Springer 2006

Journal of Neuro-Oncology (2006) 77: 247–255 DOI 10.1007/s11060-005-9045-5

Laboratory Investigation

Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines Katsu Mizukawa, Atsufumi Kawamura, Takashi Sasayama, Kazuhiro Tanaka, Masahito Kamei, Masato Sasaki and Eiji Kohmura Department of Neurosurgery, Kobe University Graduate School of Medicine, Chuo-ku, Kobe, Japan

Key words: apoptosis, etoposide, glioblastoma, Smac/DIABLO, synthetic Smac peptide, XIAP Summary Smac/DIABLO is a mitochondrial protein released into cytosol during the progression of apoptosis. Smac/DIABLO promotes apoptosis by neutralizing the inhibitory effect of the inhibitor of apoptosis proteins (IAPs) on the processing and activity of the effecter of caspase. Here, we generated synthetic Smac peptide which possesses an IAP-binding domain and Drosophila antennapaedia penetration sequence, and examined whether it enhances the effect of the chemotherapeutic agent etoposide in the human glioblastoma cell line. Cellular uptake of Smac peptide in several glioma cell lines was most prominent at 6–12 h after addition. Caspase activity assay showed that our peptide successfully increased the activity of caspase-3 and caspase-9 in etoposide-induced apoptosis. In addition, Smac peptide increased the amount of cleaved PARP (poly ADP-ribose polymerase), but control peptides did not. Moreover, the addition of z-VAD-fmk, a caspase inhibitor, counterbalanced the effect of Smac peptide. Finally, we demonstrated that Smac peptide could enhance the growth inhibition effect of etoposide compared with control peptides. These results suggest that synthetic Smac peptide may be a new molecular targeting anti-tumor therapy for human glioblastoma.

Introduction Glioblastoma multiform is the most common malignant primary brain tumor in adults. Despite advances in intensive multimodality treatment, including surgical resection, irradiation, and chemotherapy, the 5-year survival rate of this tumor has remained less than 10% in the past decade [1]. According to recent reports, resistance to the current strategy protocols includes the problem that target tumor cells resist apoptosis [2,3]. The correction of defects in the apoptosis programs of malignant tumor cells may contribute to the improvement of tumor progression and treatment resistance [2,4–6]. Apoptosis is a distinct form of cell death essential for the correct development, homeostasis, host defense and suppression of oncogenesis, and is provoked by various stimuli, including chemotherapeutic agents or irradiation. The pathway of apoptosis may be initiated through two different entry sites, death receptors or mitochondria resulting in the activation of effecter caspases [7–10]. The stimulation of death receptors of the tumor necrosis factor (TNF) receptor superfamily, such as CD95 (APO-1/Fas) or TNF-related apoptosis-inducing ligand (TRAIL) receptors, results in caspase-8 activation, which initiates the direct cleavage of downstream effecter caspases [7,8]. The mitochondrial pathway is initiated by the release of apoptogenic factors such as cytochrome c, apoptosis-inducing factor (AIF), or a second mitochondria-derived activator of caspase

(Smac)/direct IAP-binding protein with low pl (DIABLO) from mitochondria to the cytosol [11]. These factors activate caspase-3, -7, -9 and induce apoptosis. On the other hand, this pathway is tightly regulated by a family of polypeptides known as inhibitors of apoptosis proteins (IAPs) [12–21]. In mammals, eight IAP family members are currently known, all of which contain 1–3 Baculoviral IAP repeat (BIR) domains [22–25]. Among these, XIAP, cIAP1, and cIAP2 have been shown to inhibit caspases-3, -7, and -9 [26–28]. Conversely, Smac was recently identified as a mitochondria protein that is released into the cytosol where it is able to promote cell death by preventing the IAP inhibition of caspase [29–31]. Its 55 N-terminal residues encode the mitochondrial-targeting sequence, which is proteolytically removed after the apoptotic stimuli [29]. This cleavage results in the exposure of four hydrophobic amino acids, Ala-Val-Pro-Ile (AVPI) at the N-terminus of mature Smac. This tetrapeptide represents the founding member of a family of IAP-binding motifs in mammals and fruit flies [32]. The co-crystal structure of mature Smac in complex with the XIAP BIR3 domain shows that Smac N terminus interacts with a groove formed on the BIR3 surface [33,34]. Some articles have reported that the overexpression of Smac protein induced apoptosis, and that synthetic Smac peptide enhanced the effect of chemotherapeutic agents in various types of malignant tumor cells by interacting with IAPs and preventing their inhibition of caspase [35–39]. However, there is no report concerning the

248 additive or synergistic cytotoxicity of synthetic Smac peptide in the drug-induced apoptosis of glioblastoma mediated by the mitochondrial pathway, and not triggered by the stimulation of death receptors. In this study, we examined whether synthetic Smac peptide could promote the activity of caspases and enhance the effect of the chemotherapeutic agent etoposide in the human glioblastoma cell line. We also showed that synthetic Smac peptide increased the activity of caspase9 and caspase-3 by 22 and 17%, respectively, and could reduce about 7–10% less tumor cell survival than etoposide alone.

