Danger Signaling Protein HMGB1 Induces a Distinct Form of Cell Death Accompanied by Formation of Giant Mitochondria

Published OnlineFirst October 19, 2010; DOI: 10.1158/0008-5472.CAN-10-0204 Published OnlineFirst on October 19, 2010 as 10.1158/0008-5472.CAN-10-0204

Prevention and Epidemiology Cancer Research Danger Signaling Protein HMGB1 Induces a Distinct Form of Cell Death Accompanied by Formation of Giant Mitochondria

Georg Gdynia1,2, Martina Keith1,2, Jürgen Kopitz2, Marion Bergmann2, Anne Fassl1, Alexander N.R. Weber1, Julie George1, Tim Kees1, Hans-Walter Zentgraf1, Otmar D. Wiestler1, Peter Schirmacher2, and Wilfried Roth1,2

Abstract Cells dying by necrosis release the high-mobility group box 1 (HMGB1) protein, which has immunostimu- latory effects. However, little is known about the direct actions of extracellular HMGB1 protein on cancer cells. Here, we show that recombinant human HMGB1 (rhHMGB1) exerts strong cytotoxic effects on malignant tumor cells. The rhHMGB1-induced cytotoxicity depends on the presence of mitochondria and leads to fast depletion of mitochondrial DNA, severe damage of the mitochondrial proteome by toxic malondialdehyde adducts, and formation of giant mitochondria. The formation of giant mitochondria is independent of direct nuclear signaling events, because giant mitochondria are also observed in cytoplasts lacking nuclei.

Further, the reactive oxygen species scavenger N-acetylcysteine as well as c-Jun NH2-terminal kinase block- ade inhibited the cytotoxic effect of rhHMGB1. Importantly, glioblastoma cells, but not normal astrocytes, were highly susceptible to rhHMGB1-induced cell death. Systemic treatment with rhHMGB1 results in significant growth inhibition of xenografted tumors in vivo. In summary, rhHMGB1 induces a distinct form of cell death in cancer cells, which differs from the known forms of apoptosis, autophagy, and senescence, possibly representing an important novel mechanism of specialized necrosis. Further, our findings suggest that rhHMGB1 may offer therapeutic applications in treatment of patients with malignant brain tumors. Cancer Res; 70(21); 8558–68. ©2010 AACR.

Introduction In addition to the established role of HMGB1 in inflammation, there is growing evidence for its contribution to cancer High-mobility group box 1 (HMGB1) is an architectural (7, 8). Breast cancer patients with a TLR4 single-nucleotide transcription factor present in most eukaryotic cells (1), polymorphism had a worse clinical outcome due to the lack but it can also act as a cytokine when released from ne- of immunoadjuvant effects of HMGB1 released from dying crotic cells into the extracellular space (2). HMGB1 exerts tumor cells (9). Interaction of glioblastoma-derived HMGB1 pleiotropic biological effects by binding to several mem- with TLR2 of dendritic cells was crucial for initiating an ef- bers of the Toll-like family such as receptor for advanced fective antitumor immune response in an intracranial glioma glycation end products (RAGE) or Toll-like receptor 2 xenograft mouse model (10). Studies in an orthotopic synge- (TLR2) and TLR4 (1), thereby promoting inflammatory pro- neic intracranial glioma rat model showed that the efficacy of cesses as shown in a mouse model of sepsis (3). Interest- an immunotherapy with adenoviruses expressing the cyto- ingly, HMGB1 can also function in an antimicrobial kine Flt3L and thymidine kinase was dependent on circulat- manner. Phosphorylated recombinant HMGB1 and native ing HMGB1 (11). In contrast, HMGB1 may also elicit direct HMGB1 extracted from tumor cells inhibit eukaryotic antitumor activity. Endogenous HMGB1 influences the closed circular DNA replication in vitro (4). Similarly, responsiveness of cancer cells to cisplatin by shielding the recombinant and human adenoid-derived HMGB1 revealed drug-induced DNA adducts from excision repair, thus pro- antimicrobial activity (5, 6). moting apoptosis specifically in tumor cells (12, 13). In this study, we examined the potential of exogenous HMGB1 to induce cell death in cancer cells in vitro and Authors' Affiliations: 1German Cancer Research Center; 2Institute of in vivo. We describe a recombinant human HMGB1 Pathology, University of Heidelberg, Heidelberg, Germany (rhHMGB1)–dependent novel form of cell death, which is Note: Supplementary data for this article are available at Cancer accompanied by the formation of vacuolated giant mito- Research Online (http://cancerres.aacrjournals.org/). chondria and a rapid depletion of mitochondrial DNA Corresponding Author: Wilfried Roth, Molecular Neuro-Oncology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 (mtDNA). Thus, the HMGB1-induced signaling pathway Heidelberg, Germany. Phone: 49-6221-56-2647; Fax: 49-6221-42-3630; might be a distinct mechanism of how tumor cell death E-mail: [email protected]. evolves in vivo, thereby contributing in the formation doi: 10.1158/0008-5472.CAN-10-0204 and expansion of nonvital tissue areas classically viewed ©2010 American Association for Cancer Research. as tumor “necrosis.”

