Role of miRNA in Micro-Environment Mediated Drug Resistance in Acute Promyelocytic Leukemia to Arsenic Trioxide

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5125-5125 ◽  
Author(s):  
Saravanan Ganesan ◽  
Vairavan Lakshmanan ◽  
Hamenth Kumar Palani ◽  
Nithya Balasundaram ◽  
Ansu Abu Alex ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Arsenic Trioxide ◽  
Rna Sequencing ◽  
Acute Promyelocytic Leukemia ◽  
Small Rna ◽  
Stromal Cells ◽  
Promyelocytic Leukemia ◽  
Small Rna Sequencing ◽  
Nb4 Cells

Abstract Role of stromal microenvironment in drug resistance has been extensively reported for several cancers. We have demonstrated earlier that there is significant micro-environment mediated drug resistance (EM-DR) to arsenic trioxide (ATO) in acute promyelocytic leukemia (APL) and that this was predominantly driven by upregulation of the NF-ⱪB pathway in the malignant cell. In our current study we have probed the molecular mechanism of ATO resistance in further detail. The role of microRNA (miRNA) in mediating this cross talk, if any, has not been reported on. We undertook a study to evaluate the potential role played by miRNA in EM-DR to ATO in APL. Using NGS based small RNA sequencing we identified two miRNA's that were differentially regulated in NB4 cells upon co-culture with HS-5 stromal cells (FDR corrected p values < 0.05). The two miRNAs were hsa-miR-23a-5p (downregulated) and hsa-miR-125a-3p (upregulated)](Fig 1a). These miRNAs have also been previously reported to be involved in NF-kB regulation, specifically miR125a-3p has been reported to be involved in activation of the NF-kB pathway and miR-23a- 5p can be repressed by the same pathway. These results were consistent with our earlier reported observations that NF-kB pathway is dysregulated and enhances drug resistance to ATO. We also observed miR-23a-5p mimics were able to restore the sensitivity of NB4 cells to ATO even in the presence of stromal cells (Fig 1b). Consistent with the above small RNA sequencing and our previously reported microarray data, using quantitative proteomics approach we have identified that both NFkB signaling and metallothionein 2A (MT-2A) levels are upregulated in leukemic cells upon stromal co-culture. MT 2A is a known target for hsa-mir-23a-5p. MT's are known to sequester heavy metals such as arsenic and could potentially reduce their cytotoxic effect. The role of metallothionein in ATO resistance in APL and possibly other leukemia's needs further evaluation. This data along with that reported earlier by us illustrates multiple levels of regulation of the NF-kB pathway and resistance to ATO by stromal cell co-culture. Disclosures No relevant conflicts of interest to declare.

Dissecting The Mechanism and Functional Role Of Silenced Microrna-125a In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3803-3803
Author(s):  
Melanie L Ufkin ◽  
Maria Trissal ◽  
Heather Driscoll ◽  
Christine W Duarte ◽  
Daniel C Link ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Functional Role ◽  
Cell Cycle Progression ◽  
Small Rna Sequencing ◽  
Nb4 Cells ◽  
Hematopoietic Malignancies ◽  
Rna Expression Profiling ◽  
Altered Cell

