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

Blood Cells, Molecules and Diseases 56 (2016) 46–52

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Tanshinone IIA inhibits acute promyelocytic leukemia cell proliferation and induces their apoptosis in vivo Kaiji Zhang a, Jian Li b, Wentong Meng c, Hongyun Xing d, Yiming Yang b,⁎ a

Department of Hematology, Guizhou Medical University Affiliated Hospital, Guiyang 550004, Guizhou Province, China Department of Hematology, West China Medical School, Sichuan University, Chengdu 610041, Sichuan Province, China Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, Sichuan Province, China d Department of Hematology, Sichuan Medical University affiliated hospital, Luzhou 646000, Sichuan Province, China b c

a r t i c l e

i n f o

Article history: Submitted 6 September 2015 Revised 9 October 2015 Accepted 26 October 2015 Available online 27 October 2015 Editor: Mohandas Narla Keywords: Tanshinone IIA Acute promyelocytic leukemia Mice Apoptosis Proliferation

a b s t r a c t 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 APLbearing mice. Our data indicate that TanIIA has potent anti-APL activity with little adverse effect. Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved.

1. Introduction Acute promyelocytic leukemia (APL) is characterized by a balanced reciprocal translocation between chromosomes 15 and 17, which results in the fusion between the promyelocytic leukemia (PML) gene and retinoic acid receptor α (RARa). Previously, APL had high mortality. Therapeutic strategy to induce the differentiation of APL cells by all trans retinoic acid (ATRA) and arsenic trioxide (ATO) has shifted APL from a highly fetal disease to a highly curable one [1]. Previous studies have shown that ATRA can induce APL cell terminal differentiation and ATO can induce APL cell apoptosis by rapid degradation of PML/PML-RARa. However, APL patients with ATRA and ATO treatment develop severe side effects and 48% of patients may develop the ATRA-related differentiation syndrome [2–6]. Furthermore, some patients with ATO treatment may display Q–Tc prolongation to N 500 ms [7]. In addition, treatment with 7.5–10 μg/g ATO results in hepatic necrosis in animals [8]. Hence, the safety of long-term application of ATRA and ATO is concerned. Therefore, discovery of new safe and effective reagents to induce APL terminal differentiation will be of great significance. Tanshinone IIA (TanIIA) is an extract of Danshen, a traditional Chinese medicine, which has been used for many years in the clinic. ⁎ Corresponding author. E-mail address: [email protected] (Y. Yang).

http://dx.doi.org/10.1016/j.bcmd.2015.10.007 1079-9796/Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved.

Previous studies have demonstrated that TanIIA has potent antioxidant and anti-inflammatory activities [9,10]. Importantly, TanIIA is a relatively safe reagent with few side effects [11,12]. Our previous study has shown that TanIIA can induce ATRA-sensitive APL cell differentiation by upregulating CEBPβexpression and trigger ATRA-resistant APL cell apoptosis in vitro [13]. In this study, we further evaluated the effect of TanIIA on APL in a human leukemia xenograft model. 2. Methods 2.1. Reagents TanIIA was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). A TanIIA stock solution [50 mM in dimethyl sulfoxide (DMSO); Sigma, St. Louis, MO] was prepared and diluted to working concentration immediately before use. ATO solution (0.1%) for intraperitoneal administration was purchased from Harbin Yita Pharmaceutical (Heilongjiang, China). 2.2. Cell culture NB4 cells with chromosomal translocation t(15;17) were originally isolated from long-term cultures of leukemia blast cells on bonemarrow stromal fibroblasts by Lanotte et al. [14], and were provided

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by Ruijing Hospital, Shanghai. NB4 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a saturated humidified incubator of 5% CO2. The cells in logarithmic growth phase were used for further experiments.

2.3. Animals All animal studies were approved by the Animal Care and Use Committee of Sichuan University. An APL mouse model was established, as described previously [15]. Briefly, female NOD/scid mice (4 weeks old) were obtained from the Medical Institute of Experimental Animals of Chinese Academy of Medical Sciences, Beijing and were housed in a specific pathogen-free (SPF) facility at the Lab Animal Center of Sichuan University. The mice were injected intraperitoneally (i.p) with NB4 cells (1 × l06), randomized at 7 days later, and treated i.p with saline (control), 5 mg/kg ATO (ATO), 10 mg/kg TanIIA (10 mg TanIIA) or 100 mg/kg TanIIA (100 mg TanIIA) daily for 14 consecutive days (n = 10 per group). The mice were closely monitored for their body weights (every 5 days) up to 80 days post-inoculation and sacrificed if any signs of approaching death appeared.

