Leukemia & Lymphoma, 2015; Early Online: 1–6 © 2015 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2014.981179
ORIGINAL ARTICLE: RESEARCH
Ceramide participates in lysosome-mediated cell death induced by type II anti-CD20 monoclonal antibodies Yuzhi Liu1, Ling Shu2 & Jingjing Wu3 1Department of Otolaryngology, Tianjin Medical University General Hospital, Tianjin, China, 2Department of Oncology,
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Yancheng City No. 1 People’s Hospital affiliated to Nantong Medical Collage, Yancheng, China and 3Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
issue is enhanced understanding of the lymphoma-killing mechanisms of anti-CD20 mAbs. Currently, anti-CD20 mAbs can be generally classified into two types, with distinct tumor killing mechanisms. Type I (rituximab-like) anti-CD20 mAbs can redistribute CD20 into membrane lipid rafts and potently mediate complement-dependent cytotoxicity (CDC), whereas type II (tositumomab-like) mAbs are less effective in activating complement, but are more potent in inducing programmed cell death (PCD) [2,11]. Nevertheless, both types can efficiently mediate antibody-dependent cell-mediated cytotoxicity (ADCC) [2,11]. Increasing evidence has revealed that the limitations of rituximab can be mostly ascribed to the exhaustion and/or disability of complement and Fc-γ-receptor-expressing effector cells such as macrophages and natural killer (NK) cells [12–15]. Otherwise, some in vitro studies have demonstrated that antigenic modulation might be another major limitation of rituximab-based immunotherapy [16]. As a result, increasingly studies are devoted to developing novel mAbs with potent PCD-inducing ability while reducing the antigen modulation after antibody binding [6,8,17–19]. Morschhauser et al. developed a novel humanized typed II anti-CD20 mAb (GA101), which can directly induce superior PCD and enhanced ADCC in malignant B cells and showed excellent anti-lymphoma efficacy in phase I/II clinical trials [6,20,21]. In addition, in vitro studies have demonstrated that unlike rituximab, type II mAbs elicit little modulation of CD20 from the cell surface, the observation of which has strengthened the case for focusing on type II mAbs in pre-clinical and clinical studies [16]. Ivanov et al. first reported that a type II mAb (tositumomab) can elicit strong homotypic adhesion (HA), followed by a swelling and/or collapse of the lysosomal compartment in human lymphoma and leukemia cells [22]. However, no detailed pathways concerning the relationships between mAb-evoked HA and specific lysosome leakage have been reported to date.
Abstract Considering the variable and often modest therapeutic efficacy of rituximab for a substantial proportion of patients suffering from non-Hodgkin lymphomas (NHLs), various type II anti-CD20 monoclonal antibodies (mAbs) with excellent ability in inducing programmed cell death (PCD) are currently being developed for their enhanced therapeutic index. Although homotypic adhesion (HA) and lysosome leakage are proven to be of vital importance in type II mAb-induced PCD in NHL cells, the detailed relationship between them remains unclear. Herein, for the first time we discovered that improved intracellular ceramide level is an important mediator between HA and lysosome leakage in tositumomab-induced cell death. Further experimental results revealed that the generation of intracellular ceramide acts as the outcome of HA and major cause of lysosome leakage. The clarification of ceramide involvement in type II anti-CD20 mAb-induced PCD may provide new ideas on CD20-based immunotherapy against NHLs. Keywords: Non-Hodgkin lymphoma, CD20, programmed cell death, ceramide, homotypic adhesion, lysosome leakage
Introduction Although clinical applications have proved the unprecedented success of rituximab in treating a range of CD20positive non-Hodgkin lymphomas (NHLs), a substantial proportion of patients fail to achieve a complete remission (CR) [1,2]. Moreover, tumor relapse is inevitable for most patients [3,4]. The limitations of rituximab call for the development of improved treatments for these patients. Given the fact that CD20 is one of the most ideal therapeutic targets of NHL due to its high and relatively specific expression on the surface of malignant B cells [3,5], recently various therapeutic approaches have been devoted to exploiting novel antiCD20 monoclonal antibodies (mAbs) aimed at improving therapeutic efficacy [5–10]. To this end, the most important
Correspondence: Jingjing Wu, Department of Hematology, Institute of Clinical Medicine, the First Affiliated Hospital of Zhengzhou University, 1st East Jianshe Road, Zhengzhou, Henan Province, China, 450052. Tel: 0371-66295562. Fax: 0371-66295562. E-mail: [email protected] Received 1 July 2014; Revised 14 October 2014; accepted 19 October 2014
1
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2 Y. Liu et al. Previous studies revealed that ceramide plays an important role in PCD induced by a variety of chemotherapeutic drugs and cytokines (such as tumor necrosis factor-α and interleukin-1β, etc.) [23–28]. It is worth mentioning that sphingolipids are important constituents of cell membranes, which can either serve as substrates for or be produced by enzymes activated in response to cellular stress. Ceramide can be generated during sphingomyelin catabolism by various sphingomyelinases during the responses to these stresses. Ceramide acts as a second messenger in the sphingomyelin signaling pathway, which has been identified as an important mediator in cell differentiation, survival and apoptosis [23,29,30]. Further, ceramide can be produced by de novo synthesis, responding to similar agents identified for the activation of sphingomyelinases [28,29,31,32]. In this study, we encouragingly found that ceramide also takes part in lysosome-mediated PCD induced by type II anti-CD20 mAbs. In addition, the relationships among mAbmediated HA, lysosome leakage and ceramide generation were comprehensively investigated. A thorough understanding of type II anti-CD20 mAb-induced PCD may provide the basis for developing more effective methods for curing NHLs.
