The proteasome inhibitor bortezomib promotes mitochondrial ... - Nature

Leukemia (2003) 17, 2036–2045 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu

MOLECULAR TARGETS FOR THERAPY (MTT)

The proteasome inhibitor bortezomib promotes mitochondrial injury and apoptosis induced by the small molecule Bcl-2 inhibitor HA14-1 in multiple myeloma cells X-Y Pei1, Y Dai1 and S Grant1,2,3 1

Department of Medicine, Virginia Commonwealth University/Medical College of Virginia, Richmond, VA, USA; 2Department of Biochemistry, Virginia Commonwealth University/Medical College of Virginia, Richmond, VA, USA; and 3Department of Pharmacology, Virginia Commonwealth University/Medical College of Virginia, Richmond, VA, USA

Interactions between the small molecule Bcl-2 inhibitor HA14-1 and proteasome inhibitors, including bortezomib (Velcadet; formerly known as PS-341) and MG-132, have been examined in human multiple myeloma cells. Sequential (but not simultaneous) exposure of MM.1S cells to bortezomib or MG-132 (10 h) followed by HA14-1 (8 h) resulted in a marked increase in mitochondrial injury (loss of DWm, cytochrome c, Smac/DIABLO, and apoptosis-inducing factor release), activation of procaspases-3, -8, and -9, and Bid, induction of apoptosis, and loss of clonogenicity. Similar interactions were observed in U266 and MM.1R dexamethasone-resistant myeloma cells. These events were associated with Bcl-2 cleavage, Bax, Bak, and Bad accumulation, mitochondrial translocation of Bax, abrogation of Mcl-1, Bcl-xL, and XIAP upregulation, and a marked induction of JNK and p53. Bortezomib/HA14-1 treatment triggered an increase in reactive oxygen species (ROS), which, along with apoptosis, was blocked by the free radical scavenger N-acetyl-L-cysteine (L-NAC). L-NAC also opposed bortezomib/HA14-1-mediated JNK activation, upregulation of p53 and Bax, and release of cytochrome c and Smac/DIABLO. Finally, bortezomib/HA14-1-mediated apoptosis was unaffected by exogenous IL-6. Together, these findings indicate that sequential exposure of myeloma cells to proteasome and small molecule Bcl-2 inhibitors such as HA14-1 may represent a novel therapeutic strategy in myeloma. Leukemia (2003) 17, 2036–2045. doi:10.1038/sj.leu.2403109 Keywords: apoptosis; myeloma; Bcl-2; HA14-1; ROS; mitochondrial injury

Introduction Over the last decade, considerable attention has focused on the control of apoptosis, a genetically regulated, evolutionarily conserved program of cell suicide associated with caspase activation that is involved in neoplastic transformation as well as in the response of neoplastic cells to diverse cytotoxic drugs.1 Two major apoptotic pathways have been described: the extrinsic pathway, which is triggered by binding of members of the tumor necrosis factor family to cell surface receptors,2 and the intrinsic or mitochondrial pathway, which is primarily activated by events that induce mitochondrial injury.3 MitoCorrespondence: Dr S Grant, Division of Hematology/Oncology, Medical College of Virginia, Virginia Commonwealth University, MCV Station Box 230, Richmond, VA 23298, USA; Fax: þ 1 804 828 8079 Received 2 May 2003; accepted 30 June 2003

chondrial damage results in loss of mitochondrial membrane potential (DCm), and release of multiple proapoptotic proteins (eg cytochrome c, Smac/DIABLO, apoptosis-inducing factor, AIF)3–6 into the cytoplasm, where they trigger caspase activation and degradation of diverse cellular constituents. Release of cytochrome c leads to activation of the so-called apoptosome, consisting of apoptotic protease activating factor-1 (apaf-1), dATP, cytochrome c, and procaspase-9, which induces cleavage and activation of the executioner procaspase-3.7 Apoptosis is regulated by an expanding family of Bcl-2-related proteins, of which some (eg Bcl-2, Bcl-xL, A1, and Mcl-1) block cell death, while others (eg Bax, Bak, Bim, and Bid) promote lethality.8 Although the mechanism(s) by which Bcl-2 opposes apoptosis remains the subject of controversy, recent evidence suggests that it largely acts by interfering with the ability of proapoptotic proteins such as Bax and Bak to initiate mitochondrial injury.9 The importance of Bcl-2 in promoting neoplastic cell survival has prompted the search for strategies capable of overcoming its cytoprotective activities. One such strategy involves the administration of Bcl-2 antisense oligonucleotides (eg G3139; Genasense), which diminish levels of the Bcl-2 protein.10 An alternative approach involves the use of small molecules that bind to and inhibit the biologic functions of Bcl-2. An example of this class of compounds is HA14-1, a small molecule Bcl-2interacting agent that induces mitochondrial injury, caspase activation, and apoptosis.11 Multiple myeloma is an incurable plasma cell dyscrasia characterized by dysregulation of diverse signal transduction pathways and impairment in apoptosis.12 Over the last several years, a number of novel agents have shown therapeutic potential in myeloma, including angiogenesis inhibitors such as thalidomide13 and arsenic trioxide, an agent that kills cells through a redox-related mechanism.14 Among the most promising of these new agents is the dipeptidyl boronic acid proteasome inhibitor bortezomib (Velcadet; previously known as PS-341).15 Proteasome inhibitors interfere with the function of the 26S proteasome complex, thereby disrupting the degradation and/or function of diverse cell cycle-, signaling-, and survival-related proteins, including NF-kB, p53, Bax, and stressactivated kinases, among others.16,17 For reasons that remain unclear, proteasome inhibitors are lethal to tumor cells, while relatively sparing toward their normal counterparts.18 In preclinical studies, proteaseome inhibitors such as bortezomib disrupt multiple signaling pathways and potently induce apoptosis in multiple myeloma cells.19 In accord with these

