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Clinical Cancer Research Vol. 11, 8186-8194, November 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Inhibition of Nuclear Translocation of Nuclear Factor-{kappa}B Despite Lack of Functional I{kappa}B{alpha} Protein Overcomes Multiple Defects in Apoptosis Signaling in Human B-Cell Malignancies

Roman K. Thomas1,2, Martin L. Sos1, Thomas Zander1, Özlem Mani1, Alexey Popov1, Daniel Berenbrinker1, Sigrun Smola-Hess3, Joachim L. Schultze1,2 and Jürgen Wolf1,2

Authors' Affiliations: 1 Molecular Tumor Biology and Tumor Immunology, Department I for Internal Medicine, 2 Center for Molecular Medicine Cologne, and 3 Institute of Virology, University of Cologne, Cologne, Germany

Requests for reprints: Jürgen Wolf, Molecular Tumor Biology and Tumor Immunology, Department I for Internal Medicine, University of Cologne, Joseph-Stelzmann-Str. 9, 50931 Cologne, Germany. E-mail: Juergen.Wolf{at}medizin.uni-koeln.de.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Defective apoptosis signaling is a typical feature of classic Hodgkin's lymphoma, multiple myeloma, and activated B-cell-like diffuse large B-cell lymphoma. In these malignancies, the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) is a critical mediator of apoptosis resistance and oncogenic growth, making it an attractive therapeutic target. Here, we sought to determine how to overcome apoptosis resistance experimentally in these malignancies by targeting NF-{kappa}B.

Experimental Design: We investigated the effect of different inhibitors of NF-{kappa}B on classic Hodgkin's lymphoma, multiple myeloma, and activated B-cell-like diffuse large B-cell lymphoma cell lines harboring different molecular defects in apoptosis signaling both quantitatively and qualitatively.

Results: The cyclopentenone prostaglandin, 15-deoxy-{Delta}12,14-prostaglandin J2, a known inhibitor of NF-{kappa}B, induced caspase-dependent apoptosis; it restored mitochondrial apoptotic signaling by down-regulation of X-linked inhibitor of apoptosis protein and heat shock protein 27 and led to breakdown of the mitochondrial membrane potential and, finally, cleavage of caspase-3 irrespective of I{kappa}B{alpha} mutational status. Surprisingly, 15-deoxy-{Delta}12,14-prostaglandin J2 and the I{kappa}B kinase inhibitor curcumin both reduced nuclear levels of p65 in cell lines lacking I{kappa}B{alpha}, suggesting that inhibition of nuclear translocation of NF-{kappa}B can occur in the absence of I{kappa}B{alpha}. Finally, a synthetic peptide that specifically abrogates the assembly of the I{kappa}B kinase complex killed I{kappa}B{alpha}-defective cells by induction of apoptosis, paralleled by reduction of nuclear NF-{kappa}B.

Conclusions: These results show that molecular defects in apoptotic signaling, such as I{kappa}B{alpha} mutations, can be circumvented by targeting NF-{kappa}B through inhibition of the I{kappa}B kinase complex followed by induction of apoptosis in classic Hodgkin's lymphoma, multiple myeloma, and activated B-cell-like diffuse large B-cell lymphoma. Thus, targeting I{kappa}B kinases may represent an attractive therapeutic approach against these malignancies regardless of the mutational status of I{kappa}B{alpha}.

Resistance to apoptosis is a hallmark of various germinal center–derived B-cell malignancies, such as classic Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (1–3). The germinal center is a microanatomic site of stringent immune surveillance. B cells enter the germinal center with an activated apoptotic program that is only prevented from execution in those cells displaying favorable B-cell receptors (4). Thus, the outgrowth of transformed B cells in the germinal center represents a breakdown of such control mechanisms. It has therefore been postulated that acquisition of an apoptosis-resistant phenotype is intimately connected with malignant transformation in germinal center–derived B-cell lymphomas (5, 6). In a therapeutic strategy that aims at the primary transforming event, apoptosis resistance may thus also be regarded as the central phenotype to be targeted in B-cell malignancies.

Altered functional states of genes involved in different apoptotic pathways have been reported to be associated with apoptosis resistance in classic Hodgkin's lymphoma, multiple myeloma, and activated B-cell-like DLBCL (ABC-DLBCL). High-level expression of c-FLIP was shown to correlate with apoptosis resistance in classic Hodgkin's lymphoma and multiple myeloma (1, 7–10). c-FLIP is also highly expressed by ABC-DLBCL, implying a role for c-FLIP also in this lymphoma entity (2). Within the mitochondrial apoptosis pathway, defective Bax activation was reported in classic Hodgkin's lymphoma that was unrelated to Bcl-2 overexpression (11). Furthermore, a defect in caspase-3 activation was detected in classic Hodgkin's lymphoma apparently due to overexpression of X-linked inhibitor of apoptosis protein (XIAP; ref. 12). In multiple myeloma, overexpression of heat shock protein 27 (Hsp27) was shown to inhibit cytochrome c release from mitochondria and subsequent execution of mitochondrial apoptosis (13).

