Investigational New Drugs

, Volume 30, Issue 5, pp 1830–1840

The cytotoxic activity of Aplidin in chronic lymphocytic leukemia (CLL) is mediated by a direct effect on leukemic cells and an indirect effect on monocyte-derived cells

Authors

  • Pablo E. Morande
    • Laboratory of Immunology, IIHEMANational Academy of Medicine
  • Samanta R. Zanetti
    • Laboratory of Immunology, IIHEMANational Academy of Medicine
  • Mercedes Borge
    • Laboratory of Immunology, IIHEMANational Academy of Medicine
  • Paula Nannini
    • Laboratory of Immunology, IIHEMANational Academy of Medicine
  • Carolina Jancic
    • Laboratory of Immunology, IIHEMANational Academy of Medicine
  • Raimundo F. Bezares
    • Department of HematologyHospital Teodoro Alvarez
  • Alicia Bitsmans
    • Department of HematologyHospital Ramos Mejía
  • Miguel González
    • Laboratory of Immunology, IIHEMANational Academy of Medicine
  • Andrea L. Rodríguez
    • Laboratory of Immunology, IIHEMANational Academy of Medicine
  • Carlos M. Galmarini
    • Cell Biology DepartmentPharmaMar SAU
  • Romina Gamberale
    • Laboratory of Immunology, IIHEMANational Academy of Medicine
    • Department of Microbiology, Parasitology and Immunology, School of MedicineUniversity of Buenos Aires
    • Laboratory of Immunology, IIHEMANational Academy of Medicine
    • Department of Microbiology, Parasitology and Immunology, School of MedicineUniversity of Buenos Aires
    • Laboratorio de Inmunología OncológicaIIHEMA, Academia Nacional de Medicina
PRECLINICAL STUDIES

DOI: 10.1007/s10637-011-9740-3

Cite this article as:
Morande, P.E., Zanetti, S.R., Borge, M. et al. Invest New Drugs (2012) 30: 1830. doi:10.1007/s10637-011-9740-3
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Summary

Aplidin is a novel cyclic depsipeptide, currently in Phase II/III clinical trials for solid and hematologic malignancies. The aim of this study was to evaluate the effect of Aplidin in chronic lymphocytic leukemia (CLL), the most common leukemia in the adult. Although there have been considerable advances in the treatment of CLL over the last decade, drug resistance and immunosuppression limit the use of current therapy and warrant the development of novel agents. Here we report that Aplidin induced a dose- and time-dependent cytotoxicity on peripheral blood mononuclear cells (PBMC) from CLL patients. Interestingly, Aplidin effect was markedly higher on monocytes compared to T lymphocytes, NK cells or the malignant B-cell clone. Hence, we next evaluated Aplidin activity on nurse-like cells (NLC) which represent a cell subset differentiated from monocytes that favors leukemic cell progression through pro-survival signals. NLC were highly sensitive to Aplidin and, more importantly, their death indirectly decreased neoplasic clone viability. The mechanisms of Aplidin-induced cell death in monocytic cells involved activation of caspase-3 and subsequent PARP fragmentation, indicative of death via apoptosis. Aplidin also showed synergistic activity when combined with fludarabine or cyclophosphamide. Taken together, our results show that Aplidin affects the viability of leukemic cells in two different ways: inducing a direct effect on the malignant B-CLL clone; and indirectly, by modifying the microenvironment that allows tumor growth.

Keywords

AplidinPlitidepsinTumor microenvironmentChronic lymphocytic leukemiaMonocytesMyeloid cells

Introduction

Aplidin (Plitidepsin), originally isolated from the Mediterranean tunicate Aplidium albicans, exhibits strong cytotoxic effects against a variety of cancer cell types [13]. These effects are related to the induction of early oxidative stress and the sustained activation of JNK and p38 MAPK [1, 4, 5]. Aplidin has shown promising results in various neoplastic diseases and is currently in Phase II/III clinical trials for solid malignancies and multiple myeloma [68].

In this study we investigated the effect of Aplidin in chronic lymphocytic leukemia (CLL), the most common form of leukemia among older adults in Western countries. Over the past 20 years, more effective therapies have substantially improved response rates and overall survival in CLL [9, 10]. Currently, the most effective treatment consists of a combination of the nucleoside analog fludarabine, the alkylating agent cyclophosphamide and rituximab, an anti-CD20 monoclonal antibody [9, 11]. Despite the encouraging results obtained, CLL remains incurable and the majority of patients relapse following the first-line therapy. New treatments and therapeutic strategies are needed, especially for the small but challenging subgroup of high risk patients whose life expectancy is less than 3 years.

