BMCActa Medica Okayama2051-5960812020Annexin A2-STAT3-Oncostatin M receptor axis drives phenotypic and mesenchymal changes in glioblastoma42ENYujiMatsumotoDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesTomotsuguIchikawaDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesKazuhikoKurozumiDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesYoshihiroOtaniDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesAtsushiFujimuraDepartment of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKentaroFujiiDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesYusukeTomitaDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesYasuhikoHattoriDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesAtsuhitoUnedaDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesNobushigeTsuboiDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesKeisukeKanedaDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesKeigoMakinoDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesIsaoDateDepartment of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesGlioblastoma (GBM) is characterized by extensive tumor cell invasion, angiogenesis, and proliferation. We previously established subclones of GBM cells with distinct invasive phenotypes and identified annexin A2 (ANXA2) as an activator of angiogenesis and perivascular invasion. Here, we further explored the role of ANXA2 in regulating phenotypic transition in GBM. We identified oncostatin M receptor (OSMR) as a key ANXA2 target gene in GBM utilizing microarray analysis and hierarchical clustering analysis of the Ivy Glioblastoma Atlas Project and The Cancer Genome Atlas datasets. Overexpression of ANXA2 in GBM cells increased the expression of OSMR and phosphorylated signal transducer and activator of transcription 3 (STAT3) and enhanced cell invasion, angiogenesis, proliferation, and mesenchymal transition. Silencing of OSMR reversed the ANXA2-induced phenotype, and STAT3 knockdown reduced OSMR protein expression. Exposure of GBM cells to hypoxic conditions activated the ANXA2-STAT3-OSMR signaling axis. Mice bearing ANXA2-overexpressing GBM exhibited shorter survival times compared with control tumor-bearing mice, whereas OSMR knockdown increased the survival time and diminished ANXA2-mediated tumor invasion, angiogenesis, and growth. Further, we uncovered a significant relationship between ANXA2 and OSMR expression in clinical GBM specimens, and demonstrated their correlation with tumor histopathology and patient prognosis. Our results indicate that the ANXA2-STAT3-OSMR axis regulates malignant phenotypic changes and mesenchymal transition in GBM, suggesting that this axis is a promising therapeutic target to treat GBM aggressiveness.No potential conflict of interest relevant to this article was reported.Springer International PublishingActa Medica Okayama2193-180122013Gene expression profiling of the anti-glioma effect of Cilengitide160ENManabuOnishiKazuhikoKurozumiTomotsuguIchikawaHiroyukiMichiueKentaroFujiiJojiIshidaYosukeShimazuE AntonioChioccaBalveenKaurIsaoDateCilengitide (EMD121974), an inhibitor of the adhesive function of integrins, demonstrated preclinical efficacy against malignant glioma. It is speculated that cilengitide can inhibit tumor growth, invasion, and angiogenesis. However, the effects of cilengitide on these processes have not been sufficiently examined. In this study, we investigated the anti-glioma effect of cilengitide using DNA microarray analysis. U87ΔEGFR cells (human malignant glioma cell line) were used for this experiment. The cells were harvested after 16 h of cilengitide treatment, and mRNA was extracted. Gene expression and pathway analyses were performed using a DNA microarray (CodeLink™Human Whole Genome Bioarray). The expression of 265 genes was changed with cilengitide treatment. The expression of 214 genes was up-regulated by more than 4-fold and the expression of 51 genes was down-regulated by more than 4-fold compared to the controls. In pathway analysis, "apoptotic cleavage of cellular proteins" and "TNF receptor signaling pathway" were over-represented. Apoptotic-associated genes such as caspase 8 were up-regulated. Gene expression profiling revealed more detailed mechanism of the anti-glioma effect of cilengitide. Genes associated with apoptosis were over-represented following cilengitide treatment.No potential conflict of interest relevant to this article was reported.Elsevier Science IncActa Medica Okayama1944-7124722014Integrin Inhibitor Suppresses Bevacizumab-Induced Glioma Invasion292302ENJojiIshidaManabuOnishiKazuhikoKurozumiTomotsuguIchikawaKentaroFujiiYosukeShimazuTetsuoOkaIsaoDateGlioblastoma is known to secrete high levels of vascular endothelial growth factor (VEGF), and clinical studies with bevacizumab, a monoclonal antibody to VEGF, have demonstrated convincing therapeutic benefits in glioblastoma patients. However, its induction of invasive proliferation has also been reported. We examined the effects of treatment with cilengitide, an integrin inhibitor, on bevacizumab-induced invasive changes in glioma. U87 Delta EGFR cells were stereotactically injected into the brain of nude mice or rats. Five days after tumor