TRAIL Overcome Chemoresistance

Article pubs.acs.org/molecularpharmaceutics

Liposomes Decorated with Apo2L/TRAIL Overcome Chemoresistance of Human Hematologic Tumor Cells Diego De Miguel,† Gorka Basáñez,‡ Diego Sánchez,† Patricia Galán Malo,† Isabel Marzo,† Luis Larrad,§ Javier Naval,† Julián Pardo,†,∥,⊥ Alberto Anel,*,†,# and Luis Martinez-Lostao*,†,# †

Departamento de Bioquímica, Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain Unidad de Biofísica, Universidad del País Vasco-Consejo Superior de Investigaciones Científicas (UPV/EHU-CSIC), Bilbao, Spain § Unidad de Investigación en Inmunología y Cáncer, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain ∥ Fundación Aragón I+D (ARAID), Gobierno de Aragón, Zaragoza, Spain ⊥ Instituto de Nanociencia de Aragón, Universidad de Zaragoza, Zaragoza, Spain ‡

S Supporting Information *

ABSTRACT: Human Apo2-ligand/TRAIL is a member of the TNF cytokine superfamily capable of inducing apoptosis on tumor cells while sparing normal cells. Besides its antitumor activity, Apo2L/TRAIL is also implicated in immune regulation. Apo2L/TRAIL is stored inside activated T cells in cytoplasmic multivesicular bodies and is physiologically released to the extracellular medium inserted in the internal membrane vesicles, known as exosomes. In this study we have generated artificial lipid vesicles coated with bioactive Apo2L/ TRAIL, which resemble natural exosomes, to analyze their apoptosis-inducing ability on cell lines from hematological tumors. We have tethered Apo2L/TRAIL to lipid vesicles by using a novel Ni2+-(N-5-amino-1-carboxylpentyl)-iminodiacetic acid, NTA)-containing liposomal system. This lipidic framework (LUVsApo2L/TRAIL) greatly improves Apo2L/TRAIL activity, decreasing by around 14-fold the LC50 on the T-cell leukemia Jurkat. This increase in bioactivity correlated with the greater ability of LUVs-Apo2L/TRAIL to induce caspase-3 activation and is probably due to the increase in local concentration of Apo2L/TRAIL, improving its receptor cross-linking efficiency. More important, liposome-bound Apo2L/TRAIL overcame the resistance to soluble recombinant Apo2L/TRAIL exhibited by tumor cell mutants overexpressing Bcl-xL or by a Bax and Bak-defective Jurkat cell mutant (Jurkat-shBak) and are also effective against other hematologic tumor cells. Jurkat-Bcl-xL and Jurkat-shBak cells are resistant to most chemotherapeutic drugs currently used in cancer treatment, and their sensitivity to LUVs-Apo2L/TRAIL could have potential clinical applications. KEYWORDS: Apo2L/TRAIL, cancer, liposomes, therapy, chemoresistance

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INTRODUCTION Targeting of extrinsic apoptosis pathway for anticancer therapy is uniquely attractive for several reasons: (i) death receptors are widely expressed in tumors, (ii) some oncogenes increase the sensitivity to death ligand-induced-apoptosis, and (iii) proapoptotic receptors activate caspases and induce apoptosis regardless of the p53 status of cancer cells.1 Apoptosis ligand 2/ TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the TNF cytokine family2,3 potentially useful as anticancer agent. Apo2L/TRAIL interacts with 5 different receptors: DR4 (TRAILR1), DR5 (TRAILR2), DcR1, DcR2, and osteoprotegerin (OPG). Only DR4 and DR5 can transduce a death signal.4 Apo2L/TRAIL is able to induce apoptosis in a wide variety of tumor cells while sparing most normal cells. Loss-of-function studies in mice suggest that Apo2L/TRAIL is important for the immune control of tumor growth and metastasis.5,6 All these findings support the potential usefulness of Apo2L/TRAIL as a novel anticancer agent, and indeed, Apo2L/TRAIL-based therapies have been developed.4,7 © 2013 American Chemical Society

Apo2L/TRAIL is currently being used in several phase II/III clinical trials on a wide variety of human cancers.8 There are currently two main Apo2L/TRAIL-based therapeutic strategies: (i) the use of recombinant human Apo2L/TRAIL9,10 or (ii) agonistic monoclonal antibodies directed against DR4 or DR5 receptors.11−14 Apo2L/TRAIL is being tested as monotherapy in tumors that are particularly sensitive to its action ex vivo but also in combination with other drugs. Such combinatorial therapies include monoclonal antibodies and inhibitors of receptor-tyrosine kinases, proteasome, or serin/theonin kinases.15−19 The major purpose of these combination regimens is to improve therapeutic response. In these cases, conventional or targeted drugs induce apoptosis through the intrinsic pathway and/or facilitate Apo2L/TRAIL action. Received: Revised: Accepted: Published: 893

May 9, 2012 December 20, 2012 January 18, 2013 January 18, 2013 dx.doi.org/10.1021/mp300258c | Mol. Pharmaceutics 2013, 10, 893−904

