Proliferation Of Acute Promyelocytic Leukemia Cells Research Articles

Tanshinone IIA inhibits acute promyelocytic leukemia cell proliferation and induces their apoptosis in vivo

Tanshinone IIA (TanIIA) is a traditional Chinese agent and has been widely used for treatment of cardiovascular diseases. Our previous study has shown that TanIIA can induce the differentiation of acute promyelocytic leukemia (APL) cells by increasing C/EBPβexpression and induce APL cell apoptosis in vitro. In this study, we evaluated the activity of TanIIA against APL in vivo. We found that treatment with TanIIA prevented APL-mediated reduction in body weights. Treatment with TanIIA inhibited the proliferation of APL cells and triggered APL cell apoptosis and differentiation in vivo. Treatment with TanIIA significantly prolonged the survival of APL-bearing mice. Our data indicate that TanIIA has potent anti-APL activity with little adverse effect.

Preclinical Studies of the IAP Antagonist Debio 1143 in Combination with Cytarabine or Doxorubicin in a Mouse Model of AML

Background: Relapse and drug resistance remains the major cause of therapy failure in Acute Myeloid Leukemia (AML). Inhibitors of apoptosis proteins (IAPs) are a family of proteins that suppress proapoptotic signaling and represent attractive targets to overcome chemotherapy resistance. Debio 1143 (a.k.a. AT-406) is an orally-active IAP antagonist able to promote apoptosis alone and/or in combination with chemotherapy. We recently reported the results of a Phase 1 study of Debio 1143 in combination with daunorubicin and cytarabine (araC) in poor-risk AML patients (DiPersio et al. J Clin Oncol 32:5s, 2014, a7029). Although Debio 1143 showed good tolerability and could be safely combined with chemotherapy, 62% (8/13) of patients with complete remissions relapsed within the study period. Here, we used a preclinical AML model to begin examining the effects of BM stroma on the efficacy of Debio 1143 combined with araC or doxorubicin (dox).Methods: We generated murine acute promyelocytic leukemia (APL) cell lines that are resistant to araC and dox both in vitro and in vivo by chronically exposing APL cells to increasing concentrations of drug in vitro. This APL model was previously generated by knocking in the human PML-RARa cDNA into the cathepsin G locus (Westervelt et al. Blood, 2003). We used WST-1 cell proliferation assays and bioluminescence imaging (BLI) to evaluate the efficacy of Debio 1143 to induce apoptosis alone and in combination with araC or dox in vitro in chemotherapy sensitive and resistant APL cells in the presence and absence of M2-10B4 stromal cells. Dox or araC was provided for (a) a 6h period initiated 24h before Debio 1143 (i.e., with an intervening 18h washout of the chemotherapy), (b) simultaneously with Debio 1143, or (c) 6h after Debio 1143. To assess Debio 1143 in vivo, we injected APL cells into syngeneic mice. In our first experiment, mice (n=5/cohort) were treated with PBS, araC (500 mg/kg, s.c.), dox (50 mg/kg, s.c.), Debio 1143 (100 mg/kg, p.o.) or their combination on days 5, 6, 12, and 13 after APL injection. In our second experiment, mice (n=8/cohort) were treated with PBS, araC (200 mg/kg, s.c.), dox (10 mg/kg, s.c.), Debio 1143 (100 mg/kg, p.o.) or their combination on days 3 to 7 and 10 to 16 after APL injection. In both experiments, Debio 1143 was given 2 hours prior to chemotherapy.Results: Figure 1 shows that coculture of APL cells with stromal cells protects them from araC-induced killing (Figure 1A) and that our araC-resistant cells are indeed resistant to araC (Figure 1B). Treatment with Debio 1143 alone has no effect on parental or araC-resistant APL survival in the presence or absence of stroma cells. However, Debio 1143 in combination with araC killed the parental APL cells and overcame the stroma cell-mediated chemotherapy protection (Figure 1A). Although combination therapy partially inhibited araC-resistant APL cell proliferation in the absence of stroma, it had no effect when the cells were cultured in the presence of stroma (Figure 1B). We obtained similar results with dox and dox-resistant cells (data not shown). This in vitro data with the parental APL cell line was confirmed in vivo. Combination therapy with chemotherapy (araC or dox) and Debio 1143 for 4 days (EXP#1) or 10 days (EXP#2) significantly prolonged the overall survival of mice by 4 to 7 days compared to single agent therapy (Debio 1143, araC, or dox) in this aggressive tumor model (Table 1). Preliminary in vitro data suggests that pretreatment of parental or araC-resistant APL cells with chemotherapy (araC or dox) enhances their killing by Debio 1143 compared to simultaneous treatment.Conclusions: Debio 1143 combined with chemotherapy inhibits parental and chemotherapy-resistant APL growth in vitro and in vivo. Future mechanistic studies are required to determine how Debio 1143 can overcome stroma-mediated resistance. [Display omitted] [Display omitted] DisclosuresRettig:Debiopharm International SA: Research Funding. Vuagniaux:Debiopharm International SA: Employment. Zanna:Debiopharm International SA: Employment. DiPersio:Debiopharm International SA: Research Funding.