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Materials and Methods Intracellular localization of 125I-labeled rhHMGB1 For iodination of rhHMGB1, 100 μg protein was incubat- Cell culture ed at room temperature for 15 minutes in 50 mmol/L Human cancer cell lines were purchased from the American phosphate (pH 7.0) with 74 MBq carrier-free Na125I (Hart- Type Culture Collection, expanded, and frozen in aliquots mann Analytic) in the presence of one Iodobead (Pierce). within 4 weeks of purchase. For the experiments described U251MG cells were seeded on 10-cm culture dishes. After here, the cells were thawed and cultured for no more than treatment of cells with 80 nmol/L of 125I-coupled rhHMGB1 eight passages. Nonimmortalized human astrocytic cells were for 0, 4, 16, and 24 hours, the cells were detached carefully purchased from ScienCell and immediately used for the by incubation with cell dissociation buffer (30 minutes). experiments described. For detailed information, see Supple- The radioactivity of each subcellular fraction was measured mentary Materials and Methods. in a liquid scintillation counter (Packard TriCarb 2900). For detailed information, see Supplementary Materials Animal studies and Methods. Six-week-old male athymic CD1 nude mice (n =8; Charles River) were injected s.c. with 7.5 × 106 U251MG Cellular fractionation cells in 100 μL PBS in the right flank using a 30-gauge Enrichment of mitochondrial fractions was performed needle. After 3 weeks, treatment of the xenograft tumors using the ApoAlert Cell Fractionation kit (Clontech) as de- was initiated with 10 μg rhHMGB1 in 500 μLPBSor scribed previously (14). Briefly, after several centrifugation PBS only (control group) by daily i.p. injections at the con- steps, U251MG cells were homogenized with a Dounce tissue tralateral side for 6 weeks. For detailed information, see grinder, and the homogenate was centrifuged at 700 × g for Supplementary Materials and Methods. 10 minutes at 4°C and the resulting supernatant at 10,000 × g for 25 minutes at 4°C. One-dimensional/two-dimensional electrophoresis For large-format two-dimensional (2-D) electrophoresis Immunofluorescence and confocal microscopy and 2-D Western blots, isoelectric focusing was performed U251MG cells were plated onto 8-well chamber slides (Nunc) as described previously using dry polyacrylamide gel or grown confluently in 6-cm culture dishes containing round strips (IPG, 24 cm) with an immobilized pH gradient glass cover plates, permeabilized as described (14), and incu- (pH 3–10) and the IPGphor1 Isolectric Focusing System bated with monoclonal rabbit fluoro-conjugated anti–COX IV (Amersham Pharmacia Biotech). For the second dimen- antibody (1:10, 3E11, Alexa Fluor 488 conjugate; Cell Signaling) sion, the ETTAN DALTsix electrophoresis system was or monoclonal mouse anti–poly-his-tag antibody (1:1,000; R&D used. Western blotting was performed as described previ- Systems) for 30 minutes at 4°C. For detailed information, see ously. For detailed information, see Supplementary Mate- Supplementary Materials and Methods. rials and Methods. Statistical analysis Enucleation of cells All data are means ± SD. The significance of differences U251MG cells (10,000) were seeded in drops of DMEM between groups was determined by Student's t test. P values (10% FCS, 1% penicillin/streptomycin) on round coverslips of <0.05 were considered statistically significant. and incubated for 24 hours at 37°C and 5% CO2. The coverslips were put upside down in a 15-mL Falcon tube containing growth medium complemented with 10 μg/mL Results cytochalasin B. After 1-hour incubation at 37°C, the 15- mL tubes were centrifuged for 1.5 to 3 hours at 37°C with rhHMGB1 exerts cytotoxic effects on cancer cells 4,000 rpm. Then, the coverslips were removed and put into To study the cellular effects of exogenous rhHMGB1, we 6-cm plates containing growth medium. After a recovering treated human glioblastoma cells with increasing concentra- period of 8 hours, the cytoplasts were treated with tions of rhHMGB1 for several days (Fig. 1A). Treatment with rhHMGB1 for 24 hours. Successful enucleation was confirmed rhHMGB1 resulted in a strong inhibition of tumor cell prolif- by immunofluorescence with 1 μg/mL 4′,6-diamidino-2- eration and induction of cell death even at nanomolar con- phenylindole. centrations. Cytotoxicity assays revealed that glioblastoma cells, but not human nonimmortalized astrocytes, were sus- Fluorescence-activated cell sorting analysis of reactive ceptible to rhHMGB1-induced cell death (Fig. 1B). Similarly, oxygen species and mitochondrial membrane potential rhHMGB1 induced a strong inhibition of clonogenic cell sur- For measurement of reactive oxygen species (ROS), 1.5 × vival of glioma cells, but not of astrocytes (Supplementary 106 U251MG cells were seeded in 6-cm plates, treated with Fig. S1). Except U251MG glioblastoma cells, the cytotoxic 40 nmol/L rhHMGB1 for 24 hours, and incubated with the effect of rhHMGB1 was also observed in other long-term fluorescent H2DCF-DA (dichlorodihydrofluorescein diace- (T98G and LN18) and primary (NCH89 and NCH82) glioblas- tate; 10 μmol/L; Invitrogen) for 1 hour at 37°C. ROS was mea- toma cell lines. In these cell lines, the following rhHMGB1 sured immediately at FL-1. For detailed information, see concentrations were required for half-maximal cell killing Supplementary Materials and Methods. (72 hours): T98G, 15 nmol/L; LN18, 15 nmol/L; NCH89,