The transcription factor nuclear factor-{kappa}B (NF-{kappa}B) induces the expression of antiapoptotic genes, such as Bcl-2, c-FLIP, and XIAP, thereby leading to disruption of apoptotic signaling pathways (14). Survival of mature B cells has been shown to depend on NF-{kappa}B activity via signaling of the I{kappa}B kinase (IKK) complex (15). Importantly, NF-{kappa}B is constitutively active in classic Hodgkin's lymphoma, multiple myeloma, and ABC-DLBCL (2, 16–19). Moreover, NF-{kappa}B is critical for the survival of classic Hodgkin's lymphoma, multiple myeloma, and ABC-DLBCL in in vitro models, because interruption of NF-{kappa}B transcriptional activity leads to induction of massive cell death (2, 18). Thus, constitutively active NF-{kappa}B seems to be a central molecular switch in apoptosis resistance shared by different germinal center–derived B-cell malignancies. In a subset of classic Hodgkin's lymphoma cases and in a DLBCL cell line, clonal deleterious mutations of the I{kappa}B{alpha} gene, the main negative regulator of NF-{kappa}B, have been reported to cause NF-{kappa}B activation (20–23). Recently, mutations in another member of the I{kappa}B family of proteins, I{kappa}B{varepsilon}, were reported in classic Hodgkin's lymphoma, which may also play a role in NF-{kappa}B activation in classic Hodgkin's lymphoma (24). Alternatively, EBV-encoded latent gene products LMP1 and LMP2a may trigger NF-{kappa}B transcriptional activity in classic Hodgkin's lymphoma (6). Other factors known to be activators of the NF-{kappa}B pathway are constitutively active IKKs, CD40, insulin-like growth factor-I activation, etc. (2, 6, 25). Taken together, multiple defects prevent transmission of apoptotic signals in human germinal center–derived B-cell malignancies, encompassing mutational inactivation and altered expression of important genes. Among these alterations, NF-{kappa}B is a central mediator of apoptosis resistance in classic Hodgkin's lymphoma, multiple myeloma, and ABC-DLBCL. Thus, the dependence of these lymphoid malignancies on NF-{kappa}B activity makes it an attractive target for novel therapeutic approaches. To date, most NF-{kappa}B-inhibitory strategies target the IKK complex. Because I{kappa}B{alpha} is a central downstream mediator of IKK function, it has been speculated that IKK-targeted therapy in patients with B-cell malignancies harboring an I{kappa}B{alpha} mutation will not be effective (21, 26).

15-Deoxy-{Delta}12,14-prostaglandin J2 (15dPGJ2) is a prostaglandin derivative characterized by a highly reactive cyclopentenone ring system that enables it to modify intracellular signaling molecules (27). 15dPGJ2 induces apoptosis in a variety of human malignancies (28, 29). In T lymphocytes, this has been recently related to activation of the mitochondrial signaling pathway and subsequent activation of executioner caspases (30). Importantly, 15dPGJ2 potently inhibits NF-{kappa}B by different modes of action: First, it inhibits IKKs, thereby preventing the phosphorylation of I{kappa}B{alpha} and, thus, nuclear entry of NF-{kappa}B (31, 32). Additionally, at high doses, 15dPGJ2 activates peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), leading to transrepression of NF-{kappa}B. Finally, by interaction of the cyclopentenone ring system with Cys38 in the p65 and with Cys62 in the p50 subunit, respectively, 15dPGJ2 directly inhibits the transcriptional activity of NF-{kappa}B (31, 33). This unique profile of proapoptotic and NF-{kappa}B-inhibitory actions makes 15dPGJ2 an ideal compound for studying apoptosis-related defects in human cancer models, particularly in those with genetically fixed constitutive activation of NF-{kappa}B. In a recent article, apoptosis was induced by 15dPGJ2 in Burkitt's lymphoma and myeloma cell lines through inhibition of NF-{kappa}B, showing the potential of this agent to study apoptosis resistance in these NF-{kappa}B-dependent malignancies (34).

Here, we investigated if 15dPGJ2 might also be able to overcome the apoptosis-resistant phenotype caused by the multiple qualitative and quantitative genetic alterations that are a hallmark of the three germinal center–derived B-cell lymphoma entities classic Hodgkin's lymphoma, ABC-DLBCL, and multiple myeloma. We were particularly interested if 15dPGJ2 may circumvent the barrier that mutations in the I{kappa}B{alpha} gene are expected to impose to therapeutic strategies aiming at inhibition of IKKs. Furthermore, we characterized the molecular sequelae of 15dPGJ2-mediated cytotoxicity in in vitro models of these neoplasms. Finally, we aimed at establishing potential preclinical strategies for inhibition of NF-{kappa}B in classic Hodgkin's lymphoma, DLBCL, and multiple myeloma by analyzing the effect of various IKK inhibitors on I{kappa}B{alpha}-mutated lymphoma cell lines.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents. Eight cell lines were used in this study: L1236 and L428 (classic Hodgkin's lymphoma); OCI-Ly3 and OCI-Ly10 (ABC-DLBCL); RC-K8 (DLBCL; ref. 2); and RPMI-8226, U266, and OPM-2 (multiple myeloma). Jurkat cells served as a positive control in apoptosis assays (1). All cell lines, except OCI-Ly3 and OCI-Ly10, were grown in RPMI 1640 (Life Technologies, Karlsruhe, Germany) supplemented with 10% to 20% heat-inactivated FCS, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine at 37°C in an atmosphere containing 5% CO2 under sterile conditions. OCI-Ly3 and OCI-Ly10 cells were grown in Iscove's modified Dulbecco's medium (PAA, Pasching, Austria) supplemented with 20% human AB serum (OCI-Ly3; PAA) or 20% FCS (OCI-Ly10), 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mmol/L glutamine, and 55 µmol/L mercaptoethanol (OCI-Ly10). To exclude cellular cross-contamination during the experiments, Western blots were done on lysates of L428 and RC-K8 cells that confirmed the absence of I{kappa}B{alpha} protein in these cell lines. 15dPGJ2 was obtained from Biomol (Plymouth Meeting, PA) and dissolved in 30 mmol/L ethanol. Troglitazone was obtained from Alexis (Lausen, Switzerland) and dissolved in 30 mmol/L DMSO. z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-Fmk) was purchased from Bachem Biochemica (Heidelberg, Germany) and dissolved in 100 mmol/L DMSO. Curcumin (diferuloylmethane) was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in 30 mmol/L DMSO. Reagents were stored in aliquots at –80°C. NF-{kappa}B essential modulator–binding domain (NBD) peptide and the respective control peptide in which two amino acids have been substituted were obtained from Calbiochem/Merck (Darmstadt, Germany), dissolved in DMSO, diluted in medium, and immediately used in cell culture experiments.

