ANTICANCER RESEARCH 25: 3321-3326 (2005)

Proteasome Inhibitors Abolish Cell Death Downstream of Caspase Activation During Anti- Drug-induced in Leukemia Cells

KATALIN NAGY1,3, ISTVAN PETAK1,3, GERGELY IMRE1, GABOR BARNA1, MARIA GEZANE-CSORBA1, ANNA SEBESTYEN1, JANET A. HOUGHTON4, RUDOLF MIHALIK2 and LASZLO KOPPER1,2,3

1First Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest; 2Molecular Pathology Research Group, Joint Research Organization of the Hungarian Academy of Science and Semmelweis University, Budapest; 3Szentágothai János Knowledge Center, Budapest, Hungary; 4Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, U.S.A.

Abstract. Purpose: Anti-microtubule drugs and proteasome to inhibit apoptosis independently from caspase activation. In inhibitors are currently among the most intensively studied this study, MG-132 up-regulated HSP70 protein expression, anti-tumor agents, however little is known about their both in the presence or absence of nocodazole. Conclusion: pharmacological interactions at the cellular level. Materials Proteasome inhibitors decreased anti-microtubule agent- and Methods: The human promyelocytic leukemia cell line, induced apoptotic DNA fragmentation downstream of caspase- HL-60, was exposed to nocodazole or in 3 activation, possibly due to increased HSP70 expression. The combination with proteasome or caspase inhibitors. Apoptotic results indicate that combination treatment with these novel cell death was detected by flow cytometry as sub-G1 anti-tumor agents in leukemia requires careful evaluation of population. Caspase and proteasome activities were monitored their molecular interaction at the level of apoptosis induction. by the fluorogenic substrates Ac-DEVD-AMC and Suc-LLVY- AMC, respectively, in cell lysate. Heat shock protein 70 Drugs which interfere with , such as taxol, have (HSP70) expression was determined by Western blotting. been among the most successful chemotherapeutic agents of Results: Nocodazole, a microtubule inhibitor, induced caspase- recent years (1), and have been used in the treatment of many dependent apoptosis in the HL-60 cell line. At sub-cytotoxic solid tumors including breast and ovarian cancers (2, 3). concentrations, proteasome inhibitors, including MG-132 or However, considerably less is known about their therapeutic clasto-‚-lactone, decreased nocodazole-induced apoptotic value in leukemias (4, 5). Interestingly, anti-tumor cytotoxicity DNA fragmentation without affecting the induction of caspase- induced by arsenic, a novel and effective anti-leukemia agent, 3 activity. In contrast, MG-132 decreased both DNA is mediated via the inhibition of microtubule function (6). fragmentation and caspase activation induced by etoposide, a Proteasome inhibitors can affect apoptotic signaling in topoisomerase-II inhibitor. HSP70 had previously been found several ways (7). Inhibition of the proteasomal degradation of p53 can enhance apoptosis induced by chemotherapeutic drugs (8). Activation of Nuclear Factor-Î B (NF-Î B) requires proteolytic degradation of Inhibitor-Î B (I-Î B), Abbreviations: Ac-DEVD-AMC, Ac-Asp-Glu-Val-Asp-7-amino-4- methyl-coumarin; CBL, Galsto-‚-lactone; DMSO, dimethylsulfo- xide; therefore proteasome inhibitors can abolish the NF-Î B- Eto, etoposide; HSP70, heat shock protein 70 kD; MG-132, induced up-regulation of several anti-apoptotic proteins benzyloxycarbonyl-Leu-Leu-Leu-aldehyde; Noc, nocodazole; Z-VAD- such as Inhibitor of Apoptosis Protein (IAP-1/2) and X FMK, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluorome- thylketone. chromosome linked IAP (XIAP) by preventing degradation of I-Î B (9). This could also be the molecular rationale for Correspondence to: Dr. Rudolf Mihalik, 1st Dept. of Pathology, the anti-tumor activity of proteasome inhibitors in tumors Semmelweis University, Budapest, 1085, Ülloi út 26, Hungary. Tel: with high constitutive NF-Î B activity. Further, this is the (36) (1) 4591500 ext: 4440, Fax: (36) (1) 2660451, e-mail: [email protected] molecular basis of the synergistic interaction between proteasome inhibitors and death ligands, which induce Key Words: Nocodazole, etoposide, MG-132, clasto-‚-lactone, HSP70. NF-Î B activation in parallel to the activation of the caspase