Bortezomib and HA14-1 in multiple myeloma X-Y Pei et al

2037 findings, results from early clinical studies indicate that bortezomib exhibits impressive activity in patients with multiple myeloma, including those refractory to prior therapy.20 Interestingly, bortezomib has been shown to induce apoptosis through a Bcl-2-independent mechanism, at least in human leukemia cells.21 Whether similar events occur in multiple myeloma cells is not yet known. While combinations of small molecule Bcl-2 inhibitors and conventional cytotoxic drugs may lead to enhanced tumor cell killing,22 interactions between the former agents and proteasome inhibitors remain largely unexplored. To address this issue, we have examined interactions between HA14-1 and bortezomib in human multiple myeloma cells. Our results indicate that sequential exposure of these cells to bortezomib followed by HA14-1 results in a marked increase in mitochondrial injury, caspase activation, and apoptosis, accompanied by multiple perturbations in signal transduction- and survival-related proteins. Moreover, enhanced lethality of bortezomib and HA14-1 toward multiple myeloma cells appears to operate through a free radical-related mechanism. Together, these findings suggest a potential role for combining small molecule Bcl-2 antagonists with proteasome inhibitors such as bortezomib in multiple myeloma and possibly other hematologic malignancies.

Materials and methods

or to the sequence HA14-1 followed by proteasome inhibitors. In some studies, cells were pretreated with L-NAC for 3 h prior to proteasome inhibitors. IL-6 was added concurrently with proteasome inhibitors. After drug treatment, cells were harvested and subjected to further analysis as described below.

Assessment of apoptosis The extent of apoptosis was evaluated by assessing Wright– Giemsa-stained cytospin slides under light microscopy and scoring the number of cells exhibiting classic morphological features of apoptosis. For each condition, 5–10 randomly selected fields per slide were evaluated, encompassing at least 800 cells. To confirm the results of morphologic analysis, in some cases cells were also evaluated by Annexin V-FITC staining. Briefly, 1 106 cells were washed twice with cold PBS and then resuspened in 1 binding buffer (10 mM HEPES/ NaOH, pH 7.4, 140 mM NaOH, 2.5 mM CaCl2). The cells were incubated with Annexin V-FITC (BD PharMingen, San Diego, CA, USA) and 5 mg/ml propidium iodide (PI), and incubated for 15 min at room temperature in the dark as per the manufacturer’s instructions. The samples were analyzed by flow cytometry within 1 h to determine the percentage of cells displaying Annexin V staining (early apoptosis) or both Annexin V and PI staining (late apoptosis).

Cells and reagents The human multiple myeloma cell line U266 was purchased from ATCC. The dexamethasone-sensitive (MM.1S) and -resistant (MM.1R) human MM cell lines were kindly provided by Dr Steven T Rosen.23 Cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 200 U/ml penicillin, 200 mg/ml streptomycin, minimal essential vitamins, sodium pyruvate, and glutamine as previously described.24,25 The specific proteasome inhibitor bortezomib was kindly provided by Sarah Waywell, Millenium Inc. (Cambridge, MA, USA). A cell-permeable, low-molecular-weight Bcl-2 inhibitor HA14-1 and the proteasome inhibitor MG-132 were purchased from Biomol (Plymouth Meeting, PA, USA), and JNK inhibitor SP600125 from Calbiochem (San Diego, CA, USA). They were dissolved in DMSO as a stock solution, stored at 201C. N-acetylL-cysteine (L-NAC, Calbiochem, San Diego, CA, USA) was prepared in sterile water immediately before use. Arsenic trioxide (As2O3), dexamethasone (Dex), and recombinant human IL-6 were supplied by Sigma Chemicals (St Louis, MO, USA). As2O3 was dissolved in 1.65 M NaOH at 50 mM as a stock solution. Dexamethasone was dissolved in DMSO, aliquoted and stored at 201C. Recombinant human IL-6 was rehydrated in phosphatebuffered saline (PBS) containing 0.1% bovine serum albumin (BSA), aliquoted and stored at 801C. In all experiments, the final concentration of DMSO did not exceed 0.1%.

Experimental format All experiments were performed utilizing logarithmically growing cells (4–6 105 cells/ml). Cell suspensions were placed in sterile FALCON tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ, USA), and incubated with either bortezomib or MG-132 for 10 h at 371C. At the end of this period, HA14-1 was added to the suspension, and the dishes placed in a 371C, 5% CO2 incubator for various intervals. Alternatively, cells were exposed to proteasome inhibitors and HA14-1 simultaneously,

Mitochondrial membrane potential (DCm) assay After drug treatment, 2 105 cells were incubated with 40 nM 3,3-dihexyloxacarbocyanine (DiOC6, Molecular Probes Inc., Eugene, OR, USA) in PBS at 371C for 20 min, and then analyzed by flow cytometry. The percentage of cells exhibiting low level of DiOC6 uptake, which reflects loss of mitochondrial membrane potential, was determined using Becton-Dickinson FACScan (Becton-Dickinson, San Jose, CA, USA).

