Tanshinone IIa Induces Autophagy and Apoptosis via PI3K/Akt/mTOR Axis in Acute Promyelocytic Leukemia NB4 Cells

Tanshinone IIa (TanIIa), an ingredient of Radix Salviae Miltiorrhizae, has an anticancer effect on various solid tumors with high efficiency and low toxicity. Nonetheless, the underlying role of TanIIa in acute promyelocytic leukemia (APL) remains unclear. Here, we revealed that TanIIa drastically inhibited NB4 cell viability with an IC50 value of 31.25 μmol/L. Using flow cytometry apoptosis assay, we identified that TanIIa dose-dependently exacerbated NB4 cell apoptosis. Mechanistically, TanIIa upregulated apoptotic factor levels, namely, cleaved-caspase 9, cleaved-caspase 3, and cleaved-PARP-1. Moreover, we noticed that TanIIa dose-dependently suppressed the PI3K/Akt/mTOR axis. This axis not only functions as an essential antiapoptotic modulator but also serves as a suppressant regulator of autophagy. Correspondingly, we detected the levels of autophagic marker, namely, LC3B, which were increased after the TanIIa treatment. Furthermore, the autophagy inhibitor Baf-A1 could effectively reverse the TanIIa-induced apoptosis, manifesting that TanIIa eliminated NB4 cells in an autophagy-dependent manner. In conclusion, tanshinone IIa exerts anti-APL effects through triggering autophagy and apoptosis in NB4 cells.

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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 ◽

10.1182/blood.v110.11.2836.2836 ◽

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

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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 ◽

10.1182/blood.v122.21.3858.3858 ◽

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.

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

Blood ◽

10.1182/blood.v124.21.5217.5217 ◽

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.

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

Blood ◽

10.1182/blood.v97.1.264 ◽

2001 ◽

Vol 97 (1) ◽

pp. 264-269 ◽

Cited By ~ 153

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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CD44 Ligation Induces Apoptosis Via Caspase- and Serine Protease-Dependent Pathways in Acute Promyelocytic Leukemia Cells.

Blood ◽

10.1182/blood.v106.11.4432.4432 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4432-4432

Author(s):

Eliane Maquarre ◽

Cedric Artus ◽

Zeineb Gadhoum ◽

Claude Jasmin ◽

Florence Smadja-Joffe ◽

...

Keyword(s):

Serine Protease ◽

Acute Promyelocytic Leukemia ◽

Caspase 3 ◽

Death Receptor ◽

Promyelocytic Leukemia ◽

Caspase 8 ◽

Cytochrome C Release ◽

Nb4 Cells ◽

Effector Caspase ◽

Induced Apoptosis

Abstract We have recently reported that ligation of the CD44 cell surface antigen with A3D8 monoclonal antibody (mAb) triggers incomplete differentiation and apoptosis of the acute promyelocytic leukemia (APL)-derived NB4 cells. The present study characterizes the mechanisms underlying the apoptotic effect of A3D8 in NB4 cells. We show that A3D8 induces activation of both initiator caspase -8 and -9, and effector caspase-3 and -7 but only inhibition of caspase-3/7 and caspase-8 reduces A3D8-induced apoptosis. Moreover, A3D8 induces mitochondrial alterations (decrease in mitochondrial membrane potential ΔΨm and cytochrome c release) which are reduced by caspase-8 inhibitor suggesting that caspase-8 is primarily involved in A3D8-induced apoptosis of NB4 cells. However, the apoptotic process is independent of TNF-family death receptor signalling. Interestingly, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) decreases A3D8-induced apoptosis and when combined with general caspase inhibitor displays an additive effect resulting in complete prevention of apoptosis. These results suggest that both caspase-dependent and serine protease-dependent pathways contribute to A3D8-induced apoptosis. Finally, A3D8 induces apoptosis in ATRA-resistant NB4-derived cells and in APL primary blasts, characterizing the A3D8 anti-CD44 mAb as a novel class of apoptosis-inducing agent in APL.

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Anti-Leukemic Effect of Tetra-Arsenic Oxide in Acute Promyelocytic Leukemia Cells in Vitro and In Vivo

Blood ◽

10.1182/blood.v118.21.4306.4306 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4306-4306

Author(s):

So Young Chong ◽

Moon Ju Jang ◽

Sun Ju Lee ◽

Ji Young Huh ◽

Jeehyeon Bae ◽

...

