Otology & Neurotology 36:714Y719 Ó 2014, Otology & Neurotology, Inc.
Proteomic Analysis of Vestibular Schwannoma: Conflicting Role of Apoptosis on the Pathophysiology of Sporadic Vestibular Schwannoma *Jae-Hyun Seo, *Kyoung-Ho Park, *Eun-Ju Jeon, *Ki-Hong Chang, †Heejin Lee, †Weonsun Lee, and *Yong-Soo Park *Department of OtolaryngologyYHead and Neck Surgery, College of Medicine, The Catholic University of Korea, Seoul; and ÞClinical Medicine Research Institute of Bucheon St. Mary’s Hospital, The Catholic University of Korea, Bucheon, Republic of Korea
Hypothesis: In this study, we investigated the pathophysiology and mechanism underlying sporadic forms of vestibular schwannoma (VS) by comparing VS tissue with normal nerve tissue using proteomics. Background: Proteomic analysis by two-dimensional electrophoresis and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry facilitates identification and characterization of specific proteins related to the pathogenesis of various diseases. Methods: Proteins were extracted from two vestibular nerve specimens and two VS specimens and analyzed in parallel using two-dimensional electrophoresis. We then analyzed 29 spots that were differentially expressed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry.
Upregulated proteins associated with apoptosis were confirmed by Western blot analysis and immunohistochemistry. Results: Twenty-nine proteins showing significant changes in the expression level between VS tissue and normal nerve tissue were identified. Of these, seven proteins were related to apoptosis. Conclusion: Our findings indicate that apoptosis is associated in a complex manner with the pathophysiology of VS. The suppression of apoptosis is presumably involved in tumor occurrence and, conversely, increased apoptotic expression may contribute to the slow tumor growth rate and may be correlated with the Antoni type B area. Key Words: ApoptosisV ProteomicsVVestibular schwannoma.
A vestibular schwannoma (VS) is a benign tumor arising from the vestibular branch of Cranial Nerve VIII, which accounts for 8% to 10% of all intracranial tumors (1). Most cases of VS occur as a solitary and sporadic form, which exhibits the characteristics of slow-growing tumors, whereas 5% of tumors assume the form of bilateral Neurofibromatosis Type 2 (NF2), with faster growth rates (2). When the sporadic form of VS leads to a range of neurologic symptoms or increases in size, it may be removed through surgery or radiation treatment. However, because of its slow-growing characteristic, the wait-and-see approach is often used for treatment of the tumor, during which its growth is monitored regularly. The mutation or deletion of the NF2 gene, a tumor suppressor gene contributing to the expression of the
protein merlin (also called schwannomin), plays a role in the occurrence of VS. However, the etiopathogenic mechanism of VS has yet to be identified, and few studies have investigated the factors influencing the slow growth rate of VS (3,4). Despite the success of the human genome project, gaining information regarding the functionality of, and relationship between, proteins on a cellular level remains challenging because the posttranslational status of the genome cannot be predicted from the nucleotide sequence (5). In this regard, proteomics, a comprehensive study of proteins, can play a critical role in understanding cell mechanisms. The word proteome is a combination of the words protein and genome and refers to all proteins expressed by a living organism at a certain time and under specific circumstances (6). Proteomics typically involves two-dimensional electrophoresis (2-DE) and matrixassisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS); popular research topics include protein expression, posttranslational modification, and protein-protein interactions.
Otol Neurotol 36:714Y719, 2015.
Address correspondence and reprint requests to Yong-Soo Park, M.D., Ph.D., Department of OtolaryngologyYHead and Neck Surgery, Incheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 56 Dongsu-ro, Bupyeong-gu, Incheon, 403-720, Republic of Korea; E-mail: [email protected] The authors disclose no conflicts of interest.
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Copyright © 2015 Otology & Neurotology, Inc. Unauthorized reproduction of this article is prohibited.
PROTEOMIC ANALYSIS OF VESTIBULAR SCHWANNOMA In this study, we investigated the pathophysiology and mechanism underlying VS by comparing VS tissue with normal nerve tissue using proteomics. MATERIALS AND METHODS Sample Preparation for Proteomics This study was approved by the institutional review board of Bucheon St. Mary’s Hospital (HC13SISI0085), and informed patient consent was obtained. Noncystic, solid, unilateral VS tissue was obtained from two patients after tumor removal via the translabyrinthine approach. Normal vestibular nerves from each VS were also removed at the time of surgery. Approximately onehalf of the tumor and the entire vestibular nerve tissue were obtained immediately after surgical excision and stored at j70-C before use. The remaining tumor tissue underwent histopathologic evaluation and immunohistochemical staining. Because of the limited amount of normal vestibular nerve, a normal peripheral sensory nerve was dissected from the neck region of a different patient for Western blot analysis.
2-D Electrophoresis VS and vestibular nerve tissues were suspended in a sample buffer containing 40 mmol/L Tris, 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 100 mmol/L 1,4-dithioerythritol, and protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). The suspensions were sonicated for approximately 30 seconds and centrifuged at 100,000 g for 45 minutes. Isoelectric focusing was performed using the IPG phor system (Amersham Biosciences, Uppsala, Sweden). A 110-Kg protein sample was mixed with a sufficient volume of rehydration buffer (7 mol/L urea, 2 mol/L thiourea, 4.5% CHAPS, 100 mmol/L DTE, 40 mmol/L Tris, pH 8.8). Samples were applied to 18-cm immobilized pH 3 to 10 nonlinear gradient strips (Amersham Biosciences). Isoelectric focusing was conducted for approximately 80,000 Vh as follows: 500 V for 1 hour, 1,000 V for 1 hour, and 8,000 V for 10 hours. Immediately before loading the focused IPG strips, the second dimension gels were incubated for 15 minutes with equilibration buffer 1 (50 mmol/L Tris-HCl, pH 8.8, 6 mol/L urea, 30% glycerol, 2% sodium dodecyl sulfate, and 65 mmol/L dithiothreitol), followed by equilibration buffer 2 (50 mmol/L TrisHCl, pH 8.8, 6 mol/L urea, 30% glycerol, 2% sodium dodecyl sulfate, and 260 mmol/L iodoacetamide) for 15 minutes. The second dimension used 9% to 17% linear gradient polyacrylamide gels (18 cm 20 cm 1.5 mm) at constant 40 mA per gel for approximately 5 hours. After fixation in 40% methanol and 5% phosphoric acid for 1 hour, gels were stained with Coomassie brilliant blue G250 (Bio-Rad, Hercules, CA, USA) for 12 hours. Gels were destained with H2O, scanned in a Bio-Rad (Richmond, CA, USA) GS710 densitometer, and analyzed using the Image Master Platinum 5.0 image analysis software (Amersham Biosciences, Uppsala, Sweden).