Western blot assay After drug treatment, whole-cell pellets were lysed by sonication in 1 sample buffer (62.5 mM Tris base, pH 6.8, 2% SDS, 50 mM DTT, 10% glycerol, 0.1% bromophenol blue, and 5 mg/ ml each chymostatin, leupeptin, aprotitin, pepstatin, and soybean trypsin inhibitor) and boiled for 5 min. For analysis of protein phosphorylation, 1 mM each of Na vanadanate and Na pyrophosphate was added to 1 sample buffer. Protein samples were harvested as the supernatant following centrifugation of the samples at 12 800 g for 5 min, and the amount of protein quantified using Coomassie Protein Assay Reagent (Pierce, Rockford, IL, USA). Equal amounts of protein (25 mg) were separated by SDS-PAGE and electrotransferred onto nitrocellulose membrane. For blotting phosphoproteins, no SDS was included in the transfer buffer. The blots were blocked with 5% milk in PBS–Tween 20 (0.1%) at room temperature for 1 h and probed with the appropriate dilution of primary antibody in 5% BSA/PBS–Tween 20 overnight at 41C. The membranes were washed twice in PBS–Tween 20 for 30 min and then incubated with a 1:2000 dilution of HRP-conjugated secondary antibody (Kirkegaard and Perry, Gaithersburg, MD, USA) in 5% milk/ PBS–Tween 20 at room temperature for 1 h. After washing twice in PBS–Tween 20 for 30 min, the blots were visualized by Western Blot Chemiluminescence Reagent (NEN Life Science Products, Boston, MA, USA). For blots of phosphoproteins, TBS Leukemia


2038 was used instead of PBS throughout. Where indicated, the blots were reprobed with antibodies against a-actin or b-tubulin (BD PharMingen) to ensure equal loading and transfer of proteins. The following antibodies were used as primary antibodies: PARP antibody (mouse monoclonal, Biomol), anticaspase-3 (rabbit polyclonal, PharMingen, San Diego, CA, USA), anticaspase-9 (rabbit polyclonal, PharMingen), anticaspase-8 (mouse monoclonal, Alexis, San Diego, CA, USA), Bid antibody (rabbit polyclonal, Santa Cruz, Santa Cruz, CA, USA), Bax antibody (rabbit polyclonal, Santa Cruz), Bak antibody (rabbit polyclonal, Santa Cruz), Bad antibody (rabbit polyclonal, Santa Cruz), anti-human Bcl-2 oncoprotein (mouse monoclonal, Dako, Carpinteria, CA), XIAP antibody (rabbit polyclonal, Cell Signaling, Beverly, MA, USA), Mcl-1 antibody (mouse monoclonal, PharMingen), phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (rabbit polyclonal, Cell Signaling), phospho-p38 MAP kinase (Thr180/Tyr182) antibody (rabbit polyclonal, Cell Signaling), phospho-JNK (Thr183/Tyr185) antibody (mouse monoclonal, Santa Cruz), JNK1 antibody (mouse monoclonal, Santa Cruz), JNK2 antibody (mouse monoclonal, Santa Cruz), antip21CIP/WAF1 (mouse monoclonal, Transduction Lab.), antip27KIP1 (mouse monoclonal, PharMingen), and p53 antibody (mouse monoclonal, Santa Cruz).

Analysis of cytosolic cytochrome c, Smac/DIABLO, and AIF After drug treatment, 4 106 cells were washed in PBS and lysed by incubating them for 30 s in lysis buffer (75 mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mM EDTA, and 350 mg/ml digitonin). The lysates were centrifuged at 12 000 g for 1 min, and the supernatant was collected in an equal volume of 2 sample buffer.24 The proteins were quantified, separated by 15% SDS-PAGE, and subjected to Western blot as described above. Cytochrome c antibody (mouse monoclonal, PharMingen) and Smac/DIABLO antibody (rabbit polyclonal, Upstate Biotechnology, Lake Placid, NY, USA), and AIF antibody (mouse monoclonal, Santa Cruz) were used as primary antibody.

scored using an Olympus Model CK inverted microscope, and colony formation for each condition calculated in relation to values obtained for untreated control cells.

Measurement of cellular ROS production Dichlorodihydrofluorescein (DCF), which is nonfluorescent in its dihydro form but becomes highly fluorescent upon reaction with reactive oxygen species (ROS), was used to monitor production of cellular ROS.26 Briefly, 2 105 cells were incubated with 10 mM acetooxymethyl ester of dihydro-DCF (Molecular Probes) in PBS at 371C for 30 min, and then analyzed by flow cytometry. The production of ROS was determined by comparing increased intensity of DCF for drug-treated vs untreated control cells.

Statistical analysis For morphological assessment of apoptotic cells, Annexin V analysis, analysis of DCm, analysis of ROS production, and clonogenic assays, experiments were repeated at least three times. Values represent the means7s.d. for at least three separate experiments performed in triplicate. The significance of differences between experimental variables was determined using the Student’s t-test. For analysis of interactions between bortezomib and HA14-1, median dose effect analysis was employed using a commercially available software program (Calcusyn; Biosoft; Ferguson, M, USA).27 In this method, cells are exposed to each agent alone and in combination over a range of concentrations at a fixed ratio (eg HA14-1:bortezomib ¼ 3000:1). For each fraction affected (FA), corresponding to the percentage of cells that have undergone cell death, a combination index (CI) value is calculated. CI values o1.0 correspond to synergistic interactions, CI values 41.0 reflect antagonistic interactions, and CI values equal to 1.0 denote additive interactions.

Results

Analysis of Bax translocation After drug treatment, 4 106 cells were washed in PBS and lysed in digitonin lysis buffer as described above in the analysis of cytosolic cytochrome c, Smac/DIABLO, and AIF. After centrifugation at 12 000 g for 1 min, the supernatant was collected in an equal volume of 2 sample buffer (cytosolic fraction), and the pellet was lysed by sonication in 1 sample buffer as described in the Western blot assay (pellet fraction). For both cytosolic and pellet fractions, the proteins were quantified, separated by 15% SDS-PAGE, and subjected to Western blot using Bax antibody as primary antibody.