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Figure 1. Cytotoxic effects of rhHMGB1 on human glioblastoma cells. A, U251MG glioma cells were treated with rhHMGB1 at concentrations as indicated. Points, mean (n = 4); bars, SD. Cell numbers were determined by counting in a Neubauer chamber. B, U251MG glioma cells and nonneoplastic astrocytes were treated with rhHMGB1 for 72 h. Points, mean (n = 3); bars, SD. Cellular survival was assessed by crystal violet staining. C, cotreatment of U251MG glioma cells with rhHMGB1 (4 nmol/L) and TRAIL (24 h, n = 3) or temozolomide (72 h, n = 3). Points, mean; bars, SD. D, systemic (i.p.) rhHMGB1 treatment of CD1 nude mice bearing U251MG xenograft tumors was started 3 wk after inoculation of tumor cells (time point 0). Control mice were injected with saline. The tumor volumes were determined at regular intervals. Points, mean (n = 4); bars, SD.

30 nmol/L; and NCH82, 30 nmol/L. Tumor cell lines of other treatment of glioma cells with rhHMGB1 and the death ligand tissue origins were also susceptible to rhHMGB1-induced TRAIL [tumor necrosis factor (TNF)–related apoptosis- cytotoxicity, although to a somewhat lower extent (IC50 con- inducing ligand] or the anticancer drug temozolomide, which centrations of rhHMGB1: HeLa cells, 35 nmol/L; MCF7 cells, is the first-line chemotherapy for glioblastomas, resulted in 60 nmol/L; HCT116 cells, 65 nmol/L; SW480 and RKO cells, a strong synergistic induction of cell death (Fig. 1C). In >160 nmol/L; Supplementary Fig. S2). Further, combined contrast, astrocytes were not susceptible to the synergistic