CLL is characterized by the progressive accumulation of B lymphocytes in the blood, bone marrow and lymphoid tissues [12, 13]. An increasing number of studies have emphasized the importance of non-malignant cells for the survival and proliferation of CLL cells [1416]. Thus, it is now clear that the expansion of the malignant clone depends not only on its intrinsic characteristics such as the expression of anti-apoptotic molecules, but also on stimulating signals delivered from stromal, myeloid and lymphoid cells in the microenvironment. We have previously shown that circulating monocytes from CLL patients play an important role in leukemic cell survival [14]. In addition, monocytes can differentiate in vitro into large, adherent cells that protect leukemic cells from spontaneous and drug-induced apoptosis. These cells have been called nurse-like cells (NLC) and reside in lymphoid tissues where they presumably protect CLL cells from apoptosis [17, 18]. Taking these data into account, we decided to evaluate the effect of Aplidin not only on circulating CLL cells but also on non-malignant leukocytes from peripheral blood. In ex vivo experiments, we demonstrate that Aplidin exerts a dual cytotoxic activity in CLL: a direct effect on primary leukemic cells and an indirect effect by targeting NLCs. In fact, we have found that monocytic cells are particularly sensitive to Aplidin and their death indirectly impairs leukemic cells viability, problably through the disruption of prosurvival interactions.

Material and methods

Chemicals

Ficoll-Hypaque solution (Lymphoprep, Nycomed Pharma, Oslo, Norway), dextran (GE Healthcare, Munich, Germany). Recombinant human GM-CSF (rhGM-CSF) was from Laboratorios Gautier (Buenos Aires, Argentina). RPMI 1640 and PBS were purchased from HyClone Laboratories Inc. (Logan, UT, USA). Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Invitrogen Life Technologies (Grand Island, NY, USA). Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- and peridin chlorophyll (PerCP)-conjugated monoclonal antibodies specific for human CD3, CD19, CD14, CD56, CD25 and control antibodies with irrelevant specificities (isotype control) were purchased from BD Biosciences, USA. FITC conjugated rabbit anti-active caspase-3 mAb and anti-cleaved PARP were from BD Pharmingen, USA. Recombinant human (rh)GM-CSF, rhIL-4, rhIL-2, phytohemagglutinin (PHA), acridine orange, ethidium bromide, propidium iodide, 7-amino-actinomycin D (7-AAD), Annexin V-FITC, Ebselen and 2′,7′-dichlorofluorescein diacetate (2′,7′-DCFH-DA) were purchased from Sigma-Aldrich (St Louis, MO, USA). IntraStain kit was from Dako (Denmark). Aplidin® is manufactured by PharmaMar S.A. (Madrid, Spain). Fludarabine (9-β-d-arabinosyl-2-fluoroadenine-monophosphate) was obtained from Genzyme Argentina (Buenos Aires) and 4-OH-cyclophosphamide was obtained from Asta Medica (Frankfurt am Main, Germany).

Cell isolation and culture

Studies were performed in peripheral blood samples obtained from patients diagnosed with CLL according to standard clinical and laboratory criteria [19]. Blood samples were obtained after informed consent in accordance with the Declaration of Helsinki and with Institutional Review Board approval from the National Academy of Medicine, Buenos Aires. At the time of the analysis all patients were free from clinically relevant infectious complications and were either untreated or had not received treatment for a period of at least 6 months before investigation. The prognostic markers CD38 and ZAP-70 were evaluated by flow cytometry as previously described [20]. Patients were considered positive for CD38 when at least 30% of CD19+CD5+ cells express CD38 and positive for ZAP-70 when at least 20% of CD19+CD5+ express ZAP-70.

Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over a Ficoll-Hypaque layer, washed twice with saline and resuspended in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin (complete medium) to a cell concentration of 3 × 106/ml. Aliquots of 0.150 ml were cultured with different doses Aplidin, fludarabine, 4-OH cyclophosphamide or vehicle (0.01% DMSO) for the periods indicated.

Nurse-like cells (NLC) from peripheral blood of CLL patients were obtained by culturing PBMC during 2 weeks with complete medium on 24- or 48- well flat-bottomed microtiter to allow differentiation of large, round, adherent cells. In some experiments, nonadherent cells (>90% leukemic B cells) were removed by vigorous pipetting and NLC were harvested with trypsin [17]. Peripheral blood monocytes were obtained from healthy donors. Purification was performed by positive selection using anti-CD14–conjugated magnetic microbeads according to manufacturer’s instructions (Miltenyi Biotec, Auburn, CA).

The purity of the different cells population was checked using specific antibodies and flow cytometry analysis using a FACSCalibur™, BD Biosciences. Data was analyzed with CELLQuest™ software.

Quantitation of cell death

Total cell death of the different populations was determined by evaluating membrane permeability to the fluorescent DNA-binding probe, 7-AAD. To this aim, 100 μl of the cell culture was collected at the indicated time points and transferred to polypropylene tubes containing 10 μg/ml of 7-AAD. The cells were incubated at room temperature for 10 min and immediately analyzed by flow cytometry using a FACSCalibur (BD). Fluorescence was recorded at 600 nm.