Clonogenic assays Colony-forming ability following drug treatment was evaluated using a soft-agar cloning assay as described previously.25 Briefly, cells were washed three times with serum-free RPMI medium. Subsequently, 500 cells/well were combined with RPMI medium containing 20% FBS and 0.3% agar, and plated on 12-well plates (three wells per condition). The plates were then maintained in a 371C/5% CO2, fully humidified incubator. After 15 days of incubation, colonies consisting 450 cells were Leukemia

To evaluate interactions between bortezomib and HA14-1 in myeloma cells, MM. 1S and U266 cells were simultaneously exposed to 10 mM HA14-172.2 nM bortezomib for 18 h or 12.5 mM HA14-172.3 nM for 24 h, respectively, after which apoptosis was assessed. As shown in Figure 1a and b, the drugs administered individually were minimally toxic, and coadministration of both agents resulted in only a small increase in cell death. Moreover, similar results were obtained in each cell line when cells were exposed to HA14-1 (10 h) followed by bortezomib (MM.1S, 8 h; U266, 14 h). However, when cells were preincubated with bortezomib for 10 h followed by an exposure to HA14-1 (MM.1S, 8 h; U266, 14 h), a very substantial increase in apoptosis (ie to levels approaching 70%) was observed. In addition, concordant results were obtained when loss of mitochondrial membrane potential (DCm) was monitored (data not shown). These results indicate that sequential (but not simultaneous) exposure of multiple myeloma cells to bortezomib followed by the small molecule Bcl-2 inhibitor HA14-1 (but not the reverse sequence) results in a marked increase in mitochondrial damage and apoptosis. Based on these findings, the experimental protocol for subsequent studies involved proteasome inhibitor pretreatment followed by HA14-1 exposure.


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Figure 1 Bortezomib potentiates the lethality of HA14-1 in MM cells in a sequence-dependent manner. MM.1S (a) or U266 (b) cells were treated either (1) simultaneously with bortezomib (btzmb; MM.1S: 2.2 nM; U266: 2.3 nM) þ HA14-1 (HA; MM.1S: 10 mM; U266: 12.5 mM) for 18 h (MM.1S) or 24 h (U266); (2) sequentially with btzmb (10 h) followed by HA (MM.1S: 8 h; U266: 14 h); or (3) sequentially with HA14-1 (10 h) followed by btzmb (MM.1S: 8 h; U266: 14 h). At the end of this period, the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright–Giemsa-stained cytospin preparations. Values represent the means7s.d. for three separate experiments performed in triplicate.

Interactions between HA14-1 and bortezomib vs another proteasome inhibitor, MG-132, were then compared (Figure 2). Exposure of MM.1S cells to either bortezomib (2.2 nM) or MG132 (120 nM; 10 h each) followed by 10 mM HA14-1 resulted in very similar increases in the percentage of cells exhibiting loss of DCm (Figure 2a) and Annexin V/PI positivity (Figure 2b). Comparable results were obtained when loss of DCm was monitored in U266 multiple myeloma cells (Figure 2c) as well as in a dexamethasone-resistant multiple myeloma cell line (MM.1R; data not shown). Finally, median dose effect analysis of apoptosis induction by bortezomib followed by HA14-1 (administered over a range of concentrations at a fixed ratio of 1:3000) was examined. CI values for each FA were considerably less than 1.0, corresponding to synergistic interactions (Figure 2d).

Figure 2 Bortezomib promotes mitochondrial injury and apoptosis induced by HA14-1 in MM cells. (a) MM.1S cells were incubated for 10 mh with 2.2 nM bortezomib (btzmb) or 120 nM MG-132 (MG) followed by treatment for 8 h with 10 mM HA14-1 (HA). At the end of this interval, the percentage of cells exhibiting reduced mitochondrial membrane potential (DCm) was determined by monitoring DiOC6 uptake. (b) Alternatively, the percentage of apoptotic cells was monitored by Annexin V-FITC staining and flow cytometry. Annexin V þ /PI corresponds to ‘early apoptotic cells’; Annexin V þ /PI þ corresponds to ‘late apoptotic cells’. (c) U266 cells were pretreated for 10 h with of 2.3 nM btzmb or 120 nM MG-132 (MG) followed by incubation for 14 h with 12.5 mM HA. At the end of this period, the percentage of cells exhibiting reduced DCm was determined as above. For (a)–(c), results represent the means7s.d. for three separate experiments performed in triplicate. (d) MM.1S cells were exposed to a range of btzmb and HA14-1 concentrations alone and in combination at fixed ratios (eg 1:3000) (btzmb, 10 h-HA, 8 h). At the end of this period, the percentage of cells exhibiting apoptotic morphology was determined for each condition; FA values were determined by comparing results to those of untreated controls, and median dose effect analysis employed to characterize the nature of the interaction. CI values less than 1.0 denote a synergistic interaction. Two additional studies yielded equivalent results.