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interaction of rhHMGB1 and temozolomide (Supplementary metabolic pathways is essential for the cytotoxic activity Fig. S3). Rat recombinant HMGB1 protein, which exhibits of rhHMGB1. a high homology to human HMGB1 (>98%; ref. 15), showed similar cytotoxic effects as rhHMGB1 (Supplementary rhHMGB1 localizes to the mitochondria and induces Fig. S4). Digestion of rhHMGB1 with 100 μg/mL proteinase the formation of giant mitochondria K before treatment completely abrogated the cell death Given the mitochondria-dependent effects of rhHMGB1, (data not shown). To test the anticancer activity of we investigated whether rhHMGB1 induces morphologic rhHMGB1 in vivo, we systemically treated CD1 athymic changes in mitochondria. Because treatment of glioma cells nude mice bearing subcutaneous glioma xenograft tumors with rhHMGB1 resulted in the formation of large vacuolated with rhHMGB1. After 6 weeks, the rhHMGB1-treated ani- organelles as observed by phase-contrast microscopy (data mals showed significantly smaller tumors than the control not shown), we performed electron microscopy of cells trea- group (Fig. 1D). ted with rhHMGB1. The ultrastructure of the vacuoles revealed a double-membrane and rudimentary cristae rhHMGB1-dependent increase in radical oxygen species (Fig. 3), confirming the mitochondrial nature of the vacuolat- leads to c-Jun NH2-terminal kinase activation ed organelles. The size of these giant mitochondria increased Next, we examined the intracellular signaling events lead- in a time- and concentration-dependent manner after incu- ing to rhHMGB1-induced cell death. rhHMGB1 treatment re- bation with rhHMGB1 (Supplementary Fig. S9). To further sulted in a strong generation of ROS, which was partially characterize the rhHMGB1-dependent mitochondrial prevented by the ROS scavenger N-acetylcysteine (NAC; changes, we performed immunofluorescence studies using Fig. 2A, left). Cotreatment of the cells with NAC substantially the mitochondrial marker enzyme COX IV. These experi- inhibited rhHMGB1-induced cell death (Fig. 2A, right). ments unambiguously identified the vacuolated organelles Similarly, both the rhHMGB1-induced increase in ROS and as extremely enlarged spherical mitochondria in 100% of the rhHMGB1-dependent cell death were significantly inhib- cells treated with rhHMGB1 (Fig. 4A). In contrast, tumor cells ited by PEG-superoxide dismutase (SOD) and PEG-catalase devoid of intact mtDNA (ρ0 cells) that were highly resistant as well as by overexpression of mitochondrially targeted to the cytotoxic actions of rhHMGB1 (Fig. 2C, right) did not manganese-SOD (MnSOD) and (to a lesser extent) catalase form giant mitochondria (Supplementary Fig. S10). Given the (Supplementary Fig. S5). The rhHMGB1-induced ROS gener- chromatin-binding function of rhHMGB1, we next investigat- ation occurred as early as 6 hours after treatment began. ed whether the nuclear localization of rhHMGB1 is required In contrast, loss of plasma membrane integrity as assessed for the formation of giant mitochondria (e.g., by nucleomito- by lactate dehydrogenase release was a late event observed chondrial translocation). Therefore, we generated U251MG after ∼36 hours of rhHMGB1 treatment (Supplementary cells devoid of nuclei, the so-called cytoplasts. Treatment of Fig. S6). Moreover, rhHMGB1 caused a continuous and cytoplasts with rhHMGB1 resulted in the formation of giant dose-dependent phosphorylation of c-Jun NH2-terminal ki- mitochondria to a similar extent and in a similar time frame nase 1/2 (JNK; Fig. 2B, left). The activation of JNK contributes compared with nucleus-containing control cells (Fig. 4B), to rhHMGB1-dependent cytotoxicity because coincubation showing that nuclear events are not required for the gener- with the JNK inhibitor SP600125 not only reduced the ation of giant mitochondria. To address the question of how amount of phosphorylated JNK (Supplementary Fig. S7) but exogenous HMGB1 can induce such marked mitochondrial also partially inhibited cell death induced by rhHMGB1 (Fig. effects, we investigated the subcellular localization of 2B, right). The NAC-dependent inhibition of ROS prevented rhHMGB1. When U251MG glioma cells were incubated with the activation of JNK after rhHMGB1 treatment (Fig. 2C, left), 125I-labeled rhHMGB1, a time-dependent increase of radioac- indicating that rhHMGB1-induced ROS generation occurs tivity was observed in the mitochondrial fraction (Fig. 4C), upstream of JNK activation. To investigate the functional im- suggesting that after penetration of the cell membrane, a portance of intact mitochondria in rhHMGB1-induced cyto- substantial amount of rhHMGB1 translocated to the mito- toxicity, we generated cells devoid of intact mtDNA, so-called chondria. Translocation of rhHMGB1 to the mitochondria pseudo rho zero cells (ρ0 cells; ref. 16). Importantly, ρ0 cells was also confirmed by confocal immunofluorescence micros- were almost completely resistant to rhHMGB1-induced copy after costaining with a mitochondrial marker (Fig. 4D). cell death (Fig. 2C, right), whereas the apoptotic cell death signaling cascade was still intact in these cells (Supplemen- The rhHMGB1-induced cellular effects define a distinct tary Fig. S8). These results suggested that mitochondria mode of cell death represent a main target for cytotoxic actions of rhHMGB1. To further characterize the rhHMGB1-mediated cyto- This hypothesis was further confirmed by the observation toxicity, we examined whether rhHMGB1-induced cell that rhHMGB1-induced cell death in U251MG glioma cells death shares the phenotypic characteristics of apoptosis, was substantially inhibited by adding uridine to the culture autophagy, senescence, or classic necrosis. First, after medium (Fig. 2C, right). Whereas ρ0 cells devoid of intact rhHMGB1 treatment, the typical morphologic features of mtDNA are pyrimidine auxotrophs (16, 17), normal cells apoptosis, such as membrane blebbing, formation of essentially depend on mitochondrial-derived uridine. There- apoptotic bodies, or chromatin condensation, were absent. fore, the uridine-mediated protection from rhHMGB1-induced In addition, activation of caspase-3 or release of cytochrome cell death indicates that the disturbance of mitochondrial c was not observed after rhHMGB1 treatment (Fig. 5A), and