Catechins induced acute promyelocytic leukemia cell apoptosis and triggered PML-RARα oncoprotein degradation.

BackgroundIt has recently been reported that the extracts of green tea polyphenol have cancer preventive effects. In this study, we investigated the effect of the natural composition from green tea leaves Catechins on acute promyelocytic leukemia (APL).MethodsIn vitro, APL cell lines NB4, retinoic acid-resistant NB4-R1 and NB4-R2 were treated with different concentrations of Catechins. Cell viability and cell apoptosis were analyzed using MTT assay and flow cytometric assay, respectively. Expression of proteins related to apoptosis and PML-RARα oncoprotein were assessed by Western blot. In vivo anti-tumor activity of Catechins was examined in nude mice xenografted with NB4 cells and in situ cell apoptosis was detected by terminal deoxytransferase-catalyzed DNA nick-end labeling assay.ResultsCatechins at micromolar concentration levels significantly inhibited APL cell proliferation and induced cell apoptosis, in association with mitochondria damage, ROS production and caspase activation. The anti-apoptotic Bcl-2 family member Bcl-xL was down regulated, with pro-apoptotic member Bax remaining unchanged. Moreover, Catechins induced the degradation of PML-RARα oncoprotein. Catechins-mediated apoptotic effect was also observed in primary APL cells without affecting normal hematopoietic progenitor cells. In the murine xenograft model, Catechins remarkably inhibited tumor growth and induced in situ leukemic cell apoptosis.ConclusionsCatechins might be a potential candidate for APL treatment by activating intrinsic apoptotic pathway and targeting PML-RARα oncoprotein.

Targeting Smad4 links microRNA-146a to the TGF-β pathway during retinoid acid induction in acute promyelocytic leukemia cell line

The expression pattern of microRNAs (miRNAs) and their potential target genes were investigated in acute promyelocytic leukemia (APL) cell line NB4 cells during all-trans-retinoid acid (ATRA) treatment by using a miRNA microarrays platform and real-time quantitative PCR (RTQ-PCR). MiR-146a as one of the miRNAs down-regulated by ATRA during APL differentiation was identified. Direct interaction between miR146a and its predictive target gene Smad4 were confirmed by Luciferase assay. Down-regulation of miR-146a and upregulation of Smad4 at protein levels were demonstrated. These data suggested that miR-146a might influence proliferation of APL cells through TGF-beta1/Smad signal transduction pathway during ATRA induction.

The Activation of STAT3 Contributes to the Effect of Autocrined G-CSF On the Proliferation of HL60 Cells Induced by ATRA.