0250-7005/2005 $2.00+.40 3321 ANTICANCER RESEARCH 25: 3321-3326 (2005) cascade (10). However, in certain cell types, NF-Î B can be the caspase inhibitor: Z-VAD-FMK (100 ÌM). In separate pro-apoptotic by up-regulating death ligands themselves in experiments, cells were treated with etoposide (10 ÌM) for 6 h, response to chemotherapeutic drugs (11). It has also been with or without 30 min pretreatment with the same protease inhibitors. Control samples were exposed to either DMSO (as reported that proteasome inhibitors can up-regulate the vehicle) or protease inhibitors only. Finally, the cells were expression of HSP70, (12, 13) which, in turn, can inhibit cell harvested and fixed in 70% ethanol (–20ÆC) for 30 min at room death by interacting with various components of the death temperature, and stored at –20ÆC until analysis. Fragmented DNA pathways (14, 15). was extracted with alkaline buffer (200 mM di-sodiumphosphate, Both microtubule and proteasome inhibitors are valuable pH 7.8, adjusted with 200 mM citric acid) supplemented with targets for the development of novel anti-leukemic drugs. In 100 Ìg/ml RNase A. Samples were left at room temperature for the current experiments, HL-60 leukemia cells were treated 30 min followed by the addition of 5 ml ethidium bromide (final concentration 10 Ìg/ml). After a further 15 min, samples were with the microtubule inhibitor nocodazole, either alone or in measured with a flow cytometer (FACScan, Becton-Dickinson) and combination with the proteasome inhibitors MG-132 or clasto- the and proportion of sub-G1 (apoptotic) cells were ‚-lactone. Combinations of cytotoxic agents may be synergistic calculated (17). Gating was evaluated as described previously (18). or antagonistic at the level of apoptosis induction (16). In this In the case of nocodazole, the presented percentage of sub-G1 cells study, nocodazole was found to induce apoptosis in leukemia represents the difference between the results obtained after 20-h cells, but low concentrations of proteasome inhibitors and 12-h treatment. interfered with this microtubule inhibition-induced apoptosis, downstream of effector caspase activation but upstream of Caspase and proteasome activity assays. Assays were performed as described earlier (19), with slight modifications. The cells were apoptotic DNA fragmentation. These findings indicate that treated as described above, and subsequently washed twice in PBS certain combinations of microtubule and proteasome-inhibitors containing 5 mM glucose (PBS+G), then resuspended in 100 Ìl of in the treatment of leukemia can be antagonistic. protease activity buffer (PAP) composed of PBS+G and 10 mM dithiothreitol (DTT). Caspase activity was detected with Materials and Methods Ac-DEVD-AMC (50 ÌM final concentration) added to the cell suspension in addition to 100 Ìl PAP containing 1% Triton X-100. Materials. The materials were purchased from the following vendors: Proteasome activity was detected by Suc-LLVY-AMC (50 ÌM final Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-FMK) from Enzyme System concentration) added to the cell suspension in 100 Ìl PAP Products (Livermore, CA, USA); nocodazole (Noc), etoposide containing 1% Triton X-100 and 5 mM EGTA. Lysates were (Eto), MG-132 (MG), clasto-‚-lactone (CBL), Ac-DEVD-AMC incubated for 3 min at room temperature, and