Time course studies in MM.1S cells preincubated with bortezomib for 10 h revealed a major increase in apoptosis 8 h after addition of HA14-1, and a modest increase thereafter (Figure 3a). Dose–response studies revealed that bortezomib concentrations as low as 1 nM significantly increased the lethal effects of a minimally toxic concentration of HA14-1 (ie 10 mM), and that higher bortezomib concentrations resulted in further lethality (Figure 3b). Moreover, subsequent administration of HA14-1 at concentrations X5.0 mM to bortezomib-pretreated cells resulted in a significant increase in apoptosis, which occurred in the large majority of cells at HA14-1 concentrations X10.0 mM (Figure 3c). Finally, in view of the evidence that loss of clonogenic survival may not correlate with apoptosis,28 clonogenic assays were performed in MM.1S cells exposed to HA14-17bortezomib. While HA14-1 alone was minimally toxic, and bortezomib administered alone only slightly more so, sequential exposure to bortezomib followed by HA14-1 resulted in a highly significant reduction in clonogenic survival (ie B7% of controls, Figure 3d). Leukemia


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Figure 3 Bortezomib and HA14-1 interact in a time- and dosedependent manner to induce apoptosis in MM cells and result in a marked reduction in colony formation. (a) After 10 h preincubation with 2.2 nM bortezomib (btzmb), MM.1S cells were treated with 10 mM HA14-1 (HA). At the indicated time points, the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright–Giemsa-stained cytospin preparations. (b) MM.1S cells were pretreated with a range of btzmb concentrations for 10 h, after which they were exposed to 10 mM HA for an additional 8 h. (c) Alternatively, MM.1S cells were pretreated with 2.2 nM btzmb for 10 h followed by an exposure to the indicated HA concentration for an additional 8 h. In each case, the percentage of cells exhibiting apoptotic morphology was determined as above. (d) MM.1S cells were treated for 8 h with 10 mM HA following a 10 h preincubation with 2.2 nM btzmb. The cells were washed free of drug and plated in soft agar as described in Materials and methods. After 15 days of incubation, colonies, consisting of groups of 450 cells, were scored, and colony formation for each condition expressed relative to untreated control cells. Results represent the means7s.d. for three separate experiments performed in triplicate.

Western blot analysis was employed to assess the effects of sequential exposure of MM1.S and U266 cells to bortezomib and HA14-1 on mitochondrial damage and caspase activation (Figure 4a and b). HA14-1 administered alone had minimal effects on cytosolic release of the proapoptotic proteins cytochrome c, Smac/DIABLO, and AIF, whereas bortezomib induced relatively modest degrees of mitochondrial damage in both cell lines. However, sequential administration of bortezomib followed by HA14-1 induced extensive cytosolic release of these proteins. Consistent with these findings, combined treatment with bortezomib followed by HA14-1 resulted in a significant increase in cleavage/activation of procaspases-3, -8, and -9, Bid, and PARP (Figure 4a and b). These findings indicate that sequential exposure of cells to bortezomib followed by HA14-1 potently induces mitochondrial injury and caspase activation in multiple myeloma cells. Effects of sequential exposure of multiple myeloma cells to bortezomib and HA14-1 were then examined in relation to the expression and intracellular disposition of several apoptotic regulatory proteins. As shown in Figure 5a, treatment of MM.1S cells with bortezomib for 10 h7HA14-1 for 2 h resulted in an increase in the levels of the proapoptotic proteins Bax, Bak, and Bad. Moreover, the formation of a putatively proapoptotic Bcl-2 cleavage product29 was faintly discernible. In addition, while exposure of cells to bortezomib or HA14-1 alone resulted in a Leukemia

Figure 4 Sequential exposure to bortezomib and HA14-1 induces release of proapoptotic molecules from mitochodria and activation of caspases in MM cells. (a) MM.1S cells were incubated for 8 h with 10 mM HA14-1 (HA) following a 10 h pretreatment with 2.2 nM bortezomib (btzmb). (b) Alternatively, U266 cells were treated for 14 h with 12.5 mM HA after 10 h pretreatment with 2.3 nM btzmb. The cells were then lysed and subjected to Western blot analysis using the indicated primary antibodies. CF ¼ cleavage fragment. Alternatively, cytosolic (S-100) fractions were obtained as described in Materials and methods, and expressions of cytochrome c, Smac/DIABLO, and AIF were monitored by Western blot. Each lane was loaded with 25 mg of protein; the blots were stripped and reprobed with anti-actin or antitubulin antibody for equal loading and transfer. Two additional studies yielded equivalent results.

modest redistribution of Bax from the cytosol to the mitochondria, reflected by an increase in Bax expression in the pelleted fraction, combined treatment with both agents was associated with a very significant increase in Bax expression in the cell pellet, corresponding to extensive mitochondrial translocation of this protein. When MM.1S cells were exposed to bortezomib (10 h) followed by HA14-1 for 8 h, expression of the Bcl-2 cleavage product was substantially more pronounced than that observed after 2 h (Figure 5b). Moreover, cells exposed to bortezomib alone exhibited increased expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and XIAP. However, these increases were largely abrogated in cells subsequently treated with HA14-1. Thus, subsequent exposure of bortezomib-pretreated multiple myeloma cells to HA14-1 increased expression of a proapoptotic cleavage product of Bcl-2 and abrogated enhanced expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and XIAP. The effects of bortezomib and HA14-1 were then examined in relation to the expression of several signaling proteins (Figure 6a). Exposure of MM.1S cells to HA14-1 (8 h) alone resulted in a small increase in phosphorylation (activation) of JNK, whereas bortezomib (18 h) was more effective in this regard, consistent with previous reports.17 However, sequential exposure of cells to bortezomib followed by HA14-1 resulted in a substantial increase in JNK activation. Cells exposed to bortezomib7HA14-1 exhibited a modest increase in the levels of phospho-p38 MAPK, but no changes in the levels of total or phospho-ERK. As previously reported,16,30 treatment of cells with the proteasome inhibitor (7HA14-1) resulted in the induction of p53, although to a greater extent in cells exposed to both agents. Notably, exposure of cells to HA14-1 alone