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous disease marked by a highly variable clinical course and response to therapy. microRNA's (miR) have entered the spotlight for being involved in pathogenesis of numerous diseases, including hematopoietic malignancies. miR-125a-5p previously was identified to be decreased in AML, however its functional role leading to the pathogenesis of AML is unknown leading us to dissect its functional role in AML within these studies. Results Small RNA sequencing illustrated that miR-125a is decreased in many AML FAB subtypes at time of diagnosis and relapse (p<0.05) beyond the previously reported cytogenetically normal AML (Figure 1A&B). miR-125a expression in comparison to clinical aspects (167 AML patients) was conducted using data from the National Cancer Institute the Cancer Genome Atlas (TCGA) Data Portal to support small RNA sequencing findings. The majority of patients had low miR-125a expression (1-250 RPM), which is significantly decreased if compared to the small RNA sequencing results for healthy CD34+ cells (∼5000RPM). Interestingly, there is no difference between gender and miR-125a expression. Though not significant, there was a trend of low miR-125a expression towards low survival rate. To dissect the pathways affected when miR-125a expression is restored in AML, acute promyelocytic leukemia (APL) NB4 cells containing t (15; 17), which had the most significantly decreased expression when AML lines were screened, were utilized for studies. Previously we reported that miR-125a was epigenetically silenced in AML and caused altered cell proliferation, cell cycle progression, and apoptosis, however through RNA expression profiling we now know the potential players altered leading to these altered biological pathways. Among the significant decreased genes were FLT1, MMP-9, IL-32Rα and HIP-1 while several increased genes were of interest such as cathepsin-G, EPX, and SPARC. Though all of these were interesting due to their previous implications within cancer or AML, they are not predicted targets of miR-125a. Therefore, we focused our analysis on identifying a potential target of miR-125a within AML. From profiling results, Trib2 was significantly decreased (p=0.0003, Figure 2) when miR-125a expression was restored in NB4 cells and is a predicted target. RT-qPCR and 3'UTR luciferase confirmed that Trib2 was a target of miR-125a. Trib2 has been implicated in AML in several contexts including inhibition of C/EBPα causing decreased cell differentiation and its ability to interact with HoxA9 to aid in the progression of AML. Although Trib2 has been implicated in cancer, inhibitors are not developed currently but a necessity has been demonstrated. Therefore, we focused our studies on identifying a pathway with known inhibitors. Several reports demonstrate enhanced ErbB2 expression when miR-125a is decreased leading us to test Mubritinib, which selectively inhibits ErbB2 phosphorylation. Previously we demonstrated the profound affect on inhibition of cell cycle progression and altered cell proliferation, differentiation, and apoptosis in NB4 cells. Most strikingly was the lack of affect of the inhibitor on HL60 cells, which do not have decreased miR-125a expression like NB4 cells suggesting that inhibition of the ErbB pathway would be specific for low miR-125a AML. Excitingly, ANOVA pathway analysis revealed the ErbB pathway was significantly altered, such as decreased ErbB receptors (ErbB1 and ErbB3) and downstream effectors (PI3K, AKT, and Stat5). After establishing the role of the ErbB pathway in NB4 cells we tested the effect of Mubritinib on retinoic acid resistant NB4 cells (NB4-LR1), which is indicating that Mubritinib could potentially be utilized for a new therapeutic treatment in NB4-LR1 cells in addition to NB4 cells. Conclusion Decreased miR-125a within AML may give leukemic blasts an advantage in multiple AML subtypes. Through RNA expression profiling, Trib2 was identified as a target of miR-125a within AML. By utilizing pathway analysis in we have identified a potential new therapeutic, Mubritinib, for miR-125a low AML, which is now being tested in vivo. From our strong preliminary work ErbB inhibitors currently being utilized for treating ErbB overexpressing epithelial cancers could be tested in hematopoietic malignancies in addition to identifying a potential role of Trib2 in miR-125a low AML. Disclosures: No relevant conflicts of interest to declare.

Evolving Role of Arsenic Trioxide in Acute Promyelocytic Leukemia

2006 ◽  
Vol 1 (1) ◽  
pp. 36-40 ◽  
Author(s):  
Neeta K. Venepalli ◽  
Jessica K. Altman ◽  
Martin S. Tallman
Keyword(s):  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia

scholarly journals Arsenic Trioxide Promotes Histone H3 Phosphoacetylation at the Chromatin ofCASPASE-10in Acute Promyelocytic Leukemia Cells

2002 ◽  
Vol 277 (51) ◽  
pp. 49504-49510 ◽  
Author(s):  
Ji Li ◽  
Peili Chen ◽  
Natasha Sinogeeva ◽  
Myriam Gorospe ◽  
Robert P. Wersto ◽  
...  
Keyword(s):  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Histone H3 ◽  
Gene Expression Profiles ◽  
Promyelocytic Leukemia ◽  
Therapeutic Effects ◽  
Acid Therapy ◽  
Nb4 Cells