2.4. Peripheral blood cells Peripheral blood samples were collected from individual mice at days 7, 21, 28 and 35 post-inoculation and then smeared on glassslides. The cells were stained with Wright's solution and examined under a microscope.

2.5. Histological and cytological analyses At the end of the experiment, the liver, spleen, and lung of individual mice were dissected, fixed in 4% formaldehyde, and embedded in paraffin. The tissue sections at 5 μm were stained with hematoxylin/eosin or May–Grünwald–Giemsa and examined under a microscope. The frequency of apoptotic cells in the liver, spleen, and lung of individual mice was determined by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assays using the in situ cell death detection kit, according to the manufacturers' instruction (Boehringer Mannheim). The proliferation rates of cells in the liver, spleen, and lung of individual mice were determined by immunohistochemistry using anti-Ki67 staining. Briefly, the liver sections (5 μm) were treated with 3% H2O2 in methanol to block endogenous peroxidase activity, and subjected to antigen retrieval in a 0.1 M sodium citrate buffer. The sections were incubated with an anti-Ki67 monoclonal antibody (Santa Cruz Biotech, Santa Cruz, USA) at 4 °C overnight. After being washed, the bound antibodies were detected with HRP-conjugated second antibodies, and visualized with DAB solution. The percentages of proliferative or apoptotic cells in five high fields (magnification ×400) were counted in a blinded manner.

2.6. Differentiation and apoptosis analyses Ascites cells were collected from mice at 30 days, smeared on glass-slides. The cells were stained with Wright's solution and examined under a microscope. The differentiation and apoptosis of ascites cells were determined by flow cytometry. Briefly, ascites cells (5 × 105/tube) were stained in duplicate with PE-anti-CD11b and the percentages of CD11b + differentiated APL cells were determined by flow cytometry on FACSAria ((Becton Dickinson, San Jose, USA). In addition, ascites cells (5 × 105/tube) were stained in duplicate with FITC-anti-Annexin-V and PI in the dark for 30 min at room temperature. The percentages of apoptotic cells were determined by flow cytometry.

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2.7. Statistical analysis Data are expressed as the mean ± 95% confidence interval (C.I.). The survival of different groups of mice was estimated by the Kaplan–Meier method and analyzed by the log-rank test. The difference in the percentage of CD11b +, TUNEL + and Ki67 + cells in the different groups of mice was analyzed by one­way ANOVA. Statistical analysis was performed by using the SPSS 16.0 software (SPSS, Chicago). A two-side P-value of b 0.05 was considered statistically significant.

3. Results 3.1. TanIIA prolongs the survival of APL-bearing mice To test the safety of TanIIA treatment, NOD/SCID mice were injected i.p with NB4 cells to induce APL and treated with or without, 10, 100 mg/kg TanIIA, or ATO beginning on day 7 for two weeks. Analysis of peripheral blood at 0 day indicated that APL cells with typical morphological characteristics were present in all mice (Fig. 1A). However, there was no significant difference in the frequency of circulating APL cells among the different groups of mice (data not shown). These indicated the establishment of APL model. Treatment with TanIIA and ATO significantly prolonged the survival of APL-bearing mice and the mean survival time of the control mice was 37 days, which was significantly shorter than 45 days, 48 days, and 51 days of the mice treated with 10 mg TanIIA, 100 mg TanIIA and ATO, respectively (P N 0.05, Fig. 1C). The mean survival time of the mice treated with 10 mg TanIIA seemed shorter than that of the mice with 100 mg TanIIA or ATO, but there is no statistically significant difference (P N 0.05). It was notable that the some mice in the TanIIA and ATO treatment groups survived more than 80 days. Peripheral blood leukemic cells in these mice disappeared and WBC and platelet counts as well as hemoglobin level returned to normal. Furthermore, there was no detectable leukemia cells in the liver and spleen of mice. Together, these data indicated that treatment with TanIIA and ATO resulted in leukemia remission in mice.