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Figure 2. Role of mitochondria, ROS generation, and JNK activation in rhHMGB1-induced cell death. A, left, ROS emission after treatment with rhHMGB1 was measured in U251MG cells by DCF fluorescence and fluorescence-activated cell sorting analysis. Columns, mean (n = 3); bars, SD. P < 0.05. auto, autofluorescence control. Right, survival of U251MG cells after 48-h treatment with rhHMGB1 and/or NAC (1 mmol/L) as determined by crystal violet analysis. B, left, Western blot analysis of JNK1/2 phosphorylation in cells treated with rhHMGB1 (24 h). TNF-α served as a positive control for the induction of JNK1/2 phosphorylation. Right, survival of U251MG cells after 48-h treatment with rhHMGB1 in the presence or absence of the JNK inhibitor SP600125 (10 μmol/L). C, left, effect of the ROS scavenger NAC on the phosphorylation of JNK1/2 in cells treated with rhHMGB1 (40 nmol/L). Right, survival of U251MG cells after 48-h treatment with rhHMGB1 and/or uridine (5 mg/mL). Pseudo rho zero cells were highly protected from rhHMGB1-induced cell death as assessed by crystal violet staining.

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Figure 3. rhHMGB1-induced formation of giant mitochondria. Electron microscopy of U251MG cells after treatment with rhHMGB1 (24 h). Left, in control cells, the ultrastructure of mitochondria is maintained with numerous cristae and a dense matrix. Middle and right, in contrast, treatment with rhHMGB1 results in the formation of massively enlarged mitochondria with pale matrix and a nearly complete lack of cristae. Asterisks, double membranes. C, rudimentary cristae. Scale bars, 1 μm; scale bars in inset, 200 nm. rhHMGB1-induced cell death was not blocked by the caspase DNA double-strand break repair enzyme H2A.X (18) was inhibitor zVAD-fmk (Supplementary Fig. S11A). Electron mi- activated by rhHMGB1. However, H2A.X phosphorylation croscopy showed neither autophagosomes characteristic for was not observed on rhHMGB1 treatment (Fig. 5B). Se- autophagic cell death nor disintegration and rupture of the nescence was also excluded by analyzing β-galactosidase outer cell membrane indicative of necrotic cell death (Fig. 3). activity after treatment with rhHMGB1 (Supplementary Moreover, autophagy was excluded by a lack of formation of Fig. S11D). Similarly, the necrosis-associated BCL2/adenovi- LC3-GFP dots (Supplementary Fig. S11B) and by a lack of rus E1B 19-kDa interacting protein 3 (BNIP3) was not upre- LC3I/II conversion (Supplementary Fig. S11C) after incuba- gulated after treatment of cells with rhHMGB1 (Fig. 5B). tion with rhHMGB1. The rhHMGB1-induced cell death was Thus, the rhHMGB1-mediated cell death lacks the typical not blocked by the autophagy inhibitor 3-methyladenine or features of apoptosis, autophagy, or classic necrosis. the necroptosis inhibitor necrostatin (Supplementary Fig. To show that the rhHMGB1-induced formation of giant 11A). Next, we examined whether the senescence-associated mitochondria does not only occur in cultured tumor cells

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but also in vivo, we examined the tumor tissue of xeno- associated cell death might represent a mechanism com- grafted nude mice treated systemically with rhHMGB1 monly occurring in the setting of tumor necrosis. To test (Fig. 1D). After immunohistochemical staining of COX IV, this hypothesis, we examined a panel of 25 human malig- numerous giant mitochondria were observed in the xeno- nant tumors of diverse tissue origin containing necrotic grafted tumor cells of rhHMGB1-treated animals (42% of areas by COX IV immunohistochemistry to detect giant cells; Fig. 5C, arrows). However, giant mitochondria were mitochondria. In fact, in the majority of cases (80%), to a lesser extent also visible in perinecrotic areas in un- tumor cells with giant mitochondria surrounding nonvital treated tumors (16% of perinecrotic cells; Fig. 5C, asterisk). tissue areas were observed, especially in glioblastomas and Therefore, we hypothesized that giant mitochondria– squamous cell carcinomas (Fig. 5D).