Arsenic trioxide (As2O3) is highly effective for the treatment of acute promyelocytic leukemia, even in patients who are unresponsive to all-trans-retinoic acid therapy. As2O3is believed to function primarily by promoting apoptosis, but the underlying molecular mechanisms remain largely unknown. In this report, using cDNA arrays, we have examined the changes in gene expression profiles triggered by clinically achievable doses of As2O3in acute promyelocytic leukemia NB4 cells.CASPASE-10expression was found to be potently induced by As2O3. Accordingly, caspase-10 activity also substantially increased in response to As2O3treatment. A selective inhibitor of caspase-10, Z-AEVD-FMK, effectively blocked caspase-3 activation and significantly attenuated As2O3-triggered apoptosis. Interestingly, the treatment of NB4 cells with As2O3markedly increased histone H3 phosphorylation at serine 10, an event that is associated with acetylation of the lysine 14 residue. Chromatin immunoprecipitation assays revealed that As2O3potently enhances histone H3 phosphoacetylation at theCASPASE-10locus. These results suggest that the effect of As2O3on histone H3 phosphoacetylation at theCASPASE-10gene may play an important role in the induction of apoptosis and thus contribute to its therapeutic effects on acute promyelocytic leukemia.

scholarly journals Arsenic trioxide inhibits ATRA-induced prostaglandin E2 and cyclooxygenase-1 in NB4 cells, a model of acute promyelocytic leukemia

Leukemia ◽  
2008 ◽  
Vol 22 (6) ◽  
pp. 1125-1130 ◽  
Author(s):  
A Habib ◽  
E Hamade ◽  
R Mahfouz ◽  
M S Nasrallah ◽  
H de Thé ◽  
...  
Keyword(s):  
Prostaglandin E2 ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nb4 Cells ◽  
Cyclooxygenase 1

scholarly journals Arsenic Trioxide Potentiates Extracellular Traps-Formation Cell Death in Acute Promyelocytic Leukemia through mTOR-Regulated Autophagy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1575-1575
Author(s):  
Tao Li ◽  
Muhua Cao ◽  
Ruishuang Ma ◽  
Xiaoming Wu ◽  
Lu Zhao ◽  
...  
Keyword(s):  
Cell Death ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Extracellular Dna ◽  
Apoptosis Resistance ◽  
Dna Complexes ◽  
Nb4 Cells ◽  
Wide Range

Abstract Background:Emerging clinical data shows that arsenic trioxide (ATO) exerts selective cytotoxicity against acute promyelocytic leukemia (APL) without severe side effects that mainly ascribed to nonspecific induction of apoptosis. It is attractive to speculate whether other uncovered APL cell death exists which can be specifically sparked by ATO administration. We have recently demonstrated that APL cells can undergo a previously unrecognized pathway for death by releasing extracellular DNA traps (ETs), termed ETosis (Ma R et al, Cell Death Dis 2016). However, besides apoptosis, whether ATO induces this APL-specific ETotic cell death remains to be explored. We wereto investigated the effects of a wide range of concentrations of ATO on ETotic death in APL cells and elucidate the underlying molecular mechanisms. Methods: Bone marrow samples were obtained from sixteen APL patients before and after the continuous administration of ATO for two weeks. APL cells were isolated and cultured in the presence and absence of ATRA for 3 days. We used confocal microscopy to image ET formation by APL cells and the percentage of ETotic cells was simultaneously quantified. ELISA was used to measure the concentration of myeloperoxidase (MPO)-DNA complexes in the supernant. We also assessed the effects of a wide range of concentrations (0.1, 0.25, 0.5, 0.75, 1.0, 2.0 μM) of ATO treatment for 24, 48 and 72 hours on ETosis in APL-cell line NB4 cells in vitro. Autophagy activation and leukemia-initiating cell (LIC) activity were evaluated by immunoblotting and imaged by immunostaining. LICs were analyzed using colony-forming unit (CFU) assays, and identified and quantified by flow cytometry. Results: APL cells isolated from ATO-treated APL patients underwent significantly increased spontaneous (P = 0.005) and ATRA-stimulated (P = 0.002) ETosis compared to those from newly diagnosed patients (n = 16). In vitro ATO treatment showed that the percentage of ETotic NB4 cells dramatically increased at 0.5 μM (8 ± 1.6%), peaked at 0.75 μM (15 ± 2.4%) and was gradually suppressed at higher concentrations. The concentration of MPO-DNA complexes, an indirect marker of ETosis, parallelled the dose-dependent change in the percentage of ETotic cells. Interestingly, inhibition experiments indicated that ATO caused concentration-dependent APL cell death: ATO primarily triggered ETosis at moderate concentrations (0.5 to 0.75 μM) and switched to apoptosis at relatively high doses (1.0 to 2.0 μM). Furthermore, We found that ATO induced ETosis through mammalian target of rapamycin (mTOR)-regulated autophagy. Surprisingly, inhibition of ETosis spared LIC activity from ATO reduction, while combined treatment with ATO and rapamycin further increased ETosis-mediated LIC loss (~3.5-fold). Conclusion s : This is the first study to show that ATO potentiates ETotic death in APL cells through mTOR-regulated autophagy. Importantly, further investigation suggests that ATO specifically targets the APL LICs to ETosis. This implies that, in combination with ATO, therapy-triggered ETosis by targeting the corresponding signaling pathways could be a novel and effective strategy to improve a long relapse-free survival through LIC clearance, avoid lethal apoptosis-related complications and overcome apoptosis resistance. Conflict of interest statement: None. Disclosures No relevant conflicts of interest to declare.