3¢-TUNEL analysis for the assessment of apoptosis For the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) experiments, cells were briefly rinsed in PBS and fixed for 20 min in 4% paraformaldehyde in PBS. Cells were then permeabilized with 0.5% Triton X-100 in PBS for 10 min and washed three times with water. TUNEL reaction was performed by a MEBSTAIN apoptosis Kit II (Medical & Biological Laboratories Co., Ltd. Nagoya, Japan), according to the manufacturer’s instructions. Analysis of DNA fragmentation

Materials and methods Cells and drugs used in this study We performed all examinations using the T98G glioblastoma cell line, which was reported by our institute to undergo apoptosis only at high concentrations of etoposide [40]. In addition, we also used other glioblastoma cell lines (A172, U251, U87) for some experiments. Tumor cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing glutamine, 10% fetal bovine serum, and penicillin/streptomycin. Cells were grown at 37 C in a 5% CO2 incubator. Etoposide (a kind gift from Nippon Kayaku Co., Ltd.) was dissolved in DMSO as a 50 mg/ml stock solution. The final concentration of DMSO in the culture medium was less than 0.1%, according to a previous report by our institute [40]. We also used benzyloxycarbonyl-Val-Ala-Asp (OME) fluoromethylketone (z-VAD-fmk) (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) caspase inhibitor to investigate caspase-dependent apoptosis [41]. Construction of synthesized Smac peptide and control peptides We generated synthetic Smac peptide with the first eight N-terminal amino acids of Smac, the Drosophila antennapaedia penetration sequence [42] and fluorescein isothiocynate (FITC). We prepared two different control peptides and their constructions are shown in Figure 2b. Our peptides were prepared by a private institute (Peptide Institute Inc., Osaka, Japan). All peptides were dissolved in distilled water as a 25 mg/ml stock solution.

DNA fragmentation was detected using an Apoptosis Ladder Detection Kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). After incubation for 48 h after the addition of etoposide, the harvested cells (1.0106) were centrifuged and washed twice with cold PBS. DNA was extracted according to the manufacturer’s instructions and gently dissolved in TE buffer. The samples were electrophoresed on 2% agarose gel and DNA was visualized using SYBRTM Green (Molecular Probes, Inc., Oregon). Evaluation of cell survival Cell survival inhibition by etoposide with or without synthetic peptides in T98G cells or A172 cells was quantified using a modified methylthiazol tetrazolium (MTT) colorimetric assay. Cells were seeded at 104 cells/ well in 96-well flat-bottomed plates and incubated for 48 h at 37 C in a 5% CO2 incubator. At first, we evaluated the cytotoxity of synthetic peptides without etoposide (Figure 4b). Next, various concentrations of etoposide (40 or 80 lg/ml) were added to the wells (Figure 4c). We used 16 wells for each concentration’s group. Synthetic peptides were added as 10 lg/ml in medium, 6 h before etoposide treatment. After incubation, 0.01 ml of MTT reagent (5 mg/ml in PBS) was added to each well for an additional 4 h of incubation at 37 C. Isopropanol (0.11 ml with 0.04 N HCl) was added to dissolve the precipitates, and then absorbance at 570 nm was measured using an automated plate reader. The cell survival ratio was calculated as follows: cell survival (%) = (the absorbance of treated cells with medium – absorbance of only medium)/(absorbance of non-treated cells with medium – absorbance of only medium)  100. The statistical significance of the findings was assessed using the Mann–Whitney U test.

Flow cytometric analysis Western blot assay Cells were grown in medium containing 10% fetal bovine serum for 48–72 h. To prepare samples for flow cytometric analysis, cells were treated with trypsin and fixed with 70% ethanol, and then were stained for DNA by incubating in phosphate-buffered saline (PBS) containing 50 lg/ml propidium iodide (PI) and 40 U/ml of RNase for 30 min at 37 C. Samples were analyzed by the fluorescence-activated cell sorter (FACS) using the CellQuest software (Beckton Dickinson).