Cytotoxicity assay. Screening for cytotoxicity was done on individual cell lines by trypan blue exclusion. For assessment of several cell lines, cytotoxicity was assessed using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Roche, Mannheim, Germany) following the instructions of the manufacturer. Cells were seeded in 96-well plates and incubated with different concentrations of stimuli for different times. Controls included medium and vehicle as negative controls and one setup containing 10% DMSO as a positive control for killing. Viability was calculated as percentage of vehicle control after subtraction of background control values. Negative values as a result of subtraction were set to zero. Statistical significance was calculated using ANOVA and two-sided t tests.

Flow cytometric assessment of apoptosis. Cells were incubated in 24-well plates with various stimuli for the indicated times as described (1). Briefly, apoptosis was detected by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany) using FITC-coupled Annexin V (Becton Dickinson) and propidium iodide. At least 10,000 events were recorded. Analyses were done using CellQuest software version 3.3 (Becton Dickinson).

Flow cytometric analysis of mitochondrial membrane potential. Cells were incubated in 24-well plates with various stimuli. At the indicated time points, cells were harvested and incubated for 20 minutes in culture medium containing 5 mg/mL JC-1 (Molecular Probes, Eugene, OR). Cells were analyzed on a FACSCalibur cytometer. At least 10,000 events were recorded. Analyses were done using CellQuest software. A disruption of mitochondrial membrane potential ({Delta}{psi}m) was indicated by a decrease of events in the FL2 channel and a concomitant increase of events in the FL1 channel. Data were calculated as the ratio of the mean fluorescence intensities of the FL1 and FL2 channels.

Electrophoretic mobility shift assay. Cells were incubated in six-well plates with the respective stimuli, harvested after 6 hours, and washed twice with ice-cold PBS. Nuclear lysates were prepared as described (35). Five micrograms of each extract were analyzed by electrophoretic mobility shift assay with a 32P-labeled NF-{kappa}B probe as described (35).

Western blotting. Cells were incubated with various stimuli for indicated times, harvested, washed twice in ice-cold PBS, and lysed in 1% Triton X-100 buffer as described (30). Total cellular protein (15-75 µg) per slot was separated by PAGE. After blotting onto nitrocellulose membranes (Hybond-C Extra, Amersham Pharmacia, Freiburg, Germany), membranes were blocked in 5% nonfat dry milk for 1 hour and incubated overnight at 4°C with antibodies against the following proteins: XIAP (clone 46, 1:2,000, Becton Dickinson), Hsp27 (clone F4, 1:2,000, Santa Cruz Biotechnology, Santa Cruz, CA), caspase-3 (clone 5A1, 1:1,000, Cell Signaling Technology, Inc., Beverly, MA), I{kappa}B{alpha} (C-21, sc-371, 1:500, Santa Cruz Biotechnology), or actin (clone C4, 1:2,000, Chemicon, Hofheim, Germany). After washing, the blots were incubated with appropriate secondary antibodies coupled to horseradish peroxidase (DAKO, Hamburg, Germany) for 1 hour at room temperature. The Enhanced Chemiluminescence System (Amersham Pharmacia) was used to develop the blots.