Abstract 4565 BackgroundIn previous works, we detected several significant cytokines in the serum of patients with APL after treatment with ATRA, and the results revealed a close association of serum G-CSF and the number of promyelocytes or more mature granulocytes. Some of the results also indicate that ATRA in vitro has a conspicuous effect on the expression of some cytokines, such as IL-1β, IL-8, TNF, G-CSF, and so on. At least, it is showed that cytokines in serum or supernatant secreted by APL cells perhaps played an important role in the genesis of leukocytosis. However, the role of G-CSF in proliferation of APL cells has not yet been explained clearly. In the present study, we analyse the secretion and expression of cytokines in APL patients who has hyperleukocytosis, study the relationship between cytokines and leukocytosis, and detect the signal transduction pathway of cytokines. ObjectiveTo detect the signal pathway of IL-1β and G-CSF secreted by primary acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). MethodsPrimary APL cells from ATRA-treated patients were cultured in RPMI 1640, then the level of supernatant IL-1β and G-CSF were detected by ELISA assay. The expression of IL-1β or G-CSF mRNA was measured by RT-PCR, and the level of STAT1α and STAT3 evaluated by Western blot. ResultsTreated with ATRA, the peripheral blood leucocytes of patients with APL was obviously up-regulated, and peaked at 12 days averagely, of which mainly were myelocytes and metamyelocytes. The secretion of IL-1β or G-CSF in supernatant was up-regulated (P<0.05). RT-PCR assay confirmed that in the primary APL cells, with higher secretion of IL-1β and/or G-CSF, the expression of IL-1β and/or G-CSF was up-regulated, correspondingly. Cytokines and signal transduction factors analysis indicated that the expression of STAT1α protein increased in all the patients, irrelevant to the expression of IL-1β or G-CSF, but the increase of STAT3 expression was consistent with the expression and secretion of G-CSF. ConclusionThe higher expression of G-CSF in primary APL cells induced by ATRA contributes to the activation of STAT3. Disclosures:No relevant conflicts of interest to declare.

Rig-I Activates Expression of Interferon-Stimulated Genes (ISGs) and Inhibits the Proliferation of Acute Myeloid Leukemia Cells

Acute promyelocytic leukemia (APL) is initiated by the formation of PML/RARα oncogenic fusion protein, a potent transcriptional repressor. Retinoid acid (RA) at pharmacological dosage can physically bind to the PML/RARα protein, ushering in the unfolding of downstream programs normally regulated by the wild type RARα. However, through what particular regulatory pathways RA inhibits APL malignant hematopoiesis has remained largely obscured. Rig-I is one of the genes whose mRNA levels were highly up-regulated, along with all-trans-RA (ATRA)-induced terminal granulocytic differentiation of APL cell line NB4 cells in vitro. Based on the analysis of a Rig-I−/− mouse model, recently we have reported a critical regulatory role of Rig-I in normal granulopoiesis. To understand the functional contribution of Rig-I induction in RA-mediated leukemia cell differentiation, we converted a pair of previously reported Rig-I RNAi-duplex sequences into a miR30a-based small hairpin-encoding sequence, which was expressed under the CMV enhancer/promoter within a lentiviral vector. As expected, Rig-I shRNAmir30 infection induced a significant knockdown of Rig-I protein level, and accordingly its delivery into HL-60 cells partially inhibited ATRA-induced granulocytic differentiation, growth inhibition/cell cycle arrest and apoptosis induction, suggesting that Rig-I upregulation participates in RA-induced granulocytic differentiation of acute myeloid leukemia cells. In order to investigate the effect of Rig-I induction on the proliferation of APL cells in vivo, we transduced PML/RARα-harboring leukemic cells with vector or Rig-I-expressing retrovirus, and then transplanted these cells into the syngeneic mice. The vector-transduced APL cells readily expanded in vivo, but the proliferation of Rig-I-transduced cells was apparently prohibited. Moreover, we found that the forced expression of Rig-I induced the expression of numerous ISGs in APL cells, which was recapitulated by the transduction of the C terminal part of Rig-I, but not by the N terminal part. In line with this, during the in vitro short-term culture post-IFNγ or IFNα stimulation, Stat1 phosphorylation at p701 in Rig-I−/− granulocytes was significantly inhibited. In parallel, the induction of multiple ISGs by IFNs was also significantly impaired. In conclusion, our findings indicate that the Rig-I induction inhibited APL reconstitution potentially through up-regulating a number of ISGs via regulating Stat1Tyr701 phosphorylation.