Molecular Pharmaceutics

Article

rApo2L/TRAIL) was determined by dynamic light scattering (DLS) at 37 °C at a fixed 90° angle by using a DynaPro instrument (Wyatt Technology). Each measurement consisted of an average of 20 data points during 15−20 min. Data were analyzed by the cumulated method using the software provided by the supplier (Supplemental Figure 1B in the Supporting Information). Purified liposomes were analyzed by SEM as previously described.20 Briefly, glutaraldehyde solution was added to LUVs and LUVs-Apo2L/TRAIL suspensions up to a final concentration of 1%. A drop of these suspensions was placed onto glass microscope slides, previously treated with 3aminopropyltriethoxysilane (Sigma). Samples were then washed with KHE, fixed with 1% OsO4, washed again with KHE, dehydrated in a graded series of ethanol (25−100% by volume), dried, mounted, and coated with gold. Samples were examined in a Hitachi S-3400 N scanning electron microscope (Supplemental Figure 1C in the Supporting Information). Cell Culture and Cytotoxicity Assays. Jurkat clone E6.1 (derived from human acute T cell leukemia), U937 (derived from histiocytic lymphoma), U266 (derived from multiple myeloma), and MM.1S (derived from multiple myeloma) were obtained from ATCC. Cells overexpressing the antiapoptotic proteins CrmA (Jurkat-CrmA), Bcl-xL (Jurkat-Bcl-xL), and Bcl2 (U937-Bcl-2) were kindly provided by Dr. Daniel Johnson (University of Pittsburgh), Dr. Victor Yuste (Autonomous University of Barcelona), and Dr. François Hirsch (Centre National de la Recherche Scientifique, Villejuif) respectively. Cells lacking expression of the proapoptotic protein Bak (Jurkat-shBak) were generated in our laboratory.17,23,24 Cell lines were routinely cultured at 37 °C in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS), 2 mM Lglutamine, and penicillin/streptomycin (hereafter, complete medium). For cytotoxicity assays, cells (3 × 105 cells/mL) were seeded in 96-well plates (100 μL/well) in complete medium and treated with different concentrations 1−1,000 ng/mL) of rApo2L/TRAIL either as a soluble protein or inserted in liposomes, 5 μM doxorubicin (Sigma), or 500 ng/mL cytotoxic anti-human Fas mAb (clone CH11, Millipore) for 24 h. Human peripheral blood mononuclear cells (PBMC) were obtained from blood of healthy donors by Ficoll-Paque density centrifugation. T cell blasts were generated as follows: PBMC (2 × 106 cells/mL) were stimulated for 1 day with 5 μg/mL PHA. Then, PHA was washed out, and cells were resuspended in complete medium supplemented with 30 UI/mL IL-2 and cultured for 6 days with medium changes every 48 h. Correct activation was routinely tested analyzing standard activation parameters such as sensitivity to activation-induced cell death and increase in granzyme B expression (data not shown). Both fresh PBMC and T cell blasts were treated with 500 ng/mL or 1,000 ng/mL rApo2L/TRAIL or LUVs-Apo2L/TRAIL for 24 h in the presence of IL-2. For cell death inhibition assays, cells were preincubated for 1 h prior to the addition of rApo2L/TRAIL or LUVs-Apo2L/ TRAIL with blocking anti-human TRAIL mAb (500 ng/mL, clone RIK2, BD Biosciences), with an excess of DR5-Fc chimera (R&D Systems), with either the pan-caspase inhibitor Z-VAD-fmk (30 μM, Bachem), the caspase-8 inhibitor ZIETD-fmk (30 μM, Bachem), or the necroptosis inhibitor necrostatin-1 (25 μM, Sigma). Then, cell sensitivity to rApo2L/ TRAIL or LUVs-Apo2L/TRAIL was determined by annexin-V binding and 7-AAD staining (see below).

However, since some cancers cells respond to Apo2L/TRAIL recombinant forms currently available poorly, novel Apo2L/ TRAIL versions with increased bioactivity would be potentially useful. Previous work from our group described that death ligands FasL and Apo2L/TRAIL were stored in activated human T cells inserted in the membrane of inside microvesicles of cytoplasmic multivesicular bodies. Upon stimulation, multivesicular bodies fuse with the plasma membrane and release these bioactive molecules to the extracellular medium in the form of exosomes.20,21 Since exosomes are the physiological form of Apo2L/TRAIL release, in this study we have generated artificial lipid vesicles with a similar size and composition as natural exosomes containing membrane-bound Apo2L/TRAIL and tested their ability to induce apoptosis of human cell lines from hematological tumors. Using a novel Ni2+-(N-5-amino-1carboxylpentyl)-iminodiacetic acid, NTA)-containing liposomal system, we find that tethering Apo2L/TRAIL to the liposome membrane increases its bioactivity against cell lines compared with free soluble recombinant Apo2L/TRAIL. Moreover, Apo2L/TRAIL bound to liposomes is toxic to human leukemias resistant to chemotherapeutic drugs. This liposome-bound Apo2L/TRAIL with enhanced tumor apoptosis-inducing ability could be useful to improve Apo2L/TRAILbased anticancer therapies.