implantation, cilengitide and bevacizumab were administered intraperitoneally three times a week. At 18 days after tumor implantation, the brains were removed and observed histopathologically. Next, the bevacizumab and cilengitide combination group was compared to the bevacizumab monotherapy group using microarray analysis. Bevacizumab treatment led to increased cell invasion in spite of decreased angiogenesis. When the rats were treated with a combination of bevacizumab and cilengitide, the depth of tumor invasion was significantly less than with only bevacizumab. Pathway analysis demonstrated the inhibition of invasion-associated genes such as the integrin-mediated cell adhesion pathway in the combination group. This study showed that the combination of bevacizumab with cilengitide exerted its anti-invasive effect. The elucidation of this mechanism might contribute to the treatment of bevacizumab-refractory glioma.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0919-65443322013Bimodal anti-glioma mechanisms of cilengitide demonstrated by novel invasive glioma models162174ENManabuOnishiTomotsuguIchikawaKazuhikoKurozumiKentaroFujiiKoichiYoshidaSatoshiInoueHiroyukiMichiueE. AntonioChioccaBalveenKaurIsaoDateIntegrins are expressed in tumor cells and tumor endothelial cells, and likely play important roles in glioma angiogenesis and invasion. We investigated the anti-glioma mechanisms of cilengitide (EMD121974), an v3 integrin inhibitor, utilizing the novel invasive glioma models, J3T-1 and J3T-2. Immunohistochemical staining of cells in culture and brain tumors in rats revealed positive v3 integrin expression in J3T-2 cells and tumor endothelial cells, but not in J3T-1 cells. Established J3T-1 and J3T-2 orthotopic gliomas in athymic rats were treated with cilengitide or solvent. J3T-1 gliomas showed perivascular tumor cluster formation and angiogenesis, while J3T-2 gliomas showed diffuse single-cell infiltration without obvious angiogenesis. Cilengitide treatment resulted in a significantly decreased diameter of the J3T-1 tumor vessel clusters and its core vessels when compared with controls, while an anti-invasive effect was shown in the J3T-2 glioma with a significant reduction of diffuse cell infiltration around the tumor center. The survival of cilengitide-treated mice harboring J3T-1 tumors was significantly longer than that of control animals (median survival: 57.5 days and 31.8 days, respectively, P<0.005), while cilengitide had no effect on the survival of mice with J3T-2 tumors (median survival: 48.9 days and 48.5, P=0.69). Our results indicate that cilengitide exerts a phenotypic anti-tumor effect by inhibiting angiogenesis and glioma cell invasion. These two mechanisms are clearly shown by the experimental treatment of two different animal invasive glioma models.No potential conflict of interest relevant to this article was reported.Wiley-BlackwellActa Medica Okayama0919-65443332013Proteomics-based analysis of invasion-related proteins in malignant gliomas264275ENTomokoMaruoTomotsuguIchikawaHirotakaKanzakiSatoshiInoueKazuhikoKurozumiManabuOnishiKoichiYoshidaHirokazuKambaraMamoruOuchidaKenjiShimizuSeijiTamaruE. AntonioChioccaIsaoDateOne of the insidious biological features of gliomas is their potential to extensively invade normal brain tissue, yet molecular mechanisms that dictate this locally invasive behavior remain poorly understood. To investigate the molecular basis of invasion by malignant gliomas, proteomic analysis was performed using a pair of canine glioma subclones - J3T-1 and J3T-2 - that show different invasion phenotypes in rat brains but have similar genetic backgrounds. Two-dimensional protein electrophoresis of whole-cell lysates of J3T-1 (angiogenesis-dependent invasion phenotype) and J3T-2 (angiogenesis-independent invasion phenotype) was performed. Twenty-two distinct spots were recognized when significant alteration was defined as more than 1.5-fold change in spot intensity between J3T-1 and J3T-2. Four proteins that demonstrated increased expression in J3T-1, and 14 proteins that demonstrated increased expression in J3T-2 were identified using liquid chromatography-mass spectrometry analysis. One of the proteins identified was annexin A2, which was expressed at higher levels in J3T-1 than in J3T-2. The higher expression of annexin A2 in J3T-1 was corroborated by quantitative RT-PCR of the cultured cells and immunohistochemical staining of the rat brain tumors. Moreover, immunohistochemical analysis of human glioblastoma specimens showed that annexin A2 was expressed at high levels in the tumor cells that formed clusters around dilated vessels. These results reveal differences in the proteomic profiles between these two cell lines that might correlate with their different invasion profiles. Thus, annexin A2 may be related to angiogenesis-dependent invasion.No potential conflict of interest relevant to this article was reported.Wiley-BlackwellActa Medica Okayama0919-65443262012Role of VEGF and matrix metalloproteinase-9 in peritumoral brain edema associated with supratentorial benign meningiomas638646ENEijiIwadoTomotsuguIchikawaHiroshiKosakaShinjiOtsukaHirokazuKambaraTakashiTamiyaSeijiKondoIsaoDateAccumulating