Electrophoresis was performed for 90 min at 100 mA in a 1.0 mm-thick 10 or 15% polyacrylamide gel. The proteins were transferred electrophoretically at a constant voltage of 15 V for 30–180 min. The cells were assessed by immunoblotting using the primary antibodies for Smac/DIABLO (ZYMED Laboratories, Inc., South San Francisco, California), FITC (DakoCytomation, Denmark), a-tubulin (SIGMA), XIAP (Cell

249 Signaling Technology, Inc.), or PARP (Cell Signaling Technology, Inc.). The procedures were followed by the recommendation of ECL Advance Western Blotting detection kit (Amersham Biosciences. BCA protein-assay reagent kit (PIERCE, Rockford, IL) was used to adjust each sample to equalize the concentration of proteins. The intensities of the bands were quantified by NIH Image software. Caspase-3 and caspase-9 colorimetric assay Briefly, harvested cells, with added etoposide with or without peptides and incubated 48 h, were centrifuged and washed twice with cold PBS. The cell pellet was treated using a caspase assay kit (caspase-3/CPP32 Colorimetric Protease Assay Kit, caspase-9/Mch6 Colorimetric Protease Assay Kit, Medical & Biological Laboratories Co., Ltd. Nagoya, Japan) according to the manufacturer’s instructions. These caspase assay kits provide a simple and convenient means for assaying the activity of caspases that recognize the sequences of DVED (caspase-3) or LEHD (caspase-9). The assays are based on the spectrophotometric detection of chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA or LEHD-pNA. The samples were applied to a 96-well plate and incubated for 1–3 h at 37 C. Protease activity was detected in a microplate reader at 405 nm. Comparison of the absorbance of pNA allows the determination of fold increase in caspase activity. We constructed graphs with the indicator of caspase activities calculated as follows: relative ratio = the absorbance of each sample from treated cells/absorbance of non-treated cells  1. The statistical significance of the findings was assessed using the Mann–Whitney U test. Fluorescence microscope As our synthetic peptides were generated with conjugating FITC, we observed the uptake of peptides into the cytoplasm using a fluorescence microscope (LSM 5 PASCAL, ZWEISS), without fluorescent staining.

Results Detection of etoposide-induced apoptosis in the T98G and A172 glioblastoma cell line Etoposide is a chemotherapeutic agent, which induces apoptosis in many cancer cells, including glioma cells [40]. To determine whether etoposide induced apoptosis in T98G cells, we assessed apoptosis by two different methods, flow cytometric analysis and TUNEL assay. As shown in Figure 1a, flow cytometric analysis showed marked induction of subG1 fraction (29–35%) in T98G cells, indicating cell death by etoposide treatment. In TUNEL assay, no TUNEL-positive cells was seen in untreated cells, whereas TUNEL-positive cells could be observed remarkably in etoposide treated cells, indicating etoposide-induced apoptosis (Figure 1b). Moreover, we examined etoposide-induced apoptosis and obtained

similar results in another glioblastoma cell line A172 by with same methods (flow cytometric analysis and TUNEL assay) (data not shown). We also confirmed etoposide-induced apoptosis in A172 cells by DNA fragmentation assay (Figure 1c). Furthermore, the induction of apoptosis was detected with a Western blotting for the cleavage of PARP. PARP is one of the cleavage targets of caspases and serves as a marker of cells undergoing apoptosis [43–46]. The expression of cleaved PARP increased in a time-dependent manner until 48 h, but had almost vanished after 78 h (Figure 1d). The cleaved PARP also increased depending on the concentration of etoposide (Figure 1e). Translocation of endogenous Smac in etoposide-induced apoptosis To examine the translocation of endogenous Smac in T98G cells, mitochondria or cytosolic extracts were prepared using a mitochondria/cytosol fractionation kit (BioVision, California) and Western blot was performed. Endogenous Smac was released from the mitochondria to the cytosol fraction, induced by treatment with 40 lg/ ml etoposide in medium (Figure 2a). However, not all Smac was released into the cytosol, and there was only partial translocation of endogenous Smac induced by 40 lg/ml etoposide. Uptake of synthetic Smac peptide into the cytoplasm We generated synthetic Smac peptide with the first 4 amino acids of Smac, the IAP-binding motif, Drosophila antennapaedia penetration sequence and FITC, and two different control peptides (Figure 2b). We then assessed the cellular uptake of peptides using Western blotting and a fluorescence microscope. We administrated synthetic peptides 10 lg/ml in medium, and harvested culture cells after various hours of incubation. Our synthetic Smac peptide was successfully taken up into the cytoplasm of all glioblastoma cell lines: T98G, A172, U251, and U87. The uptake in T98G, A172, and U251 cells were most prominent after 6 h, while the uptake in U87 cells was prominent after 12 h (Figure 2d). After 48 h, Smac peptide had decreased in all cell lines. The Smac peptide in T98G decreased 72.8% of peak level after 48 h, while Smac peptide in A172 decreased 34.0% (Figures 2d, lower graph). Control peptides were taken up into cytoplasm in a similar manner (data not shown). Increasing the activities of caspases Next, we examined whether our synthetic Smac peptide increased the activity of caspases, and observed that treatment with 40 lg/ml etoposide increased caspase activity. Relative ratio of caspase-3 activity increased 4.24±0.24 fold and that of caspase-9 activity increased 2.64±0.15 fold compared with no treatment. The administration of 10 lg/ml Smac peptide with etoposide significantly enhanced the relative ratio of caspase-3 activity by 17% compared to etoposide alone (4.97 ± 0.34, P<0.05) and that of caspase-9 activity by 22% compared to etoposide alone (3.24 ± 0.21,