3.2. TanIIA affects APL growth and is safe for mice All mice with treatment had no tremor, deteriorated diet, and raised fur. The body weights of individual groups of mice were measured longitudinally (Fig. 2A). The body weights in the mice treated with either drug were significantly higher than that in the control at 30 days post-inoculation (P b 0.05). There were 8 out of 10 control mice that developed ascites and peritoneal solid tumors while only 6 out of 10 mice in the 10 mg TanIIA group, 4 out of 10 in the 100 mg TanIIA group and 6 out of 10 in the ATO group developed tumor in enterocelia. The mean tumor size was 4.97 cm3 (the control), 2.01 (TanIIA 10 mg), 1.08 (TanIIA 100 mg), and 1.85 cm3 (ATO), respectively. Treatment with TanIIA or ATO significantly decreased tumor growth rate and reduced the tumor size by 40–70% (P b 0.05, Fig. 2B). It was notably that the time when we measured the size of tumor was the mice appeared any signs of approaching death in the control group was obviously shorter than any of the treatment groups, so that the growth time of tumor in the treatment groups was longer than that of the control group. We considered that the actual effect of TanIIA and ATO on APL was greater. While ATO treatment induced some hepatocyte apoptosis, tissue edema and steatosis in the liver of mice, treatment with TanIIA at either dose did not cause any obviously morphological changes in the liver of mice. Collectively, these data indicated that treatment with TanIIA at the dose range was relatively safe in mice.

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Fig. 1. Morphological examination of APL cells. Treatment with TanIIA inhibits the progression of APL and prolongs the survival of APL-bearing mice. A. APL-like cells in the peripheral blood of mice display large size, high nuclear-cytoplasm ratio, fine chromatin structure with visible nucleoli, and basophilic cytoplasm; B. most ascites cells have typical APL morphological characteristics with pleomorphic, varied sizes with a high nuclear-cytoplasm ratio and obvious nucleoli; C. Cumulative survival of APL-bearing mice. Group 1: Controls treated with saline (blue line); group 2: the mice treated with 10 mg TanIIA (green line); group 3: the mice treated with 100 mg TanIIA (yellow line) and group 4: the mice treated with ATO (red line). All treatment groups have significantly longer periods of survival than the control (P b 0.05).

3.3. TanIIA affects APL cell infiltration To evaluate the effect of TanIIA treatment on leukemia cell infiltration, the livers and spleens of individual mice were stained with hematoxylin/eosin and observed under a microscope. There were

many leukemia infiltrates in the livers and spleens, particularly in the liver's sinusoids and the parenchyma in the control mice, accompanied by confluent liver necrosis. In contrast, there were only a few leukemia cells infiltrated not only in the livers, but also in the spleens in the TanIIA-treated mice. There was no confluent liver necrosis in the livers

Fig. 2. Treatment with TanIIA or ATO protects from loss of body weights and reduces tumor sizes in mice. A. Longitudinal measurements of body weights in mice. B. Tumor volumes in the different groups of mice. Data are expressed as the mean ± SD of each group of mice (n = 10 per group). *P b 0.05; **P b 0.05; ***P b 0.05.

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Fig. 3. TanIIA treatment reduce APL infiltrates in the liver and spleen of mice. The livers and spleens of individual groups of mice were dissected and their tissue sections were stained with H&E. Data are representative images (magnification ×400) of each group of mice (n = 10 per group). Treatment with TanIIA or ATO obviously reduced the numbers of APL infiltrates in the liver and spleen of mice.