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EXPERIMENTAL SECTION Preparation of Lipid Vesicles Coated with Soluble Recombinant Apo2L/TRAIL. Lipid vesicles mimicking the lipid composition of exosomes were prepared with a mixture of phosphatidylcholine, sphingomyelin (SM), cholesterol, and 1,2dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)-iminodiacetic acid]succinyl}(nickel salt) (DOGS-NTA-Ni) all from Avanti Polar Lipids and of the highest purity available. Preparation of large unilamellar vesicles (LUVs) with inserted recombinant Apo2L/TRAIL (rApo2L/TRAIL) was performed as described.22 In brief, mixtures of polar lipids were prepared in chloroform and dried in glass test tubes first under nitrogen gas and next under vacuum. Lipids were resuspended in 100 mM KCl, 10 mM HEPES, pH 7.0, containing 0.1 mM EDTA (KHE buffer). Lipid suspensions were freeze−thawed 10 times and extruded 10 times through two polycarbonate membranes with a pore size of 200 nm (Whatman) using an extruder (Northern Lipids) to generate large unilamellar vesicles (LUVs). LUVs containing 5% (weight/weight) of DOGSNTA-Ni and rApo2L/TRAIL were incubated in KHE buffer for 30 min at 37 °C. The mixture was ultracentrifugated at 4 °C for 5 h at 100,000 rpm. The supernatant was removed, and the pellet was resuspended in an equal volume of fresh KHE buffer. Fluorescent LUVs containing 0.25% (mol/mol) 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl chloride) (LUVs-RhoB) were prepared and used in flow cytometry assays. rApo2L/TRAIL and fluorescent LUVsRhoB were incubated and collected as previously described. Then, LUVs-Apo2L/TRAIL were incubated sequentially with either a mouse anti-human Apo2L mAb (clone 5C2, Genentech) or a mouse IgG (isotype control, Invitrogen) for 1 h at 4 °C followed by a fluorescein isothiocyanate (FITC)conjugated goat F(ab′)2 anti-mouse antibody for 30 min at 4 °C. All samples were analyzed by flow cytometry using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences; Supplemental Figure 1A in the Supporting Information). The size of LUVs (alone or coated with 894

dx.doi.org/10.1021/mp300258c | Mol. Pharmaceutics 2013, 10, 893−904


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Figure 1. Cytotoxicity assays on Jurkat cells and on apoptosis-resistant Jurkat sublines. Cells (3 × 105 cells/mL) were incubated in the presence or absence of different doses of soluble rApo2L/TRAIL and LUVs-Apo2L/TRAIL for 24 h. Graphics show the percentage of annexin-V positive cells analyzed by flow cytometry. The results shown are the mean ± SD of at least three experiments. (A) Jurkat; (B) Jurkat-CrmA; (C) Jurkat-Bcl-xL; (D) Jurkat-shBak. Apoptosis inhibition by blocking antibody RIK2. Bar diagrams show the mean ± SD of annexin-V positive cells in the controls (control and LUVs alone), in cells treated with 500 ng/mL of soluble rApo2L/TRAIL or LUVs-Apo2L/TRAIL, and in cells treated with 500 ng/mL of soluble rApo2L/TRAIL or LUVs-Apo2L/TRAIL previously preincubated with a concentration of 500 ng/mL of the anti-TRAIL blocking mAb, RIK2. Data are the mean of at least three different assays. (E) Jurkat; (F) Jurkat-CrmA; (G) Jurkat-Bcl-xL; (H) Jurkat-shBak. *p < 0.05; **p < 0.01.

Flow Cytometry. Quantitative determination of apoptosis was performed by determination of phosphatidylserine exposure on the surface of cells. Briefly, cells (3 × 105 in 200 μL) were incubated with 0.5 μg/mL annexin-V-FITC, or annexin-V-DY634 (all from Immunostep, Spain) at room temperature for 15 min in annexin-binding buffer (140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES/NaOH, pH 7.4). Cell suspension was diluted to 200 μL with binding buffer and analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences). In some experiments, cells were also stained with 7-AAD. For determination of cell surface expression of death receptors, cells (1 × 105 in 100 μL) were incubated with either anti-DR4, anti-DR5, anti-DcR1, or anti-DcR2 monoclonal antibodies or isotype, all of them PE-conjugated (eBioscience) at room temperature in PBS containing 5% FCS for 30 min and analyzed by flow cytometry. To determine caspase-3 activation, cells (2 × 105 in 100 μL) were fixed in 1% paraformaldehyde in PBS, permeabilized for 15 min with 0.1%

saponin in PBS containing 5% FCS, and then incubated with a FITC-labeled anti-active caspase-3 mAb (C92-605, BD Biosciences). Jurkat-shBak were incubated with an unlabeled anti-caspase-3 antibody and, then, with a secondary PE-Cy5.5labeled antibody. Finally, cells were resuspended in 500 μL of PBS and analyzed by flow cytometry. For determination of the intracellular expression of CrmA, Bcl-xL, and Bak, cells (2 × 105 in 100 μL) were fixed in 1% paraformaldehyde in PBS, and then incubated with the corresponding antibody: anti-CrmA (BD Biosciences), anti-Bcl-xL (Cell Signaling), or anti-Bak (Calbiochem) in PBS containing 5% FCS and 0.1% saponin at room temperature for 30 min followed by the corresponding secondary antibody labeled with Alexa 488 (Invitrogen). Western Blot Analysis. Cells (5 × 106) were lysed at 4 °C with 100 μL of a buffer containing 1% Triton X-100 and protease and phosphatase inhibitors, as previously described.25 Proteins from lysed cells (1 × 106 cells/mL) were separated by 12% SDS−PAGE and transferred to PVDF membranes. They were blocked with TBS-T buffer (10 mM Tris/HCl, pH 8.0, 895



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Figure 2. (A) DR expression on Jurkat cells and Jurkat sublines. Surface expression of DR4, DR5, DcR1, and DcR2 in Jurkat, Jurkat-CrmA, JurkatBcl-xL, and Jurkat-shBak was determined by flow cytometry. Solid line indicates DR expression; dotted line indicates isotype control. (B) Protein expression on Jurkat cells and Jurkat sublines. Expression levels of CrmA, Bcl-xL, and Bak in Jurkat sublines Jurkat-CrmA, Jurkat-Bcl-xL, and JurkatshBak respectively, in comparison with Jurkat, were analyzed by Western blot and flow cytometry. In Western blot, cell lysates (106 cells) were separated by SDS−PAGE and analyzed by immunoblotting with specific antibodies, using tubulin and actin immunoblots to control protein loading. In assays by flow cytometry, cells (2 × 105) were permeabilized with saponin and incubated with primary antibodies recognizing the proteins CrmA, Bcl-xL, and Bak. Then, cells were incubated with labeled secondary antibodies and analyzed. Solid line indicates the expression of the protein of interest in each subline, and dotted line indicates the expression of the protein of interest in Jurkat.