Death Associated Protein 5 (DAP5/p97/NAT1) Contributes to Retinoic Acid-Induced Granulocytic Differentiation and Arsenic Trioxide Induced Apotosis through Inhibition of PI3K/Akt/mTOR Pathway in Acute Promyelocytic Leukemia Cells: A Novel Mechanism.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2836-2836
Author(s):  
Bulent Ozpolat ◽  
Ugur Akar ◽  
Isabel Zorilla-Calancha ◽  
Pablo Vivas-Mejia ◽  
Gabriel Lopez-Berestein
Keyword(s):  
Retinoic Acid ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Translation Initiation ◽  
Promyelocytic Leukemia ◽  
Mtor Pathway ◽  
Granulocytic Differentiation ◽  
Induced Expression ◽  
Nb4 Cells ◽  
Induced Apoptosis

Abstract All-trans Retinoic Acid (ATRA) is a naturally occurring metabolite of retinol (vitamin A)and acts as a potent inducer of cellular differentiation and growth arrest in acute promyelocytic leukemia (APL), a type of acute myeloid leukemia (M3-AML). APL is characterized by translocation t(15;17), fusing PML (promyelocytic leukemia) and RARα (retinoic acid receptor) genes, leding to expression of PML/RARα receptor protein and differentiation block. Arsenic trioxide (ATO) induces (<0.5 μM) differentiation at low doses and apoptosis at high doses (>1 μM) in APL cells. Currently, both ATRA and ATO are successfully used in the treatment of APL in the clinic. However, the molecular mechanisms of myeloid differentiation and apoptosis induced by these agents are not fully understood. We previously reported that ATRA inhibits the translation initiation through multiple mechanisms, including upregulation of translation initiation inhibitors, DAP5/p97 and PDCD4 tumor suppressor protein. Here we investigated the role and regulation of death associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in myeloid (granulocytic and monocytic) cell differentiation and apoptosis. We found that ATRA (1 μM) induced a marked DAP5/p97 protein and mRNA expression during granulocytic differentiation of NB4 and HL60 cells but not in differentiation-resistant cells, which express very low levels of DAP5/p97. DAP5/p97 was translocated into nucleus during the differentiation of NB4 cells induced ATRA. At differentiation inducing doses, ATO, dimethysulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate-13-acetate also induced a significant DAP5/p97 expression in NB4 cells. However, ATO at apoptotic doses, but not ATRA, induced DAP5/p86, a proapoptotic form of DAP5/p97. ATRA and ATO -induced expression of DAP5/p97 was associated with inhibition of phosphaditylinositol 3-kinase (PI3K)/Akt pathway, which is known to stimulate cap-dependent translation of mRNAs. To show direct link between PI3K/Akt/mTOR pathway and DAP5 expression, we treated cell with PI3K and mTOR inhibitors LY294002 and by rapamycin, respectively. We found that inhibition of PI3K/Akt/mTOR pathway upregulated DAP5/p97 expression in NB4 cells. Finally, knockdown of DAP5/p97 expression by small interfering RNA significantly inhibited ATRA-induced granulocytic differentiation detected by expression of CD11b and ATO-induced apoptosis in NB4 cells detected by Annexin V assay (p<0.05). In conclusion, our data suggest that DAP5/p97 plays a role in ATRA-induced differentiation and ATO-induced apoptosis in APL cells. Our data demonstrated for the first time that DAP5/p97 is constitutively suppressed by of PI3K/Akt/mTOR pathway, and ATRA and ATO-induced expression of DAP5 is mediated by the inhibition of this survival pathway, suggesting a novel mechanism of DAP5 regulation and a role of translational control in induction of differentiation and apoptosis. Figure Figure