Design and Synthesis of Novel Derivatives of ATRA Demonstrate the Combined Importance of Acid Moiety and Conjugated Double Bonds in Its Binding to PMLRAR-Alpha Oncogene in Acute Promyelocytic Leukemia

All trans retinoic acid (ATRA) is the treatment of choice for Acute Promyelocytic Leukemia (APL) and binds to the transcriptional repressor PML-RAR-alpha oncogene. The binding of ATRA to RAR-alpha relieves transcriptional repression and leads to induction of genes important for myeloid differentiation. ATRA consists of an acid moiety at position 15 and is also characterized by conjugated double alkene bonds (Fig1A). The exact contributions of these structural components of ATRA in its clinically important binding to RAR-alpha receptor are not very well studied. In an effort to improve the therapeutic index and to study its binding mechanism, we synthesized novel retinoic acid analogs (retinoids). Our objective was to construct retinoids to test these structure functional correlates. 3 novel retinoids created were synthesized as follows:Compound 13a (Fig1B): consisting of modifying conjugated alkene backbone while keeping acid moiety intact.Compound 13b (Fig1C): consisting of modifying conjugated alkene backbone and converting acid to ester moiety.Compound 13c (Fig1D): consisting of modifying conjugated alkene and cycloalkene ring backbone and keeping acid moiety intact.We tested the ability of these newer retinoids to bind to RAR-alpha as well as inhibit the proliferation of PML-RAR-alpha containing acute promyelocytic leukemic cell line NB4. Reporter assays using a RAR- luciferase demonstrated that Compound 13a successfully relieved transcriptional repression by RAR-alpha and led to downstream gene induction. This was significantly less compared to wild type ATRA demonstrating the importance of C9–C10 double bonds in this binding. Compounds 13b and 13c could not relieve RAR mediated repression of gene induction demonstrating that the acid moiety plays a important role in this binding. Functional studies were consistent with these observations and showed that both ATRA and 13a were able to inhibit the proliferation of APL cells while 13b, 13c were not. Flow cytometry showed that 13a led to partial differentiation of NB4 cells, as evident from CD11b expression after five days of culture. 13b and 13c did not lead to any differentiation, while ATRA led to the greatest expression of CD11b.Our results demonstrate that both the acid moiety and conjugated double bonds are important in binding to RAR-alpha and the subsequent transcriptional repression by retinoic acid. These insights will be important in future efforts to improve the stability and efficacy of ATRA and also have implication in efforts focused on treating ATRA resistant cases of APL. [Display omitted]

Cytokine secretion in patients with acute promyelocytic leukemia after treatment with all-trans retinoic acid

Objective: To detect the modulation of cytokines production by acute promyelocytic leukemia (APL) cells before or after exposure to all-trans retinoic acid (ATRA). Methods: Diagnoses were performed according to the FAB cytological classification criteria and cytogenetic criteria. Bone marrow or blood samples from APL patients were collected in heparinized microfuge tube. Primary APL cells were separated and purified by traditional Ficoll-Hypaque density centrifugation and enriched after adherence to plastic surfaces. IL-1β, IL-6, IL-8, TNFα and G-CSF levels in the supernatants of cultured leukemia cells were estimated by ELISA method. NBT method was used to detect the differentiation of APL cells at the same time. Results: 96 h after exposure to ATRA at 10−6 M in vitro or 60 mg/day in vivo, APL cells showed a significant increase of IL-1β (P<0.05) and G-CSF (P<0.05) production, and a significant decrease of IL-6 (P<0.05) and IL-8 (P<0.05), however, there was no obvious variation of TNFα. On the other hand, the proliferation of APL cells in vitro was statistically correlated to the IL-1β secretion or G-CSF secretion. And the cell number ratio in patients with detectable IL-1β or G-CSF was higher than that without detectable IL-1β or G-CSF. Conclusion: IL-1β and G-CSF secretion may play an important role in the proliferation of APL cells after exposure to ATRA.