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RESULTS Generation of LUVs-Apo2L/TRAIL with Cytotoxic Activity. In order to confirm the binding of rApo2L/TRAIL to LUVs, soluble rApo2L/TRAIL was incubated with LUVs containing 0.25% (mol/mol) RhoB-conjugated phosphoethanolamine (LUVs-RhoB) and then with an anti-human Apo2L mAb (5C2) or with IgG isotype control. After staining with an FITC-conjugated secondary F(ab′)2 antibody, flow cytometric analysis was performed. When LUVs-Apo2L/TRAIL were incubated with 5C2, more than 95% of LUVs positive for RhoB were also positive for FITC fluorescence (Supplemental Figure 1A in the Supporting Information). To examine overall morphological changes in LUVs when rApo2L/TRAIL was bound to their surface, the size distribution of the vesicle suspension was quantitatively monitored by DLS (Supplemental Figure 1B in the Supporting Information). Binding soluble rApo2L/TRAIL to LUVs produced little changes in the size and homogeneity of the liposome population. LUVs alone showed a Z-average of 155

0.12 M NaCl, 0.1% Tween-20, 0.05% sodium azide) containing 5% skimmed milk, and incubated with anti-caspase-8 (BD Biosciences), anti-caspase-3 (Cell Signaling), or anti-β-actin (Sigma) antibodies in TBS-T containing 2% skimmed milk. Membranes were washed with TBS-T and incubated with 0.2 μg/mL of the corresponding phosphatase alkaline-labeled or peroxidase-labeled secondary antibody (Sigma). Immunoblots were revealed with the CDP-Star substrate (Merck) or with the Pierce ECL Western Blotting Substrate (Thermo Scientific), as previously described.26 Statistical Analysis. Computer-based statistical analysis was performed using SPSS 10.0 software. Results are shown as mean ± SD of at least three different experiments. Statistical significance was evaluated by using Student’s t test for nonpaired variants. A p < 0.05 value was considered to be significant. 896



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Figure 3. Cytotoxicity of CH11 on Jurkat and Jurkat-CrmA. Cells were untreated (control) or treated with 500 ng/mL of the anti-Fas/CD95 agonistic mAb CH11. After 24 h, apoptosis was analyzed by annexin-V staining (A), and the summary of all experiments performed is showed as a bar chart (B). Data are the mean ± SD of four independent experiments. Cytotoxiciy of doxorrubicin on Jurkat and on Jurkat-Bcl-xL and Jurkat shBak sublines. Cells were untreated or treated with doxorubicin 5 μM (24h), and labeled with annexin-V (C). The percentage of annexin-V positive events was recorded and the summary of the experiments performed expressed as a bar plot (D). Data are the mean ± SD of three independent experiments.

nm with a width of 61.2 nm while the Z-average of LUVsApo2L/TRAIL was 178 nm with a width of 85.3 nm. These data were confirmed by analyzing LUVs and LUVs-Apo2L/ TRAIL by SEM, observing round vesicles with a diameter size that ranged from 150 to 200 nm, with a high degree of homogeneity (Supplemental Figure 1C in the Supporting Information). LUVs-Apo2L/TRAIL Greatly Reduce Apo2L/TRAIL LC50 on the Jurkat T-Cell Leukemia and Overcome Resistance to Apoptosis of Jurkat Cell-Derived Sublines. After obtaining LUVs-Apo2L/TRAIL, we first performed dose− response toxicity assays on the acute T-cell leukemia Jurkat, compared with soluble rApo2L/TRAIL. Jurkat cells showed a moderate sensitivity to soluble rApo2L/TRAIL (Figure 1A). However, LUVs-Apo2L/TRAIL were capable of inducing apoptosis with a greater efficiency at every dose tested, reducing LC50 by around 14-fold. CrmA is a cowpox virus-derived protein which inhibits caspase-8 activity, thus preventing death receptor-induced apoptosis.27 Bcl-xL is an antiapoptotic member of the Bcl-2 family, which when overexpressed prevents the activation of the mitochondrial apoptotic pathway.28 Bcl-2 overexpression is casual factor of follicular lymphoma,29 and cancer cells that become resistant to chemotherapy usually overexpress antiapoptotic proteins from the Bcl-2 family.30 When soluble rApo2L/TRAIL was incubated at increasing doses with Jurkatderived sublines overexpressing the antiapoptotic proteins CrmA or Bcl-xL, both cell mutants showed complete resistance to apoptosis induced by this death ligand (Figure 1B,C).