250

Figure 1. Detection of etoposide-induced apoptosis in the T98G and A172 glioblastoma cell lines. (a) Etoposide-induced cell death in T98G cells. The cells were analyzed by flow-cytometory 48 h after the addition of 0, 1, 10, 100 lg/ml etoposide. (b) Apoptosis in T98G cells treated with etoposide. The cells were analyzed 48 h after the addition of 50 lg/ml etoposide. Apoptotic cells were stained by the TUNEL assay (green). Cell nuclei were stained red with propidium iodide (PI). Scale bars, 50 lm. (c) A172 cells were treated with 1, 10 or 50 lg/ml of etoposide, then cultured for 48 h. The DNA of these cells was isolated and electrophoresed on 2% agarose gel. (d, e) Western blots showing the expression of cleaved PARP (arrow) in etoposide-treated T98G cells. The cells were analyzed 24, 48 or 72 h after the addition of 40 lg/ml etoposide, or analyzed 48 h after the addition of 10 or 40 lg/ml etoposide.

P<0.05). Smac peptide alone did not increase caspase activity (caspase-3: 0.96 ± 0.04, caspase-9: 1.06 ± 0.04) (Figure 3a, c). On the other hand, control peptide-1 10 lg/ml did not affect the activity of caspase (Figure 3b, d). A similar result was obtained with control peptide-2 (data not shown). Synthetic Smac peptide enhances etoposide-induced apoptosis and cell survival inhibition We analyzed whether our synthetic peptides enhance etoposide-induced apoptosis, by comparing with the expression of cleaved PARP and by assessment using a modified methylthiazol tetrazolium (MTT) colorimetric assay. Combination with our synthetic Smac peptide and etoposide increased the cleavage of the caspase substrate PARP. But on the other hand, control pep-

tide-2 and etoposide showed no effect (Figure 4a, lanes 3, 4). We examined the effect of z-VAD-fmk, a caspase family inhibitor, which could suppress this phenomenon to reveal whether Smac peptide would promote etoposide-induced apoptosis by mediating the caspase pathway. The administration of z-VAD-fmk, 5 lM in medium, suppressed the cleavage of PARP completely (Figure 4a, lane 5). Moreover, the effect of our Smac peptide was suppressed almost completely (Figure 4a, lane 6). Finally, we confirmed the effect of Smac peptide on tumor cell survival by comparing the results of the modified MTT assay. Smac peptide or control peptide-1 alone did not reduce the survival ratio in T98G, similarly to the results of caspase activities (No treatment: 99.99 ± 1.00%, Smac peptide: 98.93 ± 1.55%, Control peptide: 96.86 ± 1.68%) (Figure 4b). Forty lg/ml and 80 lg/ml etoposide reduced T98G cell survival to

