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Cell Death by Bortezomib-Induced Mitotic Catastrophe in Natural Killer Lymphoma Cells

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Cell Death by Bortezomib-Induced Mitotic Catastrophe in Natural Killer Lymphoma Cells 3807 Cell death by bortezomib-induced mitotic catastrophe in natural killer lymphoma cells Lijun Shen,1 Wing-Yan Au,2 Kai-Yau Wong,1 higher pharmacologic concentrations of bortezomib. Norio Shimizu,3 Junjiro Tsuchiyama,4 Hence, activating mitotic catastrophe by bortezomib Yok-Lam Kwong,2 Raymond H. Liang,2 may provide a novel therapeutic approach for treating and Gopesh Srivastava1 apoptosis-resistant NK-cell malignancies and other can- cers. [Mol Cancer Ther 2008;7(12):3807–15] Departments of 1Pathology and 2Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, People’s Republic Introduction of China; 3Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; and 4Department of Pathology, According to the WHO classification scheme, natural killer Kawasaki Medical School, Kurashiki, Okayama, Japan (NK)-cell neoplasms include extranodal NK/T-cell lym- phoma (nasal type) and aggressive NK-cell leukemia (1). Theyare aggressive malignancies with poor treatment Abstract outcomes (2). The lymphoma cells are characteristically À The proteasome inhibitor bortezomib (PS-341/Velcade) is CD3 CD3q+CD56+ and are infected bythe EBV. NK-cell used for the treatment of relapsed and refractory multiple lymphomas show a geographic predilection, as they myeloma and mantle-cell lymphoma. We recently reported constitute 5% to 10% of all lymphomas in Asia and South its therapeutic potential against natural killer (NK)-cell America but are extremelyuncommon in the West. An neoplasms. Here, we investigated the molecular mecha- optimal treatment for NK-cell malignancies has yet to be nisms of bortezomib-induced cell death in NK lymphoma found (2). Radiotherapyof localized nasal NK-cell lym- cells. NK lymphoma cell lines (SNK-6 and NK-YS) and phomas maybe curative in patients with stage I disease. primary cultures of NK lymphomas treated with bortezo- For patients with stage II or more advanced disease, mib were examined for alterations in cell viability, however, treatment results are unsatisfactory. Despite the apoptosis, cellular senescence, and cell cycle status. availabilityof prognostic models (3) and accurate lympho- Bortezomib primarily induced mitochondrial apoptosis in ma load assessment byquantification of EBV DNA (4), NK-YS cells and in primary lymphoma cells at the same improvement in treatment results has not been obtained by concentration as reported in myeloma cells. Unexpectedly, conventional chemotherapy. Although allogeneic trans- SNK-6 cells required a significantly higher median inhibi- plantation mayrescue some patients, the rapid progression tory concentration of bortezomib (23 nmol/L) than NK-YS of chemorefractorydisease or the toxic side effects of high- and primary lymphoma cells (6-13 nmol/L). Apoptosis was dose chemotherapyoften preclude such an option. Thus, limited in SNK-6 cells due to the extensively delayed new therapeutic agents, preferablywithout dose-limiting turnover of Bcl-2 family members. These cells were killed effects, are urgentlywanted. To achieve meaningful clinical by bortezomib, albeit at higher pharmacologic concen- results, a more effective and less toxic agent will be critical trations, via mitotic catastrophe—a mitotic cell death to attain disease stabilization before transplantation. Re- associated with M-phase arrest, cyclin B1 accumulation, search on new drug development for this disease has been and increased CDC2/CDK1 activity. Our results suggest hampered due to the verylimited number of bona fide NK that, in addition to cell death by apoptosis at lower lymphoma cell lines that are available (5). Establishment of bortezomib concentrations, NK lymphoma cells resistant NK lymphoma cell lines has been very difficult over the to bortezomib-induced apoptosis can be killed via mitotic years. Similarly, successful development of NK lymphoma catastrophe, an alternative cell death mechanism, at primarycultures for the evaluation of therapeutics has been equallychallenging, as tumor biopsies are usuallysmall and contain significant necrosis. Tumorigenesis is generallyprevented bythe watchdog Received 7/9/08; revised 9/11/08; accepted 9/23/08. activityof tumor suppressors that trigger apoptosis. Grant support: Research Grants Council of Hong Kong Special Administrative Region, People’s Republic of China grant HKU 7627/06M Similarly, anticancer agents often work by inducing cancer (G. Srivastava and R.H. Liang). cells to undergo apoptosis (6). Cancer cells, however, may The costs of publication of this article were defrayed in part by the still escape apoptosis byenhancing the activityof