Keyword(s):

Acute Promyelocytic Leukemia ◽

Cell Lines ◽

Xenograft Model ◽

Promyelocytic Leukemia ◽

Dependent Manner ◽

Facs Analysis ◽

Arsenic Oxide ◽

Induced Apoptosis ◽

Dose Dependent

Abstract Abstract 4306 Arsenic trioxide (As2O3; ATO) has been established to be an effective agent for treating acute promyelocytic leukemia (APL). Tetra-arsenic oxide (As4O6; TAO) is a new arsenic compound which has shown anti-proliferative and apoptosis-inducing effects against human leukemic and solid tumor cells, but TAO has never been studied in APL so far. We investigated the effect of TAO in APL cell lines (NB-4), and evaluated their anti-leukemic effect in murine xenograft model. In both the XTT assay and in FACS analysis, TAO inhibited cell proliferation and induced apoptosis in NB4 cell lines as effective as ATO in dose dependent manner. In FACS analysis, TAO induced apoptosis in NB4 cell lines as effective as ATO in a dose dependent manner. However, expression of differentiation (CD11b) was unchanged. For in vivo study, we established a xenograft model in nude mice (BALB/cBy J-nu) using the NB4 cell line. 1 × 107 NB4 cells were inoculated into the flank subcutaneously. When the tumor size reached 1.0 cm3, intra-peritoneal ATO or TAO treatment (5mg/kg or 10mg/kg) was initiated and administration was performed daily. We found that TAO (10mg/kg) significantly inhibited tumor growth of the inoculated NB4 cell as effective as ATO (10mg/kg). These results suggest that TAO may have a potential for the treatment of APL. Disclosures: No relevant conflicts of interest to declare.

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2-Methoxyestradiol Is Exclusively Effective on Both Retinoic Acid-Sensitive and -Resistant Acute Promyelocytic Leukemia through the Inhibition of NF-κB.

Blood ◽

10.1182/blood.v106.11.4434.4434 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4434-4434

Author(s):

Ming-jer Huang ◽

Yunchi Cheng ◽

Shufan Lin ◽

Zhilone Chen ◽

Hsingjin E. Liu

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Promyelocytic Leukemia ◽

Leukemic Cells ◽

Caspase 9 ◽

Nb4 Cells ◽

All Trans Retinoic Acid ◽

Intracellular Ros ◽

Induced Apoptosis ◽

Mtt Assays

Abstract The superoxide dismutase inhibitor, 2-methoxyestradiol (2ME), has recently been found to have anti-leukemic activity. As both 2-ME and ATO induce apoptosis of leukemic cells through similar mechanisms- mitochondrial damage through increased intracellular ROS, we hypothesized that 2-ME, like ATO, might be particularly effective on acute promyelocytic leukemia (APL). Using MTT assays, we found that the dose of 2-ME required to inhibit 50% of cell growth (IC50=0.25 μM) was 5 times less for NB4 cells than for other types of leukemic cells such as HL-60, U937, K562, THP-1 and KG1a (Fig 1). The exclusive sensitivity correlated with the increase of cellular superoxide and the decrease of cellular glutathione upon treatment but not with the endogenous levels of superoxide or glutathione. 2ME induced apoptosis of NB4 cells through upregulation of pro-apoptotic Bax, and Bak proteins, and induced apoptosis through mitochondrial pathways, shown by the release of cytoplasmic cytochrome C, and cleavage of caspase 9 and 3, and 6. Of note, 2-ME also induced neutrophilic differentiation, confirmed by NBT assays, and the expression of CD11b. We showed that, although 2-ME did not affect the PML/RARα transcription, it decreased the levels of PML/RARα in the cells. Like ATO, 2-ME specifically inhibited the activation of NF-κB in NB4 cells but not in HL-60 cells, shown by the EMSA assay (Fig 2). The effectiveness of 2-ME on NB-4 cells could also be observed in both all-trans retinoic acid (ATRA)- resistant and ATO-resistant cells. Furthermore, a combination of 2-ME with ATO was more effective on NB4 cells than either agent alone. Therefore, 2-ME could be potentially useful for eradication of APL or as a salvage agent for ATRA-and ATO-resistant APL. Fig 1, Fig 2 Fig 1, Fig 2.

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Requirement for ERK Activity in Sodium Selenite-induced Apoptosis of Acute Promyelocytic Leukemia-derived NB4 Cells

BMB Reports ◽

10.5483/bmbrep.2007.40.2.196 ◽

2007 ◽

Vol 40 (2) ◽

pp. 196-204 ◽

Cited By ~ 16

Author(s):

Bingshe Han ◽

Wei Wei ◽

Fangyuan Hua ◽

Tingming Cao ◽

Hua Dong ◽

...

Keyword(s):

Acute Promyelocytic Leukemia ◽

Sodium Selenite ◽

Promyelocytic Leukemia ◽

Nb4 Cells ◽

Induced Apoptosis ◽

Erk Activity

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Synergic effects of arsenic trioxide and cAMP during acute promyelocytic leukemia cell maturation subtends a novel signaling cross-talk

Blood ◽

10.1182/blood.v99.3.1014 ◽

2002 ◽

Vol 99 (3) ◽

pp. 1014-1022 ◽

Cited By ~ 48

Author(s):

Qi Zhu ◽

Ji-Wang Zhang ◽

Hai-Qing Zhu ◽

Yu-Lei Shen ◽

Maria Flexor ◽

...