Immunofluorescence microscopy. Cells were incubated with different stimuli for the indicated times, washed twice with ice-cold PBS, and cytospun onto coated slides. After drying for 2 hours, cells were fixed in 4% paraformaldehyde/PBS for 10 minutes at room temperature, briefly washed in PBS, and permeabilized with 0.1% Triton X-100/PBS for 5 minutes. After brief washing, cells were blocked in blocking solution (10% goat serum, 0.1% fish skin gelatin/PBS) and incubated overnight at 4°C with the primary p65 antibody (C20, sc-372, 1:100, Santa Cruz Biotechnology). All antibodies were diluted in Antibody Diluent Reagent Solution (Zymed, San Francisco, CA). After washing twice, the slides were incubated with a secondary goat anti-rabbit antibody coupled to Cy3 for 1 hour at room temperature. After two additional washing steps, the slides were counterstained with 4',6-diamidino-2-phenylindole (1:2,000) for 5 minutes and mounted using Fluoromount-G mounting medium (Electron Microscopy Sciences, Hatfield, PA). The slides were analyzed on a Nikon Eclipse E800 fluorescence microscope (Nikon Instruments, Kanagawa, Japan). Pictures were taken using a digital camera (Nikon DXM1200F) and analyzed using Lucia GF software version 4.81 (Laboratory Imaging, Prague, Czech Republic; http://www.lim.cz). Exposure times were identical for serum and control slides. Extensive control experiments were carried out to ensure accurate nuclear staining of p65. For this purpose, Jurkat cells were incubated with either medium alone or medium containing recombinant tumor necrosis factor-{alpha} (50 ng/mL) for 30 minutes at 37°C. This treatment leads to a robust nuclear translocation of NF-{kappa}B and up-regulation of its target genes.4 The cells were cytospun as described above and subjected to various fixation and permeabilization techniques, including acetone, methanol, and the one described above. The best and most reliable results were obtained by paraformaldehyde fixation followed by permeabilization with 0.1% Triton X-100. All experimental slides were analyzed microscopically in a blinded fashion to ensure investigator independence. At least two slides per setup (control or experimental) were analyzed from at least three independent experiments.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
15-Deoxy-{Delta}12,14-prostaglandin J2 is cytotoxic to classic Hodgkin's lymphoma, diffuse large B-cell lymphoma, and multiple myeloma cell lines. Based on recent finding in other malignancies, we primarily attempted to address whether 15dPGJ2 is cytotoxic to germinal center–derived B-cell lymphomas. In a pilot experiment, L428, RC-K8, and RPMI-8226 cells derived from classic Hodgkin's lymphoma, DLBCL, and multiple myeloma were incubated with 30 µmol/L 15dPGJ2 for 72 hours and viability was determined by trypan blue staining and by XTT assay. This experiment clearly showed that 15dPGJ2 strongly reduced the viability of all three cell lines (Fig. 1A; data not shown). Further analysis of additional five B-cell lines [classic Hodgkin's lymphoma (n = 1), ABC-DLBCL (n = 2), and multiple myeloma (n = 2); Table 1] harboring characteristic defects in apoptosis signaling showed the same strong reduction of cell viability. After 72 hours of incubation, 15dPGJ2 was highly cytotoxic to all cell lines tested at a dose of 10 µmol/L, with a mean cytotoxicity of 79.5% (Fig. 1A; data not shown). Slight differences in the response to 15dPGJ2 were, however, unrelated to I{kappa}B{alpha} or p53 mutational status (Fig. 1A and B; Table 1; data not shown). At a dose of 30 µmol/L, viability of all cell lines tested was completely abrogated. To test if the cytotoxicity mediated by 15dPGJ2 results from activation of PPAR{gamma}, we determined the expression of PPAR{gamma} in the cell lines by reverse transcription-PCR. PPAR{gamma} was only inconsistently expressed (data not shown). Thus, it is unlikely that the uniformly observed 15dPGJ2-mediated cytotoxicity in classic Hodgkin's lymphoma, multiple myeloma, and DLBCL is mainly due to activation of PPAR{gamma}. To further support this observation, we determined the effect of the synthetic PPAR{gamma} agonist troglitazone on the viability of the chosen cell lines. As shown in Fig. 1C, troglitazone was only marginally toxic at concentrations up to 30 µmol/L, further supporting that PPAR{gamma} did not play a major role in 15dPGJ2-mediated effects. Similar results were obtained using the thiazolinedione ciglitazone, another PPAR{gamma} agonist (data not shown).



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  		Fig. 1. 15dPGJ2 is cytotoxic to lymphoma cell lines irrespective of their I{kappa}B{alpha} mutational status. A, L428, RC-K8, and RPMI-8226 cells derived from classic Hodgkin's lymphoma (cHL), DLBCL, and multiple myeloma (MM), respectively, were incubated with different doses of 15dPGJ2 for 72 hours and viability was determined using the XTT assay and calculated as described in Materials and Methods. Similar results were obtained with all other cell lines tested in this study (Table 1). *, P < 0.05. B, cellular lysates were prepared in log-phase growth, separated by gel electrophoresis, blotted onto membranes, and probed with antibodies to I{kappa}B{alpha} and actin as a loading control. Lysates from L1236 cells served as a positive control. C, L428, RC-K8, and RPMI-8226 cells (RPMI) were incubated with different doses of troglitazone (Tro) for 72 hours and viability was determined as described above. Similar results were obtained for all other cell lines tested in this study.

 

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  		Table 1. Characteristics of cell lines used in this study

 
15-Deoxy-{Delta}12,14-prostaglandin J2 inhibits constitutive activity of nuclear factor-{kappa}B and down-regulates antiapoptotic proteins X-linked inhibitor of apoptosis protein and heat shock protein 27. Constitutive transcriptional activity of NF-{kappa}B is known to play a central role in apoptosis resistance and proliferation of classic Hodgkin's lymphoma, ABC-DLBCL, and multiple myeloma cell lines. As 15dPGJ2 was shown in other cellular systems to inhibit NF-{kappa}B transcriptional activity by different mechanisms, we determined if NF-{kappa}B was also inhibited in our model cell lines. The classic Hodgkin's lymphoma cell line L428, the RC-K8 DLBCL cells, and the myeloma cell line RPMI-8226 were selected for these analyses, because they are derived from three different neoplastic entities. Moreover, L428 and RC-K8 cells lack expression of functional I{kappa}B{alpha} protein due to destructive mutations (Fig. 1B). These cell lines are therefore ideal models to study I{kappa}B{alpha} dependence of NF-{kappa}B-inhibitory strategies. Importantly, 15dPGJ2 strongly inhibited the DNA-binding capacity of nuclear NF-{kappa}B as determined by electrophoretic mobility shift assay after 6 hours of treatment irrespective of the mutational status of I{kappa}B{alpha} (Fig. 2A). Similar results were obtained with L1236, OCI-Ly3, and OPM-2 cells that were also analyzed by electrophoretic mobility shift assay (data not shown).



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  		Fig. 2. 15dPGJ2 inhibits constitutive DNA-binding activity of NF-{kappa}B and down-regulates antiapoptotic proteins. A, L428 cells (classic Hodgkin's lymphoma) and RC-K8 cells (DLBCL), both lacking I{kappa}B{alpha} protein, and RPMI-8226 cells (multiple myeloma) expressing I{kappa}B{alpha} were incubated for 6 hours with 15dPGJ (30 µmol/L; +) or vehicle control (ethanol; –) and nuclear extracts were prepared as described in Materials and Methods. The DNA-binding capacity of nuclear NF-{kappa}B complexes was determined by electrophoretic mobility shift assay. Arrow, shifted NF-{kappa}B bands. B, L428, RC-K8, and RPMI-8226 and U266 cells were incubated with 15dPGJ2 for the indicated times and cellular lysates were prepared at each time point. Blots were probed with antibodies recognizing XIAP, Hsp27, and actin as a loading control. Note that the upper band on the XIAP blots corresponds to a nonspecific cross-reacting nuclear protein as determined by using cytosolic extracts only.

