◽

Glucocorticoid Resistance ◽

Down Regulation

Abstract Abstract 1809 Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including acute lymphoblastic leukemia (ALL). However, patients often develop resistance to GC on relapse. Resistance to GC in ALL can be associated with defects in apoptosis machinery, but not in the GC receptor. Thus, targeting downstream molecules may lead to the development of new therapeutic strategies. GC-induced apoptosis is through the intrinsic mitochondria-dependent pathway. The BCL-2 family proteins are central regulatory proteins in this pathway. We hypothesized that targeting anti-apoptotic MCL-1 might be effective among the BCL-2 family proteins, since (1) we recognized that treatment with dexamethasone (Dex) in CCRF-CEM or Molt-4 T-ALL cells slightly induce MCL-1 and the expression level of MCL-1 is higher in Dex-resistant ALL cells compared with that in Dex-sensitive cells; (2) recent studies have demonstrated that increased expression of MCL-1 associates with GC resistance. In support of our hypothesis, down-regulation of MCL-1 by shRNA enhances Dex-induced cell death. We then pharmacologically inactivate MCL-1 function by GX15-070 (obatoclax), a BH3 mimetic small molecule that targets anti-apoptotic BCL-2 family proteins including BCL-2, BCL-XL, and MCL-1. Treatment with GX15-070 in both Dex-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases Annexin V-positive population, indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from BAK/MCL-1 complex following GX15-070 treatment. Consistently, down-regulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, down-regulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Down-regulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy. Enhanced anti-apoptotic BCL-2 family protein expression has been observed in several types of tumors. Targeting these proteins is therefore an attractive strategy for restoring the apoptosis process in tumor cells. Among the small molecule BCL-2 inhibitors, ABT-737 and its analog ABT-263 are the leading compounds currently in clinical development. However, these molecules have an affinity only with BCL-2 and BCL-XL, but not with MCL-1. Thus, ABT-737 can not be effective as a single agent therapeutic for ALL when MCL-1 is overexpressed. In contrast, GX15-070 can overcome the resistance conferred by high level of MCL-1. Our results suggest that GX15-070 could be useful as a single agent therapeutic against ALL and that the activity/expression of anti-apoptotic proteins could be a biomarker to determine the treatment strategy to ALL patients. (Supported by NIH R01CA134473 and the William Lawrence and Blanche Hughes Foundation) Disclosures: No relevant conflicts of interest to declare.

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Inhibitory effect of hydnocarpin D on T-cell acute lymphoblastic leukemia via induction of autophagy-dependent ferroptosis

Experimental Biology and Medicine ◽

10.1177/15353702211004870 ◽

2021 ◽

pp. 153537022110048

Author(s):

Siyue Lou ◽

Huanwu Hong ◽

Liwaliding Maihesuti ◽

Hang Gao ◽

Zhihui Zhu ◽

...

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Inhibitory Effect ◽

Cell Acute Lymphoblastic Leukemia ◽

Pre Treatment ◽

Interesting Compound ◽

Induced Apoptosis

Hydnocarpin D (HD) is a bioactive flavonolignan compound that possesses promising anti-tumor activity, although the mechanism is not fully understood. Using T cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat and Molt-4 as model system, we found that HD suppressed T-ALL proliferation in vitro, via induction of cell cycle arrest and subsequent apoptosis. Furthermore, HD increased the LC3-II levels and the formation of autophagolysosome vacuoles, both of which are markers for autophagy. The inhibition of autophagy by either knockdown of ATG5/7 or pre-treatment of 3-MA partially rescued HD-induced apoptosis, thus suggesting that autophagy enhanced the efficacy of HD. Interestingly, this cytotoxic autophagy triggered ferroptosis, as evidenced by the accumulation of lipid ROS and decrease of GSH and GPX4, while inhibition of autophagy impeded ferroptotic cell death. Our study suggests that HD triggers multiple cell death processes and is an interesting compound that should be evaluated in future preclinical studies.

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Phenyhexyl Isothiocyanate, a Dual Inhibitor of Histone Deacetylation and Methylation, Inhibit Acute Lymphoblastic Leukemia Cells through Epigenetic Modulation.

Blood ◽

10.1182/blood.v108.11.4532.4532 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4532-4532

Author(s):

Yiqun Huang ◽

Xudong Ma ◽

Dicky J.W. Chiao ◽

Delong Liu

Keyword(s):