Keyword(s):

Retinoic Acid ◽

Arsenic Trioxide ◽

Acute Promyelocytic Leukemia ◽

Cross Talk ◽

Fusion Gene ◽

Leukemia Cell ◽

Promyelocytic Leukemia ◽

Dependent Manner ◽

Induced Differentiation

Abstract Acute promyelocytic leukemia (APL) is characterized by the specific chromosome translocation t(15;17) with promyelocytic leukemia-retinoic acid receptor-α (PML-RARA) fusion gene and the ability to undergo terminal differentiation as an effect of all-trans retinoic acid (ATRA). Recently, arsenic trioxide (As2O3) has been identified as an alternative therapy in patients with both ATRA-sensitive and ATRA-resistant APL. At the cellular level, As2O3 triggers apoptosis and a partial differentiation of APL cells in a dose-dependent manner; both effects are observed in vivo among patients with APL and APL animal models. To further explore the mechanism of As2O3-induced differentiation, the combined effects of arsenic and a number of other differentiation inducers on APL cell lines (NB4 and NB4-R1) and some fresh APL cells were examined. The data show that a strong synergy exists between a low concentration of As2O3 (0.25 μM) and the cyclic adenosine monophosphate (cAMP) analogue, 8-CPT-cAMP, in fully inducing differentiation of NB4, NB4-R1, and fresh APL cells. Furthermore, cAMP facilitated the degradation of As2O3-mediated fusion protein PML-RARα, a process considered to play a key role in overcoming the differentiation arrest of APL cells. On the other hand, cAMP could significantly inhibit cell growth by modulating several major players in G1/S transition regulation. Interestingly, H89, an antagonist of protein kinase A, could block the differentiation-inducing effect of As2O3potentiated by cAMP. These results thus support the existence of a novel signaling cross-talk for APL maturation, which may deepen understanding of As2O3-induced differentiation in vivo, and thus furnish insights for new therapeutic strategies.

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Retinoic Acid Induces Aggregation of the Acute Promyelocytic Leukemia Cell Line NB-4 by Utilization of LFA-1 and ICAM-2

Blood ◽

10.1182/blood.v90.7.2747.2747_2747_2756 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2747-2756 ◽

Cited By ~ 3

Author(s):

Richard S. Larson ◽

David C. Brown ◽

Larry A. Sklar

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Promyelocytic Leukemia ◽

Leukemia Cell Line ◽

High Avidity ◽

Dependent Manner ◽

Adhesive Properties ◽

Dose Dependent ◽

Β2 Integrins ◽

Retinoic Acid Syndrome

All-trans retinoic acid (tRA) is a potent differentiation agent that is effective therapy for acute promyelocytic leukemia (APL). However, 5% to 25% of patients develop retinoic acid syndrome, a potentially life-threatening complication in which the pathogenesis relates to adhesive alterations of APL cells. Therefore, we investigated the relationship between tRA-induced differentiation and the adhesive properties of APL cells. After confirming differentiation-related morphological changes of NB-4 cells in response to tRA, we showed that homotypic aggregation of NB-4 cells grown in tRA for 72 hours is dose-dependent with a median effective dose of approximately 50 nmol/L. Maximal aggregation occurred at mean and peak therapeutic serum concentrations (100 and 1,000 nmol/L, respectively). Aggregation also increased with the length of tRA exposure over 168 hours. Aggregation was inhibited by neutralizing antibodies against LFA-1 and ICAM-2. Notably, antibodies directed against VLA-4, other β2 integrins (Mac-1 and p150), or other potential LFA-1 counterstructures that were expressed on the cell surface (ICAM-1 and ICAM-3) did not block aggregation. Aggregation occurred with similar kinetics regardless of the presence of phorbol ester or the “activating” monoclonal antibody (MoAb) KIM 185, suggesting that the avidity of LFA-1 is not modulated on NB-4 cells in a manner similar to other leukocytes. Consistent with the prompt clinical effectiveness of methyl prednisolone sodium succinate (MPSS) in retinoic acid syndrome, MPSS rapidly inhibited homotypic aggregation in a dose-dependent manner. Thus, tRA alters the adhesive properties of APL cells by inducing the expression of high-avidity β2 integrins, aggregation is inhibited by LFA-1 and ICAM-2 MoAb, and tRA effects are rapidly reversible by MPSS. Taken together, our findings provide a clinically relevant system for study of LFA-1/ICAM-2 interaction and suggest a mechanism in part for retinoic acid syndrome and the effectiveness of MPSS in ameliorating retinoic acid syndrome.

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