Effects of Novel RAR- and RXR-Selective Retinoids on Myeloid Leukemic Proliferation and Differentiation In Vitro

Retinoids such as all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA) have an important role in many aspects of proliferation and differentiation of hematopoietic cells. They exert their effects by binding to retinoic acid receptors (RARs) and/or retinoid X receptors (RXRs). We studied the effects of novel retinoids on proliferation and differentiation of HL-60 and NB4 myeloid leukemic cells, as well as acute promyelocytic leukemia (APL) cells from patients. RXR-selective SR11345 (Retinoid C) had little ability to inhibit the clonal growth and to induce the differentiation of either HL-60 or NB4 cells. However, SR11276 (Retinoid E), which activated both the RAR and RXR classes, and SR11278 (Retinoid D), which activated the RAR subtypes alpha, beta, and gamma, could inhibit clonal growth of both cell types, as well as leukemic cells from APL patients. The combination of ATRA and either SR11276 or SR11278 additively inhibited APL cell proliferation. SR11302 (Retinoid A), with reported anti-AP-1 activity and no activation of RARs and RXR and SR11363 (Retinoid B), which selectively activated RARbeta and gamma, were inactive. The clonal proliferation of both HL-60 and NB4 cells that were pulse-exposed to 10(-9) mol/L ATRA, SR11276, SR11278, or SR11345 for 3 days, washed, and plated in methylcellulose culture were inhibited by 0%, 51%, 21%, and 1% for HL-60 cells and 43%, 41%, 35%, and 1% for NB4, respectively, compared with nontreated control cells. When the HL-60 cells were pulse-exposed to 10(-9) mol/L of either SR11278 or SR11276, plus 10(-9) mol/L ATRA for 3 days, colony numbers were reduced by 46% and 64%, respectively. Induction of leukemic cell differentiation as determined by the nitroblue tetrazolium (NBT) assay showed that the combination of 10(-7) mol/L of either SR11278 or SR11276 with 10(-7) mol/L ATRA had additive effects on HL-60 cells, NB4 cells, and fresh APL cells. Induction of CD11b expression on both HL-60 and NB4 cells occurs during their differentiation. Expression of this antigen was synergistically augmented by the combination of either 10(-7) to 10(-8) mol/L SR11278 or 10(-7) to 10(-9) mol/L SR11276 with 10(-9) mol/L ATRA compared with either analog alone in HL-60 cells. Expression of the novel myeloid specific transcription factor C/EBPepsilon was increased by SR11278 and SR11276 in both the HL-60 and NB4 cell lines. We conclude that retinoids or combination of retinoids with specificities for both RAR and RXR may markedly enhance the ability of ATRA to inhibit clonal growth and induce differentiation of HL-60 and NB4 leukemic cells. This occurs in the absence of continuous contact with retinoids.

Granulocyte‐colony Stimulating Factor and Retinoic Acid Cooperatively Induce Granulocytic Differentiation of Acute Promyelocytic Leukemia Cells in vitro

The interaction of granulocyte‐colony stimulating factor (G‐CSF) and retinoic acid (RA) in proliferation and differentiation of acute promyelocytic leukemia (APL) cells was examined. G‐CSF stimulated proliferation of APL cells at concentrations of 0.1 to 50 ng/ml in a dose dependent manner. More than 10−8M RA induced granulocytic differentiation of APL cells. Although G‐CSF induced lysozyme activities in APL cells, it alone did not induce terminal differentiation of APL cells. G‐CSF significantly enhanced the RA‐induced granulocytic differentiation of APL cells in vitro. Enhancement by G‐CSF was not due to the prolongation of survival of RA‐induced differentiated cells, but the differentiation‐inducing effects of G‐CSF might be evident only in the presence of RA. Since G‐CSF has a potential to induce the granulocytic differentiation of myeloid leukemia cells, G‐CSF in combination with RA may be applicable in differentiation induction therapy for some types of myeloid leukemia.