251

Figure 2. The movement of endogenous Smac by etoposide treatment and cellular uptake of the synthetic Smac peptide. (a) Subcellular fractionation analysis of endogenous Smac distribution in untreated and etoposide-treated T98G cells. Cells were treated with 40 lg/ml etoposide for 24 h. Mitochondrial and cytosolic fractions were prepared and subjected to immunoblotting with anti-Smac antibody. Abbreviations: Mito: mitochondrial fraction, Cyto: cytosolic fraction. (b) Schematic representation of the Smac peptide and two control peptides. The Drosophila antennapaedia penetration sequence is boxed. * indicates FITC, which is conjugated at the C-terminal. (a) Synthetic Smac peptide. AVPI is the first 4 N-terminal amino acids of mature Smac. (b) Control peptide-1. (c) Control peptide-2. (c) Fluorescence microscope images showing the uptake of peptides in cytoplasm (400). T98G cells were fixed 3, 6, 12 or 24 h after the addition of 10 lg/ml peptides in medium and analyzed using a fluorescence microscope. (d) Western blots showing the cellular uptake of the synthetic Smac peptide. Tumor cells were treated with 10 lg/ml of Smac peptide for 6, 12, 24 or 48 h. Total cell extracts were subjected to immunoblotting with anti-FITC antibodies. The intensities of the Smac peptide’s bands were quantified by NIH Image software.

50.2 ± 1.8% and 40.1 ± 1.2% after 72 h. The addition of Smac peptide resulted in a further reduction of T98G cell survival to 40.2 ± 1.4% (P <0.01) and 34.3 ± 0.7% (P <0.01) (Figure 4c, left panel). Control peptide-1 could not affect the reduction of absorbance (40 lg/ml etoposide: 51.9 ± 2%, 80 lg/ml etoposide: 40.4 ± 0.8%). In addition, because Smac peptide was more stable in A172 cells than in T98G cells (Figure 2d), we examined the effect of Smac peptide on A172 cell survival by the same methods. While 40 and 80 lg/ml etoposide reduced A172 cell survival to 82.6 ± 2.1% and 78.0 ± 2.9% respectively, the addition of Smac peptide resulted in a further reduction of A172 cell survival to 75.9 ± 1.0% (P <0.05) and 70.2 ± 0.9% (P <0.05) respectively (Figure 4c, right panel).

Discussion Smac protein is expressed in most adult human tissues including the brain, heart, liver, lung and spleen and several types of human cancer [47], however, the

expression of Smac protein in human glioma cells has not been reported. We first examined the expression of Smac protein and confirmed that it is released from mitochondria to cytosol after etoposide treatment. As in previous reports, endogenous Smac protein released from mitochondria was not complete, and a significant amount of endogenous Smac protein was detected in mitochondria extracts after etoposide treatment in T98G cells (Figure 2a). Based on previous results that the N-terminal 4 peptide of mature Smac binds to and inhibits XIAP protein [33, 34], we generated a synthetic Smac peptide with a Drosophila antennapaedia penetration sequence. The synthetic Smac peptide was successfully taken up into the cytoplasm of established glioblastoma cell lines (Figure 2c and d), and the amount of cytoplasmicaccumulated Smac peptide in most of cell lines peaked at 6 h after addition, then Smac peptide was degraded. Reduction of Smac peptide in T98G was rapid, while Smac peptide in A172 was reduced gradually (Figure 2d). Recent studies have revealed that endogenous Smac protein is rapidly degraded after being released

252

Figure 3. Graphs showing the effects of synthetic Smac peptide on the activation of caspases during etoposide-induced apoptosis. T98G cells were treated with 10 lg/ml of Smac or control peptides, and 6 h later, the cells were treated with 40 lg/ml of etoposide. After 48 h of incubation, the cells were analyzed using a colorimetric protease assay kit. (a, b) Graphs of caspase-9. (c, d) Graphs of caspase-3. *: P<0.05 relative to etoposide alone (n = 16). Abbreviations: Smac: Synthetic Smac peptide, Control: Control peptide-1, NS: not significant.

from mitochondria by proteasome, and XIAP can function as an E3 ligase and promotes the degradation of Smac during apoptosis [48]. Thus, it could be considered that XIAP would promote the degradation of synthetic Smac peptide in glioma cells. But, it was not clarified the difference of the degradation among T98G and A172 cells. In this study, Smac peptide increased the activity of caspase-9 and caspase-3 by about 22 and 17%, respectively, in the procedure of etoposide administration (Figure 3). In addition, the Smac peptide promoted the expression of cleaved PARP, and the addition of z-VAD-fmk inhibited the effect of the Smac peptide (Figure 4a), indicating that our synthetic Smac peptide induces caspase-dependent apoptosis. Several papers have demonstrated the anti-neoplastic effect of Smac in various tumor cells. Fulda, et al. [35] reported that the ectopic expression of cytosolic Smac without a mitochondrial targeting sequence sensitized neuroblastoma, glioma, and breast carcinoma cell lines for apoptosis induced by TRAIL, anti-CD95 or doxorubicin in vitro. In addition, synthetic Smac peptide enhanced the anti-tumor activity of Apo-2L/TRAIL in an intracranial malignant glioma xenograft model in vivo [35]. The N-terminus of Smac peptide also enhanced the effects of chemotherapeutic agents, paclitaxel, etoposide, 7-ethyl-10-hydroxycamptothecin (SN-38) and doxorubicin in breast-cancer cell lines [36]. Further-