However, whereas Jurkat-CrmA cells were still relatively resistant to LUVs-Apo2L/TRAIL, Jurkat-Bcl-xL cells became sensitive to LUVs-Apo2L/TRAIL, this increase in sensitivity being statistically significant from 100 ng/mL. We previously generated a Jurkat-derived cell subline with the proapoptotic member of the Bcl-2 family Bak silenced (Jurkat-shBak).17,23,24 Since Jurkat cells are naturally deficient in the expression of the proapoptotic protein Bax, Jurkat-shBak cells are double Bax and Bak mutants and are highly resistant to intrinsic apoptosis. As expected, Jurkat-shBak cells were completely resistant to soluble rApo2L/TRAIL. However, LUVs-Apo2L/TRAIL were capable of overcoming this resistance, leading to an induction of apoptosis of about 55% at a dose of 1,000 ng/mL (Figure 1D). To verify that the cell death observed in Jurkat cells and mutants was due indeed to the action of rApo2L/TRAIL, the cells were preincubated with the neutralizing anti-TRAIL antibody RIK2. In all experiments, RIK2 completely abrogated apoptosis induced by either soluble rApo2L/TRAIL or LUVs-Apo2L/TRAIL confirming the specificity of the interaction (Figure 1F−H). In this line, preincubation with a DR5-Fc chimera also prevented apoptosis induced by both formulations of rApo2L/TRAIL (soluble and associated with liposomes) indicating that response to LUVsApo2/TRAIL is dependent on DR5 (Supplemental Figure 2 in the Supporting Information). The distinct sensitivity to rApo2L/TRAIL found in Jurkat cells versus its derived sublines was not due to differences in plasma membrane expression of death receptors. As shown in Figure 2A, the expression of DR4 was undetectable in all cells 897



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Figure 4. Caspase activation in Jurkat cells and Jurkat sublines. For Jurkat, cells were untreated (control), treated with LUVs alone, or treated with 50 ng/mL of rApo2L/TRAIL (either soluble or associated with LUVs) for 24 h. For the sublines Jurkat-CrmA, Jurkat-Bcl-xL, and Jurkat-shBak, 1,000 ng/mL of rApo2L/TRAIL was used. Furthermore, the same doses of soluble rApo2L/TRAIL and LUVs-Apo2L/TRAIL were used over cells preincubated with RIK2 (500 ng/mL), Z-VAD-fmk (30 μM), Z-IETD-fmk (30 μM), and necrostatin-1 (25 μM), as indicated. After 24 h, cells were lysed for Western blot analysis or labeled with annexin-V and analyzed by flow cytometry. (A) Cells corresponding to the first four points of each experiment were counted and lysed (106 cells), and cytosolic fractions were separated by SDS−PAGE. Levels of pro-caspase-8, cleaved caspase-8, pro-caspase-3, and cleaved caspase-3 in cell extracts were analyzed by Western blot using specific antibodies. β-Actin levels were determined in the same blots as a control for equal protein loading. Protein localization and molecular weights are indicated with arrows. The blot shown is representative of three independent experiments. (B) Representative annexin-V histograms of one of at least three experiments analyzed by flow cytometry. (C) Bar plot showing annexin-V data means ± SD of at least three experiments analyzed by flow cytometry.

TRAIL receptors was analyzed by flow cytometry and shows no difference with Jurkat cells (Supplemental Figure 3C in the Supporting Information). Thus, the resistance to soluble rApo2L/TRAIL-induced apoptosis exhibited by Jurkat-CrmA, Jurkat-Bcl-xL, and Jurkat-shBak was specifically due to the differential expression of antiapoptotic (CrmA or Bcl-xL) or proapoptotic (Bak) proteins rather than to unspecific effects elicited by the plasmid vectors. Caspase Activation by LUVs-Apo2L/TRAIL. After assessing the increased cytotoxicity of LUVs-Apo2L/TRAIL compared with soluble rApo2L/TRAIL in Jurkat cells and mutants, we next analyzed the activation of the main caspases implicated in the extrinsic apoptotic pathway. Activation of initiator caspase-8 and effector caspase-3 was performed with analysis by Western blot analysis of Jurkat cells and Jurkatderived sublines. The concentration of soluble rApo2L/TRAIL and LUVs-Apo2L/TRAIL used for each cell type was LC50

and the expression level of DR5 was similar in parental Jurkat cells and in its derived sublines. Expression of decoy receptors, DcR1 and DcR2, was also undetectable in all cases (Figure 2). Assessment of the increased resistance of apoptosis of Jurkatderived cell lines was performed by carrying out specific cytotoxic assays. Overexpression of Bcl-xL or silencing of Bak conferred complete protection against doxorubicin, a cytotoxic drug currently used in cancer chemotherapy, which induces apoptosis through the mitochondrial pathway (Figure 3C,D). On the other hand, expression of CrmA abrogated apoptosis induced by the anti-Fas mAb CH11, demonstrating an effective caspase-8 inhibition (Figure 3A,B). Both Jurkat-Neo and Jurkat-pHTLV sublines, used as control of Jurkat transfectants, showed similar behavior with respect to soluble rApo2L/TRAIL or associated with liposomes as parental Jurkat cells (Supplemental Figure 3A,B in the Supporting Information). Furthermore, expression of Apo2L/ 898



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Figure 5. Analysis of early and late apoptosis and caspase-3 activation in time-course assays. For Jurkat cells and mutants, cells were untreated (control) or treated with 1,000 ng/mL of soluble rApo2L/TRAIL or with LUVs-Apo2L/TRAIL. Cells were collected at different times in duplicate; for each time, one sample was labeled with annexin-V and the other one was probed to check caspase-3 activation by flow cytometry. Annexin-V data were presented as bars (left panels), and caspase-3 activation as line diagrams (right panels). The experiment shown is representative of three different experiments. (A) Jurkat; (B) Jurkat-CrmA; (C) Jurkat-Bcl-xL; (D) Jurkat-shBak.