Figure 4. rhHMGB1 localizes to mitochondria and induces the formation of giant mitochondria. A, formation of giant mitochondria is shown by immunofluorescence labeling of COX IV in U251MG cells treated with his-tagged rhHMGB1 (80 nmol/L) for 24 h. Scale bars, 5 μm; scale bars at the right, 1 μm. B, the formation of giant mitochondria is maintained in enucleated U251MG cytoplasts. Eight hours after enucleation, the cytoplasts were treated with rhHMGB1 (80 nmol/L) for 24 h. Scale bars, 10 μm. C, subcellular fractionation of U251MG cells treated with 125I-labeled HMGB1. The mitochondrial fraction showed a time-dependent increase in radioactivity. 125I-labeled rhHMGB1 also accumulated in the nucleus (∼1.5 mega cpm in 4 h; data not shown). D, confocal immunofluorescence imaging of U251MG cells shows a colocalization of rhHMGB1 (40 nmol/L for 24 h) with MitoTracker (50 nmol/L for 30 min) accumulating in vital mitochondria. Scale bar, 10 μm.

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Figure 5. rhHMGB1 does not trigger apoptotic signaling. Morphologic features of giant mitochondria–associated cell death. A, subcellular fractionation and Western blot analysis from whole-cell lysates of U251MG cells (25 μg loaded). Left, release of cytochrome c into the cytosolic fraction (C) was detected after treatment of cells with the death ligand TRAIL (200 ng/mL, 3 h), but not after treatment with rhHMGB1 (80 nmol/L, 24 h), although rhHMGB1 at this concentration induces substantial cell death after 48 h. M, mitochondrial fraction. Right, similarly, cleavage of caspase-3 (casp3) is observed after TRAIL treatment, but not after rhHMGB1 treatment. B, expression levels of the proteins phospho-H2A.X and BNIP3 by Western blot analysis. Whole-cell lysates of U251MG cells (25 μg loaded) were generated after treatment with rhHMGB1 (24 h). Treatment with TNF-α (100 ng/mL, 1 h) served as a positive control. C, the formation of giant mitochondria was visualized by staining of COX IV in U251MG xenograft tissue (after a 6-wk treatment with rhHMGB1). Arrows and asterisks point to giant mitochondria. Scale bars, 10 μm. D, human tumor tissue with extensive necrosis: basaloid squamous cell carcinoma of the lung (top four panels) and glioblastoma (bottom two panels). N, necrotic area; T, viable tumor tissue. Scale bars, 20 μm. Tumor cells in close vicinity to the necrosis contain giant mitochondria as visualized by H&E staining (top two panels) and COX IV immunohistochemistry (bottom four panels). rhHMGB1 rapidly depletes mtDNA and severely showed a rhHMGB1-dependent substantial and rapid deple- damages the mitochondrial proteome tion of mtDNA (Fig. 6A). The rapid mtDNA depletion after To further characterize the nature of the rhHMGB1- rhHMGB1 treatment was accompanied by a severe depletion mediated mitochondrial damage, we examined the mtDNA of the isoelectric-neutral proteins and an increase of predom- content of cells after rhHMGB1 treatment. Dot blot analysis inantly alkaline proteins (Fig. 6B). Moreover, 2-D Western

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Figure 6. Effects of rhHMGB1 on mtDNA and proteins. A, effect of rhHMGB1 on mtDNA: U251MG cells were treated with rhHMGB1 (24 h) followed by isolation of mtDNA. In the dot blot analysis, the indicated amounts of DNA were loaded in a volume of 1 μLof double-distilled water. B, the mitochondrial protein fractions (100 μg) from U251MG cells treated with rhHMGB1 (80 nmol/L, 24 h) or left untreated were visualized by 2-D gel electrophoresis and subsequent silver staining. Successful subcellular fractionation was confirmed by Western blot (data not shown). C, mitochondrial protein fractions were subjected to a 2-D gel electrophoresis. Then, the membrane was stained with anti-malondialdehyde antibody.