of all treatment groups of mice, although some apoptotic hepatocytes, tissue edema and steatosis were observed in these mice (Fig. 3). These data indicated that TanIIA treatment inhibited leukemia cell infiltration. 3.4. Treatment with TanIIA inhibits NB4 cell proliferation and triggers NB4 cell apoptosis in mice The percentages of apoptotic and proliferative cells in the liver and spleen of different groups of mice were determined by TUNEL and immunohistochemistry. There were varying numbers of apoptotic cells in different groups of mice and apoptotic cells covered 7.7% to 55.3% of

the tissue areas in the liver and spleen sections. Treatment with either TanIIA or ATO significantly increased the percentages of apoptotic NB4 cells in the livers and spleen of APL-bearing mice. The percentage of apoptotic cells in the liver of the control mice was 7.7%, and was significant less than 29.2%, 54.3%, and 55.3% in the TanIIA 10 mg, 100 mg, and ATO groups of mice (P b 0.05). Similarly, the percentage of apoptotic cells in the spleen of the control mice was 6.2%, and was significantly less than 42.1%, 58.8%, and 60.8% in the TanIIA 10 mg, 100 mg, and ATO groups of mice, respectively. The percentages of apoptotic cells in the ATO group were slightly higher than that in the TanIIA 100 mg group and there was no significant difference between these two groups (P N 0.05,

Fig. 4. TanIIA treatment inhibits APL proliferation and induces APL cell apoptosis. All of these data were representative images (magnification ×200) from each group of mice (n = 10 per group). The liver and spleen tissue sections were subjected to TUNEL (A and B). The data showed that percentages of TUNEL+ cells increased significantly in treatment with TanIIA or ATO than that in the control group. The liver and spleen tissue sections were stained by anti-KI67 staining (C and D). Percentages of Ki67 + cells decreased obviously in treatment with TanIIA or ATO, as compared with that in the control group.

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Fig. 5. TanIIA treatment induces APL cell differentiation and apoptosis. A. Quantitative analysis of the percentages of apoptotic cells in the livers of mice by counting TUNEL+ cells in the total number of cells in the five high power fields. B. Quantitative analysis of the percentages of apoptotic cells in the spleens of mice by counting TUNEL+ cells in the total number of cells in the five high power fields. C. Quantitative analysis of the percentages of proliferative cells in the livers of mice using similar method described above. D. Quantitative analysis of the percentages of proliferative cells in the spleens of mice. E. Flow cytometry analysis of differential CD11b+ and annexin V+ apoptotic ascites cells. Data are representative FACS histograms and expressed as the mean ± SEM of each group (n = 10) of mice *P b 0.05; **P b 0.05; ***P b 0.05.

Figs. 4A, B and 5A, B). To understand the therapeutic action of TanIIA, the percentages of proliferative cells in the livers of different groups of mice were determined by immunohistochemistry using anti-Ki67 staining. There were many positive anti-Ki67 stained cells in the livers and spleens of the control mice while there were a few positive anti-Ki67 stained cells in the livers and spleens of the TanIIA and ATO groups of mice (Figs. 4C, D and 5C, D). Quantitative analyses indicated that treatment with TanIIA or ATO significantly reduced the frequency of proliferative NB4 cells in the livers and spleens of the APL-bearing mice. Interestingly, the anti-KI67 stained cells in the TanIIA 10 mg group have a lowest rate and there was no significant difference in the percentages of KI67 + cells between the ATO and TanIIA treated groups of mice (P N 0.05). Hence, treatment with TanIIA, especially with 10 mg TanIIA inhibited the proliferation of NB4 cells, and with 100 mg TanIIA triggered their apoptosis in vivo.

3.5. TanIIA affects APL cell differentiation and apoptosis Finally, the percentages of differentiated CD11b + and apoptotic NB4 cells in ascites cells were determined by flow cytometry. Ascites cells were collected from individual mice at 30 days post-inoculation. We observed that most cells had typical APL morphological characteristics with pleomorphic, varied sizes and a high nuclear-cytoplasm ratio and obvious nucleoli (Fig. 1B). Analysis of NB4 cell differentiation indicated that a significant increase in the number of mature myeloid CD11b + cells appeared in the drug-treated groups, especially in the mice treated with TanIIA 10 mg (control 1.7%, TanIIA 10 mg 30.8%, TanIIA 100 mg 8.2%, ATO 4.6%). Furthermore, analysis of NB4 cell apoptosis revealed that treatment with ATO and TanIIA 100 mg promoted APL cell apoptosis and the percentages of apoptotic APL cells were 0.4%, 10.4%, 19.5% and 21.6% in the control, TanIIA 10 mg, TanIIA