Additionally, cell death in the adherent NLC was corroborated by morphological criteria using phase contrast microscopy. For non-adherent subsets, cell death was corroborated in two ways: 1) by using acridine orange and ethidium bromide as previously described [21] and 2) by comparing FSH and SSC parameters by flow cytometry analysis.

The percentage of overall apoptosis in monocytes was quantified by flow cytometry using Annexin V-FITC and 7-AAD. Briefly, 100 μl of cell suspension were labeled with Annexin-V FITC for 20 min at 4°C and then 7-AAD was added for a further 10 min incubation, before evaluation by flow cytometry. Results are depicted as percentage of Annexin-V-positive cells.

Drug combination effects in PBMC samples were determined using the Chou and Talalay method based on the median effect equation [22]. Combination index (CI) values of 0 to 0.89 indicate synergism, CI values of 0.9 to 1.1 indicate additive effects and CI values higher than 1.1 indicates antagonism. Data were analysed using the Calcusyn concentration-effect analysis software (Biosoft, Cambridge, UK).

Analysis of caspase-3 activation. PARP fragmentation and ROS production by flow cytometry

For detection of caspase-3 activation, monocytes (0.5 × 106) were cultured in the presence Aplidin (10 and 100 nM) for 4 h at 37°C. Then, cells were washed twice, fixed and permeabilized using IntraStain kit following the manufacturer’s instructions. After incubating with an specific FITC-labeled antibody for 30 min, cells were washed and analyzed by flow cytometry. To evaluate PARP fragmentation, fixed and permeabilized cells were incubated with an specific mouse anti-cleaved PARP antibody for 30 min, washed twice and labeled with an FITC anti-mouse IgG, as previously described [21]. Flow cytometry data was analyzed with CellQuest software.

For measurement of intracellular ROS levels, the fluorescent dye 2′,7′-DCFH-DA was used. Purified monocytes were incubated with Aplidin (10 or 100 nM) for 30 min at 37°C, washed with PBS and labeled in medium containing 2 μM 2′,7′-DCFH-DA for an additional 15 min before evaluation by flow cytometry. For ROS scavenging, PBMC were incubated with Esbselen at 0.5 or 5 μg/ml, and Aplidin (10 or 100 nM) was added 30 min later. The percentage of CD14+ cells was determined at 24 h of culture by flow cytometry.

Confocal microscopy

PBMC (3 × 106/ml) from CLL patients were seeded in chambered coverglass slides (Lab-Tek, Nunc, USA) and incubated for 14 days at 37°C to allow differentiation of NLC. Non-adherent cells were removed and kept in culture at 37°C. NLC were exposed to Aplidin or vehicle for 24 h before re-adding non-adherent cells. After 72 h incubation, cells were stained using the fluorescent nucleic acid-binding dye acridine orange (100 μg/ml) to visualize cell morphology in an inverted confocal scanning microscope. The images were acquired by sequentially scanning with settings optimal for acridin orange (488 nm excitation with argon laser line and detection of emitted light between 505 and 525 nm; pseudocoloured green), using a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan) equipped with a Plapon 603/1.42 oil immersion objective.

Statistical analysis

Statistical significance was determined using the nonparametric Wilcoxon matched pairs test or the Friedman test to compare data sets of paired groups. All calculations were performed using GraphPad Prism 5 for Windows (GraphPad Software, San Diego, USA).

Results

Aplidin is cytotoxic against primary CLL cultures

Table 1 summarizes the clinical and phenotypic features of CLL samples included in this study (n = 15). We first evaluated the in vitro effect of Aplidin on peripheral blood mononuclear cells (PBMC) from CLL patients using the fluorescent DNA-binding probe, 7-AAD, as a marker of disruption of plasma membrane integrity. Incorporation of 7-AAD was followed by flow cytometry analysis. Representative dot plots showing 7-AAD-positive cells after 48 h of incubation with increasing doses of Aplidin are depicted in Fig. 1a. We found that the cytotoxic effect of Aplidin on CLL samples was dose-and time-dependent (Fig. 1b). Of note, there was a marked difference in Aplidin-sensitivity among samples. Thus, percentages of cell death induced by Aplidin 100 nM ranged from 24% to 92% at 24 h and from 31% to 96% at 48 h (Fig. 1b and Table 1). However, we could not find any obvious correlation between sensitivity to Aplidin and CLL staging, progressive disease, prior treatments or poor prognostic factors such as CD38 and ZAP-70 (Table 1).
Table 1

CLL patients characteristics and percentages of cell death induced by Aplidin

Pt ID

Gender

Age

WBC (× 109/L)

RAI/Binet stage

Lymphocytes (%)

Haemoglobin (g/dL)

Platelets (× 109/L)

ZAP-70 (%)