blot analysis of the mitochondrial proteome revealed a sub- recombinant HMGB1 act as immune adjuvants, enabling stantial increase in covalently bound malondialdehyde in the dendritic cells and cytotoxic T cells to attack specifically tu- alkaline range after treatment with rhHMGB1 (Fig. 6C). Sim- mor cells (7, 10). Moreover, HMGB1 signaling through TLR2 ilarly, rhHMGB1 induced the formation of hydroxynonenal and TLR4 is crucial for an effective immune response medi- (19) adducts (Supplementary Fig. S12A). HMGB1 also altered ating tumor regression in humans (9) and in rodents (10, 11). the ratio of glutathione (GSH) to glutathione disulfide (GSSG) However, the giant mitochondria–associated rhHMGB1- shown by a significant increase in GSSG and a decrease in induced cell death we describe is independent of TLR2, GSH after 48 hours of rhHMGB1 treatment (Supplementary TLR4, or RAGE signaling, because the TLR4, TLR2, and Fig. S12B). Taken together, these results show that rhHMGB1 RAGE agonists lipopolysaccharide (LPS), PAM3CSK4, and induces lipid peroxidation, severe oxidative stress, and inhi- AGE did not induce formation of giant mitochondria or cell bition of mtDNA replication. death in glioma cells (Supplementary Fig. S13). Moreover, overexpression of the receptors TLR2, TLR4, RAGE, or their Discussion intracellular transducers MyD88 and TRIF, or the small inter- fering RNA–mediated downregulation of TLR4 or RAGE did In this study, we describe that rhHMGB1 protein induces not alter the susceptibility of glioma cells to the rhHMGB1- a novel form of cell death that is characterized by the for- induced cell death (Supplementary Fig. S14; data not shown). mation of giant mitochondria and a severe damage to Recently, it was shown that endogenous HMGB1 can mtDNA and proteins. It has been described that both endog- translocate from the nucleus to the mitochondria, resulting enous HMGB1 that is released from dying tumor cells and in reorganization of the mitochondria in endothelial cells

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(20). In our study, we show that rhHMGB1 enters the mito- increase and cell death on rhHMGB1 treatment were inhib- chondria, which is followed by the formation of giant mito- ited by NAC as well as by specific ROS scavengers such as chondria. However, the mechanisms that enable HMGB1 to PEG-catalase and PEG-SOD. Similarly, a ROS increase and pass the cell membrane and the mitochondrial membranes higher intramitochondrial concentrations of its toxic lipid still have to be clarified. Interestingly, HMGB1 has been used peroxidation product malondialdehyde were measured dur- to potentiate the transfection of cells with compacted DNA, ing the formation of megamitochondria in hepatocytes yielding greater transfection rates compared with convention- from rats fed with the megamitochondria-inducing com- al techniques (21). This might be due to the polybasic penetra- pound hydrazine (22, 27), which resulted in the induction tin-like domain of HMGB1, common to proteins such as of apoptosis in this cell model (28). Accordingly, our experi- Antennapedia, lactoferrin, and the HIV protein Tat, which ments revealed high concentrations of intramitochondrial can transport DNA or RNA across cell and organelle mem- toxic malondialdehyde protein adducts after treatment with branes (1). In this context, it is interesting that the uptake of rhHMGB1, resulting in a severe damage of the mitochon- rhHMGB1 in glioma cells was not inhibited by the endocytosis drial proteome. The mitochondria seem to be the main inhibitor brefeldin A and that several endocytosis inhibitors mediators of the rhHMGB1-induced cell death, because nu- did not alter the extent of HMGB1-induced cell death (Supple- clear signaling events are not required for giant mitochon- mentary Fig. S15), suggesting that endocytosis does not play dria formation as shown in glioma cells devoid of nuclei. an essential role for the internalization of rhHMGB1. With respect to both morphology and signal transduction Our results suggest that rhHMGB1 is the first known events, the rhHMGB1-induced cell death is different from mammalian protein that specifically induces the generation apoptosis, autophagocytotic cell death, and senescence. It is of giant mitochondria. The giant mitochondria observed in also different from classic necrosis, which is characterized our study are morphologically similar to megamitochon- by cytoplasmic swelling, rupture of the plasma membrane, dria, which can be induced by several chemical com- and mild swelling (but not fusion) of cytoplasmic organelles pounds, including ethanol, chloramphenicol, hydrazine, (29). Moreover, the giant mitochondria–associated cell death and ethidium bromide (22). However, in the glioblastoma observed in our study clearly differs from the recently de- cell model we used, the giant mitochondria–associated cell scribed “necroptosis,” a form of programmed necrosis that death was not observed after treatment with compounds depends on the kinase RIP1 (30). Giant mitochondria have that are reportedly known to induce megamitochondria thus far not been reported to be associated with necroptosis, and depletion of mtDNA in other cell systems (22), such and necrostatin-1 had no effect on rhHMGB1-induced cell as high doses of ethidium bromide or ethanol (data not death (Supplementary Fig. S11A), which excludes an essential shown). Moreover, we show that even very low concentra- role for the RIP1 kinase activity. Because cells with giant tions of rhHMGB1 are capable of inducing giant mitochon- mitochondria are present at perinecrotic areas in human dria. Once translocated to mitochondria, rhHMGB1 leads tumor tissue and because the HMGB1 protein is released to a rapid depletion of mtDNA. A similarly rapid but re- during necrosis, the HMGB1-induced, giant mitochondria– versible and less effective depletion of mtDNA is described associated cell death might represent an important novel for ethanol in the liver cells of an alcoholic binge mouse mechanism of specialized necrosis. model (23). Importantly, the rhHMGB1-dependent effects The molecular basis of giant mitochondria formation is on mitochondria are absent in pseudo ρ0 cells harboring still not understood. Similarly, further studies are needed mutated and intercalated mtDNA instead of intact, wild- to elucidate why exogenous HMGB1 protein is much more type mtDNA. In this context, it is of interest that the hu- effective in inducing giant mitochondria than the endo- man HMGB1 protein can inhibit closed circular plasmid genously expressed HMGB1 protein. Although giant replication in vitro (24) and that the yeast homologue of mitochondria formation is observed on transfection and HMGB1, the mitochondrial HM protein, is required for overexpression of hHMGB1 (Supplementary Fig. S6), the the maintenance and expression of the yeast mitochondrial extent of giant mitochondria generation is larger after ex- genome. In contrast, a deficiency of HM under certain cir- ogenous treatment with HMGB1. Thus, we hypothesize cumstances leads to loss of mtDNA, which can be pre- that exogenous HMGB1 undergoes a modification when vented by overexpression of NHP6A, the yeast nuclear it passes the cell membrane, for example, as described variant of HM (25, 26). Therefore, HMGB1 or its yeast for serine-phosphorylated HMGB1 that is directed toward homologues are important regulatory proteins that can secretion (31). Tyrosine residue modification seems not maintain or inhibit the expression of the mitochondrial to play a crucial role, as the covalent binding of 125Ito genome. We postulate that the interaction of rhHMGB1 tyrosine phosphorylation sites did not interfere with with mtDNA mediates the deleterious effects of rhHMGB1 rhHMGB1-induced giant mitochondria formation and cell on the mitochondrial genome and proteome as well as the death (data not shown). giant mitochondria–associated cell death. Finally, the application of rhHMGB1 might be a prom- What are the downstream effectors of rhHMGB1-induced ising new approach in the therapy of patients with malig- cell death? Our data indicate an important role of ROS, nant tumors. Because glioblastoma cells, but not normal which rapidly rise after treatment of cells with rhHMGB1, astrocytic cell cultures, were highly susceptible to whereas the loss of plasma membrane integrity is a rather rhHMGB1-induced cell death, the rhHMGB1-mediated late event in rhHMGB1-induced cytotoxicity. Both the ROS mitochondrial effects might be tumor specific. However,