processing of pro-caspase-8 in Jurkat cells (Figure 4A). Similarly, only when cells were treated with LUVs-Apo2L/ TRAIL, high levels of processed caspase-3, the main effector caspase, were detected (Figure 4A). Similar results were observed in Jurkat mutants. In all cases, soluble rApo2L/TRAIL barely induced apoptosis, while an equivalent amount of this same amount of protein linked to liposomes clearly overcame the resistance offered by overexpression of Bcl-xL or by silencing of Bak and partially that provided by CrmA (Figure 4B,C). In all cases, cell death was inhibited when cells were preincubated with RIK2 antibody or the caspase inhibitors Z-VAD-fmk or Z-IETD-fmk (Figure 4B,C) but not with the necroptosis inhibitor necrostatin-1. Furthermore, the processing pattern of caspase-8 and caspase-3 in these resistant mutants as analyzed by Western blot was similar to that observed for Jurkat cells. Only in cells treated with LUVs-Apo2L/TRAIL, activation of pro-caspase-8 and procaspase-3 and the consequent appearance of the processed fragments were detected (Figure 4A).

observed in the corresponding dose−response curves previously obtained. Since in Jurkat-CrmA LC50 was higher than 1 μg/mL, we used the highest dose of Apo2L/TRAIL tested in the study. Therefore, 50 ng/mL was used for Jurkat, and 1,000 ng/mL for Jurkat-CrmA, Jurkat-Bcl-xL, and Jurkat-shBak cells. Jurkat cells incubated with rApo2L/TRAIL in soluble form showed low toxicity (barely 20% of annexin-V-positive cells), but this greatly increased when rApo2L/TRAIL was bound to liposomes (about 70% of annexin-V positive cells, Figure 4C). Cell death induced by either soluble rApo2L/TRAIL or LUVsApo2L/TRAIL was inhibited by the neutralizing mAb RIK-2, by the general caspase inhibitor Z-VAD-fmk, and by the caspase-8 inhibitor Z-IETD-fmk (Figure 4B,C). These data strongly suggest that cell death induced by LUVs-Apo2L/ TRAIL followed the canonical death receptor-induced, caspasedependent apoptotic pathway. According to this, the necroptosis inhibitor necrostatin-1 had no effect on cell death of Jurkat cells and Jurkat-derived mutants incubated with either soluble rApo2L/TRAIL or LUVs-Apo2L/TRAIL (Figure 4B,C). Treatment with LUVs-Apo2L/TRAIL caused a clear 899



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Figure 6. Cytotoxicity assays on other human hematologic tumor cell lines. Cells (3 × 105 cells/mL) were incubated in the presence or absence of different doses of soluble rApo2L/TRAIL and LUVs-Apo2L/TRAIL for 24 h. Graphics show the percentage of annexin-V positive cells analyzed by flow cytometry. The results shown are the mean ± SD of at least three experiments. (A) U937; (B) U937-Bcl-2; (C) U266; (D) MM.1S. Apoptosis inhibition by blocking antibody RIK2. Bar diagrams show the mean ± SD of annexin-V positive cells in the controls (control and LUVs alone), in cells treated with 500 ng/mL of soluble rApo2L/TRAIL or LUVs-Apo2L/TRAIL, and in cells treated with 500 ng/mL of soluble rApo2L/TRAIL or LUVs-Apo2L/TRAIL previously preincubated with a concentration of 500 ng/mL of the anti-TRAIL blocking mAb RIK2. Data are the mean of at least three different assays. (E) U937; (F) U937-Bcl-2; (G) U266; (H) MM.1S.

We also studied the kinetics of cell death induced by LUVsApo2L/TRAIL in comparison with the soluble form of this protein. Jurkat and Jurkat-derived cell lines were incubated with 1,000 ng/mL rApo2L/TRAIL for different times and phosphatidylserine exposure and caspase-3 activation analyzed at each time by flow cytometry. As shown in Figure 5, at early time points, both molecular forms of Apo2L/TRAIL (soluble rApo2L/TRAIL and LUVs-Apo2L/TRAIL) similarly induced a low amount of caspase-3 activation and apoptosis on Jurkat cells. However, at longer incubation times (15 and 24 h), LUVs-Apo2L/TRAIL induced a considerable amount of cell death, concomitant with caspase-3 activation, on Jurkat, JurkatBcl-xL, and Jurkat-shBak, while soluble rApo2L/TRAIL did not

induce a significant apoptosis on Jurkat-CrmA, Jurkat-Bcl-xL, and Jurkat-shBak cells. Soluble rApo2L/TRAIL did not induce further caspase-3 activation and apoptosis on Jurkat cells at longer incubation times than that observed at short times (Figure 5A−C). LUVs-Apo2L/TRAIL Are Effective against Several Human Hematologic Tumor Cell Lines. Finally, we have expanded our study to other human tumor cell lines derived from other hematologic malignancies. We have tested the effectiveness of LUVs-Apo2L/TRAIL on the tumor cell line U937 (derived from histiocytic lymphoma), U937-Bcl2 (cell subline overexpressing the antiapoptotic protein Bcl-2), MM.1S, and U266 (both derived from multiple myeloma). 900