Materials and methods Cell lines and reagents A human B cell lymphoma cell line, Raji, was obtained from the American Type Culture Collection (ATCC). Cells were maintained in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO). Rituximab (C2B8) was purchased from Roche, and tositumomab (B1, trade name: Bexxar) was obtained from GlaxoSmithKline (GSK, Brentford, UK). C2-ceramide (N-acetylsphingosine) and fumonisin Bl (FB1) were purchased from Sigma-Aldrich.
was dissolved in chloroform (100 μL). The solution was reacted at ⫺ 20°C for 3 h with 10 μL of 100 mM (⫹)-6-methoxyα-methyl-2 naphthaleneacetic acid (NAP; Sigma-Aldrich, St Louis, MO), 100 mM N,N’-dicyclohexylcarbodiimide (DCC; Sigma-Aldrich) and 100 mM 4-dimethylaminopyridine (DMAP, 4-dimethylaminopyridine; Sigma-Aldrich). After evaporation, the residue was suspended in chloroform (15 μL), followed by mixing with 2 mL of hexane and centrifugation (800g ⫻ 10 min). The supernatant was vigorously mixed with 5 mL of methanol/water (v/v ⫽ 4:1). After centrifugation, the upper phase was collected and filtered using a 0.45 μm membrane filter (Millipore, Massachusetts, MN). A 20 μL portion of the filtrate was injected into an Econosphere CN 5U column (4.6 ⫻ 250 mm; Alltech, Chicago, IL). The derivatized ceramide was separated from by-products with 3% 2-propanol in n-hexane as the mobile phase with a flow rate of 2.0 mL/min. The effluent was monitored fluorometrically at a wavelength of 230/352 nm (excitation/emission).
Cathepsin B release assessment Released cathepsin B in cytoplasm was detected by confocal microscopy following the method of Ivanov et al. [22]. Briefly, harvested cells were placed onto poly-d-lysine (Sigma-Aldrich) coated sterilized microscope slides, fixed in 4% paraformaldehyde and permeabilized by 0.5% Triton X-100. Then each sample was incubated with 5 μg/mL cathepsin B mouse mAb (Invitrogen) at 4°C overnight and stained by Alexa Fluor-555 Rabbit Anti-Mouse imunoglobulin G (IgG) (H ⫹ L) secondary antibodies (Invitrogen). After washing, samples were observed using a confocal microscope (Zeiss, Jena, Germany).
Lysosomal permeability assessment Harvested cells were labeled with 75 nM LysoTracker Red DND (Invitrogen) at 37°C for 30 min and assessed by FCM and confocal microscopy. Unlabeled cells were used as a background control [22].
Annexin V/propidium iodide assays Raji cells that experienced PCD were assessed by flow cytometry (FCM). Briefly, cells were incubated with 10 μg/ mL rituximab, tositumomab or different concentrations of C2-ceramide for 12 h. After washing with phosphate buffered saline (PBS), cells were stained with Alexa Fluor-488 annexin V and propidium iodide (PI) (Invitrogen, Carlsbad, CA) and analyzed by FCM (FC500; Beckman Coulter, Brea, CA). For PCD inhibition assays, a caspase inhibitor (Z-VAD-FMK; Promega) or FB1 at different concentrations was added 1 h prior to the addition of mAb.
Determination of cellular ceramide levels The content of cellular ceramide was determined by high performance liquid chromatography (HPLC) following the method of Soeda et al. [30]. Briefly, cells were washed, suspended in 200 μL of 0.25 M sucrose in PBS and disrupted by sonication. After centrifugation (800g ⫻ 10 min), the cell lysate pellet was washed, resuspended in 200 μL of PBS and mixed with 4.0 mL of chloroform/methanol (2:1) for 30 min. Subsequently the sample was added to 1 mL of Mini-Q water, vortexed and centrifuged. The lower phase was collected and evaporated to dryness under a nitrogen stream. The residue
Homotypic adhesion measurement Cells were incubated with 10 μg/mL tositumomab or different concentrations of ceramide for 4 h and cell morphology was observed by light microscopy. For HA inhibition assessment, FB1 was added before the addition of tositumomab for HA inhibition assays.
Statistical analysis Statistical analysis involving two groups was performed by Student’s unpaired t-test, whereas analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was used for comparison among three or more groups. All data were processed with SPSS 10.0 software. p ⬍ 0.05 was considered statistically significant in all calculations.
Results Type II but not type I anti-CD20 mAbs can induce caspase-independent cell death in NHLs To determine the PCD-inducing ability of anti-CD20 mAbs, Raji cells were respectively incubated with 10 μg/mL tositumomab or rituximab, and analyzed by FCM. Figure 1(a)
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Ceramide- and tositumomab-evoked PCD 3
Figure 1. Tositumomab can induce caspase-independent cell death in Raji cells. (a) Raji cells were incubated with 10 μg/mL rituximab or tositumomab for 12 h and cell death was analyzed using annexin V/PI assay. Not treated (NT) cells were used as a negative control. (b) Z-VAD-FMK (a caspase inhibitor) over a range of 0–30 μM was incubated with Raji cells 1 h before the addition of tositumomab and rituximab. Cell death was analyzed after 12 h using annexin V/PI assay. Data are expressed as mean ⫾ SD (n ⫽ 3), **p ⬍ 0.01.
demonstrates that tositumomab could evoke significantly stronger PCD (28.32⫾4.16%) compared with no treatment (NT, 8.62 ⫾ 1.45%, p ⫽ 0.000), while rituximab only weakly induced PCD in CD20 positive Raji cells (10.62 ⫾ 1.64%). In order to investigate the involvement of caspase activation in anti-CD20 mAb-induced PCD, a cell-permeable caspase inhibitor, Z-VAD-FMK, was employed in PCD inhibition assays. Figure 1(b) indicates that Z-VAD-FMK was unable to prevent both tositumomab and rituximab-induced PCD up to 30 μM. We can conclude from these results that a type II (tositumomab) but not type I (rituximab) anti-CD20 mAb can evoke remarkable PCD in NHL cells in a caspase-independent manner.