Cell Cycle ◽

Acute Lymphoblastic Leukemia ◽

Caspase 3 ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Caspase 8 ◽

Dependent Manner ◽

Caspase 9 ◽

Dual Inhibitor

Abstract We have shown that phenyhexyl isothiocyanate(PHI) is a novel histone deacetylase inhibitor and can also modulate histone methylation. In this study we investigated the effect of PHI on human Acute Lymphoblastic leukemia cell line Molt-4 in vitro. The viability of Molt-4 cells was determined by MTT method. Apoptosis and cell cycle arrest were measured by flow cytometry. The expression of bcl-2, caspase-9, caspase-8, caspase-3, PRAP protein, acetylated H3 and H4, methylated H3K9 and H3K4 were detected by Western Blotting. The results showed that PHI inhibited the cell growth and decreased viability of Molt-4 cells. Cell cycle analysis indicated an arrest in G0/G1 phase. The expression of bcl-2, caspase-9, caspase-3, and PRAP was inhibited by PHI in a dose and time dependent manner. In contrast, there was no significant change in the expression of Caspase-8. PHI also significantly increased the level of acetylated histone H3 as well as H4. Interestingly, PHI increased the level of methylated H3k4, but decreased methylated H3K9. These data suggest that ALL cells are sensitive to the novel HDAC inhibitor, and PHI may become a novel agent in ALL therapy.

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Methanolic Extract from Aerial Parts of Artemisia Annua L. Induces Cytotoxicity and Enhances Vincristine-Induced Anticancer Effect in Pre-B Acute Lymphoblastic Leukemia Cells

International Journal of Hematology-Oncology and Stem Cell Research ◽

10.18502/ijhoscr.v13i3.1271 ◽

2019 ◽

Author(s):

Pargol Mashati ◽

Somayeh Esmaeili ◽

Nasrin Dehghan-Nayeri ◽

Davood Bashash ◽

Mina Darvishi ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Real Time ◽

Real Time Pcr ◽

Caspase 3 ◽

Artemisia Annua ◽

Practical Implication ◽

Lymphoblastic Leukemia ◽

Pcr Analysis ◽

Dependent Manner ◽

Reh Cells

Background: Nowadays, remarkable attention has been drawn towards the effective therapeutic characteristic of natural products targeting cancerous cells. This study aimed to investigate the anti-cancer effect of Artemisia annua extract (AAE), a Chinese herbal medicine alone and in combination with a microtubule binding agent used in ALL treatment, vincristine (VCR), in B-Acute lymphoblastic leukemia (ALL) Nalm-6 and Reh cells. Materials and Methods: Cytotoxic activity of AAE and VCR was determined using MTT assay in Nalm-6, and Reh cell lines and synergism was evaluated using the CompuSyn software. Caspase 3 activity and Annexin/PI staining were performed for apoptosis assessment. The expression level of apoptosis-related genes, caspase 3, Bax and Bcl-2 were determined using real time-PCR. One-way ANOVA and post hoc Tukey multiple comparisons were used for statistical analysis. Results: Our findings revealed that a single administration of AAE exerted an anti-leukemic effect in both ALL-derived cells in a time- and dose-dependent manner. Interestingly, the growth inhibitory activity of the extract was more potentiated when combined with 0.1 and 1 nM VCR through caspase 3-dependent apoptosis. Moreover, real-time PCR analysis showed that VCR-induced cytotoxicity was augmented by AAE through alteration of Bax, and Bcl-2 mRNA expression. Conclusion: Overall, owing to the nontoxic nature of AAE and its explicit role in enhancing VCR effectiveness, our study provided new insight into the development of a novel combinatorial approach in ALL using natural herbs. The practical implication of the research requires further investigation through clinical trials, opening avenues for forthcoming treatment improvements.

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Sphingosine Kinase Inhibition Has Pre-Clinical Activity in Acute Lymphoblastic Leukemia,

Blood ◽

10.1182/blood.v118.21.3573.3573 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3573-3573

Author(s):