fluorescence was (acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarine), Suc-LLVY- detected by a fluorescence plate reader (Fluoroskan Ascent, AMC (succinyl-Lys-Lys-Val-Tyr-AMC) from Sigma (St. Louis, MO, Labsystems, Thermo Electron Corp., www.thermo.com) at 380 nm USA). Stock solutions were made from all these compounds by excitation and 460 nm absorption for 20 min. All activity was linear DMSO (dimethylsulfoxide, from Sigma) at the concentration that in this time range. Activity was calculated as released AMC/min/106 the final dilutions contained less than 0.1% DMSO. Ethidium cells, calibrated by free AMC dilutions. bromide from Calbiochem (San Diego, CA, USA); Triton X-100 from Serva (Heidelberg, Germany); D(+) glucose from Reanal Western blot analysis. 1 x 106 HL-60 cells were treated with drugs, as (Budapest, Hungary); FCS (fetal calf serum) from Gibco described above. At the end of the incubation, the cells were (Invitrogen, Carlsbad, CA, USA); plastic materials from Sarstedt washed twice in PBS and pelleted (300 xg, 3 min). The pellet was (Nümbrecht, Germany); mouse monoclonal antibody to human resuspended in 40 Ìl ice-cold water then 10 Ìl 6x lysis buffer HSP70 from Transduction Laboratories (Becton Dickinson, San (300 mM Tris-HCl pH 6.8, 600 mM DTT, 12% SDS, 0.6% Jose, CA, USA); HRPO-conjugated goat anti-mouse IgG1 from bromphenol blue and 60% glycerol) was added. Each sample was Southern Biotechnology (Birmingham, AL, USA); Vectastain ABC boiled for 3 min, and the protein content was determined by the Kit from Vector Laboratories (Burlingame, CA, USA); DAB (3,3’- Bradford method. Electrophoresis of total protein (20 Ìg) was diaminobenzidine) from DAKO (Carpinteria, CA, USA); all other performed in a 12.5% polyacrylamide gel. Mouse monoclonal compounds, salts or solutions were purchased from Sigma. antibody was used to detect both inducible and constitutive HSP70. Horseradish peroxidase-conjugated goat anti-mouse IgG1 was Cell culture. The HL-60 promyelocytic leukemia cell line was obtained employed as secondary antibody. Detection was performed using from Dr. Balázs Sarkadi (National Medical Center, Budapest, the Vectastain ABC Kit with DAB as the chromogen substrate. Hungary) and was maintained in RPMI-1640 medium supplemented with 10% FCS, L-glutamine and penicillin-streptomycin, at 37ÆC, in Statistical analysis. All experiments were repeated at least three an atmosphere of 95% air and 5% CO2. For all experiments, cells times and presented as mean±SD. Statistical analysis was growing in the exponential phase were used. performed by unpaired, two-tailed Student’s t-test, and p values <0.05 were considered significant. The inhibition percentage of Apoptosis assay. HL-60 cells were plated at a density of 5x105 caspase activity by protease inhibitors in anti-tumor drug-induced cells/ml in 24-well plates. The cells were treated overnight (usually samples was calculated by the following formula: (value of caspase 12 h) with nocodazole (200 nM;) and were subsequently further activity in the drug-treated sample – value of caspase activity in the incubated for 8 h in the presence or absence of either of the drug+protease inhibitor treated samples)/(value of caspase activity protease inhibitors: MG-132 (3 ÌM); clasto-‚-lactone (10 ÌM) or in the drug-treated sample) x 100.