Oxidation Of Peroxiredoxins By Thioredoxin Inactivation Is Mediated In Arsenic Trioxide-Induced Reactive Oxygen Species Formation and Its Cytotoxicity In Acute Promyelocytic Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3858-3858
Author(s):  
Yeung-Chul Mun ◽  
Jee-Young Ahn ◽  
Eun-Sun Yoo ◽  
Jungwon Huh ◽  
Kyoung Eun Lee ◽  
...  
Keyword(s):  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Ros Generation ◽  
Oxygen Species ◽  
Redox Enzymes ◽  
Nb4 Cells ◽  
Mitochondrial Ros ◽  
Thioredoxin 1 ◽  
Induced Apoptosis

Abstract Backgrounds The Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia (APL). ATO exerts its effect mainly raising oxidative stress. However, not only the mechanisms of reactive oxygen species (ROS) generation by ATO but involvement of redox enzymes including peroxiredoxin (PRX) and thioredoxin (TRX) remains elusive. Aim of current study is to elucidate the mechanism of redox enzymes to elevate ROS during ATO-induced apoptosis in APL-derived NB4 cells. Methods NB4 cell line, which is one of the human acute promyelocytic leukemia cell lines, was cultured in RPMI-1640 medium supplemented with 10% FBS in CO2 humidified atmosphere at 37°C. NB4 cells were cultured with 2 μM arsenic trioxide to induce apoptosis for 16-48 hours. Apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) with flow cytometry. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial ROS. SO2 form for PRX I, PRX II, and PRX III was detected by western blot assay using PRX SO2 form-specific antibody. Monomer/Dimer assay for PRX I, PRX II, PRX III, and TRX I was performed by western blot using non-reducing gel. Results Intracellular ROS of NB4 cells was increased significantly after 16 hour of ATO but decreased after 24 hour of ATO. Mitochondrial ROS of NB4 cells was increased significantly after 39 hour of ATO. Apoptosis of NB4 cell after ATO treatment was increased as time elapsed (24% on 16hr, 26% on 24hr, 48% on 39hr, and 60% on 48hr). Monomer, indicated active and reduced form, of peroxiredoxins was decreased and cysteine sulfinic acid (CP–SO2H) peroxiredoxins, indicated inactive and oxidized peroxiredoxins, was increased in NB4 cells after ATO treatment as time goes by. Similarily, monomer of thioredoxin-1 (active thioredoxin) was decreased and multimer of thioredoxin-1 (inactive thioredoxin) was increased in NB4 cells after ATO treatment as time elapsed. Conclusions Our data showed inactivation of peroxiredoxins by oxidation was developed during ATO-induced ROS generation and APL cell apoptosis. These peroxiredoxins oxidation was probably due to increment of reduced thioredoxin in NB4 cells after ATO treatment. These findings suggest ATO-induced anti-leukemic activity is more likely due to a TRX system-mediated cellular redox changes. Our study may provide the insights for finding novel targets in the development of new therapies, which potentiate ATO-induced apoptosis in APL cells. Disclosures: No relevant conflicts of interest to declare.