Tositumomab-induced cell death correlates with enhanced generation of intracellular ceramide Given that ceramide plays an important role in PCD induced by a variety of chemotherapeutic drugs and cytokines [23–28], we subsequently investigated the correlation between tositumomab-induced cell death and ceramide generation in Raji cells. To evaluate the effect of anti-CD20 mAbs on intracellular ceramide generation, Raji cells were respectively incubated with 10 μg/mL tositumomab or rituximab for 12 h. The intracellular ceramide level was determined by HPLC. As shown in Figure 2(a), tositumomab but not rituximab could significantly enhance the intracellular ceramide to approximately 189.1 ⫾ 12.96 ng/105 cells, which was about 1.95-fold more than that in normal cells (p ⫽ 0.000). Moreover, ceramide over a range of concentrations from 5 to 40 μM could evoke obvious PCD in Raji cells in a dose-dependent manner [Figure 2(b)]. Based on the abovementioned results, we assumed that tositumomab-induced PCD might be correlated with intracellular ceramide generation. To verify this hypothesis, we determined tositumomab-induced PCD in the absence or presence of FB1, which acts as an inhibitor of dihydroceramide synthase (a key enzyme regulating the conversion of sphinganine to dihydroceramides in the synthesis of cellular ceramide) [29,33]. Figure 2(c) demonstrates that the increased ceramide levels evoked by tositumomab could
Figure 2. The involvement of ceramide in tositumomab-induced programmed cell death (PCD). (a) Raji cells were incubated with 10 μg/mL tositumomab and rituximab for 12 h and intracellular ceramide levels were determined by high performance liquid chromatography (HPLC). Not treated (NT) cells were used as a background control. (b) Raji cells were incubated with different concentrations of ceramide for 12 h and cell death was analyzed using annexin V/PI assay. (c, d) Fumonisin Bl (FB1) over a range of 0–40 nM was incubated with Raji cells for 1 h before the addition of tositumomab. After 12 h, intracellular ceramide levels were determined by HPLC (c) and cell death was analyzed using annexin V/PI assay (d). Data are expressed as mean ⫾ SD (n ⫽ 3), **p ⬍ 0.01.
be significantly reduced by FB1 in a dose-dependent manner. Approximately 85% of the enhancement was decreased at the concentration of 40 nM [Figure 2(c)]. Furthermore, tositumomab-mediated PCD could also be significantly prevented by FB1 in a dose-dependent manner [Figure 2(d)]. All these results support our hypothesis that the ceramide pathway is a major driving force in the signaling of tositumomabmediated PCD.
Increase of intracellular ceramide content acts as outcome but not cause of HA evoked by tositumomab Previous studies demonstrated that type II anti-CD20 mAbmediated cell death is associated with HA of target cells and is executed by lysosomes which disperse their contents into the cytoplasm and the surrounding environment [6,22]. In order to identify the intrinsic relationship between HA, lysosome leakage and ceramide generation, we designed further experiments to investigate whether FB1 can negatively influence tositumomab-evoked HA and lysosome alteration. In HA inhibition analysis, 40 nM FB1 was incubated with Raji cells for 1 h before the addition of tositumomab, and cell morphology was observed by low-magnification light microscopy 4 h after treatment. In keeping with previous publications, Raji cells underwent more pronounced HA following treatment with tositumomab compared with rituximab, which could not be disrupted by FB1 (Figure 3). Furthermore, compared with untreated Raji cells, no obvious HA was observed after treatment with ceramide up to 40 μM (Figure 3), at which concentration considerable PCD could
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4 Y. Liu et al.
Figure 3. The effect of ceramide on homotypic adhesion (HA) induced by tositumomab. Raji cells were incubated with rituximab (10 μg/mL), tositumomab (10 μg/mL) or C2-ceramide (20-40 μM) for 4 h and cell morphology was observed by light microscopy. For HA inhibition assays, FB1 at different concentrations was incubated for 1 h before the addition of tositumomab. Magnification: 20 ⫻ 20.
be successfully evoked [Figure 2(B)]. Considering the observation that FB1 could significantly inhibit the generation of ceramide but not the formation of HA, we can deduce that the increased intracellular ceramide level is the outcome of rather than the reason for tositumomab-evoked HA.
Increase of intracellular ceramide content acts as reason for but not result of lysosome leakage evoked by tositumomab It has been reported that enlargement of the lysosomal compartment and subsequent lysosome membrane
permeabilization play a vital role in type II anti-CD20 mAbinduced PCD [6,22]. Hence, after determining the causal relationship between tositumomab-induced HA and intracellular ceramide generation, we subsequently focused on investigating whether ceramide generation operates upstream or downstream of lysosome leakage. For this purpose, a lysosome tracker was employed to label the lysosome compartment. As shown in Figure 4(a), FCM results revealed that the distribution of LysoTracker fluorescence (FL-2, Red) in Raji cells experienced a visible increase and decrease after treatment with tositumomab (green histograms in left panel)
Figure 4. Effect of ceramide on lysosome leakage induced by Tositumomab. (a, b) Raji cells were incubated with tositumomab with the absence/ presence of FB1 or treated with ceramide at different concentrations for 12 h. After labeling with LysoTracker, total lysosomal volume in cells was detected using FCM (a) and confocal microscopy (b). (c) Confocal microscopy of cathepsin B staining (red). DNA was counterstained with DAPI (blue).