evidence indicates that VEGF and matrix metalloproteinase-9 (MMP-9) play a central role in the development of peritumoral brain edema (PTBE) associated with human brain tumors. However, the roles of these proteins, particularly of MMP-9, in PTBE associated with benign meningiomas have not been elucidated. We investigated the association between clinical features and biological factors, such as VEGF and MMP-9, and the incidence of PTBE and edema index (EI) in 60 patients with benign meningiomas. In this study, supratentorial lesions were examined for evaluating the extent of PTBE in the surrounding normal brain tissue. VEGF and MMP-9 expression was immunohistochemically examined. Multivariate analysis revealed that the presence of pial blood supply (odds ratio [OR] 12.250; P = 0.0096) and VEGF (OR 7.683; P = 0.0155), but not MMP-9 (OR 1.178; P = 0.8113), expression are significant factors that independently predict the incidence of PTBE and influence EI. VEGF (P = 0.0397) and MMP-9 (P = 0.0057) expression correlates with the presence of pial blood supply. Moreover, tumors with high VEGF and MMP-9 expression had higher EIs than those with high expression of either (P = 0.030). Our findings suggest that MMP-9 expression was positively related to VEGF expression and pial blood supply and promoted the occurrence of PTBE by inducing the disruption of the arachnoid membrane and formation of pial blood supply.No potential conflict of interest relevant to this article was reported.Nature Publishing GroupActa Medica Okayama0929-19031982012Therapeutic effect of suicide gene-transferred mesenchymal stem cells in a rat model of glioma572578ENHKosakaTIchikawaKKurozumiHKambaraSInoueTMaruoKNakamuraHHamadaIDateWe evaluated a new therapeutic strategy for malignant glioma, which combines intratumoral inoculation of mesenchymal stem cells (MSCs) expressing cytosine deaminase gene with 5-fluorocytosine (5-FC) administration. For in vitro and in vivo experiments, MSCs were transfected with adenovirus carrying either enhanced green fluorescent protein gene (AdexCAEGFP) or cytosine deaminase gene (AdexCACD), to establish MSC-expressing EGFP (MSC-EGFP) or CD (MSC-CD). Co-culture of 9L glioma cells with MSC-CD in a medium containing 5-FC resulted in a remarkable reduction in 9L cell viability. The migratory ability of MSC-EGFP toward 9L cells was demonstrated by double-chamber assay. For the in vivo study, rats harboring 9L brain tumors were inoculated with MSC-EGFP or MSC-CD. Immunohistochemistry of rat brain tumors inoculated with MSC-EGFP showed intratumoral distribution of MSC-EGFP. Survival analysis of rats bearing 9L gliomas treated with intratumoral MSC-CD and intraperitoneal 5-FC resulted in significant prolongation of survival compared with control animals. In conclusion, molecular therapy combining suicide gene therapy and MSCs as a targeting vehicle represents a potential new therapeutic approach for malignant glioma, both with respect to the antitumor potential of this system and its neuroprotective effect on normal brain tissue.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6312009Protein Transduction Method for Cerebrovascular Disorders17ENTomoyukiOgawaShigekiOnoTomotsuguIchikawaSeijiArimitsuKeisukeOnodaKojiTokunagaKenjiSugiuKazuhitoTomizawaHidekiMatsuiIsaoDateReview10.18926/AMO/31858<p>Many studies have shown that a motif of 11 consecutive arginines (11R) is one of the most effective protein transduction domains (PTD) for introducing proteins into the cell membrane. By conjugating this "11R", all sorts of proteins can effectively and harmlessly be transferred into any kind of cell. We therefore examined the transduction efficiency of 11R in cerebral arteries and obtained results showing that 11R fused enhanced green fluorescent protein (11R-EGFP) immediately and effectively penetrated all layers of the rat basilar artery (BA), especially the tunica media. This method provides a revolutionary approach to cerebral arteries and ours is the first study to demonstrate the successful transductionof a PTD fused protein into the cerebral arteries. In this review, we present an outline of our studies and other key studies related to cerebral vasospasm and 11R, problems to be overcome, and predictions regarding future use of the 11R protein transduction method for cerebral vasospasm (CV).</p>No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155812032008XIV 悪性神経膠腫に対する multimodality treatment307312ENTomotsuguIchikawaKazuhikoKurozumiIsaoDateNo potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030155811832007蛋白導入システム“Protein Transduction System”を利用したプロテインセラピーの発展と現状について― 悪性脳腫瘍に対する蛋白導入法の利用を中心に ―205208ENNo potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama003015581202200811Rを用いた脳血管に対する新しい蛋白質導入法129133ENTomoyukiOgawaShigekiOnoTomotsuguIchikawaSeijiArimitsuKeisukeOnodaKojiTokunagaKenjiSugiuKazuhitoTomizawaHidekiMatsuiIsaoDateNo potential conflict of interest relevant to this article was reported.Acta Medica Okayama2001In vivo efficacy and toxicity of 5-fluorocytosine/cytosine deaminase gene therapy for malignant gliomas mediated by adenovirusENNo potential conflict of interest relevant to this article was reported.