Protein Identification using MALDI-TOF MS Protein spots were excised from gels with a sterile scalpel and placed into Eppendorf tubes. Proteins were destained, reduced, alkylated, and then digested using trypsin (Promega, Madison, WI, USA). For MALDI-TOF MS analysis, the tryptic peptides were concentrated by a POROS R2, Oligo R3 column (Applied Biosystems, Foster City, CA, USA). After washing the column with 70% acetonitrile, 100% acetonitrile, and then 50 mmol/L ammonium bicarbonate, samples were applied to the R2 and R3 columns and eluted with cyano-4-hydroxycinamic
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acid (Sigma, St. Louis, MO, USA) dissolved in 70% acetonitrile and 0.1% trifluoroacetic acid before MALDI-TOF MS analysis. Mass spectra were acquired on a 4700 Proteomics Analyzer (Applied Biosystems) operated in MS and MS/MS modes. Peptide fragmentation in MS/MS mode was by collision-induced dissociation using atmosphere as the collision gas. The instrument was operated in reflectron mode and calibrated using the 4700 calibration mixture (Applied Biosystems), and each sample spectrum was additionally calibrated using trypsin autolysis peaks. Peptide mass fingerprinting was performed using the Mascot search engine included in the GPS Explorer software, and mass spectra used for manual de novo sequencing were annotated using the Data Explorer software (Applied Biosystems).
Western Blot Analysis We selected several upregulated proteins related to the pathogenesis of VS and performed Western blot analysis to confirm their identities. Tissue lysates were prepared from tumor tissues and peripheral sensory nerve by homogenization in lysis buffer. To detect zeta polypeptide (YWHAZ), Annexin A4 (ANXA4), Annexin A2 (ANXA2), rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA), heat shock protein 27 (HSP27), peroxiredoxin 6, and Annexin V, total protein extracts of tissues were obtained using RIPA buffer (Pierce, Thermo Scientific, Rockford, IL, USA). Protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Protein samples were separated using 10% sodium dodecyl sulfateYpolyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride Western blotting membranes (Roche Applied Science, Mannheim, Germany). The membranes were preincubated with 5% skim milk in Tris-buffered saline (TBS) for 2 hours at room temperature. Primary antibodies diluted 1:1,000 in 1 TBS with 0.1% Tween 20 (TBST) against YWHAZ, ANXA4, ANXA2, ARHGDIA, HSP27, peroxiredoxin 6, Annexin V, and A-actin (Cell Signaling Technology, Beverly, MA, USA) were added, and the samples were incubated overnight at 4-C. The membranes were washed four times with TBST, horseradish peroxidase (HRP)Yconjugated secondary antibodies were added, and the membranes were incubated for 1 hour at room temperature. The samples were washed in TBST, and the hybridized bands were detected with an ECL detection kit (Thermo Scientific) and Chemidoc XR image analyzer (Bio-Rad).
Immunohistochemistry Tumor tissue samples were routinely fixed in formalin and embedded in paraffin. Immunohistochemistry was performed on3-Km paraffin sections using an automated immunohistochemical stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). Sections were deparaffinized using EZ Prep (Ventana Medical Systems) solution. Deparaffinized tissue sections were pretreated with cell conditioning solution (Ventana Medical Systems) at 95-C for 60 minutes. To block the endogeneous hydroperoxidase activity, UV INHIBITOR step was performed at 37-C for 4 minutes before the detection of the primary antibody. The primary antibodies for YWHAZ, Annexin V, and HSP27 were diluted in Dako Antibody Diluent (Dako Cytomation, Glostrup, Denmark) with background-reducing components and were used at the following dilutions: YWHAZ (1:100 dilution; Cell Signaling Technology, Beverly, MA, USA), Annexin V (1:2,000 dilution; Cell Signaling Technology), and HSP27 (1:300 dilution; Cell Signaling Technology.). Then, the primary antibodies were incubated for 32 minutes at 37-C, whereas HRPlabeled secondary antibody was incubated for 8 minutes at 37-C. To visualize the signal for protein, the HRP-labeled secondary Otology & Neurotology, Vol. 36, No. 4, 2015
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FIG. 1. Images of proteomic analysis for human VS (A) and normal vestibular nerve (B) reveal 29 spots that were differentially expressed between them.
antibody was exposed to UV DAB with UV DAB H2O2 for 8 minutes and UV COPPER for 4 minutes (UV COPPER changes the DAB color to a reddish brown). Lastly, the slides were counterstained with Hematoxylin II (Ventana Medical Systems) for 4 minutes and Bluing Reagent (Ventana Medical Systems) for 4 minutes.
RESULTS Proteins were extracted from two control specimens and two VS specimens and analyzed in parallel using 2-DE TABLE 1. Spot no.
with Coomassie brilliant blue staining. Only spots that were expressed the most or more focalized were chosen. In both specimens, the amount of protein present in each spot was densitometrically measured and statistically evaluated. Representative Coomassie brilliant blueYstained 2-DE gel images from control and VS are shown in Figure 1, and the differentially expressed protein spots are marked in the images. To create a map of the proteins extracted from VS specimens, we identified the spots selected for statistical
Protein spots that were differentially expressed between vestibular schwannoma and the control Protein name
Upregulated proteins 11 Tumor rejection antigen (gp96) 1 12 BiP protein 13 Calreticulin precursor 14 Prolyl 4-hydroxylase, beta subunit 15 Hypothetical protein 16 Nonspecific 17 Aldehyde dehydrogenase 1A1 18 Chain, Annexin V (Lipocortin V, Endonexin Li, Placental Anticoagulant Protein) 19 Tropomyosin 4 20 YWHAZ protein 21 ANXA4 protein 22 Annexin A2 isoform 23 Rho GDP dissociation inhibitor alpha 24 Heat shock protein 27 25 Heat shock protein 27 26 Nonspecific Downregulated proteins 27 Nonspecific 28 Fructose-bisphosphate aldolase C 29 Brain creatine kinase 30 Unnamed protein product 31 Nonspecific 32 Nonspecific 33 Peroxiredoxin 6 Proteins only expressed in vestibular schwannoma 37 Nonspecific 38 Nonspecific 39 Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1 Proteins only expressed in control 34 Nonspecific 35 GFAP protein 36 Brain creatine kinase
Mass (Da) Mascot score Accession no.