 
XIAP and Hsp27 were shown recently to interfere with apoptotic signaling in classic Hodgkin's lymphoma and multiple myeloma, respectively (12, 13). Furthermore, we have observed high-level expression of XIAP protein throughout a panel of DLBCL cell lines, including ABC-DLBCL, arguing for a role of this protein also in apoptosis resistance in this lymphoma subtype.5 Additionally, XIAP is a known NF-{kappa}B target gene, and the HSPB1 gene encoding Hsp27 owns a noncanonical NF-{kappa}B-binding site in the promoter region.5 As 15dPGJ2 was strongly cytotoxic to the cell lines tested here and inhibited NF-{kappa}B, we were interested if 15dPGJ2 also leads to down-regulation of these proteins. We therefore analyzed the effect of 15dPGJ2 on the expression of XIAP and Hsp27 by Western blotting. Importantly, 15dPGJ2 strongly down-regulated the XIAP protein in all cell lines tested (Fig. 2B, top; data not shown). In most cells, this effect could already be observed at 6 hours of treatment, whereas at the 12-hour time point XIAP levels were down-regulated in all cell lines. Additionally, Hsp27 levels were also strongly diminished after 12 hours of treatment with 15dPGJ2 in RPMI-8226, U266, or OPM-2 multiple myeloma cells (Fig. 2B, bottom; data not shown). Taken together, XIAP and Hsp27 proteins, both known to be centrally involved in apoptosis resistance in B-cell malignancies, are down-regulated by treatment with 15dPGJ2.

15-Deoxy-{Delta}12,14-prostaglandin J2 induces apoptosis in germinal center–derived B-cell malignancies. To investigate if the cytotoxic effect of 15dPGJ2 is due to induction of apoptotic cell death, cells were incubated with 15dPGJ2 for 24 hours and apoptosis was measured by Annexin V binding and propidium iodide staining on a flow cytometer. 15dPGJ2 induced massive apoptosis in all cell lines tested (Fig. 3A; data not shown). To further exclude unspecific cytotoxicity of 15dPGJ2 and to further characterize the molecular mechanisms of 15dPGJ2-mediated apoptosis induction, all cell lines were preincubated with the pan-caspase inhibitor zVAD-Fmk followed by a 24-hour incubation with various doses of 15dPGJ2. Preincubation with zVAD-Fmk strongly abrogated apoptosis induction by 15dPGJ2 in all cell lines tested (data not shown). Taken together, 15dPGJ2 induces caspase-dependent apoptosis in different germinal center–derived B-cell lymphoma cell lines irrespective of their subtype and molecular defects in apoptosis signaling.



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  		Fig. 3. 15dPGJ2 induces apoptosis in B-cell malignancy-derived cell lines. A, L428, RC-K8, and RPMI-8226 cells were incubated with 15dPGJ2 (30 µmol/L) or vehicle control [ethanol (EtOH)] for 24 hours and apoptosis was determined by Annexin V binding and propidium iodide (PI) uptake on a flow cytometer. Similar results were obtained for other cell lines tested in this study (data not shown). B, L428, RC-K8, and RPMI-8226 cells were incubated with 15dPGJ2 (+) or vehicle control (–) for 24 hours and the effect on the {Delta}{psi}m was analyzed by JC-1 staining on a flow cytometer as described in Materials and Methods. Data are expressed as ratios of the mean fluorescence intensities of the FL1 and FL2 channels. Similar results were obtained for other cell lines tested in this study (data not shown). C, cellular lysates were prepared from L428, RC-K8, and RPMI-8226 cells that had been incubated with 15dPGJ2 (+) or vehicle control (–) for 12 hours and cleavage of caspase-3 was determined by Western blotting using an antibody specifically recognizing the 17- and 19-kDa cleavage products of caspase-3. Blots were reprobed for actin as a loading control.

 
Apoptotic stimuli may induce divergent signal transmission pathways within a cell (i.e., receptor-associated or mitochondria-associated pathways; ref. 36). Because multiple blocks in mitochondrial signaling have been described in diverse B-cell malignancies, we assessed if, in our model cell lines, 15dPGJ2-mediated apoptosis involves the disruption of the {Delta}{psi}m, an essential step in mitochondrial apoptosis. To this end, cells were incubated for 24 hours with 15dPGJ2 and the effect on {Delta}{psi}m was determined by flow cytometry assessing JC-1 staining. JC-1 forms potential-dependent aggregates that cause a fluorescence shift from red to green when excited with an argon-ion laser at 488 nm in cells undergoing apoptotic cell death. When treated with 15dPGJ2 for 24 hours, all cell lines tested exhibited a disruption of the {Delta}{psi}m indicated by a shift from the FL2 channel to the FL1 channel (Fig. 3B; data not shown). An important end point in mitochondrial signaling is cleavage of the effector caspase (caspase-3). Because this step is prevented in myeloma or lymphoma cells expressing Hsp27 or XIAP (12, 13), we investigated if caspase-3 was cleaved during 15dPGJ2-mediated apoptosis. Importantly, caspase-3 was cleaved after 12 hours of treatment with 15dPGJ2 as indicated by the detection of caspase-3 cleavage products in treated but not untreated L428, RC-K8, and RPMI-8226 cells (Fig. 3C). Thus, although germinal center–derived B-cell malignancies harbor multiple defects in mitochondrial apoptosis signaling, 15dPGJ2-mediated apoptosis involves the mitochondrial pathway in in vitro models of these neoplasms; it leads to disruption of the {Delta}{psi}m and, finally, cleavage of the effector caspase caspase-3.