Src Family Kinase Inhibitor PP2 Enhances Differentiation Of Acute Promyelocytic Leukemia Cell Line Induced By Combination Of ATRA and Arsenic Trioxide

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4911-4911
Author(s):  
Kyoung Ha Kim ◽  
Hee-Jeong Cheong ◽  
Sook-Ja Kim ◽  
Jina Yoon ◽  
Han Jo Kim ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Similar Data ◽  
Src Family Kinase ◽  
Nb4 Cells

Abstract All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia. For subsequent similar data, NCCN guidelines indicate that ATRA plus ATO is an alternative for patients who cannot tolerate anthracycline therapy. We demonstrated that SFK (Src Family Kinase) inhibitor PP2 enhanced acute promyelocytic leukemia (APL) cell differentiation when combined with either ATRA or ATO with difference in activation of RA-induced genes. In this study, we investigated SFK inhibitor PP2 could enhances the differentiation of NB4 APL cells when combined with ATRA and ATO and the changes in the expression of intercellular adhesion molecule-1 (ICAM-1) derived from the retinoic acid receptor (RAR) target gene. Treatment of NB4 cells with 1 nM of ATRA, 0.5 uM of ATO, or 10 uM of PP2 for 72 hours induced expression of CD11b-positive cells by 13.01%, 11.53% or 13.28%, respectively. However, the combination of ATRA and ATO and the combination of three agents (ATRA, ATO, and PP2) led to a significant higher expression of CD11b-positive cells (30.96% and 63.17%, respectively). The synergistic effect of the combination of three agents was more significant than the combination of ATRA and ATO. These results were confirmed by NBT staining. These effects were not related with apoptosis. Annexin-V-fluorescene staining revealed that combination of ATRA and ATO and combination of three agents did not induced apoptosis in NB4 cells. The expression of ICAM-1 was markedly increased in cells treated with the combination of three agents. These findings suggest that the SFK inhibitor can enhances differentiation of APL cells combined with ATRA and ATO. FDA approved SFK inhibitors, such as dasatinib and bosutinib, may be beneficial for the treatment of APL in combination with ATRA and ATO. Disclosures: No relevant conflicts of interest to declare.

The Role of Peroxiredoxin III and Sulfiredoxin for Mitochondrial ROS Production to Arsenic Trioxide in Acute Promyelocytic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5217-5217
Author(s):  
Yeung-Chul Mun ◽  
Jee-Young Ahn ◽  
Eun-Sun Yoo ◽  
Kyoung Min Cho ◽  
Kyoung Eun Lee ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Caspase 3 ◽  
Promyelocytic Leukemia ◽  
Ros Generation ◽  
Down Regulation ◽  
Nb4 Cells ◽  
Mitochondrial Ros ◽  
Induced Apoptosis