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Figure 5 Sequential administration of bortezomib and HA14-1 results in perturbations in expressions of pro- and antiapoptotic proteins. After 10 h preincubation with 2.2 nM bortezomib (btzmb), MM.1S cells were treated with 10 mM HA14-1 (HA) for 2 h (a) or 8 h (b), after which expression of antiapoptoic proteins (eg Bcl-2, Bcl-xL, XIAP, and Mcl-1) and proapoptotic proteins (eg Bax, Bak, and Bad) were monitored by Western blot. CF ¼ cleavage fragment. Each lane was loaded with 25 mg of protein; the blots were stripped and reprobed with anti-actin antibody to ensure equal loading and transfer. Alternatively, translocation of Bax was analyzed by monitoring expression in the cytosolic and pelleted fractions by Western blot assay as described in Materials and methods (a; lower panels). The results were representative of three separate experiments.

resulted in a very modest induction of p53. Treatment of cells with bortezomib7HA14-1 was accompanied by upregulation of the cyclin-dependent kinase inhibitors p21CIP1 and p27KIP1. Cleavage of p21CIP1 was observed in cells exposed to both bortezomib and HA14-1, whereas p27KIP1 cleavage was observed in cells treated with both agents or with bortezomib alone. Lastly, a clear increase in phosphorylation of JNK in bortezomib/HA14-1-treated cells was noted as early as 30 min after HA14-1 administration, and was very pronounced following 14 h of drug administration (Figure 6b). Similarly, exposure to HA14-1 significantly increased p53 expression in bortezomib-pretreated cells within 30 min of drug administration, and this became even more evident after 14 h. As impairment in Bcl-2 function is known to result in mitochondrial dysfunction,31 the effects of bortezomib and

Figure 6 Sequential exposure to bortezomib and HA14-1 induces activation of JNK and upregulation of p53. (a) MM.1S cells were incubated for 8 h with 10 mM HA14-1 (HA) following a 10 h exposure to 2.2 nM bortezomib (btzmb). Cells were then lysed and subjected to Western blot analysis using the indicated primary antibodies. CF ¼ cleavage fragment. (b) MM.1S cells were treated with 10 mM HA following pretreatment with 2.2 nM btzmb as described above, after which Western blot samples were harvested at the indicated intervals to monitor time-dependent changes in expressions of JNK phosphorylation and p53 expression. For (a) and (b), each lane was loaded with 25 mg of protein; the blots were stripped and reprobed with anti-actin antibody to ensure equal loading and transfer. Two additional studies yielded equivalent results.

HA14-1 on generation of ROS and loss of mitochondrial membrane potential were examined. To this end, the free radical scavenger L-NAC (10 mM) was employed. As shown in Figure 7a, treatment of U266 cells with bortezomib (10 h) or HA14-1 (30 min) individually had little effect on ROS generation. However, exposure of bortezomib-pretreated cells to HA14-1 (ie 30 min following addition of HA14-1) resulted in a significant increase in the percentage of cells displaying an increase in ROS species (Po0.02 vs values obtained for either agent alone). As anticipated, coadministration of L-NAC abrogated the increase in ROS generation. Consistent with these findings, coadministration of L-NAC substantially diminished bortezomib/HA14-1-mediated loss of DCm in U266 cells (Figure 7b). As a control, L-NAC also reduced As2O3-mediated mitochondrial injury (data not shown), as previously Leukemia


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Figure 7 L-NAC blocks ROS production and attenuates mitochondrial injury induced by sequential exposure to bortezomib and HA141in MM cells. (a) U266 cells were incubated for 30 min with 12.5 mM HA14-1 (HA) following a 10 h pretreatment with 2.3 nM bortezomib (btzmb)7preincubation with 10 mM L-NAC (3 h). At the end of this period, the percentage of cells exhibiting increased ROS production was determined by monitoring dihydro-DCF staining by flow cytometry. (b) Alternatively, the percentage of cells exhibiting reduced mitochondrial membrane potential DCm was determined by monitoring DiOC6 uptake in cells exposed to btzmb-HA7L-NAC as above at the indicate intervals. Results represent the means7s.d. for three separate experiments performed in triplicate. n ¼ significantly higher ((a) Po0.07) or lower ((b) Po 0.01) than values for cells treated with each agent alone (a) or btzmb-HA in the absence of L-NAC (b).

described.32 Thus, sequential exposure of multiple myeloma cells to bortezomib followed by HA14-1 resulted in an increase in ROS generation and mitochondrial injury. Moreover, both these effects were attenuated by the free radical scavenger LNAC. Attempts were then made to determine what effect L-NAC would exert on bortezomib/HA14-1-mediated lethality in MM.1S cells. As shown in Figure 8a, coadministration of LNAC substantially blocked apoptosis triggered by the bortezomib/HA14-1 regimen in MM.1S cells, as well as that induced by As2O3 (data not shown). As illustrated in Figure 8b, L-NAC also attenuated bortezomib/HA14-1-mediated increases in PARP degradation, JNK activation, Bax accumulation, and p53 induction. Furthermore, cytosolic release of cytochrome c and Smac/DIABLO induced by the bortezomib/HA14-1 regimen was substantially attenuated in L-NAC-treated cells. Taken together with the preceding observations, these findings suggest that upstream perturbations in redox state play an important functional role in mediating bortezomib/HA14-1-related mitochondrial injury, JNK activation, and lethality in multiple myeloma cells. To shed light on the functional role of JNK activation in bortezomib/HA14-1-mediated lethality, U266 and MM1.S cells Leukemia