CD38 (%)

CD19*CD5* (%)

Previous treatment

% death Apl 100nM 48 h

1

f

67

16.2

0/A

69

13.9

219

54

9

69

none

79

2

f

77

26.9

0/A

89

15.0

154

28

39

82

none

88

3

m

74

12.9

0/A

44

13.5

191

59

98

47

none

75

4

f

65

28.3

0/A

86

12.9

166

9

2

88

none

31

5

m

46

15.8

0/A

72

14.5

221

3

1

80

none

46

6

f

68

9.6

0/A

56

14.0

219

76

38

27

none

50

7

m

81

36.9

I/A

76

12.8

258

22

2

59

Flu

66

8

m

71

10.6

I/A

49

14.1

180

24

1

72

CLB

74

9

m

68

52.9

II/A

86

13.7

139

1

<1

90

none

68

10

m

54

29.1

II/B

81

12.1

167

8

1

74

none

73

11

f

72

29.7

II/B

85

11.4

143

18

55

93

none

78

12

f

76

25.8

II/B

79

13.9

245

26

2

55

none

54

13

m

70

36.2

II/B

80

11.6

119

27

23

73

Flu-C, R-CLB

74

14

m

68

23.8

II/B

77

11.6

182

10

5

58

CLB-R

77

15

m

73

250.3

IV/C

95

9.0

61

80

99

92

none

96

Flu fludarabine; CLB clorambucil; C cyclophosphamide; R rituximab

https://static-content.springer.com/image/art%3A10.1007%2Fs10637-011-9740-3/MediaObjects/10637_2011_9740_Fig1_HTML.gif
Fig. 1

Sensitivity of peripheral blood mononuclear cells (PBMC) from CLL patients to Aplidin-induced cell death. PBMC (3 × 106/ml) were cultured for the indicated times with different doses of Aplidin alone (n = 15), or in combination with fludarabine or 4-OH-cyclophosphamide. DMSO at 0.01% was used as vehicle (Control). After incubation, cells were washed and labeled with 7-AAD to determine the percentages of cell death by flow cytometry. a Representative dot plots of PBMC exposed to Aplidin for 24 h showing forward-scattered light (FSC-H) versus 7-AAD fluorescence. The percentages of 7-AAD positive cells (dead cells) are indicated. b Percentages of cell death induced by Aplidin. Each symbol represents an individual patient and dark lines represent averages. c Percentages of cell death obtained when using Aplidin at 10 nM for 24 h, alone or in combination with fludarabine (Fluda) at 2.5 μg/ml or cyclophosphamide (4-OH-C) at 5 μg/ml. Each symbol represents an individual patient and dark lines represent averages

Drug combinations for CLL treatment induce higher response rates compared with single-agent treatment. As mentioned before, fludarabine plus cyclophosphamide with or without Rituximab is currently considered the standard combination therapy for CLL [11]. Taking this into account, we next evaluated whether the combination of Aplidin with fludarabine or 4-hydroxy-cyclophosphamide could be synergistic. For this purpose, PBMC were concomitantly incubated with different doses of Aplidin and both drugs for 24 h or 48 h. Results obtained after 24 h of incubation are shown in Fig. 1c and supplementary Fig. 1. As depicted in Table 2, the combinations of Aplidin with fludarabine or 4-hydroxy-cyclophosphamide were synergistic in almost all the samples and conditions tested. The stronger synergism was observed for the Aplidin 100 nM and 4-hydroxy-cyclophosphamide (all concentrations) combination after exposure of 48 h. At this exposure time, CI values were located in the strong synergism range (lower than 0.3).
Table 2

Ex vivo cytotoxic effects of Aplidin combined with Fludarabine or 4-OH-cyclophosphamide on CLL samples

 

Aplidin

24 h

48 h

10 nM

100 nM

10 nM

100 nM

CIvalue

CI value

CI value

CI value

Fludarabine

0.5 μg/ml

1.32

0.70

0.67

0.91

2.5 μg/ml

0.87

0.66

0.59

0.99

10 μg/ml

0.59

0.68

0.60

0.85

4-HO-C

1 μg/ml

0.93

1.05

0.30

0.26

5 μg/ml

0.81

1.06

0.43

0.27

10 μg/ml

0.52

0.85

0.42

0.21

For combination studies, PBMC form CLL samples were concomitanly incubated with different doses of Aplidin and Fludarabine or 4-OH-cyclophosphamide, both drugs during 24 h (n = 5) or 48 h (n = 6). Combination Index (CI) values were obtained using the Calcusyn software

Different susceptibility to Aplidin-induced death of PBMC subpopulations from CLL samples