15-Deoxy-{Delta}12,14-prostaglandin J2 inhibits nuclear translocation of nuclear factor-{kappa}B without requiring I{kappa}B{alpha}. Given the dependence of classic Hodgkin's lymphoma, DLBCL, and multiple myeloma cell lines on NF-{kappa}B transcriptional activity, we were interested in the functional mechanisms that underlie 15dPGJ2-mediated inhibition of NF-{kappa}B. Inhibitors of the IKK complex are currently in preclinical and early clinical development. These inhibitors are generally thought to require I{kappa}B{alpha} that is absent in a subset of B-cell-derived lymphomas. We were thus interested if nuclear translocation of the NF-{kappa}B subunit p65 that is negatively regulated by I{kappa}B{alpha} in physiologic conditions might be altered in a setting of experimental inhibition of the IKK complex. To this end, we analyzed the amount of nuclear p65 during 15dPGJ2-mediated killing of lymphoma cell lines. L428 and RC-K8 cells were chosen for these analyses because both lack I{kappa}B{alpha} protein (Fig. 1B). Immunofluorescence experiments were done on cytospins prepared from L428 and RC-K8 cells treated with 15dPGJ2. Surprisingly, the staining intensity of nuclear p65 was reduced in the 15dPGJ2-treated specimens compared with the specimens treated with vehicle control (Fig. 4, top). As expected, nuclear translocation of p65 occurred in Jurkat cells treated with tumor necrosis factor-{alpha} that served as a positive control in these experiments (Fig. 4, bottom). Thus, nuclear translocation of NF-{kappa}B is inhibited by 15dPGJ2 in L428 and RC-K8 lymphoma cells despite the lack of functional I{kappa}B{alpha} protein.



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  		Fig. 4. Nuclear translocation of NF-{kappa}B is inhibited by 15dPGJ2. L428 and RC-K8 cells, both lacking functional I{kappa}B{alpha} protein, were incubated with 15dPGJ2 (30 µmol/L) or vehicle control for 8 hours and spun onto glass slides. After fixation and permeabilization, cells were stained with an antibody recognizing the p65 subunit of the NF-{kappa}B complex and a secondary Cy3-labeled antibody yielding red fluorescence. Nuclei were counterstained with 4',6-diamidino-2-phenylindole yielding blue fluorescence. Slides were analyzed on a fluorescence microscope in a blinded manner to ensure investigator independence and images were taken using a digital camera. Overlays of recordings of red fluorescence (p65 protein) and blue fluorescence (nuclei). Jurkat cells incubated with tumor necrosis factor-{alpha} (TNF{alpha}; 50 ng/mL) for 30 minutes served as a control for nuclear entry of p65 in these experiments (bottom).

 
Inhibition of the I{kappa}B kinase complex is cytotoxic to cell lines with constitutively active nuclear factor-{kappa}B that lack functional I{kappa}B{alpha} protein. Our results imply that reversion of nuclear translocation of NF-{kappa}B can occur in the absence of I{kappa}B{alpha}. We therefore tested the hypothesis that curcumin, a compound that has also been shown to inhibit IKKs, might in consequence also be able to abrogate viability of L428 and RC-K8 cells via inhibition of the nuclear translocation of NF-{kappa}B (37). Confirming our hypothesis, curcumin strongly abrogated the viability of L428 and RC-K8 cells in doses reported previously to kill I{kappa}B{alpha} wild-type U266 and RPMI-8226 cells (Fig. 5A; ref. 37). To investigate if this cytotoxicity is due to inhibition of nuclear translocation of NF-{kappa}B, immunofluorescence experiments were carried out on cytospin specimens of L428 and RC-K8 cells treated with curcumin. Again, these experiments clearly showed a decrease in the amount of nuclear p65 in the curcumin-treated samples compared with those treated with vehicle control (Fig. 5B). Taken together, the IKK inhibitor curcumin inhibits nuclear translocation of NF-{kappa}B and is strongly cytotoxic to lymphoma cells lacking functional I{kappa}B{alpha} protein.



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  		Fig. 5. Inhibition of IKKs is cytotoxic to lymphoma cells lacking I{kappa}B{alpha}. A, L428 and RC-K8 cells lacking functional I{kappa}B{alpha} protein were incubated with different doses of the IKK inhibitor curcumin (black columns) or vehicle control (DMSO; white columns) for 72 hours. Viability was determined using the XTT assay and calculated as described in Materials and Methods. *, P < 0.05. B, L428 and RC-K8 cells, both lacking functional I{kappa}B{alpha} protein, were incubated with curcumin or vehicle control for 8 hours and analyzed by immunofluorescence microscopy as described above. Overlays of recordings of red fluorescence (p65 protein) and blue fluorescence (nuclei). C, L428 cells were incubated for 72 hours with either the negative control peptide (NBD–; white column) or the NBD-inhibitory peptide (NBD+; black column). Live cells were enumerated by trypan blue exclusion. D, L428 cells were treated with either NBD– or NBD+ for 12 hours and analyzed by immunofluorescence microscopy as described above. Overlays of recordings of red fluorescence (p65 protein) and blue fluorescence (nuclei). E, L428 cells were treated with NBD– or NBD+ for 24 hours and apoptosis was measured by Annexin V binding and propidium iodide uptake on a flow cytometer.