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100 mg and ATO groups of mice, respectively (Fig. 5E and F). These results were very similar to our previous studies in vitro, and indicated that treatment with lower dose of TanIIA preferably induced APL differentiation while with a higher dose of TanIIA promoted APL cell apoptosis. 4. Discussion ATRA and ATO are highly efficient in treatment of APL, but they are associated potentially with severe side-effects. Retinoic acid-related differentiation syndrome (DS) is the most severe side-effect of ATRA. Other ATRA's side effects include Sweet's syndrome, vasculitis, hypercalcemia, bone marrow necrosis and fibrosis, thromboembolic events, erythema nodosum, granulomatous proliferation, some pulmonary complications, and renal failure [16,17]. ATO has similar concerns, and ATO can damage cardiac structure and functions, such as ventricular arrhythmia [18–20]. TanIIA is a traditional Chinese agent and widely used in treatment of cardiovascular diseases. Clinical reports indicate that it has low toxicity and few side effects [21,22]. Furthermore, some researchers report that it has a potential anti-tumor effect [23–25]. Yang et al. [26] reported that treatment with TanIIA resulted in CR in one refractory APL patient. In our previous studies, we found that TanIIA induced APL cell differentiation and apoptosis in vitro. In this study, we found that intraperitoneal injection with TanIIA was well-tolerated in mice. Treatment with TanIIA did not cause animal death although treatment with ATO caused some side effects in mice. Arsenic is hepatotoxic and can induce moderate edema and steatosis in the liver of mice. However, there was no significant loss of body weights in the ATO-treated mice. These data agreed with a previous report [27]. Treatment with 10 mg or 100 mg TanIIA did not cause severe adverse effects, such as hepatotoxicity and liver damage. The TanIIAtreated mice maintained their body weights, even with 100 mg TanIIA. We did not find any edema in the 10 mg TanIIA-treated group of mice, especially contrasted with that in the ATO group. Our data support the notion that TanIIA has benefits to many organs, such as the liver and renal function [28–30]. We concluded that TanIIA was much safer than ATO in mice. In this study, we found that TanIIA prolonged the survival of APLbearing mice. Compared with that in the control group, treatment with either dose of TanIIA prolonged the survival periods of APLbearing mice. The effect of treatment with 100 mg TanIIA or ATO was better than that with 10 mg TanIIA although there was no statistically significant difference. Treatment with TanIIA, similar to treatment with ATO, also decreased the tumor volumes by approximately 70%, relative to the control mice. Our previous studies have shown that treatment with TanIIA induces APL cell apoptosis and differentiation in vitro [13,31]. Now, we found that treatment with TanIIA induced APL cell apoptosis and differentiation in vivo. In our previous study, we found that treatment with a low dose, but not a high dose, of TanIIA promoted APL cell differentiation, although treatment with TanIIA induced APL apoptosis in NB4 cells. Similar to treatment with 1 μg/L ATO, TanIIA can induce APL cell apoptosis through the intrinsic pathway, which inhibits the MMP, leading to the increased release of cytosolic cyto-c, and the extrinsic apoptosis pathway by increasing TNF-α expression and caspase-8 activation. Indeed, TanIIA has been shown to induce APL cell apoptosis by activating caspase 8 and then caspase 3 [31]. In this study, histological examination of the spleen and liver indicated that treatment with TanIIA reduced leukemia cell infiltration in these organs, where some leukemia cells underwent apoptosis. Furthermore, treatment with 100 mg TanIIA induced APL cell apoptosis, and the apoptotic cells accounted for 40–60% of the sectioned tissue areas, particularly in the liver. The efficacy of TanIIA in inducing APL cell apoptosis in the liver of mice was similar to treatment with ATO. Analysis of ascetic cells revealed that treatment with 100 mg TanIIA induced 19.5% of cells undergoing apoptosis. These data