Species
92, 567 71, 002 48, 283 57, 480 71, 353 8, 586 55, 454 35, 839 28, 619 30, 100 33, 759 38, 808 23, 250 22, 427 22, 427 50, 062
195 152 66 94 148 61 106 192 101 133 168 145 73 102 102 51
gi|61656607 gi|6470150 gi|4757900 gi|20070125 gi|51476390 gi|37987958 gi|21361176 gi|999926 gi|4507651 gi|49119653 gi|39645467 gi|4757756 gi|4757768 gi|662841 gi|662841 gi|1581788
Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens
50, 062 39, 830 42, 902 49, 533 36, 769 27, 160 25, 133
51 134 187 86 63 38 180
gi|105990539 gi|4885063 gi|21536286 gi|34536332 gi|13786847 gi|45592963 gi|4758638
Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens
44, 072 43, 665 42, 829
61 47 104
gi|30354665 gi|30089997 gi|13489087
Homo sapiens Homo sapiens Homo sapiens
53, 416 49, 776 42, 902
55 214 151
gi|763431 gi|38566198 gi|21536286
Homo sapiens Homo sapiens Homo sapiens
Otology & Neurotology, Vol. 36, No. 4, 2015
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PROTEOMIC ANALYSIS OF VESTIBULAR SCHWANNOMA
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was downregulated, in contrast to the other six upregulated proteins. The Western blot analyses of YWHAZ, Annexin A4, Annexin A2, ARHGDIA, HSP27, and Annexin V revealed that all six proteins were expressed at higher levels in VS than in normal cervical plexus (Fig. 2). Immunohistochemical staining of the tumor tissues for YWHAZ and Annexin V revealed negative immunostaining in the cytoplasm of the Antoni type A area and positive immunostaining in the cytoplasm of the Antoni type B area (Fig. 3, A and B). On the contrary, there is positive staining in the Antoni type A area and negative staining in the Antoni type B area for HSP27 (Fig. 3C).
DISCUSSION FIG. 2. Western blot analysis of the six apoptosis-associated upregulated proteins. A-Actin is used as the loading control. N1 and N2 indicate normal peripheral nerve; T1 and T2, vestibular schwannoma.
analysis by peptide mass fingerprinting. Spots were excised from both control and VS gels and digested with trypsin. Peptide masses were used to query the database. The identification results obtained with Mascot analysis are shown in Table 1; 29 proteins were differentially expressed in VS tissues compared with controls. Of the 29 proteins differentially expressed between the VS and controls, 13 proteins were significantly upregulated in VS compared with controls, and four proteins, fructosebiphosphate aldolase C (spot 28), brain creatine kinase (spot 29, 36), peroxiredoxin 6 (spot 33), and unnamed protein product (spot 30), were significantly downregulated in VS compared with controls. Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), and member 1 (spot 39) were expressed only in VS, and human albumin (spot 34) and GFAP protein (spot 35) were expressed only in controls. Eight spots were nonspecific. Seven of the 29 expressed proteins, Annexin V, Annexin A4, Annexin A2 isoform 2, YWHAZ protein, ARHGDIA, HSP27, and peroxiredoxin 6 were associated with apoptosis. Among the seven apoptosis-related proteins, peroxiredoxin 6
Proteomics is a useful approach to investigating protein expression during the pathogenesis of a disease (7). However, this methodology has several limitations. First, only a limited number of protein spots are visible on a single gel and only the most highly expressed proteins are recognized, whereas some proteins cannot always be adequately resolved. Second, the range of pI values can be a limitation. Although nonlinear pH gradients are commonly used, proteins with extreme pI values cannot be focused. In addition, hydrophobic membrane proteins are not easily resolved (8). Nevertheless, despite the limitations, proteomics is a powerful and effective method of obtaining information regarding posttranslational modifications, protein-protein interactions, the ramifications of alternate protein structure splicing and function, and relative protein abundance, which cannot be achieved using other modalities, including genomics (9). Proteomics typically uses 2-DE and MS for the separation and identification of proteins, respectively. In this research, proteomics combined with two different techniques was conducted to evaluate protein expression in VS. As a result, 29 differentially expressed proteins were identified, 13 were upregulated, four were downregulated, and one was found only in VS and two only in normal vestibular nerves. Moreover, seven of the 29 proteins, Annexin V, Annexin A4, Annexin A2 isoform 2, YWHAZ protein,
FIG. 3. Immunohistochemical analysis of three upregulated proteins in VS. For YWHAZ (A) and Annexin V (B), there is negative immunostaining in the cytoplasm of the Antoni type A area and positive immunostaining in the cytoplasm of the Antoni type B area. On the contrary, there is positive immunostaining in the Antoni type A area and negative immunostaining in the Antoni type B area for HSP27 (C). Original magnification, 400. Otology & Neurotology, Vol. 36, No. 4, 2015
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ARHGDIA, HSP27, and peroxiredoxin 6 were associated with apoptosis. Among the seven apoptosis-related proteins, peroxiredoxin 6 was downregulated, in contrast to the other six upregulated proteins. Annexins are highly conserved calcium-binding proteins. Annexin V, in particular, the most abundant annexin in human platelets, has proven useful as an indicator of apoptosis and platelet activation (10). Furthermore, Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine. Consequently, Annexin V can be used as a sensitive probe for phosphatidylserine in cell membranes (11). Translocation of phosphatidylserine to the external cell surface is observed in apoptosis. Annexin A4, an early marker of apoptotic cell death, is translocated selectively from the nucleus to the cytosol during apoptosis (12). The protein-tyrosine kinase substrate Annexin A2 is a growth-regulated gene and shows increased expression in several human cancers, although the mechanism is unclear. However, Annexin A2 appears to be related to multiple physiologic activities, such as cell proliferation and DNA synthesis (13). The 14-3-3 proteins (tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein) play a role in controlling the cell cycle, transcription, and apoptosis. Despite their unknown functions, they are involved in the subcellular localization of proteins and serve