3322 Nagy et al: Proteasome in the Nocodazole-induced Apoptosis

Figure 1. Nocodazole induced G2/M cell cycle block and apoptotic DNA fragmentation in the HL-60 leukemia cell line. HL-60 cells were treated with nocodazole (200 nM) for increasing incubation times. Fragmented DNA of apoptotic cells was extracted in alkaline buffer and the percentage of cells with decreased DNA content (sub-G1 cells) was determined by flow cytometry after ethidium bromide staining. A: Representative flow cytometric DNA histograms of control and nocodazole-treated cells (inserted data: percentage of sub-G1 cells). B: Time kinetics of nocodazole-induced cell death. Apoptosis rate increased substantially after 12-h treatment. Values indicate the mean ± SD of three determinations.

Figure 2. Effect of protease inhibitors on apoptotic DNA fragmentation induced by nocodazole or etoposide. HL-60 cells with apoptotic DNA fragmentation (sub-G1 cells) were detected by flow cytometry. A: Cells were pre-incubated with nocodazole (Noc, 200 nM) for 12 h and subsequently incubated for an additional 8 h in the absence or presence of the indicated protease inhibitors. B: Cells were treated with etoposide (Eto, 10 ÌM) for 6 h in the presence or absence of protease inhibitors with 30 min preincubation time. Cont: control (DMSO); MG: MG-132 (3 ÌM); CBL: clasto-‚- lactone (10 ÌM); VAD: Z-VAD-FMK (100 ÌM). Values represent the mean±SD of three determinations.