Figure 8 L-NAC attenuates bortezomib/HA14-induced apoptosis, JNK activation, upregulation of Bax and p53, and release of proapoptotic mitochondrial molecules in MM cells. (a) MM.1S cells were sequentially treated with 2.2 nM bortezomib (btzmb; 10 h) followed by 10 mM HA14-1 (HA; 8 h)73 h preincubation with 10 mM L-NAC. The percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright–Giemsa-stained cytospin preparations at the indicated intervals. Results represent the means7s.d. for three separate experiments performed in triplicate. n ¼ significantly lower than values for cells treated with btzmb-HA in the absence of L-NAC (Po0.01). (b) At 1 h after adding HA, cells were lysed and subjected to Western blot analysis to monitor cleavage of PARP, phosphorylation of JNK, and expression of Bax and p53 (upper panels). Alternatively, cytosolic fractions were obtained as described in Materials and methods, and expression of cytochrome c and Smac/ DIABLO were monitored by Western blot (lower panels). Each lane was loaded with 25 mg of protein; blots were stripped and reprobed with anti-actin or -tubulin antibody to ensure equal loading and transfer. Two additional studies yielded equivalent results.

were exposed to bortezomib and HA14-1 as above in the presence or absence of the pharmacologic JNK inhibitor SP600125 (20 mM).33 As shown in Figure 9a and c, coadministration of SP600125 blocked the marked activation of JNK induced by bortezomib/HA14-1 in both U266 and MM.1S cells. This was accompanied by a highly significant reduction in bortezomib/HA14-1-mediated apoptosis (Figure 9b and d; Po0.01 in each case) as well as PARP degradation (Figure 9a and b). For each condition, total JNK levels remained constant. These findings indicate that the striking increase in JNK activation in myeloma cells exposed to the bortezomib/HA141 regimen plays a significant functional role in enhanced lethality.


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Figure 9 The JNK inhibitor SP600125 blocks bortezomib/HA14-1induced JNK activation, PARP cleavage, and apoptosis in MM cells. U266 (a) or MM.1S (c) cells were incubated for 10 h with 2.3 or 2.2 nM bortezomib (btzmb) followed by either 12.5 mM for 14 h (U266) or 10 mM HA14-1 for 8 h (MM.1S) in the presence or absence of 20 mM SP600125, after which cell lysates were obtained and Western blots performed to monitor expression of PARP, phospho-JNK, and total cellular JNK as described in Materials and methods. Each lane was loaded with 25 mg of protein; blots were stripped and reprobed with anti-actin or -tubulin antibody to ensure equal loading and transfer. Two additional studies yielded equivalent results. Alternatively, U266 (b) and MM.1S (d) cells were treated as above, after which the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright–Giemsa-stained cytospin. Results represent the means7s.d. for three separate experiments performed in triplicate. n ¼ significantly lower (Po0.01) than values for cells treated with btzmb-HA in the absence of SP600125.

Lastly, in view of evidence implicating IL-6 in myeloma cell survival and drug resistance,34 an attempt was made to determine whether exogenous IL-6 could protect myeloma cells from cell death induced by sequential exposure of cells to bortezomib followed by HA14-1. As shown in Figure 10, administration of IL-6 (100 ng/ml) significantly reduced dexamethasone (10 mM)-induced apoptosis in MM.1S cells (Po0.02). In contrast, IL-6 failed to diminish apoptosis induced by the combination of HA14-1 and either bortezomib or MG132. These findings indicate that the lethal effects of the HA141/proteasome inhibitor regimen in myeloma cells operate independently of IL-6-related survival signaling pathways.

Discussion The present results indicate that in multiple myeloma cells, combined exposure to proteasome inhibitors, including bortezomib, and the small molecule Bcl-2 inhibitor HA14-1 results in a synergistic increase in mitochondrial injury (eg loss of DCm, release of cytochrome c, Smac/DIABLO, AIF), caspase activation, and apoptosis. Furthermore, these events occur in dexamethasone-resistant cells, are not attenuated by IL-6, an important survival factor in multiple myeloma,35 and are accompanied by a substantial loss of clonogenic potential. Previous studies have demonstrated that bortezomib is a potent inducer of apoptosis in multiple myeloma cells,15–17 and that it

Figure 10 IL-6 protects MM cells against cell death induced by dexamethasone but not by sequential administration of bortezomib and HA14-1. MM.1S cells were incubated for 8 h with 10 mM HA14-1 (HA) following a 10 h pretreatment of 2.2 nM bortezomib (btzmb) or 120 nM MG-132 (MG) in either the presence or absence of 100 ng/ml IL-6. At the end of this period, the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright–Giemsastained cytospin preparations. Results represent the means7s.d. for three separate experiments performed in triplicate. For comparison, cells were exposed to 10 mM dexamethasone for 24 h in the presence or absence of IL-6. n ¼ not significantly different compared with values for cells treated with btzmb or MG-HA in the absence of IL-6 (P40.05). nn ¼ significantly less than values for dexamethasonetreated cells in the absence of IL-6 (Po0.02).