as adaptor molecules, a stimulant to interprotein reactions. The 14-3-3 proteins beta polypeptide (YWHAB), gamma polypeptide (YWHAG), and YWHAZ are highly abundant in the brain (14). RhoGDI, a GDP dissociation inhibitor of Rho protein, is essential for the control of many cellular functions, based on its interactions with Rho family GTPases including Rac1, Cdc42, and RhoA. RhoGDI is often overexpressed in human tumors and chemoresistant cancer cell lines, which suggests involvement of RhoGDI in the development of drug-resistant cancer cells (15). The expression of HSP27, a component of a large and heterogeneous group of chaperone proteins, protects against oxidative stress. The major functions of this protein include inhibition of apoptosis and prevention of actin intermediate filament aggregation (16). Although the suppression of apoptosis is generally considered the focal mechanism of tumorigenesis and tumor proliferation, no correlation between VS and apoptotic markers has been reported to date. There are many reports that merlin inhibits several intracellular signals implicated in cell proliferation and tumor formation (1Y4). It was also reported that c-JUN N-terminal kinase activity could either promote tumor cell proliferation in VS or increase apoptosis in normal Schwann cells (17,18). The present study verified increased expression of six proteins related to apoptosis. Among these six proteins, Annexin V, Annexin A4, Anenxin A2, and YWHAZ are likely to accelerate apoptosis, whereas RhoGDI and HSP27 tend to suppress apoptosis. These conflicting characteristics are assumed to be related to the nature of VS. That is, the slow growth rate of VS is thought to originate from two conflicting mechanisms: the suppression of apoptosis
and the acceleration of cell death (19). This hypothesis is supported by our results. Furthermore, the formation of Antoni types A and B, a distinctive pathologic finding of VS, is known to be interconnected with each other; type B is formed by the apoptosis of type A (20). In the immunostaining result of our study, immunoreactivities related to the acceleration of apoptosis were strongly expressed in the Antoni B area; whereas markers related to suppression of apoptosis were more strongly detected in the Antoni A area than in the Antoni B area. Taking these into consideration, it is probable that the overexpression of apoptosis-associated proteins is closely associated with the degeneration of Antoni type A. There are several limitations of our study. First, we chose an imperfect control tissue for proteomic analysis. We removed uninvolved vestibular nerves that VS had not originated from, but this adjacent vestibular nerve might not be a normal nerve because there may have already been genetic changes in Schwann cells of uninvolved vestibular nerve. It would have been better if we could use normal vestibular nerve specimens from vestibular neurectomy of intractable Me´nie`re’s disease. Second, we used normal peripheral sensory nerves from the neck as control tissue for Western blot analysis because of the limited amount of normal vestibular nerve. Third, the number of samples is not sufficient to prove our hypothesis. This study was a preliminary study to find the pathophysiology of the sporadic form of VS, so we have a plan to undertake the follow-up experiments. Finally, we could not confirm all the proteins associated with apoptosis; six upregulated proteins of seven proteins were confirmed by Western blot analysis, and merely three proteins, which were representative to accelerate or suppress apoptosis, were detected by immunohistochemistry. Despite these limitations, the results of this study suggested that the conflicting role of apoptosis may explain the natural history of the sporadic form of VS. The suppression of apoptosis could play an important role in controlling VS development from normal vestibular nerves. However, the increased apoptosis is thought to be correlated with an increase in the degenerative area or the slow growth rate of the tumor. Accordingly, our findings regarding a possible correlation between the conflicting role of apoptosis and the pathophysiology of VS highlight the necessity of further research. CONCLUSION In conclusion, we used a proteomics methodology to examine two VSs and identified differentially expressed proteins. A large number of proteins upregulated and downregulated in VS were related to apoptosis. These results indicate that apoptosis is potentially associated in a complex manner with the pathophysiology of VS. That is, the suppression of apoptosis is involved in tumor occurrence and, conversely, an increase in apoptosis may contribute to the slow tumor growth rate and may be correlated with the Antoni type B area.
Otology & Neurotology, Vol. 36, No. 4, 2015
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PROTEOMIC ANALYSIS OF VESTIBULAR SCHWANNOMA REFERENCES 1. Bian LG, Tirakotai W, Sun QF, et al. Molecular genetics alterations and tumor behavior of sporadic vestibular schwannoma from the People’s Republic of China. J Neurooncol 2005;73:253Y60. 2. Neff BA, Welling DB, Akhmametyeva E, Chang LS. The molecular biology of vestibular schwannomas: dissecting the pathogenic process at the molecular level. Otol Neurotol 2006;27: 197Y208. 3. Roche PH, Bouvier C, Chinot O, Figarella-Branger D. Genesis and biology of vestibular schwannomas. Prog Neurol Surg 2008;21: 24Y31. 4. Welling DB, Lasak JM, Akhmametyeva E, Ghaheri B, Chang LS. cDNA microarray analysis of vestibular schwannomas. Otol Neurotol 2002;23:736Y48. 5. von Eggeling F, Davies H, Lomas L, et al. Tissue-specific microdissection coupled with ProteinChip array technologies: applications in cancer research. Biotechniques 2000;29:1066Y70. 6. Govorun VM, Archakov AI. Proteomic technologies in modern biomedical science. Biochemistry (Mosc) 2002;67:1109Y23. 7. Alberio T, Fasano M. Proteomics in Parkinson’s disease: an unbiased approach towards peripheral biomarkers and new therapies. J Biotechnol 2010;156:325Y37. 8. BassoM Giraudo S, Corpillo D, Bergamasco B, Lopiano L, Fasano M. Proteome analysis of human substantia nigra in Parkinson’s disease. Proteomics 2004;4:3943Y52. 9. Kim JL, Jung HH. Proteomic analysis of cholesteatoma. Acta Otolaryngol 2004;124:783Y8. 10. Tzima E, Walker JH. Platelet annexin V: the ins and outs. Platelets 2000;11:245Y51.