Once established that PBMC from CLL samples were sensitive to Aplidin cytotoxicity, we asked if the non-malignant leukocyte subpopulations and the leukemic cells could differentially respond to Aplidin cytotoxicity. To that aim, we used fluorescent monoclonal antibodies directed to CD3, CD56 or CD14 to discriminate T lymphocytes, NK cells and monocytes in PBMC from CLL samples exposed to different concentrations of Aplidin (10–100 nM) for 24 h. Figure 2a shows representative dot plots corresponding to viable cells (7-AAD negative) from control or Aplidin (10 nM) cultures labeled with each lineage-specific antibodies. We found no major differences in the proportion of T, NK or CLL cells between control and Aplidin-treated cultures at any of the doses tested (Fig. 2b). On the other hand, and to our surprise, the population of monocytes (CD14+ cells) was profoundly affected by incubation with Aplidin, even at the very low dose of 10 nM (Fig. 2c).
https://static-content.springer.com/image/art%3A10.1007%2Fs10637-011-9740-3/MediaObjects/10637_2011_9740_Fig2_HTML.gif
Fig. 2

Differential sensitivity of monocytes and lymphocytes from CLL samples to Aplidin-induced cell death. PBMC from 7 CLL patients were incubated for 24 h with Aplidin at the indicated concentration and then cells were labeled with 7-AAD dye plus specific antibodies directed to T lymphocytes, NK cells, monocytes and CLL cells. a Representative dot plots showing FSC-H versus 7-AAD fluorescence under control or Aplidin 10 nM conditions. Cells negative for 7-AAD (viable cells) were further discriminated in leukocytes populations (dot plots in the lower part of the panel). The percentage of viable CD19+ cells (>98% CLL cells), CD3+ cells (T lymphocytes), CD56+ cells (NK cells) and CD14+ (monocytes) are indicated. b Shown is the proportion of viable T lymphocytes, NK cells, CLL cells and monocytes after incubation of PBMC with different concentrations of Aplidin, n = 7. c Percentages of viable monocytes after incubating PBMC from CLL samples (n = 7) with the indicated doses of Aplidin for 24 h

Nurse-like cells from CLL patients are sensitive to Aplidin-induced cell death

As mentioned before, NLC differentiated from circulating monocytes protect CLL cells from spontaneous and drug-induced apoptosis [17, 18]. Given the high sensitivity of CD14+ cells to Aplidin-induced cytotoxicity, we decided to determine whether Aplidin affects NLC survival, which indirectly could result in leukemic cells death. To this aim, we incubated PBMC from 5 CLL samples for 2 weeks to allow the differentiation of NLC. Outgrowth of adherent NLC was assessed by phase-contrast microscopy and corroborated by flow cytometry (high FSC and SSC values) as previously described [17, 23] (Suppl Fig. 2). Once NLC were fully differentiated, non-adherent cells were removed and Aplidin was added at 100 nM for 24 h. NLC death was evaluated by flow cytometry using 7-AAD dye. As shown in Fig. 3a, NLC from the 5 evaluated samples were highly sensitive to Aplidin, with approximately 90% of cells showing positivity for 7-AAD staining at this dose. Incubation with lower doses of Aplidin showed that NLC sensitivity to the drug was slightly lower to that of monocytes (not shown). Freshly isolated leukemic B cells from these 5 samples were also sensitive to the cytotoxic effect of Aplidin though to a lesser extent compared to NLC (Fig. 3b).
https://static-content.springer.com/image/art%3A10.1007%2Fs10637-011-9740-3/MediaObjects/10637_2011_9740_Fig3_HTML.gif
Fig. 3

Sensitivity of Nurse-like cells (NLC) derived from chronic lymphocytic leukemia (CLL) patients to Aplidin-induced cell death. a PBMC from 5 CLL patients were cultured for 2 weeks to allow differentiation of NLC. Non-adherent cells were removed and NLC were incubated with Aplidin (100 nM) or 0.01% DMSO (Control) for 24 h. Cells were tripsinized and labeled with 7-AAD to evaluate cell death by flow cytometry. b Freshly isolated PBMC (>85% leukemic cells) from the same 5 patients were incubated with Aplidin (100 nM) or 0.01% DMSO for 24 h, before evaluating cell death. c Purified NLC obtained as described in (A) were treated with Aplidin (100 nM) or 0.01% DMSO for 24 h while the corresponding non-adherent cells were kept at 37°C for the same period. Then Aplidin and vehicle were thoroughly washed out and non-adherent cells were re-added to the each well. After an additional 72 h in culture, cell death was evaluated in the CD19+ population by using 7-AAD and flow cytometry. d Representative fluorescent micrographies obtained at the time of revealing the experiment detailed in panel C. In the control condition, NLC are intact and in close contact with CLL cells. In contrast, when NLC had been cultured with Aplidin, many were lost in the extensive washes and the remaining showed an apoptotic phenotype, seen as well in the neoplasic CLL cells. White bars = 10 μm