onco- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to proteins or inactivating the tumor suppressors. These indicate this fact. mechanisms contribute to poor drug responses. Accumu- Requests for reprints: Gopesh Srivastava, Lymphoma Research Laboratory, lating evidence indicates that, if apoptosis is abnormally Department of Pathology, The University of Hong Kong, Queen Mary suppressed, cancer cells maybe eliminated byother Hospital, Pokfulam, Hong Kong, People’s Republic of China. Phone: 852-2855-4875; Fax: 852-2872-5197. mechanisms (7). Among these mechanisms is mitotic E-mail: [email protected] catastrophe, a type of cell death that involves abnormal Copyright C 2008 American Association for Cancer Research. mitosis. When assaulted bydrugs, cancer cells tend to doi:10.1158/1535-7163.MCT-08-0641 bypass the mitotic checkpoints and prematurely reenter the Mol Cancer Ther 2008;7(12). December 2008 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. 3808 Bortezomib-Induced Mitotic Catastrophe cell cycle, that is, they divide asymmetrically, leading to (BD Biosciences). The NK cells were purified from the cytogenetic chaos and mitotic catastrophe (8–10). New harvested cells with the NK Cell Negative Isolation Kit routes to successful cancer treatment maytherefore be (Dynal Biotech ASA) and maintained in the same medium discovered byinvestigating the factors that interrupt as described above. In the second patient with bone competent cell division. marrow involvement, mononuclear cells were isolated by Normal cellular activities are dependent on the orderly the Ficoll-Hypaque method from the patient’s bone degradation of obsolete cellular proteins bythe ubiquitin- marrow sample before anytreatment, and NK cells were proteasome system. The targeted proteins participate in a purified and maintained as described above. wide varietyof essential cellular activities, such as signal Antibodies and Reagents transduction, transcriptional regulation, stress response, Primaryantibodies against the following proteins were and cell cycle control (11). The process is tightly controlled used in this study: caspase-9, caspase-3, poly(ADP-ribose) bya network of coordinated enzymes: E 1 ubiquitin- polymerase, Mcl-1, Bcl-xL, Bcl-2, Bak, Bax, ubiquitin, cyclin 15 activating enzyme, E2 ubiquitin-conjugating enzyme, and B1, phospho-CDC2 (Tyr ), and CDC2 from Cell Signaling h E3 ubiquitin ligase. The ubiquitinated proteins are targeted Technology; -actin from Santa Cruz Biotechnology; Bax for proteolysis by the proteasome. Bortezomib (PS-341/ (clone 6A7) and Bak (clone TC-100) from Calbiochem; and Velcade) is a synthetic small molecule that specifically and MPM-2 from Upstate. Bortezomib was kindlyprovided by reversiblyinactivates the proteasome (12). It has been used Millennium Pharmaceuticals. Propidium iodide was to treat relapsed and refractorymultiple myelomaand obtained from Sigma, 4¶,6-diamidino-2-phenylindole (DAPI) mantle-cell lymphoma (11). We have described previously was from Roche Applied Science, and chloromethyl- constitutive activation of nuclear factor-nBinNK-cell X-rosamine (MitoTracker Red) was from Molecular Probes. lymphoma (13). Given the involvement of nuclear factor- Viable Cell Number Assay nB with tumorigenesis and the action of bortezomib against The viable cell number was measured with the AQueous nuclear factor-nB (11), we have recentlyinvestigated and One Solution MTS assay(Promega). Briefly,cells were reported its therapeutic potential for NK-cell neoplasms incubated in a 96-well plate at a densityof 2 Â 105 per well (14). In this study, we have examined the molecular in 100 AL culture medium. Theywere treated with various mechanisms of bortezomib-induced cell death in NK concentrations of bortezomib for 1 day. For each well, 20 AL lymphoma cell lines and primary lymphoma cells. Our MTS reagent was added and the plate was incubated at results suggest that, in addition to cell death byapoptosis at 37jC for 3 h. Absorbance was measured at 492 nm with the lower bortezomib concentrations, NK lymphoma cells that Vmax 96-well plate reader (Molecular Devices). are resistant to apoptosis via bortezomib can be killed via DNA Fragmentation Assay mitotic catastrophe, an alternative cell death mechanism, at The DNA fragmentation assaywas done as described higher pharmacologic concentrations of bortezomib. As the previously(17). Briefly,cells were harvested after 1 dayof apoptosis pathwayis often impaired in relapsed and exposure to bortezomib, washed once, and resuspended in chemorefractoryNK-cell malignancyand other cancers, 200 AL cold PBS. Theywere lysedbyadding 20 AL lysis activation of mitotic catastrophe bybortezomib may buffer [0.2 mol/L EDTA, 0.05 mol/L Tris-HCl (pH 8.0), and provide a novel therapeutic approach for the treatment
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