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11. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995;184:39Y51. 12. Herzog A, Kuntz S, Daniel H, Wenzel U. Identification of biomarkers for the initiation of apoptosis in human preneoplastic colonocytes by proteome analysis. Int J Cancer 2004;109:220Y9. 13. Chiang Y, Rizzino A, Sibenaller ZA, Wold MS, Vishwanatha JK. Specific down-regulation of Annexin II expression in human cells interferes with cell proliferation. Mol Cell Biochem 1999;199: 139Y47. 14. Berg D, Holzmann C, Riess O. 14-3-3 proteins in the nervous system. Nat Rev Neurosci 2003;4:752Y62. 15. Zhang B, Zhang Y, Dagher MC, Shacter E. Rho GDP dissociation inhibitor protects cancer cells against drug-induced apoptosis. Cancer Res 2005;65:6054Y62. 16. Sarto C, Valsecchi C, Magni F, et al. Expression of heat shock protein 27 in human renal cell carcinoma. Proteomics 2004;4:2252Y60. 17. Yue WY, Clark JJ, Fernando A, Domann F, Hansen MR. Contribution of persistent C-Jun N-terminal kinase activity to the survival of human vestibular schwannoma cells by suppression of accumulation of mitochondrial superoxides. Neuro Oncol 2011;13:961Y73. 18. Yue WY, Clark JJ, Telisak M, Hansen MR. Inhibition of c-Jun N-terminal kinase activity enhances vestibular schwannoma cell sensitivity to gamma irradiation. Neurosurgery 2013;73:506Y16. 19. Mawrin C, Kirches E, Dietzmann K, Roessner A, Boltze C. Expression pattern of apoptotic markers in vestibular schwannomas. Pathol Res Pract 2002;198:813Y9. 20. Abe M, Kawase T, Urano M, et al. Analyses of proliferative potential in schwannomas. Brain Tumor Pathol 2000;17:35Y40.
Otology & Neurotology, Vol. 36, No. 4, 2015
Copyright © 2015 Otology & Neurotology, Inc. Unauthorized reproduction of this article is prohibited.
Proteomic Analysis of Vestibular Schwannoma: Conflicting Role of Apoptosis on the Pathophysiology of Sporadic Vestibular Schwannoma *Jae-Hyun Seo, *Kyoung-Ho Park, *Eun-Ju Jeon, *Ki-Hong Chang, †Heejin Lee, †Weonsun Lee, and *Yong-Soo Park *Department of OtolaryngologyYHead and Neck Surgery, College of Medicine, The Catholic University of Korea, Seoul; and ÞClinical Medicine Research Institute of Bucheon St. Mary’s Hospital, The Catholic University of Korea, Bucheon, Republic of Korea
Hypothesis: In this study, we investigated the pathophysiology and mechanism underlying sporadic forms of vestibular schwannoma (VS) by comparing VS tissue with normal nerve tissue using proteomics. Background: Proteomic analysis by two-dimensional electrophoresis and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry facilitates identification and characterization of specific proteins related to the pathogenesis of various diseases. Methods: Proteins were extracted from two vestibular nerve specimens and two VS specimens and analyzed in parallel using two-dimensional electrophoresis. We then analyzed 29 spots that were differentially expressed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry.
Upregulated proteins associated with apoptosis were confirmed by Western blot analysis and immunohistochemistry. Results: Twenty-nine proteins showing significant changes in the expression level between VS tissue and normal nerve tissue were identified. Of these, seven proteins were related to apoptosis. Conclusion: Our findings indicate that apoptosis is associated in a complex manner with the pathophysiology of VS. The suppression of apoptosis is presumably involved in tumor occurrence and, conversely, increased apoptotic expression may contribute to the slow tumor growth rate and may be correlated with the Antoni type B area. Key Words: ApoptosisV ProteomicsVVestibular schwannoma.
A vestibular schwannoma (VS) is a benign tumor arising from the vestibular branch of Cranial Nerve VIII, which accounts for 8% to 10% of all intracranial tumors (1). Most cases of VS occur as a solitary and sporadic form, which exhibits the characteristics of slow-growing tumors, whereas 5% of tumors assume the form of bilateral Neurofibromatosis Type 2 (NF2), with faster growth rates (2). When the sporadic form of VS leads to a range of neurologic symptoms or increases in size, it may be removed through surgery or radiation treatment. However, because of its slow-growing characteristic, the wait-and-see approach is often used for treatment of the tumor, during which its growth is monitored regularly. The mutation or deletion of the NF2 gene, a tumor suppressor gene contributing to the expression of the
protein merlin (also called schwannomin), plays a role in the occurrence of VS. However, the etiopathogenic mechanism of VS has yet to be identified, and few studies have investigated the factors influencing the slow growth rate of VS (3,4). Despite the success of the human genome project, gaining information regarding the functionality of, and relationship between, proteins on a cellular level remains challenging because the posttranslational status of the genome cannot be predicted from the nucleotide sequence (5). In this regard, proteomics, a comprehensive study of proteins, can play a critical role in understanding cell mechanisms. The word proteome is a combination of the words protein and genome and refers to all proteins expressed by a living organism at a certain time and under specific circumstances (6). Proteomics typically involves two-dimensional electrophoresis (2-DE) and matrixassisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS); popular research topics include protein expression, posttranslational modification, and protein-protein interactions.
Otol Neurotol 36:714Y719, 2015.
Address correspondence and reprint requests to Yong-Soo Park, M.D., Ph.D., Department of OtolaryngologyYHead and Neck Surgery, Incheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 56 Dongsu-ro, Bupyeong-gu, Incheon, 403-720, Republic of Korea; E-mail: [email protected] The authors disclose no conflicts of interest.
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Copyright © 2015 Otology & Neurotology, Inc. Unauthorized reproduction of this article is prohibited.