sensitizes such cells to drug-induced lethality.22 Furthermore, dipeptidyl proteasome inhibitors have been shown to induce apoptosis in human leukemia cells through a Bcl-2-independent mechanism.21 On the other hand, HA14-1 is a small-molecularweight nonpeptidic compound identified through a computer screening strategy based on the predicted structure of the Bcl-2 protein.11 Rather than acting through a Bcl-2-independent mechanism, HA14-1 binds to and prevents Bcl-2 from inhibiting activation of the apaf-1/caspase-9 complex, presumably by blocking cytoplasmic release of cytochrome c. It is worth noting that potentiation of apoptosis in proteasome inhibitor/HA14-1treated cells was only observed when the former were administered prior to but not concurrent with or following HA14-1 exposure. This argues against the possibility that HA141 promotes bortezomib-mediated lethality; instead, it suggests that proteasome inhibitors lower the threshold for HA14-1mediated mitochondrial injury and apoptosis. Interestingly, the lethal effects of HA14-1 have recently been shown to be enhanced by PK11195, an inhibitor of the peripheral benzodiazepine receptor,36 a component of the voltage-dependent anion channel.37 Whether bortezomib or other proteasome inhibitors function in a similar manner remains to be determined. The lethal effects of proteasome inhibitors have been related to multiple perturbations in signal transduction- and survivalrelated proteins, including upregulation of Bax and Bid,38,39 activation of stress-related proteins (ie JNK),16 and increased expression of p53.16,30 Consistent with these findings, exposure of myeloma cells to bortezomib resulted in increased levels of Bax, Bak, Bad, induction of p53, and activation of p38 MAPK. However, with the possible exception of p53 induction, these events were not significantly enhanced by coadministration of HA14-1, suggesting that other factors contribute to synergistic interactions between these agents. On the other hand, coadministration of HA14-1 with bortezomib did induce mitochondrial translocation of Bax, an event linked to HA14-1-mediated Leukemia


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lethality.11,36 Exposure of cells to proteasome inhibitors has also been shown to increase levels of antiapoptotic proteins,17,40 and consistent with these results, bortezomib-treated cells displayed increased expression of Bcl-2, Mcl-1, XIAP, and Bcl-xL. However, treatment of cells with both bortezomib and HA141 resulted in the formation of a Bcl-2 cleavage fragment, which purportedly exhibits proapoptotic activity.29 Cells exposed to bortezomib/HA14-1 also exhibited upregulation of p21CIP1 and p27KIP1, which are known to exert antiapoptotic effects.41 In this regard, the appearance of p21CIP1 and p27KIP1 cleavage products, which, at least in the case of p21CIP1, exert proapoptotic actions,42 may be relevant. Finally, coadministration of bortezomib and HA14-1 led to a very marked activation of the stress-related kinase JNK, which has been strongly implicated in bortezomib-mediated lethality in multiple myeloma cells.16,17 It has also been associated with the response to oxidative stress.43 In view of the reports directly linking mitochondrial damage to JNK induction,44,45 the possibility that enhanced activation of JNK in bortezomib/HA14-1-treated cells contributed to the striking increase in the release of proapoptotic mitochondrial proteins (eg cytochrome c and Smac/DIABLO) appears plausible. Analogously, recent evidence that p53 may play a direct role in triggering mitochondrial injury46 raises the possibility that induction of p53 in bortezomib/HA14-1-treated cells may also play a role in these events. Based on the present findings, it appears likely that synergistic interactions between bortezomib and HA14-1 in multiple myeloma cells involve, at least in part, enhanced generation of ROS and oxidative stress. For example, coadministration of these agents increased the percentage of cells displaying increased ROS levels; moreover, coadministration of the free radical scavenger L-NAC substantially opposed bortezomib/ HA14-1-mediated ROS generation, loss of DCm, and apoptosis. In addition to opposing cytochrome c release, Bcl-2 has also been shown to play a role in protecting cells from oxidative stress.47 It is therefore tempting to speculate that disruption of this function by HA14-1, in conjunction with one or more perturbations induced by bortezomib, combine to promote cell death. It is also worth noting that JNK activation, Bax and p53 upregulation, and cytochrome c and Smac/DIABLO release were all attenuated by L-NAC, suggesting that each of these events occurred downstream of free radical formation. In this context, it is important to note that the NF-kB pathway, which may play a key role in protecting cells from oxidative stress,48 is known to be disabled by proteasome inhibitors.49 Thus, the possibility that bortezomib-mediated disruption of NF-kB function may enhance the lethal actions of HA14-1 appears plausible. The present findings could have implications for the design of novel approaches to the treatment of multiple myeloma and possibly other hematopoietic malignancies. Preclinical studies have demonstrated the efficacy of Bcl-2 antisense approaches in leukemia50 as well as myeloma,51 and clinical trials involving Bcl-2 antisense oligonucleotides (G3139; Genasense) have recently begun.52,53 Moreover, Bcl-2 antisense oligonucleotides have been shown to enhance the lethal actions of conventional chemotherapeutic drugs.51,54 The development of small molecule inhibitors of Bcl-2 such as HA14-1, as well as inhibitors of other antiapoptotic proteins,55 represents a potentially promising alternative approach. In addition, evidence has recently been presented indicating that HA14-1 may potentiate the lethal effects of several novel signal transduction modulators, including MEK inhibitors56 and 17-AAG.57 Given evidence of impressive activity of the signal transduction modulator bortezomib in patients with advanced multiple myeloma,20 an

analogous strategy of combining this agent with small molecule inhibitors of Bcl-2 appears attractive. In this context, very recent findings demonstrating that peptide inhibitors of Bcl-2 function enhance proteasome inhibitor-induced lethality in myeloma cells provide further support for this approach.17 An improved understanding of the mechanism by which proteasome and small molecule Bcl-2 inhibitors interact synergistically in myeloma cells may facilitate translation of these findings into the clinic. Accordingly, such studies are currently underway.

Acknowledgements This work was supported by awards CA63753, CA 100866, and CA93738 from the NIH, and award 6045-03 from the Leukemia and Lymphoma Society.

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