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
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- 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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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.
KeywordsAplidinPlitidepsinTumor microenvironmentChronic lymphocytic leukemiaMonocytesMyeloid cells
Aplidin (Plitidepsin), originally isolated from the Mediterranean tunicate Aplidium albicans, exhibits strong cytotoxic effects against a variety of cancer cell types [1–3]. 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 [6–8].
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 [14–16]. 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 . 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
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 . 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 . 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 . 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  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 . 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 . 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.
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 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).
Aplidin is cytotoxic against primary CLL cultures
CLL patients characteristics and percentages of cell death induced by Aplidin
WBC (× 109/L)
Platelets (× 109/L)
% death Apl 100nM 48 h
Ex vivo cytotoxic effects of Aplidin combined with Fludarabine or 4-OH-cyclophosphamide on CLL samples
Different susceptibility to Aplidin-induced death of PBMC subpopulations from CLL samples
Nurse-like cells from CLL patients are sensitive to Aplidin-induced cell death
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
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.
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.  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)  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 . 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, 32–34]. 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 . In this regard, it was reported that monocytes are more susceptible than lymphocytes to cell death triggered by oxidative stress . 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.  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.
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.