Craig T. Wallington-Beddoe ◽

David Ho ◽

Kenneth Francis Bradstock ◽

Linda J. Bendall

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Caspase 3 ◽

Lymphoblastic Leukemia ◽

Term Survival ◽

Intracellular Location ◽

Ic50 Values

Abstract Abstract 3573 Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph+ ALL has a particularly poor prognosis. Sphingosine 1-phosphate (S1P) is a lipid mediator of diverse cellular functions, most notably of lymphocyte trafficking, angiogenesis, cell proliferation and survival. S1P is produced intracellularly by the sphingosine kinases (SK) of which there are two isoforms, SK1 and SK2. SK1 is over expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. The role of SK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SK1. Inhibition of either SK1 or SK2 is currently under investigation as a novel anti-cancer strategy and potent anti-leukemic effects are likely. Application of the combined SK1/SK2 inhibitor SKI II and the selective SK2 inhibitor ABC294640 to ALL cells produced a reduction in cellular proliferation as measured by 3H-thymidine incorporation in all cell lines (REH, NALM6, LK63, ALL1, 2070 and TOM1) tested with IC50 values of 1μM – 7μM for SKI II and <40μM for ABC294640. Viability, measured by flow cytometry using annexin V and propidium iodide (PI) staining, was also reduced in all cell lines except the Ph+ ALL1 and 2070 cells treated with SKI II with IC50 values ranging from 2μM to >10μM for SKI II and 50–60μM for ABC294640. SKI II resulted in caspase-dependent cell death, as determined by flow cytometric assessment of intracellular caspase-3 cleavage and apoptotic morphology on light microscopy, with cell death prevented by pre-incubation with 100μM of the pan-caspase inhibitor Z-VAD-FMK. However, ABC294640 induced caspase-3 cleavage at lower than expected levels and cell death was not prevented by Z-VAD-FMK. Both agents significantly reduced intracellular S1P concentrations by 24 hours as determined by ELISA, thereby confirming the ability of these compounds to inhibit SK1 or SK2 activity. A search for agents that synergized with the SK inhibitors revealed that when Ph+ ALL cells were treated with the combination of imatinib and either ABC294640 or SKI II, a further reduction in cell death occurred than with either agent alone, thereby enhancing the therapeutic effect of ABC294640 and overcoming resistance seen with SKI II alone. Furthermore, the combination of mildly cytotoxic concentrations of ABC294640 and the novel pan histone deacetylase inhibitor AR-42 were found to significantly increase leukemic cell death at 24 and 48 hours in Ph+ and negative ALL cells. In vivo assessment of the SK inhibitors was determined by injecting NOD/SCID IL2gc−/− mice with 2–5 million human B-ALL cells and treating with 100 mg/kg/day ABC294640 or vehicle by intraperitoneal injection for 21 days after which all animals were sacrificed. Assessment of leukemia in blood, bone marrow and spleen was determined by flow cytometry using antibodies to human CD19 and murine CD45. Significant reductions in the levels of leukemia in all examined tissues were found in ABC294640-treated animals using three different human ALL xenografts, including the Ph+ positive xenograft 2070. Average absolute levels of leukemia in the bone marrow of ABC294640-treated mice for xenografts 2070, 1345 and 0398 were reduced by 40% (p = 0.00007), 55% (p = 0.004) and 72% (p = 0.000001) respectively. No overt toxicity was noted. SK inhibition, resulting in reduced intracellular S1P, is an exciting novel anti-leukemic strategy potentially adding to the repertoire of non-chemotherapeutic agents for the treatment of ALL. Combinations of SK inhibitors with newer targeted agents show promise of greater leukemia reduction. Disclosures: No relevant conflicts of interest to declare.

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Sensitization of acute lymphoblastic leukemia cells for LCL161-induced cell death by targeting redox homeostasis

Biochemical Pharmacology ◽

10.1016/j.bcp.2016.01.004 ◽

2016 ◽

Vol 105 ◽

pp. 14-22 ◽

Cited By ~ 8

Author(s):

Christina Haß ◽

Katharina Belz ◽

Hannah Schoeneberger ◽

Simone Fulda

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Redox Homeostasis ◽

Leukemia Cells

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BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

Cancer Cell International ◽

10.1186/s12935-021-01908-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Shuiyan Wu ◽

You Jiang ◽

Yi Hong ◽

Xinran Chu ◽

Zimu Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Acute Lymphoblastic Leukemia ◽

Caspase 3 ◽

Lymphoblastic Leukemia ◽

Rna Seq ◽

Cell Acute Lymphoblastic Leukemia ◽

Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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Inhibition of Autophagy Enhances the Antitumor Effect of Thioridazine in Acute Lymphoblastic Leukemia Cells

Life ◽

10.3390/life11040365 ◽

2021 ◽

Vol 11 (4) ◽

pp. 365

Author(s):

Carina Colturato-Kido ◽

Rayssa M. Lopes ◽

Hyllana C. D. Medeiros ◽

Claudia A. Costa ◽

Laura F. L. Prado-Souza ◽

...

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Clinical Problem ◽

Jurkat Cells ◽

Leukemia Cells ◽

Peripheral Mononuclear Blood ◽

Induced Apoptosis ◽

Cure Rates

Acute lymphoblastic leukemia (ALL) is an aggressive malignant disorder of lymphoid progenitor cells that affects children and adults. Despite the high cure rates, drug resistance still remains a significant clinical problem, which stimulates the development of new therapeutic strategies and drugs to improve the disease outcome. Antipsychotic phenothiazines have emerged as potential candidates to be repositioned as antitumor drugs. It was previously shown that the anti-histaminic phenothiazine derivative promethazine induced autophagy-associated cell death in chronic myeloid leukemia cells, although autophagy can act as a “double-edged sword” contributing to cell survival or cell death. Here we evaluated the role of autophagy in thioridazine (TR)-induced cell death in the human ALL model. TR induced apoptosis in ALL Jurkat cells and it was not cytotoxic to normal peripheral mononuclear blood cells. TR promoted the activation of caspase-8 and -3, which was associated with increased NOXA/MCL-1 ratio and autophagy triggering. AMPK/PI3K/AKT/mTOR and MAPK/ERK pathways are involved in TR-induced cell death. The inhibition of the autophagic process enhanced the cytotoxicity of TR in Jurkat cells, highlighting autophagy as a targetable process for drug development purposes in ALL.

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