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indicated that both high dose of TanIIA and ATO shared some features to induce APL cell apoptosis. ATO has been proven to prolong the mean survival time by inducing APL cell apoptosis. Therefore, high dose of TanIIA prolonged the mean survival time of APL-bearing mice at least by inducing APL cell apoptosis. There were more complications in the 10 mg TanIIA group. Our previous studies reported that treatment with a low dose of TanIIA (1 mg/L) up-regulated C/EBPβ expression in NB4 cells in vitro, and the increased C/EBPβexpression induced APL cell differentiation [11]. We found that treatment with either dose of TanIIA, similar to treatment with ATO, significantly prolonged the mean survival time in mice. However, we observed that treatment with 10 mg TanIIA only induced low frequency of NB4 cell apoptosis, which was much weaker than treatment with a high dose of TanIIA or ATO. Analysis of APL differentiation revealed that treatment with either dose of TanIIA or ATO did not promote NB4 cell differentiation in vivo, inconsistent with the findings from in vitro studies. However, we observed that treatment with a low dose of TanIIA significantly increased the frequency of CD11b+ mature myeloid cells in ascites cells, an indicative of inducing NB4 cell differentiation in vivo. It is hard to understand why TanIIA did not induce APL cell differentiation in the liver and spleen of mice but did induce ascites cell differentiation. First, we found that treatment with TanIIA, even with a low dose, significantly inhibited APL cell proliferation although the efficacy of treatment with 10 mg TanIIA in inhibiting APL cell proliferation was weaker than that of treatment with 100 mg TanIIA or ATO. We observed fewer APL infiltrates in the liver of the TanIIA 10 mg group than that of the control group. These suggest that low dose of TanIIA through some mechanisms inhibits APL cell proliferation in vivo. It is possible that low dose of TanIIA may inhibit glucose metabolism and mTOR/p70S6K/RPS6/4E-BP1 and NF-κB signaling to down-regulate HIF-1α and VEGF expression, leading to the inhibition of APL cell proliferation [28,32,33]. However, the mechanisms by which low dose of TanIIA prolonged the survival time of mice may be more complex, and different from a high dose of TanIIA. They included that low dose of TanIIA induced APL cell apoptosis and differentiation, reduced APL growth rate and suppressed APL cell proliferation. In summary, our data indicated that TanIIA was effective against APL. Treatment with different doses of TanIIA through different mechanisms inhibited APL cell proliferation, and a low dose of TanIIA treatment may be involved in a complicated way against APL. Acknowledgments We thank Dr. Xianming Mo and the Laboratory of Stem Cell Biology for their assistance. This work was supported by grants from the National Natural Science Foundation of China (No. 0040205401122) and the National Basic Research Program of China (No. 2007CB947802). References [1] Z.Y. Wang, Z. Chen, Acute promyelocytic leukemia: from highly fatal to highly curable, Blood 111 (5) (2008) 2505–2515. [2] N. Asou, K. Adachi, J. Tamura, A. Kanamaru, S. Kageyama, et al., Analysis of prognostic factors in newly diagnosed acute promyelocytic leukemia treated with alltrans retinoic acid and chemotherapy. Japan Adult Leukemia Study Group, J. Clin. Oncol. 16 (1) (1998) 78–85. [3] G. Avvisati, F. Lo Coco, D. Diverio, M. Falda, F. Ferrara, M. Lazzarino, D. Russo, et al., AIDA (all-transretinoic acid + idarubicin) in newly diagnosed acute promyelocytic leukemia: a Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto (GIMEMA) pilot study, Blood 88 (4) (1996) 1390–1398. [4] M.S. Tallman, J.W. Andersen, C.A. Schiffer, F.R. Appelbaum, J.H. Feusner, A. Ogden, et al., All-trans-retinoic acid in acute promyelocytic leukemia, N. Engl. J. Med. 337 (15) (1997) 1021–1028. [5] P. Fenaux, C. Chastang, S. Chevret, M. Sanz, H. Dombret, E. Archimbaud, et al., Randomized comparison of all transretinoic acid (ATRA) followed by chemotherapy and ATRA plus chemotherapy and the role of maintenance therapy in newly diagnosed acute promyelocytic leukemia. The European APL Group, Blood 94 (4) (1999) 1192–1200. [6] M.A. Sanz, G. Martín, C. Rayón, J. Esteve, M. González, J. Díaz-Mediavilla, et al., A modified AIDA protocol with anthracycline-based consolidation results in high antileukemic efficacy and reduced toxicity in newly diagnosed PML/RARalpha-

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