Results the absence or presence of nocodazole for 12 h, followed by incubation in the presence of Z-VAD-FMK (100 ÌM) or Cell cycle arrest and subsequent apoptosis induced by nocodazole. proteasome inhibitors (MG-132; 3 mM, or clasto-‚-lactone; HL-60 cells were treated with nocodazole (200 nM) for 10 ÌM) for an additional 8 h. The sub-G1 fraction of cells with incubation times from 0-20 h (Figure 1B). Cells with apoptotic DNA fragmentation, determined in comparison to apoptotic DNA fragmentation were detected as a sub-G1 values measured at 12 h, revealed that nocodazole induced population by FACS analysis. In time-course experiments, the apoptosis (24%) during 8 h of incubation. Apoptosis was percentage of apoptotic cells increased sharply from 14% at completely inhibited by Z-VAD-FMK (1%, p=0.007), 12 h to 38% at 20 h. Cell cycle analysis of nocodazole-treated significantly reduced by MG-132 (7.75%, p=0.033), and cells demonstrated that most cells were in the G2/M-phase of slightly reduced, but not significantly, by clasto-‚-lactone (16%, the cell cycle at 12 h (Figure 1A). These results indicated that p=0.106; Figure 2A). The protease inhibitors were non- nocodazole-induced events can be divided into two phases, cytotoxic at the concentrations and incubation time employed. namely arrest of cells in G2/M by 12 h, with subsequent Etoposide induces double-strand breaks by inhibiting induction of apoptosis. topoisomerase II during un-winding of the DNA (20). However, proteasomal degradation of the enzyme-DNA Both proteasome and caspase inhibitors reduced nocodazole- complex is required to expose DNA damage and to induced apoptotic DNA-fragmentation. Cells were incubated in subsequently activate the apoptotic signaling pathway.

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Figure 3. Effect of protease inhibitors on caspase activity induced by nocodazole or etoposide. HL-60 cells, treated as described in Figure 2, were lysed and caspase activity was detected using DEVD-AMC as the fluorogenic substrate, as described in Materials and Methods. A: Caspase activity in nocodazole- treated samples. B: Caspase activity of etoposide-treated samples. Labels are the same as in Figure 2. Values represent the mean±SD of three determinations.

nocodazole in the presence or absence of Z-VAD-FMK or of the MG-132. Nocodazole-induced caspase-3-like (DEVD-ase) activity was completely inhibited by Z-VAD-FMK (inhibition=97%, p=0.009). However, neither MG-132 (p=0.642) nor clasto-‚-lactone (p=0.603) reduced nocodazole-induced caspase-3 activity (Figure 3A). Figure 4. Up-regulation of inducible HSP70 protein expression by inhibition In contrast, etoposide-induced caspase activity was of proteasomal activity. HL-60 cells were preincubated with nocodazole significantly reduced by both MG-132 (inhibition=53%, (200 nM) for 12 h then further incubated in the presence or absence of MG- p=0.008) and clasto-‚-lactone (inhibition=52%, p=0.004). 132 (3 ÌM) for an additional 8 h. Western blot analysis was performed, as Z-VAD-FMK also completely inhibited (by 97%, p=0.001) described in Materials and Methods. The monoclonal antibody used for Western analysis detected both the inducible HSP72 and constitutive HSP73 etoposide-induced caspase-3 activity (Figure 3B). forms of HSP70. Lanes: Cont: control (DMSO); Noc: nocodazole (20 h); MG-132 (8 h); Noc+MG: nocodazole (20 h) + MG-132 (8 h). MG-132 up-regulated the expression of the inducible HSP70 protein in HL-60 cells. Inhibition of caspase-3 directed DNA-fragmentation by HSP70 (14) and induction of HSP70 by proteasome inhibition have been previously described Inhibition of proteasomal activity blocks the initiation of (12, 13). The monoclonal antibody used in this experiment etoposide-induced apoptosis (21). In accordance with these detects both the inducible HSP72 and the constitutive results, etoposide (10 ÌM) induced rapid apoptosis in HL-60 HSP73 forms of HSP70. We found that neither nocodazole- cells (57% at 6 h; Figure 2B), while apoptosis was nor MG-132 treatment changed the expression of HSP73. completely blocked by Z-VAD-FMK (2%, p=0.002) and In contrast, HSP72 expression was up-regulated by MG-132 significantly reduced by MG-132 (16%, p=0.004), as well as in both the presence and absence of nocodazole (Figure 4). by clasto-‚-lactone (19%, p=0.004; Figure 2B). Discussion Caspase activity was not influenced by proteasome inhibitors in nocodazole-treated cells. In the following experiments, it In this study, it was demonstrated that the proteasome was determined whether inhibition of DNA fragmentation inhibitors MG-132 and clasto-‚-lactone decreased apoptosis occurred upstream or downstream of caspase activation. previously induced by the microtubule polymerization inhibitor First, protease inhibitors were examined in ex vivo activity nocodazole in the HL-60 human myeloid leukemia cell line. assays. Z-VAD-FMK specifically inhibited caspase activity, Nocodazole-treated HL-60 cells accumulated in the G2/M- while the proteasome inhibitors MG-132 and clasto-‚- phase of the cell cycle prior to the induction of the lactone inhibited proteasome activity, without affecting characteristic apoptotic DNA fragmentation, in a fashion caspase activity (data not shown). Subsequently, caspase-3 similar to cells treated with taxol (22). Proteasome inhibitors activity was determined in HL-60 cells treated with can induce cell cycle block in G1 via stabilization of the cyclin-