more, co-treatment with synthetic Smac peptide enhances epothilone B-induced or TRAIL-induced apoptosis in Jurkat cells [39]. Our results demonstrated that the synthetic Smac peptide could induce 7–10% less tumor cell survival than etoposide alone (Figure 4c). These results seem to be not so much synergistic effect as additive effect, and the effect of Smac peptide was lower than that reported previously [35–38]. There are four possible reasons for this difference: first, the expression level of XIAP protein in T98G and A172 cells is extremely high so the addition of Smac peptide would not be sufficient to inhibit the anti-apoptotic function of XIAP, and IAPs, including XIAP, are reported to be highly expressed in malignant glioma [5, 6]. Second, IAP family proteins that bind to the synthetic Smac peptide in T98G and A172 cells may have weak caspase inhibitory activity. In mammals, there are eight IAP family proteins, all of which contain BIR domains, however, the relative affinity of these IAP proteins for caspases differs greatly [26, 27]. Indeed, only XIAP, generally recognized as the most potent endogenous caspase inhibitor, has a high affinity for caspases. Third, in a report by Fulda et al. [35] the cells of the control groups suppressed the natural mitochondrial pathway with an overexpression of Bcl-2 so as to suppress the function of endogenous Smac and to evaluate the precise reaction of synthetic Smac; in other words, the distinct results came from the difference between the control samples of each

253

Figure 4. (a) Western blots showing the effect of synthetic Smac peptide, which affects the expression of cleaved PARP (arrow). T98G cells were treated with 40 lg/ml of etoposide and/or 10 lg/ml of Smac or control peptide and/or 5 lM of z-VAD-fmk, as shown in the panel. Cell extracts were subjected to immunoblot analysis with antibodies to PARP, as indicated. (b) Graph showing the cell survival ratio of T98G cells treated only with 10 lg/ml of Smac or control peptide-1, as shown in the panel. Cells were analyzed 72 h after the addition of peptides by MTT assay. Similar results were obtained in three independent experiments. Abbreviations: NS: not significant. (c) Graphs showing the cell survival ratio of T98G cells or A172 cells treated with 40 lg /ml or 80 lg /ml of etoposide and 10 lg/ml of Smac or control peptide-1, as shown in the panels (T98G: left panel, A172: right panel). Cells were analyzed 48 h (A172) or 72 h (T98G) after the addition of drugs by MTT assay. *: P <0.01 relative to etoposide alone (n = 16). **: P <0.05 relative to etoposide alone (n = 16). Similar results were obtained in three independent experiments. Abbreviations: Smac: synthetic Smac peptide, Control: Control peptide-1, NS: not significant.

experiment, including the effect of endogenous Smac peptide similar to clinical samples. Finally, there is some possibility that, in etoposide-induced apoptosis of T98G and A172 cells, the Smac-related apoptotic pathway would not be major cascade during apoptosis. It was reported that chemotherapeutic agents, including etoposide, increase the expression of death receptors, such as death receptor 4 or death receptor 5, in human glioma, resulting in the promotion of TRAIL inducedapoptosis [49]. As p53 is a mutant form in T98G cells, other apoptotic pathways except Smac cascade would be co-activated by etoposide treatment. Moreover in A172 cells which have wild type p53, co-treatment with etoposide and Smac peptide revealed a less increase of apoptosis than in T98G cells. It is possible that some

drug resistant proteins may prevent A172 cells from cell death induced by the combination of Smac peptide and etoposide. As for treatment of A172 cells with 1(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), an alkylating antitumor agent, the primary target of which has been thought to be DNA, the administration resulted in elevated expression of mRNA for multidrug resistance-associated protein (MRP) within the first 2 h after the treatment [50]. Our findings suggest that co-treatment with the N-terminus Smac peptide is an effective strategy to enhance apoptosis triggered by the mitochondrial pathway, and may improve the anti-tumor activity of chemotherapeutic agents in glioma cells.

254 Conclusions We assessed the effect of synthetic Smac peptide in the etoposide-induced apoptosis of human glioblastoma cell lines in vitro. In our studies, the cellular uptake of synthetic Smac peptides and their movement were shown. Our synthetic Smac peptide successfully promoted the caspase action and resulted in the enhancement of etoposide-induced apoptosis. Taken together, the synthetic Smac peptide could be useful to the conventional glioblastoma therapy.

Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant No.15659337).

17.

18.

19.

20.

21.

22.

References 1. The Committee of Brain Tumor Registry of Japan, The Japanese Pathological Society: General Rules for Clinical and Pathological Studies on Brain Tumors. Kanehara & Co.,Ltd., Second Edition, 2002, p 54 2. Lowe SW, Lin AW: Apoptosis in cancer. Carcinogenesis 21: 485– 495, 2000 3. Nicholson DW: From bench to clinic with apoptosis-based therapeutic agents. Nature 407: 810–816, 2000 4. Goyal L: Cell death inhibition: Keeping caspases in check. Cell 104: 805–808, 2001 5. Deveraux QL, Reed JC: IAP family proteins-suppressors of apoptosis. Genes Dev 13: 239–252, 1999 6. Wagenknecht B, Glaser T, Naumann U, Kugler S, Isenmann S, Bahr M, Korneluk R, Liston P, Weller M: Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma. Cell Death Differ 6: 370–376, 1999 7. Ashkenazi A, Dixit VM: Death receptors: signaling and modulation. Science 281: 1305–1308, 1998 8. Walczak H, Krammer PH: The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems. Exp Cell Res 256: 58–66, 2000 9. Thornberry N, Lazebnik Y: Caspases: Enemies within. Science 281: 1312–1316, 1998 10. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME: Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17: 1675–1687, 1998 11. Kroemer G, Reed JC: Mitochondrial control of cell death. Nature Med 6: 513–519, 2000 12. Crook NE, Clem RJ, Miller LK: An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J Virol 67: 2168–2174, 1993 13. Birnbaum MJ, Clem RJ, Miller LK: An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs. J Virol 68: 2521–2528, 1994 14. Hay BA, Wassarman DA, Rubin GM: Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83: 1253–1262, 1995 15. Rothe M, Pan MG, Henzel WJ, Ayres TM, Goeddel DV: The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83: 1243–1252, 1995 16. Roy N, Mahadevan MS, McLean M, Shutler G, Yaraghi Z, Farahani R, Baird S, Besner-Johnston A, Lefebvre C, Kang X, Salih M, Aubry H, Tamai K, Guan X, Ioannou P, Crawford TO,

23.

24.

25. 26. 27.

28.

29.

30.

31. 32. 33.

34.

35.

36.

37.

Jong PJ, Surh L, Ikeda JE, Korneluk RG, MacKenzie A: The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy. Cell 80: 167–178, 1995 Duckett CS, Nava VE, Gedrich RW, Clem RJ, Van Dongen JL, Gilfillan MC, Shiels H, Hardwick JM, Thompson CB: A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors. EMBO J. 15: 2685–2694, 1996 Liston P, Roy N, Tamai K, Lefebvre C, Baird S, Cherton-Horvat G, Farahani R, McLean M, Ikeda JE, MacKenzie A, Korneluk RG: Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes. Nature 379: 349–353, 1996 Uren AG, Pakusch M, Hawkins CJ, Puls KL, Vaux DL: Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors. Proc Natl Acad Sci USA 93: 4974– 4978, 1996 Fraser AG, James C, Evan GI, Hengartner MO: Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis. Curr Biol 9: 292–301, 1996 Uren AG, Beilharz T, O’Connell MJ, Bugg SJ, van Driel R, Vaux DL, Lithgow T: Role for yeast inhibitor of apoptosis (IAP)-like proteins in cell division. Proc Natl Acad Sci USA 96: 10170–10175, 1999 Miller LK: An exegesis of IAPs: salvation and surprises from BIR motifs. Trends Cell Biol 9: 323–328, 1999 Hinds MG, Norton RS, Vaux DL, Day CL: Solution structure of a baculoviral inhibitor of apoptosis (IAP) repeat. Nat Struct Biol 6: 648–651, 1999 Sun C, Cai M, Gunasekera AH, Meadows RP, Wang H, Chen J, Zhang H, Wu W, Xu N, Ng SC, Fesik SW: NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature 401: 818–822, 1999 Salvesen GS, Duckett CS: IAP proteins: blocking the road to death’s door. Nat Rev Mol Cell Biol 3: 401–410, 2002 Deveraux QL, Takahashi R, Salvesen GS, Reed JC: IAP family proteinssuppressors of apoptosis. Nature 388: 300–304, 1997 Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC: The cIAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J 16: 6914–6925, 1997 Deveraux QL, Roy N, Stennicke HR, Van Arsdale T, Zhou Q, Srinivasula SM, Alnemri ES, Salvesen GS, Reed JC: IAPs block apoptotic events induced by caspase)8 and cytochrome c by direct inhibition of distinct caspases. EMBO J 17: 2215–2223, 1998 Du C, Fang M, Li Y, Li L, Wang X: Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation during apoptosis. Cell 102: 33–42, 2000 Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, Vaux DL: Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102: 43–53, 2000 Verhagen AM, Vaux DL: Cell death regulation by the mammalian IAP antagonist Diablo/Smac. Apoptosis 7: 163–166, 2002 Shi Y: A conserved tetrapeptide motif: potentiating apoptosis through IAP-binding. Cell Death Differ 9: 93–95, 2002 Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X, Shi Y: Structural basis of IAP recognition by Smac/DIABLO. Nature 408: 1008–1012, 2000 Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, Oost T, Herrmann J, Wu JC, Fesik SW: Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 408: 1004– 1008, 2000 Fulda S, Wick W, Weller M, Debatin KM: Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo. Nat Med 8: 808– 815, 2002 Arnt CR, Chiorean MV, Heldebrant MP, Gores GJ, Kaufmann SH: Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP1 in situ. J Biol Chem 277: 44236–4243, 2002 Yang L, Mashima T, Sato S, Mochizuki M, Sakamoto H, Yamori T, Oh-Hara T, Tsuruo T: Predominant suppression of apopto-