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Gdynia et al.

the potential toxicity of rhHMGB1 will need to be inves- Rauvala (University of Helsinki, Helsinki, Finland) for plasmids encoding for RAGE, ΔRAGE, and p30 (HMGB1); Peter Krammer and Marcin Kaminski tigated thoroughly, because rHMGB1 can mediate endo- (German Cancer Research Center, Heidelberg, Germany) for plasmids toxemia in mice. Wang and colleagues showed that in encoding MnSOD and catalase and the corresponding antibodies; David μ Capper and David Reuss (Department of Neuropathology, University Hospital mice between 20 and 50 g, HMGB1 is released into Heidelberg) for providing histologic sections of glioblastomas; Arne Warth the murine circulation after stimulation with LPS, where- (Institute of Pathology, Heidelberg) for providing histologic sections of as in our xenograft mouse model 10 μgofrhHMGB1were lung cancer; and Thomas Hoffmann and Dirk Sombroek (German Cancer Research Center, Heidelberg, Germany) for generously providing the administered i.p. (3). Future studies will have to examine senescence assay kit. in detail the possibilities and limitations of a rhHMGB1- based experimental therapy. Grant Support Disclosure of Potential Conflicts of Interest Deutsche Krebshilfe (German Cancer Aid, Max Eder Program; W. Roth); No potential conflicts of interest were disclosed. and Heinrich FC Behr Foundation (G. Gdynia). 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 Acknowledgments accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Angelika Bierhaus (University Hospital of Heidelberg, Heidelberg, Received 01/22/2010; revised 08/25/2010; accepted 09/10/2010; published Germany) for generously providing rat recombinant HMGB1 and AGEs; Heikki OnlineFirst 10/19/2010.

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8568 Cancer Res; 70(21) November 1, 2010 Cancer Research

Danger Signaling Protein HMGB1 Induces a Distinct Form of Cell Death Accompanied by Formation of Giant Mitochondria

Georg Gdynia, Martina Keith, Jürgen Kopitz, et al.

Cancer Res Published OnlineFirst October 19, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-10-0204

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