 
To this point, our results suggest that inhibition of NF-{kappa}B through inhibition of the IKK complex might occur in cells with destructive mutations of the I{kappa}B{alpha} gene. We aimed to better clarify this issue using an approach that is even more specific on the molecular level. Activation of the IKK complex requires interaction with its regulatory subunit, NF-{kappa}B essential modulator or IKK{gamma}, via its amino-terminal binding domain (14, 38). A short cell-permeable peptide (the NBD peptide) has been designed that specifically interferes with this interaction, leading to potent inhibition of NF-{kappa}B in vitro and in vivo (38, 39). This peptide is extremely specific, a fact that can be monitored using a control peptide that differs by only two amino acids (38). We hypothesized that if inhibition of IKKs leads to reduction in the amount of nuclear NF-{kappa}B in the absence of I{kappa}B{alpha}, direct molecular inhibition of IKK complex assembly would also prove cytotoxic to lymphoma cells lacking functional I{kappa}B{alpha} protein. To test this hypothesis, L428 cells were incubated with NBD peptide or with the respective control peptide NBD–. Confirming our hypothesis, NBD peptide significantly inhibited the growth of L428 lymphoma cells compared with NBD– (Fig. 5C). Moreover, NBD peptide but not the negative control peptide also strongly reduced nuclear p65 in L428 cells as determined by immunofluorescence (Fig. 5D). Finally, apoptosis was potently induced in L428 treated with the NBD peptide but not in the L428 cells treated with the negative control peptide as measured by Annexin V binding and propidium iodide uptake using flow cytometry (Fig. 5E). These results confirm that direct inhibition of the IKK complex inhibits the growth of L428 cells lacking I{kappa}B{alpha} by induction of apoptosis, paralleled by reduction of nuclear translocation of NF-{kappa}B.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Using 15dPGJ2 as a model compound, we show here that induction of apoptotic cell death is achieved by inhibition of nuclear entry as well as DNA-binding activity of the transcription factor NF-{kappa}B despite the absence of functional I{kappa}B{alpha} protein in a panel of B-cell-derived malignancy cell lines. Further experiments using curcumin that also inhibits IKKs, on I{kappa}B{alpha}-mutated lymphoma cells, corroborated our findings obtained with 15dPGJ2 that even B-cell-derived cell lines lacking I{kappa}B{alpha} were effectively killed and this effect was paralleled by the reduction of nuclear p65. Moreover, even when using a most specific inhibitory strategy (i.e., inhibition of the interaction of the different IKK complex members by using the NBD-inhibitory peptide), I{kappa}B{alpha}-defective L428 lymphoma cells were effectively killed and this process was paralleled by reduction of nuclear NF-{kappa}B and massive induction of apoptosis. The 15dPGJ2 treatment led to caspase-dependent apoptosis, down-regulation of XIAP and Hsp27 proteins, disruption of the {Delta}{psi}m, and cleavage of the executioner caspase (caspase-3) in classic Hodgkin's lymphoma, DLBCL, and multiple myeloma cell lines. These results show that targeting NF-{kappa}B activity by using inhibitors of the IKK complex is able to relieve the known antiapoptotic blocks in these malignancies, thereby effectively killing these cells. Moreover, they suggest that this strategy might be clinically feasible in lymphomas exhibiting constitutive NF-{kappa}B activity regardless of their I{kappa}B{alpha} mutational status.

This observation is important because most therapeutic approaches aiming at inhibition of NF-{kappa}B are targeting IKKs (40). Thus far, inhibition of NF-{kappa}B via inhibition of IKKs has been suggested to be I{kappa}B{alpha} dependent; therefore, such approaches might not be applicable to a subgroup of patients with B-cell malignancies (20–23). For example, arsenic compounds that are in preclinical and clinical evaluation were shown recently to inhibit NF-{kappa}B and to induce apoptosis in B-cell malignancies (26, 41), but this effect strongly depended on the presence of functional I{kappa}B{alpha} in the tumor cells (26). Our findings using 15dPGJ2 as a model, however, suggest that negative regulation of p65 nuclear entry and subsequent induction of apoptosis can be achieved in the absence of I{kappa}B{alpha} protein. One explanation for this finding might be that inhibition of IKKs leads to reduced phosphorylation of other I{kappa}B proteins, such as I{kappa}Bß, which may, in this setting, substitute for I{kappa}B{alpha}, thereby negatively regulating NF-{kappa}B. Importantly, I{kappa}Bß is expressed by both L428 and RC-K8 cell lines that served as I{kappa}B{alpha}-deficient models for classic Hodgkin's lymphoma and DLBCL in this study (21, 42). Cytoplasmic accumulation of I{kappa}Bß may subsequently hinder nuclear translocation of NF-{kappa}B heterodimers by binding to their nuclear localization signals. Although 15dPGJ2 and curcumin both do not solely act through inhibition of IKKs, the fact that nuclear entry of p65 was reduced following treatment with these compounds argues in favor of our conclusion (i.e., that negative regulation of nuclear translocation of p65 can occur in I{kappa}B{alpha}-defective cells). Furthermore, the reduction of nuclear NF-{kappa}B and the induction of apoptosis that were seen in I{kappa}B{alpha}-defective L428 cells treated with the NBD-inhibitory peptide but not in cells treated with the control peptide support this conclusion.

In an article that was published during the preparation of this article, 15dPGJ2 was shown to inhibit NF-{kappa}B by inhibition of IKKs, thereby inducing apoptosis in Burkitt's lymphoma and myeloma cell lines (34). Our findings are in line with this study, as apoptosis induction was caspase dependent and paralleled NF-{kappa}B inhibition. However, we show here in a preclinical setting that IKK inhibition may also be feasible in patients with I{kappa}B{alpha}-mutated tumors, an important issue in the field of molecular targeting of lymphomas.