To answer the question of whether NLC death induced by Aplidin could affect survival of leukemic cells, we treated purified NLC with Aplidin 100 nM for 24 h, thoroughly washed out the drug and re-added CLL cells to cultures. Cell death of CD19+ cells was daily evaluated by flow cytometry for the next 4 days. Results obtained at 72 h are depicted in Fig. 3c. As expected, the percentages of spontaneous CLL cell death after 2 weeks in culture were higher than those from 24 h cultures (compare with control values in panel B). Nevertheless, previous treatment of isolated NLC with Aplidin further increased CLL cell death, indicating an indirect effect of Aplidin on leukemic cell survival. Representative fluorescent micrographies are depicted in Fig. 3d. Notice the close physical interaction of NLC and CLL cells in control cultures. Disruption of this interaction through NLC death induced by Aplidin impaired leukemic cell survival.

Together these results indicate that Aplidin might be able to induce cytotoxicity on CLL cells not only by directly affecting the leukemic clone, but also by impairing their interaction with a protective microenvironment.

Aplidin induced apoptosis through ROS production

To gain insights into the early mechanisms by which Aplidin induces its cell death effect on monocytes, we chose to continue our studies using purified CD14+ cells from healthy donors, due to the very low proportion of this subset in CLL samples. We first corroborated that monocytes from healthy donors display a comparable sensitivity to Aplidin (suppl. Fig. 3). One of the earliest features of cells undergoing apoptosis is the exposure of phosphatidylserine to the outer leaflet of the plasma membrane. Due to its strong affinity to negatively charged phospholipids, Annexin V binds phosphatidylserine as it becomes exposed in apoptotic cells [24]. At later times, the loss of membrane integrity allows the incorporation of fluorescent compounds which intercalate in DNA. Similarly, necrotic cells are also permeable to these vital dyes, like propidium iodide or 7-AAD. Therefore, we used Annexin V FITC and 7-AAD to determine whether Aplidin induced monocyte apoptosis or necrosis. Dot plots from a representative experiment are depicted in Fig. 4a (upper left panel). We found that the incubation of cells with 100 nM of Aplidin for 3 h induced that nearly 30% of the monocytes became Annexin V positive. By contrast, 7-AAD positive cells at this time were only a minor fraction of Aplidin-treated monocytes. As shown in Fig. 4a (lower panel) Aplidin induced monocyte apoptosis in a dose-dependent manner. The involvement of caspase-3 activation and subsequent PARP fragmentation during the early events that lead to Aplidin-induced cell death have been previously reported in a variety of cell lines, such as malignant epithelial cells, lymphoblastic leukemia and myeloma cell lines [1, 25, 26]. We could also observe activation of caspase-3 and PARP fragmentation by Aplidin in peripheral blood monocytes using a cytometric approach (Fig. 4b).
https://static-content.springer.com/image/art%3A10.1007%2Fs10637-011-9740-3/MediaObjects/10637_2011_9740_Fig4_HTML.gif
Fig. 4

Mechanism of Aplidin-induced cell death in monocytes. Purified monocytes from healthy donors were incubated with Aplidin at different concentrations or 0.01% DMSO as vehicle (Control) for 4 h. a Evaluation of cell apoptosis by Annexin V binding. Cells were labeled with Annexin V-FITC plus 7-AAD and immediately analyzed by flow cytometry. Representative dot plots indicating the percentage of labeled cells are shown (upper panel). A dose–response curve of Annexin V positive cells is depicted in the lower panel. Results are expressed as the mean±SEM, n = 3. b Evaluation of caspase 3 activation and PARP fragmentation induced by Aplidin. After treatment, monocytes were fixed, permeabilized and labeled with specific antibodies as described under Material and Methods. Upper panel: representative histograms of activated-caspase 3 expression in monocytes incubated with 0.01% DMSO as vehicle (Control) or Aplidin (10 or 100 nM) (n = 3). Lower panel: representative histograms of PARP fragmentation in monocytes incubated with 0.01% DMSO (Control) or Aplidin 100 nM. The percentage of cells positive for cleaved PARP is indicated. (n = 3). c Evaluation of ROS involvement in Aplidin-induced cell death. Left panel: Production of ROS by monocytes exposed to Aplidin. Monocytes were incubated with Aplidin (10 or 100 nM) or 0.01% DMSO (Control) for 30 min at 37°C, labeled with 2′,7′-DCFH-DA and analyzed by flow cytometry. Shown are representative histograms of 3 independent experiments. Right panel: Inhibition of cytotoxic effects of Aplidin by Ebselen. PBMC were incubated with Ebselen (0.5 or 5 μg/ml) or 0.01% DMSO as control for 30 min before adding Aplidin (10 or 100 nM) or vehicle for an additional 24 h. The percentages of CD14+ cells were determined by flow cytometry. Results are expressed as mean±SEM, n = 3