Abstract Backgrounds: The Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia (APL), but in some cases, APL cells are resistant to ATO treatment. ATO exerts its effect mainly raising oxidative stress. However, not only the mechanisms of reactive oxygen species (ROS) generation by ATO but involvement of redox enzymes including peroxiredoxin (PRX) during ATO-induced apoptosis and its resistance remain elusive. Recently, Rhee et al had reported that PRX III and sufiredoxin together protect mice from pyrazole-induced oxidative liver injury was found (Antioxid & Redox Signal, 2012:17:1351-1361). Aims of current study are to elucidate that the changes of redox enzyme could be a mechanism of anti-leukemia effect in APL-derived NB4 cells during ATO treatment and to find ways to potentiate the anti-leukemic effects of ATO on APL cells. Methods: NB4, one of the human acute promyelocytic leukemia cell lines, was treated with 0~10 μM arsenic trioxide to induce apoptosis for 16-48 hours in RPMI-1640 medium supplemented with 10% FBS in CO2humidified atmosphere at 37°C. Apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) with flow cytometry. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red was used to detect cellular and mitochondrial ROS. SO2 form for PRX I, PRX II, and PRX III was detected by western blot assay using PRX SO2 form-specific antibody. Sulfiredoxin (SRX) and caspase 3, 9 were also detected by western blot analysis. To evaluate the effect of SRX depletion, NB4 cells were transfected with small interfering RNA (siRNA). Results: Intracellular ROS of NB4 cells was increased significantly after 16 hour of ATO treatment but decreased after 24 hour of ATO treatment. Mitochondrial ROS of NB4 cells was increased significantly after 39 hour of ATO treatment. Apoptosis of NB4 cell after ATO treatment was increased as time elapsed (24% on 16hr, 26% on 24hr, 48% on 39hr, and 60% on 48hr). Increased cysteine sulfinic acid (Cys–SO2H) PRX III, inactive and oxidized form, was observed as a hyperoxidation reaction in NB4 cells after ATO treatment in concordance with mitochondrial ROS increment of NB4 cells. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during NB4 cell apoptosis by ATO treatment. Meanwhile, SRX expression was increased in NB4 cells after ATO treatment. Down regulation of SRX by siRNA promoted ROS generation and apoptosis in ATO-treated NB4 cells. Conclusions: Our data showed inactivation of PRX III by Cys–SO2H formation as hyperoxidation is developed during ATO-induced mitochondrial ROS generation and apoptosis process in APL cells. In addition, ATO promotes expression of SRX, which is known as reducing enzyme of Cys–SO2H PRX and which leads to down regulation of ROS accumulation in APL cells. These findings might be due to protective effect of SRX from ATO on mitochondrial oxidative stress. These findings suggest ATO-induced anti-leukemic activity could be down regulated by an enhancing PRX III reduction after ATO-induced SRX activation. Currently, the effect of down regulation of SRX by siRNA are being investigated to amplify the apoptosis in ATO-treated NB4 cells. Our study may provide the insights for finding novel targets in the development of new therapies, which potentiate ATO-induced apoptosis in APL cells. Disclosures No relevant conflicts of interest to declare.

Combined effect of all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia cells in vitro and in vivo

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 264-269 ◽  
Author(s):  
Yongkui Jing ◽  
Long Wang ◽  
Lijuan Xia ◽  
Guo-qiang Chen ◽  
Zhu Chen ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Induced Apoptosis

Abstract All-trans retinoic acid (tRA) and arsenic trioxide (As2O3) induce non–cross-resistant complete clinical remission in patients with acute promyelocytic leukemia with t(15;17) translocation and target PML-RARα, the leukemogenic protein, by different pathways suggesting a possible therapeutic synergism. To evaluate this possibility, this study examined the effect of As2O3 on tRA-induced differentiation and, conversely, the effect of tRA on As2O3-induced apoptosis. As2O3 at subapoptotic concentrations (0.5 μM) decreased tRA-induced differentiation in NB4 cells but synergized with atRA to induce differentiation in tRA-resistant NB4 subclones MR-2 and R4 cells as measured by nitroblue tetrazolium reduction and tRA-inducible genes (TTGII, RARβ, RIG-E). tRA cleaved PML-RARα into distinct fragments in NB4 but not in tRA-resistant MR-2 or R4 cells, whereas As2O3 completely degraded PML-RARα in all 3 cell lines. As2O3-induced apoptosis was decreased by tRA pretreatment of NB4 cells but not of R4 cells and was associated with a strong induction of Bfl-1/A1 expression, a Bcl-2 protein family member. Severe combined immunodeficient mice bearing NB4 cells showed an additive survival effect after sequential treatment, but a toxic effect was observed after simultaneous treatment with tRA and As2O3. These data suggest that combined As2O3 and tRA treatment may be more effective than single agents in tRA-resistant patients. Although in vitro data do not always translate to in vivo response, toxicity and potential drug antagonism may be diminished by decreasing the concentration of As2O3 when given at the same time with therapeutic levels of tRA.

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