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Ceramide- and tositumomab-evoked PCD 5 compared with normal cells (red histograms). This typical and obvious alteration was clearly explained by confocal microscopy. Figure 4(b) reveals that the cellular lysosome was labeled as a relatively small and confined organelle in normal cells, while after treatment with tositumomab, an enlargement of red fluorescence-labeled compartments and diffusion of red fluorescence in the cytoplasm was detected. The above results, along with previous publications, demonstrated that Raji cells were successively undergoing a swelling of lysosomes (enlargement of red fluorescence-labeled compartments) and collapse of the lysosomal compartment (diffusion of red fluorescence in the cytoplasm) after treatment with tositumomab. To our great interest, this distinct alteration could be significantly and effectively prevented by FB1 [blue histograms in left panel of Figure 4(a)]. Furthermore, C2-ceramide could evoke similar lysosomal changes in a dose-dependent manner at a concentration range of 10–40 μM [right panel of Figure 4(a)]. For further validation, we performed immunofluorescence staining for cathepsin B, which is known as a lysosomal component [Figure 4(c)]. In keeping with the abovementioned results, a substantial increase of cathepsin B (red fluorescence) was found throughout the cytoplasm of tositumomab and ceramide treated cells, while this cathepsin B release could be definitely prevented by FB1. From the above results, we can conclude that the increase of intracellular ceramide level is the reason for but not the result of tositumomab-evoked lysosomal leakage.
Discussion In this study, we demonstrated that ceramide, which has an important role in PCD induced by a variety of chemotherapeutic drugs and cytokines, took part in tositumomabmediated PCD. To our knowledge, this is the first study to report ceramide involvement in anti-CD20 mAb-mediated cell death. First, our present study discovered that tositumomab treatment can obviously enhance intracellular ceramide levels in target cells, while C2-ceramide can also induce significant PCD in Raji cells. These two interesting results led us to study the possible relationship between tositumomab-induced cell death and intracellular ceramide generation. For further investigation, we introduced FB1 in our study. FB1 can block sphingosine biosynthesis by inhibiting the conversion of sphinganine to dihydroceramides, thus negatively influencing the endogenous production of cellular ceramide [29,32,33]. To our encouragement, we discovered that tositumomab-induced PCD can be effectively prevented by FB1. Ivanov et al. validated that HA and the subsequent lysosome leakage play important roles in type II anti-CD20 mAbmediated cell death [22]. Based on this lysosome alteration, a novel type II anti-CD20 mAb, GA101, was designed, and exhibited exceptional anti-lymphoma abilities in both preclinical studies and clinical trials [20,21,34,35]. In order to clarify the causal relationships between ceramide generation, tositumomab-induced HA and lysosome leakage, we performed further experiments to determine the effects of FB1 and ceramide on both HA formation and lysosome
Figure 5. Signal transduction pathway of programmed cell death (PCD) induced by tositumomab.
alteration. Our results clearly indicated that ceramide can induce typical lysosome leakage but not obvious HA in Raji cells. Meanwhile FB1, which can successfully block lysosome leakage, seems incapable of preventing tositumomab-mediated HA. Considering that FB1 is a notable inhibitor in the process of ceramide generation, we can determine a detailed signaling transduction pathway of tositumomab-induced PCD, as shown in Figure 5. In this, ceramide generation acts as the outcome of tositumomab-triggered HA and the major cause of lysosome leakage of its contents, resulting in PCD in target cells. The involvement of ceramide production in anti-CD20 mAb-induced cell death may provide possible novel ways of treating NHLs. Based on our findings, ceramide might possibly be an effective bioactive reagent in the killing of malignant B cells. Beers et al. demonstrated that rituximabmediated internalization of CD20 by malignant B cells led to reduced macrophage recruitment and the degradation of CD20/mAb complexes, which limits the efficacy of CD20based immunotherapy [16]. Although traditional opinions suggested that CD20 experienced no antigen modulation after antibody binding, increasingly studies have demonstrated opposite results, and hold the opinion that internalization of CD20 may explain the differing sensitivity of B-cell malignancies to rituximab [16,36–39]. Currently, more and more studies are focusing on constructing novel anti-CD20 mAbs or mAb derivatives in combating rituximab resistance. However, the low expression of CD20 as a result of antigen modulation may strongly limit the therapeutic efficacy of such products. Because of ceramide participation in antiCD20 mAb-mediated PCD in lymphoma cells, perhaps we can directly make use of this effective bioactive substance in treating NHLs regardless of surface CD20 expression. Also, the regulation of intracellular ceramide synthesis might be a promising method for improved therapeutic efficacy in the fight against NHLs. However, although we first discovered that the improved intracellular ceramide level is an important mediator between tositumomab-induced HA and lysosome leakage, the mechanism of ceramide production is still not well understood. It has been revealed that tositumomab-induced HA is associated with membrane rafts and actin polymerization [22]. It was also confirmed that ceramide can be generated during sphingomyelin catabolism by various sphingomyelinases during the responses to different stresses [23–28]. Since sphingolipids are important constituents of membrane rafts, we considered that particular alteration in membrane rafts
6 Y. Liu et al. after tositumomab treatment might be involved with ceramide generation. The verification of this hypothesis is the subject of our ongoing research.
Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal. This work was supported by the Natural Science Foundation of China (No. 81201793).
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ORIGINAL ARTICLE: RESEARCH
Ceramide participates in lysosome-mediated cell death induced by type II anti-CD20 monoclonal antibodies Yuzhi Liu1, Ling Shu2 & Jingjing Wu3 1Department of Otolaryngology, Tianjin Medical University General Hospital, Tianjin, China, 2Department of Oncology,
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Yancheng City No. 1 People’s Hospital affiliated to Nantong Medical Collage, Yancheng, China and 3Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
issue is enhanced understanding of the lymphoma-killing mechanisms of anti-CD20 mAbs. Currently, anti-CD20 mAbs can be generally classified into two types, with distinct tumor killing mechanisms. Type I (rituximab-like) anti-CD20 mAbs can redistribute CD20 into membrane lipid rafts and potently mediate complement-dependent cytotoxicity (CDC), whereas type II (tositumomab-like) mAbs are less effective in activating complement, but are more potent in inducing programmed cell death (PCD) [2,11]. Nevertheless, both types can efficiently mediate antibody-dependent cell-mediated cytotoxicity (ADCC) [2,11]. Increasing evidence has revealed that the limitations of rituximab can be mostly ascribed to the exhaustion and/or disability of complement and Fc-γ-receptor-expressing effector cells such as macrophages and natural killer (NK) cells [12–15]. Otherwise, some in vitro studies have demonstrated that antigenic modulation might be another major limitation of rituximab-based immunotherapy [16]. As a result, increasingly studies are devoted to developing novel mAbs with potent PCD-inducing ability while reducing the antigen modulation after antibody binding [6,8,17–19]. Morschhauser et al. developed a novel humanized typed II anti-CD20 mAb (GA101), which can directly induce superior PCD and enhanced ADCC in malignant B cells and showed excellent anti-lymphoma efficacy in phase I/II clinical trials [6,20,21]. In addition, in vitro studies have demonstrated that unlike rituximab, type II mAbs elicit little modulation of CD20 from the cell surface, the observation of which has strengthened the case for focusing on type II mAbs in pre-clinical and clinical studies [16]. Ivanov et al. first reported that a type II mAb (tositumomab) can elicit strong homotypic adhesion (HA), followed by a swelling and/or collapse of the lysosomal compartment in human lymphoma and leukemia cells [22]. However, no detailed pathways concerning the relationships between mAb-evoked HA and specific lysosome leakage have been reported to date.
Abstract Considering the variable and often modest therapeutic efficacy of rituximab for a substantial proportion of patients suffering from non-Hodgkin lymphomas (NHLs), various type II anti-CD20 monoclonal antibodies (mAbs) with excellent ability in inducing programmed cell death (PCD) are currently being developed for their enhanced therapeutic index. Although homotypic adhesion (HA) and lysosome leakage are proven to be of vital importance in type II mAb-induced PCD in NHL cells, the detailed relationship between them remains unclear. Herein, for the first time we discovered that improved intracellular ceramide level is an important mediator between HA and lysosome leakage in tositumomab-induced cell death. Further experimental results revealed that the generation of intracellular ceramide acts as the outcome of HA and major cause of lysosome leakage. The clarification of ceramide involvement in type II anti-CD20 mAb-induced PCD may provide new ideas on CD20-based immunotherapy against NHLs. Keywords: Non-Hodgkin lymphoma, CD20, programmed cell death, ceramide, homotypic adhesion, lysosome leakage
Introduction Although clinical applications have proved the unprecedented success of rituximab in treating a range of CD20positive non-Hodgkin lymphomas (NHLs), a substantial proportion of patients fail to achieve a complete remission (CR) [1,2]. Moreover, tumor relapse is inevitable for most patients [3,4]. The limitations of rituximab call for the development of improved treatments for these patients. Given the fact that CD20 is one of the most ideal therapeutic targets of NHL due to its high and relatively specific expression on the surface of malignant B cells [3,5], recently various therapeutic approaches have been devoted to exploiting novel antiCD20 monoclonal antibodies (mAbs) aimed at improving therapeutic efficacy [5–10]. To this end, the most important
Correspondence: Jingjing Wu, Department of Hematology, Institute of Clinical Medicine, the First Affiliated Hospital of Zhengzhou University, 1st East Jianshe Road, Zhengzhou, Henan Province, China, 450052. Tel: 0371-66295562. Fax: 0371-66295562. E-mail: [email protected] Received 1 July 2014; Revised 14 October 2014; accepted 19 October 2014
1
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2 Y. Liu et al. Previous studies revealed that ceramide plays an important role in PCD induced by a variety of chemotherapeutic drugs and cytokines (such as tumor necrosis factor-α and interleukin-1β, etc.) [23–28]. It is worth mentioning that sphingolipids are important constituents of cell membranes, which can either serve as substrates for or be produced by enzymes activated in response to cellular stress. Ceramide can be generated during sphingomyelin catabolism by various sphingomyelinases during the responses to these stresses. Ceramide acts as a second messenger in the sphingomyelin signaling pathway, which has been identified as an important mediator in cell differentiation, survival and apoptosis [23,29,30]. Further, ceramide can be produced by de novo synthesis, responding to similar agents identified for the activation of sphingomyelinases [28,29,31,32]. In this study, we encouragingly found that ceramide also takes part in lysosome-mediated PCD induced by type II anti-CD20 mAbs. In addition, the relationships among mAbmediated HA, lysosome leakage and ceramide generation were comprehensively investigated. A thorough understanding of type II anti-CD20 mAb-induced PCD may provide the basis for developing more effective methods for curing NHLs.