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As previously shown for Jurkat cells and sublines, LUVsApo2L/TRAIL were also capable of inducing apoptosis more efficiently than soluble rApo2L/TRAIL in U937 and MM.1S cells with LC50 reduction of 4- and 15-fold respectively, and this effect was specifically induced by rApo2L/TRAIL (Figure 6A,D). In addition, LUVs-Apo2L/TRAIL also overcome the resistance to soluble rApo2L/TRAIL from U937-Bcl-2 and U266 cells (Figure 6B,C). LUVs-Apo2L/TRAIL Are Not Cytotoxic against Normal Human T Lymphocytes. We also analyzed the effect of soluble rApo2L/TRAIL and LUVs-Apo2L/TRAIL on freshly isolated peripheral blood lymphocytes (PBL) and on day-6 T cell blasts. Neither soluble rApo2L/TRAIL nor LUVs-Apo2L/ TRAIL induced cell death on PBLs after 24 h of treatment (Figure 7A). None of the Apo2L/TRAIL molecular forms were

Article

DISCUSSION

Apo2L/TRAIL is a member of the TNF cytokine superfamily which can induce apoptosis of a wide variety of human tumor cells, usually not affecting normal cells.8 Current therapeutic strategies based on Apo2L/TRAIL are based on the delivery of either agonistic antibodies or rApo2L/ TRAIL in soluble form. However, as we previously demonstrated, bioactive Apo2L/TRAIL is physiologically secreted by human immune cells as a membrane protein in lipid microvesicles or exosomes.20,21 From this observation, and trying to mimic the physiological secreted form, this death ligand, we expressed and purified a His-tagged version of rApo2L/TRAIL that was linked to the surface of large unilamellar vesicles (LUVs) resembling size and lipid composition of natural exosomes. For this purpose, a chelating lipid consisting of DOGS-NTA-Ni molecule was included in the composition of LUVs to tether rApo2L/TRAIL-His10 by the formation of a coordination complex between the histidinetag and the Ni2+ cation. This system, previously used to artificially associate members of the Bcl-2 family with liposomes,32 was extremely efficient, leading to a binding of about 95% of rApo2L/TRAIL to liposomes. Moreover, rApo2L/TRAIL associated with LUVs through the interaction of its His-tag with Ni from DOGS-NTA remained fully bioactive. On the other hand, binding of rApo2L/TRAIL did not alter the size distribution of the liposome population as shown by DLS and SEM assays. After validating the novel DOGS-NTA-Ni containing liposomes decorated with bioactive rApo2L/TRAIL on their surface, we have compared the death-inducing efficiency of LUVs-Apo2L/TRAIL versus soluble rApo2L/TRAIL by using Jurkat leukemia cells and a panel of Jurkat-derived mutants. In all conditions tested, LUVs-Apo2L/TRAIL induced apoptosis in a more efficient manner than soluble rApo2L/ TRAIL. Treatment of Jurkat cells with LUVs-Apo2L/TRAIL considerably reduced LC50, by at least 14-fold. This observation could be of potential clinical relevance since the reduction in the effective dose of rApo2L/TRAIL also reduces the possibility of hypothetical adverse effects. More interestingly, LUVs-Apo2L/TRAIL were able to overcome the resistance to soluble rApo2L/TRAIL of some Jurkat-derived cell lines with enhanced resistance to apoptosis. LUVs-Apo2L/TRAIL induced apoptosis in Jurkat sublines resistant to the cytotoxic action of soluble rApo2L/TRAIL, such as Jurkat-Bcl-xL and Jurkat-shBak. This observation could also have clinical relevance, since a frequent mechanism of tumor escape from the immune system or from chemotherapy is the increase of the expression of antiapoptotic proteins.33−35 In fact, the expression levels of antiapoptotic proteins of the Bcl-2 family, such as Bcl-2 and Bcl-xL, are considered to be reliable prognosis markers in several tumors, such as colorectal cancer,36 adult acute myeloid leukemia,37 hepatocellular carcinoma,38 nonsmall cell lung cancer,39 and follicular lymphoma.29 A previous work from our group suggested that the increased bioactivity of tethered rApo2L/TRAIL was not due to differential conformational changes when this protein was associated with liposomes.22 A more probable explanation is that the increased bioactivity be due to the increase of the local concentration of rApo2L/TRAIL bound to liposomes thus interacting with improved efficiency with its cognate receptors on target cells and leading to a more efficient receptor crosslinking. In fact, as shown in Supplemental Figure 4 in the

Figure 7. Effect of rApo2L/TRAIL and LUVs-Apo2L/TRAIL on normal human T lymphocytes. (A) Freshly isolated PBMC were incubated at 2 × 106 cells/mL with control medium (control) or LUVs alone, 500 ng/mL soluble rApo2L/TRAIL, 1,000 ng/mL soluble rApo2L/TRAIL, 500 ng/nL of LUVs-Apo2L/TRAIL, or 1,000 ng/mL LUVs-Apo2L/TRAIL during 24 h. Day-6 T cell blasts were incubated at 2 × 106 cells/mL with control medium (control) or LUVs alone, or with the same doses of soluble rApo2L/TRAIL or LUVs-Apo2L/ TRAIL above indicated during 24 h with 30 UI/mL IL-2 as indicated. Cytotoxicity was evaluated by annexin-V staining on CD4+ (B) or CD8+ T subsets (C). Results are the average ± SD of experiments performed in cells from three different donors.

cytotoxic either for CD4+ (Figure 7B) or for CD8+ T blasts (Figure 7C). These results agree with previous data indicating that soluble rApo2L/TRAIL did not induce a significant cell death on T cells, and we demonstrate here that association of Apo2L/TRAIL with liposomes did not cause cytotoxic effects on CD4+ or CD8+ T subsets.26,31 In this line, we also carried out in vivo experiments using nude mice in order to analyze the toxicity of LUVs-Apo2L/TRAIL when injected systematically and no toxicity was observed in different tissues such as liver, kidney, spleen, and testicles. In addition, no hepatotoxicity was observed in these in vivo experiments since there was not increase in transaminase (ALT and AST) levels in the sera obtained from these mice (data not shown). All these results, taken together, indicate that LUVs-Apo2L/TRAIL show an increased cytotoxicity for leukemia cells while being harmless for normal cells. 901