Given that reactive oxygen species (ROS) play a central role in Aplidin pro-apoptotic effects on malignant cells [27, 28], we determined their production in human purified monocytes. To this aim, we used the fluorescent probe DCFH-DA and directly evaluated changes in the redox state at the single-cell level. Results from a representative experiment are shown in Fig. 4c (left panel). ROS production was evident after 30 min incubation with Aplidin at doses as low as 10 nM. More importantly, the cytotoxic effect of Aplidin was significantly diminished when monocytes were pre-incubated with the antioxidant reagent Ebselen, which acts as a glutathione peroxidase mimic and is an efficient scavenger of peroxynitrite (Fig. 4c, right panel). Inhibition of cell death by Ebselen depended on its concentration and that of Aplidin. Thus, Ebselen was unable to diminish cell death when Aplidin was used at 100 nM but impaired cytotoxicity of 10 nM of Aplidin at both, 0.5 and 5 μg/ml. Taken together, our results show that Aplidin induces monocyte cell death via apoptosis being the production of ROS an early central event.

Discussion

This study was primarily undertaken to evaluate the in vitro effects of the marine-derived antineoplasic drug Aplidin on leukemic cells and non-malignant mononuclear leukocytes from CLL patients. Two main conclusions emerge from our results. First, that Aplidin exerts a direct cytotoxic effect on PBMC from CLL patients, being leukemic cells slightly more sensitive to the drug than T lymphocytes or NK cells. Secondly, that Aplidin exhibits a potent activity against monocytes, which resulted to be about one order of magnitude more sensitive to the drug than lymphoid cells. The high sensitivity of monocyte and monocyte-derived cells (e.g. NLC) to Aplidin might indirectly affect leukemic cell survival by impairing the delivery of pro-survival signals from these cell populations.

In this report, we firstly evaluated Aplidin activity on PBMC from 15 CLL patients with different clinical characteristics, such as Binet/Rai staging, relapsed/refractory disease and variable levels of the poor prognostic factors, CD38 and ZAP-70. Although there was a marked heterogeneity in Aplidin sensitivity among samples, all of them were sensitive to the drug, being the average cytotoxicity of Aplidin 100 nM at 48 h: 68.6% ± 16.9% (mean±SD). Interestingly, we found that the percentages of cell death induced by Aplidin in five of the six samples with the higher expression of both poor prognostic factors (Table 1) were above the average cytotoxicity. Moreover, the most sensitive sample to Aplidin was that from a Rai IV/Binet C patient with progressive disease (# 15), suggesting that Aplidin might be useful for patients that respond poorly to standard chemotherapy.

Since it is well-established that drug combinations are beneficial for CLL patients, we evaluated Aplidin combined with fludarabine or cyclophosphamide, both agents currently used for standard therapy in CLL. We observed that the concomitant combination of Aplidin with any of them induces higher levels of CLL cell death in vitro than each of the drugs separately. In fact, synergy has been noted (as per CI < 0.89) on most of the samples and conditions that have been tested. Such findings suggest that Aplidin lacks complete cross resistance with both drugs, at least under the experimental frame of this study.

We have also observed that, among leukocyte populations, Aplidin is particularly cytotoxic against monocytes and monocyte-derived cells. Allavena et al. [29] have previously reported that Trabectedin, another marine-derived antitumor agent with a different mechanism of action than Aplidin (for review see D’Incalci & Galmarini, 2010) [30] is highly cytotoxic to monocytes and macrophages, and impairs the production of inflammatory mediators. These effects resulted in a marked reduction of infiltrating macrophages after Trabectedin treatment in a xenograft mouse model of human myxoid liposarcoma [31]. Monocyte and monocyte-derived cells are proved to be important actors in the arising and development of several malignant diseases. It has been shown that circulating monocytes are actively recruited to tumor beds where they are “conditioned” to promote the survival of malignant cells both directly and indirectly via the suppression of host immunity [14, 17, 3234]. In addition, tumor associated macrophages secrete a variety of growth factors, cytokines and matrix enzymes that stimulate tumor proliferation, angiogenesis and invasion of the surrounding tissues. In T-cell lymphomas, it has recently been shown that monocytes are actively recruited to the tumor microenvironment where they promote malignant T-cell survival and are precluded from differentiation into dendritic cells by tumor-derived IL-10. In the context of CLL, our results show that low doses of Aplidin affects the survival of NLC, a myeloid subset derived from peripheral leukocytes of CLL patients. Several reports have shown that NLC play a crucial role for leukemic cell progression as they secrete, among other pro-survival factors, CXCL12, a chemokine which not only induces CLL cells migration to lymphoid tissues but also protects them from spontaneous and drug-induced apoptosis [17, 18]. In line with the protective function of NLC on CLL cells we found that the previous treatment of isolated NLC with Aplidin impairs leukemic cell survival.