Materials and methods Cell lines and reagents A human B cell lymphoma cell line, Raji, was obtained from the American Type Culture Collection (ATCC). Cells were maintained in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO). Rituximab (C2B8) was purchased from Roche, and tositumomab (B1, trade name: Bexxar) was obtained from GlaxoSmithKline (GSK, Brentford, UK). C2-ceramide (N-acetylsphingosine) and fumonisin Bl (FB1) were purchased from Sigma-Aldrich.
was dissolved in chloroform (100 μL). The solution was reacted at ⫺ 20°C for 3 h with 10 μL of 100 mM (⫹)-6-methoxyα-methyl-2 naphthaleneacetic acid (NAP; Sigma-Aldrich, St Louis, MO), 100 mM N,N’-dicyclohexylcarbodiimide (DCC; Sigma-Aldrich) and 100 mM 4-dimethylaminopyridine (DMAP, 4-dimethylaminopyridine; Sigma-Aldrich). After evaporation, the residue was suspended in chloroform (15 μL), followed by mixing with 2 mL of hexane and centrifugation (800g ⫻ 10 min). The supernatant was vigorously mixed with 5 mL of methanol/water (v/v ⫽ 4:1). After centrifugation, the upper phase was collected and filtered using a 0.45 μm membrane filter (Millipore, Massachusetts, MN). A 20 μL portion of the filtrate was injected into an Econosphere CN 5U column (4.6 ⫻ 250 mm; Alltech, Chicago, IL). The derivatized ceramide was separated from by-products with 3% 2-propanol in n-hexane as the mobile phase with a flow rate of 2.0 mL/min. The effluent was monitored fluorometrically at a wavelength of 230/352 nm (excitation/emission).
Cathepsin B release assessment Released cathepsin B in cytoplasm was detected by confocal microscopy following the method of Ivanov et al. [22]. Briefly, harvested cells were placed onto poly-d-lysine (Sigma-Aldrich) coated sterilized microscope slides, fixed in 4% paraformaldehyde and permeabilized by 0.5% Triton X-100. Then each sample was incubated with 5 μg/mL cathepsin B mouse mAb (Invitrogen) at 4°C overnight and stained by Alexa Fluor-555 Rabbit Anti-Mouse imunoglobulin G (IgG) (H ⫹ L) secondary antibodies (Invitrogen). After washing, samples were observed using a confocal microscope (Zeiss, Jena, Germany).
Lysosomal permeability assessment Harvested cells were labeled with 75 nM LysoTracker Red DND (Invitrogen) at 37°C for 30 min and assessed by FCM and confocal microscopy. Unlabeled cells were used as a background control [22].
Annexin V/propidium iodide assays Raji cells that experienced PCD were assessed by flow cytometry (FCM). Briefly, cells were incubated with 10 μg/ mL rituximab, tositumomab or different concentrations of C2-ceramide for 12 h. After washing with phosphate buffered saline (PBS), cells were stained with Alexa Fluor-488 annexin V and propidium iodide (PI) (Invitrogen, Carlsbad, CA) and analyzed by FCM (FC500; Beckman Coulter, Brea, CA). For PCD inhibition assays, a caspase inhibitor (Z-VAD-FMK; Promega) or FB1 at different concentrations was added 1 h prior to the addition of mAb.
Determination of cellular ceramide levels The content of cellular ceramide was determined by high performance liquid chromatography (HPLC) following the method of Soeda et al. [30]. Briefly, cells were washed, suspended in 200 μL of 0.25 M sucrose in PBS and disrupted by sonication. After centrifugation (800g ⫻ 10 min), the cell lysate pellet was washed, resuspended in 200 μL of PBS and mixed with 4.0 mL of chloroform/methanol (2:1) for 30 min. Subsequently the sample was added to 1 mL of Mini-Q water, vortexed and centrifuged. The lower phase was collected and evaporated to dryness under a nitrogen stream. The residue
Homotypic adhesion measurement Cells were incubated with 10 μg/mL tositumomab or different concentrations of ceramide for 4 h and cell morphology was observed by light microscopy. For HA inhibition assessment, FB1 was added before the addition of tositumomab for HA inhibition assays.
Statistical analysis Statistical analysis involving two groups was performed by Student’s unpaired t-test, whereas analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was used for comparison among three or more groups. All data were processed with SPSS 10.0 software. p ⬍ 0.05 was considered statistically significant in all calculations.
Results Type II but not type I anti-CD20 mAbs can induce caspase-independent cell death in NHLs To determine the PCD-inducing ability of anti-CD20 mAbs, Raji cells were respectively incubated with 10 μg/mL tositumomab or rituximab, and analyzed by FCM. Figure 1(a)
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Ceramide- and tositumomab-evoked PCD 3
Figure 1. Tositumomab can induce caspase-independent cell death in Raji cells. (a) Raji cells were incubated with 10 μg/mL rituximab or tositumomab for 12 h and cell death was analyzed using annexin V/PI assay. Not treated (NT) cells were used as a negative control. (b) Z-VAD-FMK (a caspase inhibitor) over a range of 0–30 μM was incubated with Raji cells 1 h before the addition of tositumomab and rituximab. Cell death was analyzed after 12 h using annexin V/PI assay. Data are expressed as mean ⫾ SD (n ⫽ 3), **p ⬍ 0.01.
demonstrates that tositumomab could evoke significantly stronger PCD (28.32⫾4.16%) compared with no treatment (NT, 8.62 ⫾ 1.45%, p ⫽ 0.000), while rituximab only weakly induced PCD in CD20 positive Raji cells (10.62 ⫾ 1.64%). In order to investigate the involvement of caspase activation in anti-CD20 mAb-induced PCD, a cell-permeable caspase inhibitor, Z-VAD-FMK, was employed in PCD inhibition assays. Figure 1(b) indicates that Z-VAD-FMK was unable to prevent both tositumomab and rituximab-induced PCD up to 30 μM. We can conclude from these results that a type II (tositumomab) but not type I (rituximab) anti-CD20 mAb can evoke remarkable PCD in NHL cells in a caspase-independent manner.