PROTEOMIC ANALYSIS OF VESTIBULAR SCHWANNOMA In this study, we investigated the pathophysiology and mechanism underlying VS by comparing VS tissue with normal nerve tissue using proteomics. MATERIALS AND METHODS Sample Preparation for Proteomics This study was approved by the institutional review board of Bucheon St. Mary’s Hospital (HC13SISI0085), and informed patient consent was obtained. Noncystic, solid, unilateral VS tissue was obtained from two patients after tumor removal via the translabyrinthine approach. Normal vestibular nerves from each VS were also removed at the time of surgery. Approximately onehalf of the tumor and the entire vestibular nerve tissue were obtained immediately after surgical excision and stored at j70-C before use. The remaining tumor tissue underwent histopathologic evaluation and immunohistochemical staining. Because of the limited amount of normal vestibular nerve, a normal peripheral sensory nerve was dissected from the neck region of a different patient for Western blot analysis.
2-D Electrophoresis VS and vestibular nerve tissues were suspended in a sample buffer containing 40 mmol/L Tris, 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 100 mmol/L 1,4-dithioerythritol, and protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). The suspensions were sonicated for approximately 30 seconds and centrifuged at 100,000 g for 45 minutes. Isoelectric focusing was performed using the IPG phor system (Amersham Biosciences, Uppsala, Sweden). A 110-Kg protein sample was mixed with a sufficient volume of rehydration buffer (7 mol/L urea, 2 mol/L thiourea, 4.5% CHAPS, 100 mmol/L DTE, 40 mmol/L Tris, pH 8.8). Samples were applied to 18-cm immobilized pH 3 to 10 nonlinear gradient strips (Amersham Biosciences). Isoelectric focusing was conducted for approximately 80,000 Vh as follows: 500 V for 1 hour, 1,000 V for 1 hour, and 8,000 V for 10 hours. Immediately before loading the focused IPG strips, the second dimension gels were incubated for 15 minutes with equilibration buffer 1 (50 mmol/L Tris-HCl, pH 8.8, 6 mol/L urea, 30% glycerol, 2% sodium dodecyl sulfate, and 65 mmol/L dithiothreitol), followed by equilibration buffer 2 (50 mmol/L TrisHCl, pH 8.8, 6 mol/L urea, 30% glycerol, 2% sodium dodecyl sulfate, and 260 mmol/L iodoacetamide) for 15 minutes. The second dimension used 9% to 17% linear gradient polyacrylamide gels (18 cm 20 cm 1.5 mm) at constant 40 mA per gel for approximately 5 hours. After fixation in 40% methanol and 5% phosphoric acid for 1 hour, gels were stained with Coomassie brilliant blue G250 (Bio-Rad, Hercules, CA, USA) for 12 hours. Gels were destained with H2O, scanned in a Bio-Rad (Richmond, CA, USA) GS710 densitometer, and analyzed using the Image Master Platinum 5.0 image analysis software (Amersham Biosciences, Uppsala, Sweden).
Protein Identification using MALDI-TOF MS Protein spots were excised from gels with a sterile scalpel and placed into Eppendorf tubes. Proteins were destained, reduced, alkylated, and then digested using trypsin (Promega, Madison, WI, USA). For MALDI-TOF MS analysis, the tryptic peptides were concentrated by a POROS R2, Oligo R3 column (Applied Biosystems, Foster City, CA, USA). After washing the column with 70% acetonitrile, 100% acetonitrile, and then 50 mmol/L ammonium bicarbonate, samples were applied to the R2 and R3 columns and eluted with cyano-4-hydroxycinamic
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acid (Sigma, St. Louis, MO, USA) dissolved in 70% acetonitrile and 0.1% trifluoroacetic acid before MALDI-TOF MS analysis. Mass spectra were acquired on a 4700 Proteomics Analyzer (Applied Biosystems) operated in MS and MS/MS modes. Peptide fragmentation in MS/MS mode was by collision-induced dissociation using atmosphere as the collision gas. The instrument was operated in reflectron mode and calibrated using the 4700 calibration mixture (Applied Biosystems), and each sample spectrum was additionally calibrated using trypsin autolysis peaks. Peptide mass fingerprinting was performed using the Mascot search engine included in the GPS Explorer software, and mass spectra used for manual de novo sequencing were annotated using the Data Explorer software (Applied Biosystems).
Western Blot Analysis We selected several upregulated proteins related to the pathogenesis of VS and performed Western blot analysis to confirm their identities. Tissue lysates were prepared from tumor tissues and peripheral sensory nerve by homogenization in lysis buffer. To detect zeta polypeptide (YWHAZ), Annexin A4 (ANXA4), Annexin A2 (ANXA2), rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA), heat shock protein 27 (HSP27), peroxiredoxin 6, and Annexin V, total protein extracts of tissues were obtained using RIPA buffer (Pierce, Thermo Scientific, Rockford, IL, USA). Protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Protein samples were separated using 10% sodium dodecyl sulfateYpolyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride Western blotting membranes (Roche Applied Science, Mannheim, Germany). The membranes were preincubated with 5% skim milk in Tris-buffered saline (TBS) for 2 hours at room temperature. Primary antibodies diluted 1:1,000 in 1 TBS with 0.1% Tween 20 (TBST) against YWHAZ, ANXA4, ANXA2, ARHGDIA, HSP27, peroxiredoxin 6, Annexin V, and A-actin (Cell Signaling Technology, Beverly, MA, USA) were added, and the samples were incubated overnight at 4-C. The membranes were washed four times with TBST, horseradish peroxidase (HRP)Yconjugated secondary antibodies were added, and the membranes were incubated for 1 hour at room temperature. The samples were washed in TBST, and the hybridized bands were detected with an ECL detection kit (Thermo Scientific) and Chemidoc XR image analyzer (Bio-Rad).