In regard to the cytotoxic mechanism, we found that Aplidin induced monocyte death by triggering apoptosis, as evidenced by early exposure of phosphatidylserine in the outer leaflet of plasma membrane, the activation of caspase-3 and subsequent cleavage of PARP. We also show that the production of ROS induced by Aplidin plays a central role in monocyte death, as drug-induced apoptosis was blocked when preincubated with ebselen, a compound that increases intracellular GSH levels. It is currently known that ROS and reactive nitrogen species (RNS) regulate the molecular and biochemical pathways responsible for human monocytes survival and that any disbalance in this strict regulation, for e.g. induction of oxidative stress, can be detrimental for these cells [35]. In this regard, it was reported that monocytes are more susceptible than lymphocytes to cell death triggered by oxidative stress [36]. Whether the induction of ROS by Aplidin can justify the higher sensitivity of monocytes compared to other mononuclear cells remains unknown.

Of note, Mitsiades et al. [1] have previously analyzed the activity of Aplidin on bone marrow cells isolated from 4 multiple myeloma patients using a multiparametric cytometry protocol which allowed them to distinguish myelomatous plasma cells from normal lymphoid and granulo-monocytic cells present in the same sample. Their results differ from ours in that they found a comparable and moderate cytotoxic effect of Aplidin on both normal leukocyte populations. Thus, overnight incubation with Aplidin 100 nM induced levels of apoptosis that range from 20% to 60%. By contrast, we found that this dose of Aplidin almost completely eliminates peripheral monocytes (Fig. 1c) when cultured in vitro. Although we have no simple explanation for these discrepancies, the possibility exists that bone marrow monocytic cells from multiple myeloma patients (or even healthy donors) should differ from their circulating counterparts.

In conclusion, this report suggests that Aplidin might be considered of potential benefit for CLL treatment as it exerts a direct cytotoxic effect on leukemic cells and an indirect effect through the impairment of CLL cells interactions with a protective microenvironment. Given the relevance of monocytes and NLC cells in supporting tumor growth, their sensitivity to Aplidin-induced death may contribute to its antitumoral effects.

Acknowledgements

The authors would like to thank all patients and donors for their participation in this study; Dr Analía Trevani for assistance with fluorescence microscopy; Ms Beatriz Loria and Ms Mabel Horvat for technical assistance. This work was supported by grants from Agencia Nacional de Promoción Científica (Argentina), CONICET and Fundación Florencio Fiorini.

Conflict of interest

C.M. Galmarini: employment, PharmaMar. The other authors reported no potential conflicts of interest.

Supplementary material

10637_2011_9740_MOESM1_ESM.jpg (351 kb)
Fig. 1SSensitivity of CLL cells to the combination of Aplidin with fludarabine or 4-OH-cyclophosphamide. PBMC isolated from CLL patients were incubated with Aplidin at 10, 50 or 100 nM, alone or in combination with the indicated concentrations of fludarabine (Fluda) or 4-OH-cyclophosphamide (4-OH-C). Percentages of cell death were calculated by 7-AAD dye incorporation and flow cytometry analysis. Shown are the curves obtained when performing cultures for 24 h (panel A, n = 5) or 48 h (panel B, n = 6) (JPEG 351 kb)
10637_2011_9740_MOESM2_ESM.jpg (508 kb)
Fig. 2SCharacterization of nurse-like cells (NLC) from CLL samples. PBMC from CLL patients were cultured for 10–15 days on 24- or 48- well flat-bottomed microtiter to allow the differentiation of NLC. a The outgrowth of NLC was assessed by phase-contrast microscopy. Black bars = 10 μm. b NLC were differentiated in chambered coverglass slides and labeled with phycoerythrin-anti-CD14 antibody and TO-PRO®-3 stain as a nuclear counterstain. Displayed is a representative fluorescent micrography showing CD14 expression on membrane NLC (red) and nuclei (blue) White bar = 10 μm. c Differentiation of NLC was also assessed by flow cytometry analysis. A representative dot plot of the side-angle (SSC) and forward-angle (FSC) of lymphocytes and NLC is shown (JPEG 507 kb)
10637_2011_9740_MOESM3_ESM.jpg (158 kb)
Fig. 3SMonocytes from healthy donors are highly sensitive to Aplidin-induced cell death. a PBMC from 3 healthy donors (3 × 106/ml) were cultured for 24 h with different doses of Aplidin or 0.01% DMSO as vehicle (Control). Cells were washed and labeled with anti-CD14 fluorescent antibody to determine the percentage of monocytes by flow cytometry. Shown are representative dot plots of FSC versus CD14 expression under control or Aplidin (100 nM) conditions. b Purified human monocytes were incubated with Aplidin at 10, 50 or 100 nM for 48 h. Percentages of cell death as revealed by 7-AAD dye is shown (n = 3) (JPEG 157 kb)

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© Springer Science+Business Media, LLC 2011