Tositumomab-induced cell death correlates with enhanced generation of intracellular ceramide Given that ceramide plays an important role in PCD induced by a variety of chemotherapeutic drugs and cytokines [23–28], we subsequently investigated the correlation between tositumomab-induced cell death and ceramide generation in Raji cells. To evaluate the effect of anti-CD20 mAbs on intracellular ceramide generation, Raji cells were respectively incubated with 10 μg/mL tositumomab or rituximab for 12 h. The intracellular ceramide level was determined by HPLC. As shown in Figure 2(a), tositumomab but not rituximab could significantly enhance the intracellular ceramide to approximately 189.1 ⫾ 12.96 ng/105 cells, which was about 1.95-fold more than that in normal cells (p ⫽ 0.000). Moreover, ceramide over a range of concentrations from 5 to 40 μM could evoke obvious PCD in Raji cells in a dose-dependent manner [Figure 2(b)]. Based on the abovementioned results, we assumed that tositumomab-induced PCD might be correlated with intracellular ceramide generation. To verify this hypothesis, we determined tositumomab-induced PCD in the absence or presence of FB1, which acts as an inhibitor of dihydroceramide synthase (a key enzyme regulating the conversion of sphinganine to dihydroceramides in the synthesis of cellular ceramide) [29,33]. Figure 2(c) demonstrates that the increased ceramide levels evoked by tositumomab could
Figure 2. The involvement of ceramide in tositumomab-induced programmed cell death (PCD). (a) Raji cells were incubated with 10 μg/mL tositumomab and rituximab for 12 h and intracellular ceramide levels were determined by high performance liquid chromatography (HPLC). Not treated (NT) cells were used as a background control. (b) Raji cells were incubated with different concentrations of ceramide for 12 h and cell death was analyzed using annexin V/PI assay. (c, d) Fumonisin Bl (FB1) over a range of 0–40 nM was incubated with Raji cells for 1 h before the addition of tositumomab. After 12 h, intracellular ceramide levels were determined by HPLC (c) and cell death was analyzed using annexin V/PI assay (d). Data are expressed as mean ⫾ SD (n ⫽ 3), **p ⬍ 0.01.
be significantly reduced by FB1 in a dose-dependent manner. Approximately 85% of the enhancement was decreased at the concentration of 40 nM [Figure 2(c)]. Furthermore, tositumomab-mediated PCD could also be significantly prevented by FB1 in a dose-dependent manner [Figure 2(d)]. All these results support our hypothesis that the ceramide pathway is a major driving force in the signaling of tositumomabmediated PCD.
Increase of intracellular ceramide content acts as outcome but not cause of HA evoked by tositumomab Previous studies demonstrated that type II anti-CD20 mAbmediated cell death is associated with HA of target cells and is executed by lysosomes which disperse their contents into the cytoplasm and the surrounding environment [6,22]. In order to identify the intrinsic relationship between HA, lysosome leakage and ceramide generation, we designed further experiments to investigate whether FB1 can negatively influence tositumomab-evoked HA and lysosome alteration. In HA inhibition analysis, 40 nM FB1 was incubated with Raji cells for 1 h before the addition of tositumomab, and cell morphology was observed by low-magnification light microscopy 4 h after treatment. In keeping with previous publications, Raji cells underwent more pronounced HA following treatment with tositumomab compared with rituximab, which could not be disrupted by FB1 (Figure 3). Furthermore, compared with untreated Raji cells, no obvious HA was observed after treatment with ceramide up to 40 μM (Figure 3), at which concentration considerable PCD could
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4 Y. Liu et al.
Figure 3. The effect of ceramide on homotypic adhesion (HA) induced by tositumomab. Raji cells were incubated with rituximab (10 μg/mL), tositumomab (10 μg/mL) or C2-ceramide (20-40 μM) for 4 h and cell morphology was observed by light microscopy. For HA inhibition assays, FB1 at different concentrations was incubated for 1 h before the addition of tositumomab. Magnification: 20 ⫻ 20.
be successfully evoked [Figure 2(B)]. Considering the observation that FB1 could significantly inhibit the generation of ceramide but not the formation of HA, we can deduce that the increased intracellular ceramide level is the outcome of rather than the reason for tositumomab-evoked HA.
Increase of intracellular ceramide content acts as reason for but not result of lysosome leakage evoked by tositumomab It has been reported that enlargement of the lysosomal compartment and subsequent lysosome membrane
permeabilization play a vital role in type II anti-CD20 mAbinduced PCD [6,22]. Hence, after determining the causal relationship between tositumomab-induced HA and intracellular ceramide generation, we subsequently focused on investigating whether ceramide generation operates upstream or downstream of lysosome leakage. For this purpose, a lysosome tracker was employed to label the lysosome compartment. As shown in Figure 4(a), FCM results revealed that the distribution of LysoTracker fluorescence (FL-2, Red) in Raji cells experienced a visible increase and decrease after treatment with tositumomab (green histograms in left panel)
Figure 4. Effect of ceramide on lysosome leakage induced by Tositumomab. (a, b) Raji cells were incubated with tositumomab with the absence/ presence of FB1 or treated with ceramide at different concentrations for 12 h. After labeling with LysoTracker, total lysosomal volume in cells was detected using FCM (a) and confocal microscopy (b). (c) Confocal microscopy of cathepsin B staining (red). DNA was counterstained with DAPI (blue).