Immunohistochemistry Tumor tissue samples were routinely fixed in formalin and embedded in paraffin. Immunohistochemistry was performed on3-Km paraffin sections using an automated immunohistochemical stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). Sections were deparaffinized using EZ Prep (Ventana Medical Systems) solution. Deparaffinized tissue sections were pretreated with cell conditioning solution (Ventana Medical Systems) at 95-C for 60 minutes. To block the endogeneous hydroperoxidase activity, UV INHIBITOR step was performed at 37-C for 4 minutes before the detection of the primary antibody. The primary antibodies for YWHAZ, Annexin V, and HSP27 were diluted in Dako Antibody Diluent (Dako Cytomation, Glostrup, Denmark) with background-reducing components and were used at the following dilutions: YWHAZ (1:100 dilution; Cell Signaling Technology, Beverly, MA, USA), Annexin V (1:2,000 dilution; Cell Signaling Technology), and HSP27 (1:300 dilution; Cell Signaling Technology.). Then, the primary antibodies were incubated for 32 minutes at 37-C, whereas HRPlabeled secondary antibody was incubated for 8 minutes at 37-C. To visualize the signal for protein, the HRP-labeled secondary Otology & Neurotology, Vol. 36, No. 4, 2015
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FIG. 1. Images of proteomic analysis for human VS (A) and normal vestibular nerve (B) reveal 29 spots that were differentially expressed between them.
antibody was exposed to UV DAB with UV DAB H2O2 for 8 minutes and UV COPPER for 4 minutes (UV COPPER changes the DAB color to a reddish brown). Lastly, the slides were counterstained with Hematoxylin II (Ventana Medical Systems) for 4 minutes and Bluing Reagent (Ventana Medical Systems) for 4 minutes.
RESULTS Proteins were extracted from two control specimens and two VS specimens and analyzed in parallel using 2-DE TABLE 1. Spot no.
with Coomassie brilliant blue staining. Only spots that were expressed the most or more focalized were chosen. In both specimens, the amount of protein present in each spot was densitometrically measured and statistically evaluated. Representative Coomassie brilliant blueYstained 2-DE gel images from control and VS are shown in Figure 1, and the differentially expressed protein spots are marked in the images. To create a map of the proteins extracted from VS specimens, we identified the spots selected for statistical
Protein spots that were differentially expressed between vestibular schwannoma and the control Protein name
Upregulated proteins 11 Tumor rejection antigen (gp96) 1 12 BiP protein 13 Calreticulin precursor 14 Prolyl 4-hydroxylase, beta subunit 15 Hypothetical protein 16 Nonspecific 17 Aldehyde dehydrogenase 1A1 18 Chain, Annexin V (Lipocortin V, Endonexin Li, Placental Anticoagulant Protein) 19 Tropomyosin 4 20 YWHAZ protein 21 ANXA4 protein 22 Annexin A2 isoform 23 Rho GDP dissociation inhibitor alpha 24 Heat shock protein 27 25 Heat shock protein 27 26 Nonspecific Downregulated proteins 27 Nonspecific 28 Fructose-bisphosphate aldolase C 29 Brain creatine kinase 30 Unnamed protein product 31 Nonspecific 32 Nonspecific 33 Peroxiredoxin 6 Proteins only expressed in vestibular schwannoma 37 Nonspecific 38 Nonspecific 39 Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1 Proteins only expressed in control 34 Nonspecific 35 GFAP protein 36 Brain creatine kinase
Mass (Da) Mascot score Accession no.
Species
92, 567 71, 002 48, 283 57, 480 71, 353 8, 586 55, 454 35, 839 28, 619 30, 100 33, 759 38, 808 23, 250 22, 427 22, 427 50, 062
195 152 66 94 148 61 106 192 101 133 168 145 73 102 102 51
gi|61656607 gi|6470150 gi|4757900 gi|20070125 gi|51476390 gi|37987958 gi|21361176 gi|999926 gi|4507651 gi|49119653 gi|39645467 gi|4757756 gi|4757768 gi|662841 gi|662841 gi|1581788
Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens
50, 062 39, 830 42, 902 49, 533 36, 769 27, 160 25, 133
51 134 187 86 63 38 180
gi|105990539 gi|4885063 gi|21536286 gi|34536332 gi|13786847 gi|45592963 gi|4758638
Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens
44, 072 43, 665 42, 829
61 47 104
gi|30354665 gi|30089997 gi|13489087
Homo sapiens Homo sapiens Homo sapiens
53, 416 49, 776 42, 902
55 214 151
gi|763431 gi|38566198 gi|21536286
Homo sapiens Homo sapiens Homo sapiens
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PROTEOMIC ANALYSIS OF VESTIBULAR SCHWANNOMA
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was downregulated, in contrast to the other six upregulated proteins. The Western blot analyses of YWHAZ, Annexin A4, Annexin A2, ARHGDIA, HSP27, and Annexin V revealed that all six proteins were expressed at higher levels in VS than in normal cervical plexus (Fig. 2). Immunohistochemical staining of the tumor tissues for YWHAZ and Annexin V revealed negative immunostaining in the cytoplasm of the Antoni type A area and positive immunostaining in the cytoplasm of the Antoni type B area (Fig. 3, A and B). On the contrary, there is positive staining in the Antoni type A area and negative staining in the Antoni type B area for HSP27 (Fig. 3C).
DISCUSSION FIG. 2. Western blot analysis of the six apoptosis-associated upregulated proteins. A-Actin is used as the loading control. N1 and N2 indicate normal peripheral nerve; T1 and T2, vestibular schwannoma.