255

38.

39.

40.

41.

42.

43. 44.

45.

some by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginineconjugated Smac peptide. Cancer Res 63: 831–837, 2003 Bartling B, Lewensohn R, Zhivotovsky B: Endogenously released Smac is insufficient to mediate cell death of human lung carcinoma in response to etoposide. Exp Cell Res 298: 83–95, 2004 Guo F, Nimmanapalli R, Paranawithana S, Wittman S, Griffin D, Bali P, O’Bryan E, Fumero C, Wang HG, Bhalla K: Ectopic overexpression of second mitochondria-derived activator of caspases (Smac/DIABLO) or cotreatment with N-terminus of Smac/ DIABLO peptide potentiates epothilone B derivative(BMS247550) and Apo-2L/TRAIL-induced apoptosis. Blood 99: 3419–3426, 2002 Yin D, Tamaki N, Kokunai T: Wild-type p53-dependent etoposide-induced apoptosis mediated by caspase)3 activation in human glioma cells. J Neurosurg 93: 289–297, 2000 Slee EA, Zhu H, Chow SC, MacFarlane M, Nicholson DW, Cohen GM: Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochem J 315: 21–24, 1996 Thoren PE, Persson D, Karlsson M, Norden B: The antennapedia peptide penetratin translocates across lipid bilayers – the first direct observation. FEBS Lett 482: 265–268, 2000 Satoh MS, Lindahl T: Role of poly(ADP-ribose) formation in DNA repair. Nature 356: 356–358, 1992 Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC: Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371: 346–347, 1994 Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA,

46.

47.

48.

49.

50.

Munday NA, Raju SM, Smulson ME, Yamin TT, Yu VL, Douglas K: Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37–43, 1995 Oliver FJ, de la Rubia G, Rolli V, Ruiz-Ruiz MC, de Murcia G, Murcia JM: Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. J Biol Chem 273: 33533–33539, 1998 Tikoo A, O’Reilly L, Day CL, Verhagen AM, Pakusch M, Vaux DL: Tissue distribution of Diablo/Smac revealed by monoclonal antibodies. Cell Death Differ 9: 710–716, 2002 MacFarlane M, Merrison W, Bratton SB, Cohen GM: Proteasome-mediated degradation of Smac during apoptosis: XIAP promotes Smac ubiquitination in vitro. J Biol Chem 277: 36611– 36616, 2002 Nagane M, Pan G, Weddle JJ, Dixit VM, Cavenee WK, Huang HJ: Increased death receptor 5 expression by chemotherapeutic agents in human gliomas causes synergistic cytotoxity with tumor necrosis factor-related apoptosis-inducing ligand in vitro and in vivo. Cancer Res 60: 847–853, 2000 Gomi A, Shinoda S, Masuzawa T, Ishikawa T, Kuo MT: Transient induction of the MRP/GS-X pump and gamma-glutamylcysteine synthetase by 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3- nitrosourea in human glioma cells. Cancer Res 57: 5292–5299, 1997

Address for offprints: Katsu Mizukawa, Department of Neurosurgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, 650-0017, Chuo-ku, Kobe, Japan; Tel.: +81-78-382-5966; Fax: +8178-382-5979; E-mail: [email protected].