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Ceramide- and tositumomab-evoked PCD 5 compared with normal cells (red histograms). This typical and obvious alteration was clearly explained by confocal microscopy. Figure 4(b) reveals that the cellular lysosome was labeled as a relatively small and confined organelle in normal cells, while after treatment with tositumomab, an enlargement of red fluorescence-labeled compartments and diffusion of red fluorescence in the cytoplasm was detected. The above results, along with previous publications, demonstrated that Raji cells were successively undergoing a swelling of lysosomes (enlargement of red fluorescence-labeled compartments) and collapse of the lysosomal compartment (diffusion of red fluorescence in the cytoplasm) after treatment with tositumomab. To our great interest, this distinct alteration could be significantly and effectively prevented by FB1 [blue histograms in left panel of Figure 4(a)]. Furthermore, C2-ceramide could evoke similar lysosomal changes in a dose-dependent manner at a concentration range of 10–40 μM [right panel of Figure 4(a)]. For further validation, we performed immunofluorescence staining for cathepsin B, which is known as a lysosomal component [Figure 4(c)]. In keeping with the abovementioned results, a substantial increase of cathepsin B (red fluorescence) was found throughout the cytoplasm of tositumomab and ceramide treated cells, while this cathepsin B release could be definitely prevented by FB1. From the above results, we can conclude that the increase of intracellular ceramide level is the reason for but not the result of tositumomab-evoked lysosomal leakage.
Discussion In this study, we demonstrated that ceramide, which has an important role in PCD induced by a variety of chemotherapeutic drugs and cytokines, took part in tositumomabmediated PCD. To our knowledge, this is the first study to report ceramide involvement in anti-CD20 mAb-mediated cell death. First, our present study discovered that tositumomab treatment can obviously enhance intracellular ceramide levels in target cells, while C2-ceramide can also induce significant PCD in Raji cells. These two interesting results led us to study the possible relationship between tositumomab-induced cell death and intracellular ceramide generation. For further investigation, we introduced FB1 in our study. FB1 can block sphingosine biosynthesis by inhibiting the conversion of sphinganine to dihydroceramides, thus negatively influencing the endogenous production of cellular ceramide [29,32,33]. To our encouragement, we discovered that tositumomab-induced PCD can be effectively prevented by FB1. Ivanov et al. validated that HA and the subsequent lysosome leakage play important roles in type II anti-CD20 mAbmediated cell death [22]. Based on this lysosome alteration, a novel type II anti-CD20 mAb, GA101, was designed, and exhibited exceptional anti-lymphoma abilities in both preclinical studies and clinical trials [20,21,34,35]. In order to clarify the causal relationships between ceramide generation, tositumomab-induced HA and lysosome leakage, we performed further experiments to determine the effects of FB1 and ceramide on both HA formation and lysosome
Figure 5. Signal transduction pathway of programmed cell death (PCD) induced by tositumomab.
alteration. Our results clearly indicated that ceramide can induce typical lysosome leakage but not obvious HA in Raji cells. Meanwhile FB1, which can successfully block lysosome leakage, seems incapable of preventing tositumomab-mediated HA. Considering that FB1 is a notable inhibitor in the process of ceramide generation, we can determine a detailed signaling transduction pathway of tositumomab-induced PCD, as shown in Figure 5. In this, ceramide generation acts as the outcome of tositumomab-triggered HA and the major cause of lysosome leakage of its contents, resulting in PCD in target cells. The involvement of ceramide production in anti-CD20 mAb-induced cell death may provide possible novel ways of treating NHLs. Based on our findings, ceramide might possibly be an effective bioactive reagent in the killing of malignant B cells. Beers et al. demonstrated that rituximabmediated internalization of CD20 by malignant B cells led to reduced macrophage recruitment and the degradation of CD20/mAb complexes, which limits the efficacy of CD20based immunotherapy [16]. Although traditional opinions suggested that CD20 experienced no antigen modulation after antibody binding, increasingly studies have demonstrated opposite results, and hold the opinion that internalization of CD20 may explain the differing sensitivity of B-cell malignancies to rituximab [16,36–39]. Currently, more and more studies are focusing on constructing novel anti-CD20 mAbs or mAb derivatives in combating rituximab resistance. However, the low expression of CD20 as a result of antigen modulation may strongly limit the therapeutic efficacy of such products. Because of ceramide participation in antiCD20 mAb-mediated PCD in lymphoma cells, perhaps we can directly make use of this effective bioactive substance in treating NHLs regardless of surface CD20 expression. Also, the regulation of intracellular ceramide synthesis might be a promising method for improved therapeutic efficacy in the fight against NHLs. However, although we first discovered that the improved intracellular ceramide level is an important mediator between tositumomab-induced HA and lysosome leakage, the mechanism of ceramide production is still not well understood. It has been revealed that tositumomab-induced HA is associated with membrane rafts and actin polymerization [22]. It was also confirmed that ceramide can be generated during sphingomyelin catabolism by various sphingomyelinases during the responses to different stresses [23–28]. Since sphingolipids are important constituents of membrane rafts, we considered that particular alteration in membrane rafts
6 Y. Liu et al. after tositumomab treatment might be involved with ceramide generation. The verification of this hypothesis is the subject of our ongoing research.
Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal. This work was supported by the Natural Science Foundation of China (No. 81201793).
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