analysis by peptide mass fingerprinting. Spots were excised from both control and VS gels and digested with trypsin. Peptide masses were used to query the database. The identification results obtained with Mascot analysis are shown in Table 1; 29 proteins were differentially expressed in VS tissues compared with controls. Of the 29 proteins differentially expressed between the VS and controls, 13 proteins were significantly upregulated in VS compared with controls, and four proteins, fructosebiphosphate aldolase C (spot 28), brain creatine kinase (spot 29, 36), peroxiredoxin 6 (spot 33), and unnamed protein product (spot 30), were significantly downregulated in VS compared with controls. Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), and member 1 (spot 39) were expressed only in VS, and human albumin (spot 34) and GFAP protein (spot 35) were expressed only in controls. Eight spots were nonspecific. Seven of the 29 expressed proteins, Annexin V, Annexin A4, Annexin A2 isoform 2, YWHAZ protein, ARHGDIA, HSP27, and peroxiredoxin 6 were associated with apoptosis. Among the seven apoptosis-related proteins, peroxiredoxin 6
Proteomics is a useful approach to investigating protein expression during the pathogenesis of a disease (7). However, this methodology has several limitations. First, only a limited number of protein spots are visible on a single gel and only the most highly expressed proteins are recognized, whereas some proteins cannot always be adequately resolved. Second, the range of pI values can be a limitation. Although nonlinear pH gradients are commonly used, proteins with extreme pI values cannot be focused. In addition, hydrophobic membrane proteins are not easily resolved (8). Nevertheless, despite the limitations, proteomics is a powerful and effective method of obtaining information regarding posttranslational modifications, protein-protein interactions, the ramifications of alternate protein structure splicing and function, and relative protein abundance, which cannot be achieved using other modalities, including genomics (9). Proteomics typically uses 2-DE and MS for the separation and identification of proteins, respectively. In this research, proteomics combined with two different techniques was conducted to evaluate protein expression in VS. As a result, 29 differentially expressed proteins were identified, 13 were upregulated, four were downregulated, and one was found only in VS and two only in normal vestibular nerves. Moreover, seven of the 29 proteins, Annexin V, Annexin A4, Annexin A2 isoform 2, YWHAZ protein,
FIG. 3. Immunohistochemical analysis of three upregulated proteins in VS. For YWHAZ (A) and Annexin V (B), there is negative immunostaining in the cytoplasm of the Antoni type A area and positive immunostaining in the cytoplasm of the Antoni type B area. On the contrary, there is positive immunostaining in the Antoni type A area and negative immunostaining in the Antoni type B area for HSP27 (C). Original magnification, 400. Otology & Neurotology, Vol. 36, No. 4, 2015
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ARHGDIA, HSP27, and peroxiredoxin 6 were associated with apoptosis. Among the seven apoptosis-related proteins, peroxiredoxin 6 was downregulated, in contrast to the other six upregulated proteins. Annexins are highly conserved calcium-binding proteins. Annexin V, in particular, the most abundant annexin in human platelets, has proven useful as an indicator of apoptosis and platelet activation (10). Furthermore, Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine. Consequently, Annexin V can be used as a sensitive probe for phosphatidylserine in cell membranes (11). Translocation of phosphatidylserine to the external cell surface is observed in apoptosis. Annexin A4, an early marker of apoptotic cell death, is translocated selectively from the nucleus to the cytosol during apoptosis (12). The protein-tyrosine kinase substrate Annexin A2 is a growth-regulated gene and shows increased expression in several human cancers, although the mechanism is unclear. However, Annexin A2 appears to be related to multiple physiologic activities, such as cell proliferation and DNA synthesis (13). The 14-3-3 proteins (tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein) play a role in controlling the cell cycle, transcription, and apoptosis. Despite their unknown functions, they are involved in the subcellular localization of proteins and serve as adaptor molecules, a stimulant to interprotein reactions. The 14-3-3 proteins beta polypeptide (YWHAB), gamma polypeptide (YWHAG), and YWHAZ are highly abundant in the brain (14). RhoGDI, a GDP dissociation inhibitor of Rho protein, is essential for the control of many cellular functions, based on its interactions with Rho family GTPases including Rac1, Cdc42, and RhoA. RhoGDI is often overexpressed in human tumors and chemoresistant cancer cell lines, which suggests involvement of RhoGDI in the development of drug-resistant cancer cells (15). The expression of HSP27, a component of a large and heterogeneous group of chaperone proteins, protects against oxidative stress. The major functions of this protein include inhibition of apoptosis and prevention of actin intermediate filament aggregation (16). Although the suppression of apoptosis is generally considered the focal mechanism of tumorigenesis and tumor proliferation, no correlation between VS and apoptotic markers has been reported to date. There are many reports that merlin inhibits several intracellular signals implicated in cell proliferation and tumor formation (1Y4). It was also reported that c-JUN N-terminal kinase activity could either promote tumor cell proliferation in VS or increase apoptosis in normal Schwann cells (17,18). The present study verified increased expression of six proteins related to apoptosis. Among these six proteins, Annexin V, Annexin A4, Anenxin A2, and YWHAZ are likely to accelerate apoptosis, whereas RhoGDI and HSP27 tend to suppress apoptosis. These conflicting characteristics are assumed to be related to the nature of VS. That is, the slow growth rate of VS is thought to originate from two conflicting mechanisms: the suppression of apoptosis
and the acceleration of cell death (19). This hypothesis is supported by our results. Furthermore, the formation of Antoni types A and B, a distinctive pathologic finding of VS, is known to be interconnected with each other; type B is formed by the apoptosis of type A (20). In the immunostaining result of our study, immunoreactivities related to the acceleration of apoptosis were strongly expressed in the Antoni B area; whereas markers related to suppression of apoptosis were more strongly detected in the Antoni A area than in the Antoni B area. Taking these into consideration, it is probable that the overexpression of apoptosis-associated proteins is closely associated with the degeneration of Antoni type A. There are several limitations of our study. First, we chose an imperfect control tissue for proteomic analysis. We removed uninvolved vestibular nerves that VS had not originated from, but this adjacent vestibular nerve might not be a normal nerve because there may have already been genetic changes in Schwann cells of uninvolved vestibular nerve. It would have been better if we could use normal vestibular nerve specimens from vestibular neurectomy of intractable Me´nie`re’s disease. Second, we used normal peripheral sensory nerves from the neck as control tissue for Western blot analysis because of the limited amount of normal vestibular nerve. Third, the number of samples is not sufficient to prove our hypothesis. This study was a preliminary study to find the pathophysiology of the sporadic form of VS, so we have a plan to undertake the follow-up experiments. Finally, we could not confirm all the proteins associated with apoptosis; six upregulated proteins of seven proteins were confirmed by Western blot analysis, and merely three proteins, which were representative to accelerate or suppress apoptosis, were detected by immunohistochemistry. Despite these limitations, the results of this study suggested that the conflicting role of apoptosis may explain the natural history of the sporadic form of VS. The suppression of apoptosis could play an important role in controlling VS development from normal vestibular nerves. However, the increased apoptosis is thought to be correlated with an increase in the degenerative area or the slow growth rate of the tumor. Accordingly, our findings regarding a possible correlation between the conflicting role of apoptosis and the pathophysiology of VS highlight the necessity of further research. CONCLUSION In conclusion, we used a proteomics methodology to examine two VSs and identified differentially expressed proteins. A large number of proteins upregulated and downregulated in VS were related to apoptosis. These results indicate that apoptosis is potentially associated in a complex manner with the pathophysiology of VS. That is, the suppression of apoptosis is involved in tumor occurrence and, conversely, an increase in apoptosis may contribute to the slow tumor growth rate and may be correlated with the Antoni type B area.
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