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Supporting Information, the same amount of rApo2L/TRAIL (5 μg in 0.5 mL) gives a concentration of 0.454 μM when administered as soluble protein, whereas its concentration is 170.7 μM when associated with liposomes (an increase of around 375-fold). Apo2L/TRAIL needs to be at least a trimer to induce oligomerization of its receptors. The oligomerization of DR4 and/or DR5 is necessary to induce the correct formation of death-inducing signaling complex (DISC), and consequently to trigger apoptosis. In the case of Fas/CD95, it was demonstrated that receptors preassociate on the membrane of target cells through their extracellular domains and that this preassociation was required for correct apoptosis induction.40 A similar situation was reported later for Apo2L/TRAIL receptors.41 This indicates that efficient physiological death ligand-induced apoptosis needs multimerization of preassociated receptors.42 This efficient apoptotic signal transduction is also controlled at the intracellular level, as it has been demonstrated that ubiquitination controls the increase in local caspase-8 concentration after rApo2L/TRAIL receptor ligation, and that this local accumulation is needed for proper apoptotic signaling.43 The formation of high-order multimerized receptors and the increased amount of processed caspase-8 in a small area could overcome the protection given by overexpression of antiapoptotic or deletion of proapoptotic Bcl-2 family proteins acting in the mitochondrial apoptotic pathway (Supplemental Figure 5 in the Supporting Information). As previously shown, Jurkat are type II cells for rApo2L/ TRAIL-induced apoptosis and need the mitochondrial pathway to induce efficient caspase-3 activation, since Jurkat-Bcl-xL and Jurkat-shBak are completely resistant to soluble rApo2L/ TRAIL. However, our data indicate that LUVs-Apo2L/ TRAIL shift the response to a type I-like and are able to induce apoptosis independently of the mitochondrial apoptotic pathway. In this connection, it was previously reported that a stabilized rApo2L/TRAIL trimer (leucine-zipper TRAIL), could induce apoptosis on Jurkat cells overexpressing Bcl-2 or Bcl-xL.44 Hence, bioactive versions of rApo2L/TRAIL displaying increased cross-linking efficiency will probably improve the clinical outcome of treatments based on this molecule.8 Finally, the cytotoxic effect exhibited by LUVs-Apo2L/ TRAIL was greater than that exhibited by soluble rApo2L/ TRAIL not only in Jurkat cells and mutants but also in other human tumor cells derived from other hematologic malignancies such as histiocytic lymphoma (U937, U937-Bcl-2) and multiple myeloma (MM.1S and U266). The use of liposomes in the treatment of diseases has been widely described.45−47 However, their use has been always focused as vehicle for the transport of drugs inside target cells or to prevent drug degradation. Other therapeutic approaches in order to increase rApo2L/TRAIL bioactivity have been recently described.48,49 However, to our knowledge this is the first report on the use of liposomes to increase the bioactivity of a death ligand against tumor cells. Attachment of rApo2L/ TRAIL to the LUVs surface increases its bioactivity, not only making these liposomes a simple drug vehicle but also becoming a novel antitumor therapeutic strategy that could be used to replace current formulation of rApo2L/TRAIL being tested in clinic.

Article

ASSOCIATED CONTENT

S Supporting Information *

Figures depicting preparation of lipid vesicles decorated with rApo2L/TRAIL, apoptosis inhibition by a DR5-Fc chimera, cytotoxicity assays on Jurkat and vector cells, estimation of liposome and tethered rApo2L/TRAIL concentrations, and schematic representation of the proposed model for the increase of bioactivity of LUVs-Apo2L/TRAIL. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

́ *Departamento de Bioquimica, Biologiá Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, C/Pedro Cerbuna 12, Zaragoza, 50009 Spain. Phone: 34 976 76 23 01; 34 976 12 79. Fax: 34 976 76 21 23. E-mail: lumartin@ unizar.es; [email protected]. Author Contributions #

These authors share senior authorship.

Notes

The authors declare the following competing financial interest(s): Gorka Basáñez, Luis Larrad, Javier Naval, Alberto Anel, and Luis Martinez-Lostao have filed a patent application (W02011020933) for the use of liposome-bound Apo2L/ TRAIL.

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ACKNOWLEDGMENTS We gratefully acknowledge Dr. Avi Ashkenazi (Genentech) for providing reagents and for support throughout the years. We are also indebted to Ignacio Tacchini (Instituto de Carboqui-́ mica, CSIC) for performing SEM. Finally, we gratefully acknowledge Dr. Victor Yuste for providing Jurkat-Bcl-xL. This work was supported by Grants SAF2010-15341 (AA), ISCIII-RTICC RD06/0020 (JN), SAF2008-02139 (JP) and SAF2011-25390 (JP) from Ministerio de Ciencia e Innovación (Spain). L.M.-L. was supported by a Sara Borrell Postdoctoral Contract (CD05/00082) from Instituto de Salud Carlos III (Spain). J.P. was supported by Fundación Aragón I+D (ARAID). D.D.M. and D.S. were supported by predoctoral fellowships from Gobierno de Aragón.

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REFERENCES

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TRAIL Overcome Chemoresistance

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