The finding that a subset of primary classic Hodgkin's lymphoma cases and a DLBCL cell line harbor deleterious mutations in I{kappa}B{alpha} provides a mechanistic explanation for the constitutive activation of NF-{kappa}B (6, 21). Consequently, loss-of-function of I{kappa}B{alpha} likely leads to nuclear accumulation of NF-{kappa}B. Additionally, mutations in the gene encoding I{kappa}B{varepsilon} were recently detected in classic Hodgkin's lymphoma that may also contribute to activation of NF-{kappa}B (24). However, NF-{kappa}B complexes containing p65 can be detected not only in the nucleus but also, and in high amounts, in the cytoplasm of L428 and RC-K8 cells both lacking I{kappa}B{alpha} (Figs. 4 and 5). This observation underlines the major finding in this article (i.e., that the subcellular distribution of NF-{kappa}B is not completely dysregulated in the absence of I{kappa}B{alpha}). Our findings are in line with a recent article in which it was shown that inhibition of NF-{kappa}B by the proteasome inhibitor PS-341 (bortezomib) induces apoptosis in classic Hodgkin's lymphoma cell lines (43). Importantly, PS-341-mediated apoptosis also occurred in I{kappa}B{alpha}-mutant cells and was correlated to reduction of nuclear p65. However, PS-341-mediated effects are not solely attributable to inhibition of NF-{kappa}B. Inhibition of the proteasome, the major protein turnover machinery in the cell, evokes numerous other molecular sequelae, such as induction of a p53 target gene response, up-regulation of p27, or inhibition of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase pathway (44, 45). We have, however, observed reduction of nuclear NF-{kappa}B and induction of apoptosis in I{kappa}B{alpha}-mutant cells even when using a direct and specific inhibitor of the IKK complex, the NBD-inhibitory peptide.

Whereas I{kappa}B{alpha} is involved in rapid but transient activation of NF-{kappa}B, I{kappa}Bß mediates a sustained NF-{kappa}B response (14). It is thus conceivable that inactivation of I{kappa}B{alpha} has the strongest effect on NF-{kappa}B activation; supporting this idea, I{kappa}B{alpha} is in fact a target of oncogenic inactivation in B-cell-derived malignancies (6, 21). The observation that reintroduction of wild-type I{kappa}B{alpha} is sufficient for induction of apoptosis in L428 and RC-K8 cells further corroborates the concept that I{kappa}B{alpha} gene mutations are necessary for malignant transformation in the mutated cases (2, 21). We now propose a model in which I{kappa}B{alpha} gene mutations are an integral part of malignant transformation in a subset of germinal center–derived B-cell malignancies, as they lead to inactivation of the predominant negative feedback loop of NF-{kappa}B. Nevertheless, I{kappa}Bß may still be sufficient to reduce nuclear translocation in response to inhibition of the IKK complex. Indeed, in KM-H2 cells that lack expression of I{kappa}B{alpha} protein due to a truncating mutation, the IKK complex is constitutively active, supporting the idea that for full malignant transformation the inhibitory function of I{kappa}Bß needs to be antagonized even in the absence of I{kappa}B{alpha} (42). Therefore, targeting the IKK complex seems to be a promising strategy for a molecularly targeted therapy in B-cell malignancies irrespective of the I{kappa}B{alpha} mutational status.

Taken together, our results suggest that inhibition of NF-{kappa}B via inhibition of IKKs is an attractive therapeutic approach in germinal center–derived B-cell malignancies that reverses the neoplastic apoptosis-resistant phenotype and efficiently kills the malignant cell population. Because current therapies of classic Hodgkin's lymphoma are highly toxic and given the lack of a truly curative treatment option for multiple myeloma and a high proportion of DLBCL, novel therapeutic strategies aiming directly at the molecular abnormalities that cause and sustain malignant growth are promising (46–48). It is presently unclear if 15dPGJ2 may itself be used as a therapeutic agent because of its potential toxicity. However, as was the case with staurosporine that has extensively been used for studying apoptosis pathways in cancer cells and that has now entered clinical trials as 7-hydroxystaurosporine (UCN-01), a modified 15dPGJ2 derivative may prove useful as a novel, NF-{kappa}B-targeted drug in B-cell malignancies (49). Additionally, curcumin is currently in clinical development and first results suggest that this compound is generally well tolerated (50). Finally, the NBD peptide has shown excellent activity in a preclinical in vivo model of NF-{kappa}B-driven inflammatory disease (39). At the moment, it is still unclear if this compound will also prove beneficial in human lymphomas. However, our results provide a proof-of-concept of IKK inhibition throughout a spectrum of germinal center–derived B-cell malignancies, which was active in lymphoma cells lacking functional I{kappa}B{alpha} protein. In the era of individualized targeted therapy, this means that more patients may now be eligible for such a treatment strategy.


   Acknowledgments
 
We thank Julia Claasen and Ute Sandaradura de Silva for excellent technical assistance, Dr. Ralf Küppers for providing the OCI-Ly3 and OCI-Ly10 cells, Drs. Martin Dyer and Reiner Siebert for providing the RC-K8 cells, and Dr. Carien Niessen for invaluable discussion and her steady helpfulness.


   Footnotes
 
Grant support: Deutsche Forschungsgemeinschaft grant SFB502; Center for Molecular Medicine Cologne; Alexander von Humboldt Foundation, Sofja Kovalefskaja award (J.L. Schultze); Hugo Feger Nachlass and Frauke-Weiskam Foundation fellowship (T. Zander); and Innovationsprogramm Forschung (Ministerium für Schule, Wissenschaft und Forschung Nordrhein-Westfalen) awards and Deutsche Forschungsgemeinschaft-Heisenberg program (S. Smola-Hess).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: R.K. Thomas is currently at the Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115. R.K. Thomas, M.L. Sos, and T. Zander contributed equally to this work. J.L. Schultze and J. Wolf share equal senior authorship.

4 T. Zander, unpublished results. Back

5 R.K. Thomas, unpublished results. Back

Received 2/ 2/05; revised 6/13/05; accepted 7/ 7/05.


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 Results
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