3324 Nagy et al: Proteasome in the Nocodazole-induced Apoptosis dependent kinase inhibitors p27 and p21 (7, 23, 24). and justifies the development of proteasome inhibitors for Therefore, pre-treatment with proteasome inhibitors could cancer therapy (29). However, proteasome inhibition can lead potentially interfere with G2/M cell cycle-specific drugs to the accumulation of the same anti-apoptotic proteins due to including nocodazole. In the current experiments, however, their decreased degradation. Furthermore, in certain cell types, cells were initially pretreated with nocodazole for 12 h in the NF-Î B induces the expression of pro-apoptotic molecules such absence of proteasome inhibitors, which was sufficient time as FasL (11) and TRAIL (30). Therefore, inhibition of NF-Î B for the cells to accumulate in G2/M. signaling can interfere with a death receptor-mediated The general caspase inhibitor Z-VAD-FMK effectively autocrine suicide mechanism (11). All of these anti-apoptotic inhibited nocodazole-induced DNA fragmentation, indicative mechanisms can inhibit apoptosis upstream to the activation of the role of caspase activation in nocodazole-induced of the main effector caspases including caspase-3. apoptosis. To address whether inhibition of DNA In the current study, proteasome inhibitors prevented fragmentation by proteasome inhibitors occurred upstream nocodazole-induced apoptosis downstream of caspase-3 or downstream of caspase activation, caspase activity was activation, therefore other anti-apoptotic mechanisms were measured in the lysates of cells treated with nocodazole in considered. Activation of caspase-3 is generally considered to the presence or absence of proteasome inhibitors. be the point of no return in the induction of apoptosis (31). Nocodazole-induced caspase activation was completely However, Jaattela et al. have reported that overexpression of inhibited by Z-VAD-FMK, but was not influenced by HSP70 inhibited late caspase-dependent events such as DNA proteasome inhibitors, although proteasome inhibitors fragmentation, and conferred significant protection against cell effectively inhibited apoptotic DNA fragmentation under the death induced by activation of caspase-3 by TNF-· treatment same experimental conditions. In the current study, the (14). It has been reported that proteasome inhibitors can caspase activity assay employed detected DEVD-ase activity increase the HSP72 level, the inducible form of HSP70 (13). (cleavage of the fluorescent substrate, DEVD-AMC), which In the present study, the proteasome inhibitor MG-132 correlates primarily with caspase-3 activity (19). Apoptotic induced expression of HSP72 in HL-60 cells in both the DNA fragmentation is considered to be the consequence of absence and presence of nocodazole (Figure 4). These results ICAD (Inhibitor of Caspase Activated DNase) cleavage by indicate that proteasome inhibition can indirectly interfere with caspase-3 and subsequent CAD (Caspase Activated DNase) nocodazole-induced apoptosis via the HSP70 up-regulation. activation (25). These results indicate that proteasome The concept of active cell death (apoptosis) as the molecular inhibitors prevent DNA fragmentation downstream of and cellular mechanism of anti-tumor agents has led to a caspase-3 activation during apoptosis induced by microtubule paradigm shift and opened a new field of molecular onco- polymerization inhibitors, such as nocodazole. pharmacology. However, cytotoxic agents initiate different Inhibition of apoptosis by proteasome inhibitors has been apoptotic signal transduction pathways, which often lead to described in topoisomerase-inhibitor (etoposide)-induced cell alternatives to the classic biochemical and morphological death (21). Etoposide induces double DNA breaks via mechanisms of apoptosis. These differences open the inhibition of topoisomerase II, which relaxes the DNA possibility of synergistic and antagonistic interactions of various structure by temporal strand breaks. Topoisomerase II drugs at the level of apoptosis induction. Our group has inhibitors inhibit enzyme activity at this stage, preventing the previously described another example of antagonism between annealing of the DNA strands. However, the irreversibly two cytotoxic agents at the level of apoptosis initiation. In that inhibited enzyme must be removed by proteasome-mediated study, we found that, during decomposition of BCNU (bis- degradation in order to activate the apoptotic cascade (21). In chloro-), the chloroethyl-isocyanate intermediate this process, proteasome activity therefore has a pivotal role in product very effectively inhibited caspase-3 activity. Therefore, the initiation of apoptosis. Thus, experiments with etoposide subtoxic concentrations of BCNU inhibited apoptosis induced and the same proteasome inhibitors were conducted in parallel by other drugs including etoposide (16). with experiments that incorporated nocodazole. As anticipated, In conclusion, these findings indicate that further in vitro etoposide induced high caspase activity in HL-60 cells, which and in vivo investigations are warranted before combination was inhibited by Z-VAD-FMK and the proteasome inhibitors. treatments of leukemia with microtubule polymerization Inhibition of proteasome activity can influence apoptotic inhibitors and proteasome inhibitors are applied. pathways by different mechanisms, depending on the cell type and type of apoptosis induction. The best known effect of Acknowledgements proteasome inhibition is the inhibition of NF-Î B signaling (26). R.M. was supported by OTKA T029611, OTKA T049008 and ETT The NF-Î B transcription factor can increase the expression of 197/2000 grants. L.K. was supported by OTKA T34892 ETT important anti-apoptotic molecules such as IAP-1/2, XIAP, 193/2000 grants. A.S. was supported by FKFP 0150/2001, ETT c-FLIP and Bcl-2 (9, 27, 28). This mechanism is believed to be 192/2000 grants and the Bekesi Foundation 118/2001. J.H. was an important survival strategy for several human malignancies supported by NIH Awards CA 87952, CA 32613, CA 23944, and by

3325 ANTICANCER RESEARCH 25: 3321-3326 (2005) the American Lebanese Syrian Associated Charities. I.P. was 17 Gong J, Traganos F and Darzynkiewicz Z: A selective supported by Zoltan Magyary Scholarship, Terry Fox Foundation, procedure for DNA extraction from apoptotic cells applicable ETT 145/2003, NKFP-1A/20/2002, OTKA-T046665, OTKA TS for gel electrophoresis and flow cytometry. Anal Biochem 218: 049887, TET I-48/2003, OMFB-01634/2001 grants and by the 314-319, 1994. National Office for Research and Technology (NKTH), Hungary. 18 Mihalik R, Uher F, Pocsik EE, Berczi L, Benczur M and Kopper L: Detection of drug-induced apoptosis by flow References cytometry after alkaline extraction of ethanol fixed cells. Pathol Oncol Res 2: 78-83, 1996. 19 Mihalik R, Bauer P, Petak I, Krajcsi P, Marton A, Kun E and 1 Jordan MA and Wilson L: Microtubules as a target for Kopper L: Interaction of cytocidal drugs and the inhibition of anticancer drugs. 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