Manabu Onishi

Integrin Inhibitor Suppresses Bevacizumab-Induced Glioma Invasion

Glioblastoma 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. U87ΔEGFR 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.

Bimodal anti-glioma mechanisms of cilengitide demonstrated by novel invasive glioma models

Integrins 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 αvβ3 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 αvβ3 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.

Novel animal glioma models that separately exhibit two different invasive and angiogenic phenotypes of human glioblastomas

OBJECTIVE Invasive behaviors of malignant gliomas are fundamental traits and major reasons for treatment failure. Delineation of invasive growth is important in establishing treatment for gliomas and experimental neuro-oncology could benefit from an invasive glioma model. In this study, we established two new cell line-based animal models of invasive glioma. METHODS Two cell lines, J3T-1 and J3T-2, were derived from the same parental canine glioma cell line, J3T. These cells were inoculated to establish brain tumors in athymic mice and rats. Pathologic samples of these animal gliomas were examined to analyze invasive patterns in relation to angiogenesis, and were compared with human glioblastoma samples. The molecular profiles of these cell lines were also shown. RESULTS Histologically, J3T-1 and J3T-2 tumors exhibited different invasive patterns. J3T-1 cells clustered around newly developed vessels at tumor borders, whereas J3T-2 cells showed diffuse single cell infiltration into surrounding healthy parenchyma. In human malignant glioma samples, both types of invasion were observed concomitantly. Molecular profiles of these cell lines were analyzed by immunocytochemistry and with quantitative reverse transcription polymerase chain reaction. Vascular endothelial growth factor, matrix metalloproteinase-9, hypoxia-inducible factor-1, and platelet-derived growth factor were overexpressed in J3T-1 cells rather than in J3T-2 cells, whereas integrin αvβ3, matrix metalloproteinase-2, nestin, and secreted protein acidic and rich in cysteine were overexpressed in J3T-2 cells rather than in J3T-1 cells. CONCLUSIONS These animal models histologically recapitulated two invasive and angiogenic phenotypes, namely angiogenesis-dependent and angiogenesis-independent invasion, also observed in human glioblastoma. These cell lines provided a reproducible in vitro and in vivo system to analyze the mechanisms of invasion and angiogenesis in glioma progression.

Integrin antagonist augments the therapeutic effect of adenovirus-mediated REIC/Dkk-3 gene therapy for malignant glioma

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) was identified as a gene whose expression is reduced in many human cancers. REIC/Dkk-3 expression is also downregulated in malignant glioma and regulates cell growth through caspase-dependent apoptosis. cRGD (EMD121974), an antagonist of integrins, has demonstrated preclinical efficacy against malignant glioma. In this study, we investigated the antiglioma effect of combination therapy using an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) and cRGD. Quantitative real-time reverse-transcription PCR revealed the reduction of REIC/Dkk-3 mRNA levels in malignant glioma cell lines. The reduction of REIC/Dkk-3 protein expression in malignant glioma cell lines was also confirmed with western blot analysis. After treatment with Ad-REIC and cRGD, the proliferative rate of malignant glioma cells was significantly reduced in a time-dependent manner. In vivo, there was a statistically significant increase in the survival of mice treated with Ad-REIC and cRGD combination therapy compared with Ad-REIC monotherapy. We identified an apoptotic effect following monotherapy with Ad-REIC. Moreover, cRGD augmented the antiglioma efficacy of Ad-REIC. These results may lead to a promising new approach for the treatment of malignant glioma.

Proteomics-based analysis of invasion-related proteins in malignant gliomas

One 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.

Mechanisms of tumor development and anti-angiogenic therapy in glioblastoma multiforme

Despite advances in surgical and medical therapy, glioblastoma multiforme (GBM) remains a fatal disease. There has been no significant increase in survival for patients with this disease over the last 20 years. Tumor vasculature formation and glioma cell invasion along the white matter tracts both play a pivotal role in glioma development. Angiogenesis and invasion are the major factors believed to be responsible for treatment resistance in tumors, and a better understanding of the glioma invasion and angiogenesis mechanisms will lead to the development of potential new treatments. In this review, we focus on the molecular characteristics of angiogenesis and invasion in human malignant glioma. We discuss bevacizumab and cilengitide, which are used to inhibit angiogenesis in GBM.

The integrin inhibitor cilengitide enhances the anti-glioma efficacy of vasculostatin-expressing oncolytic virus

Oncolytic viral (OV) therapy has been considered as a promising treatment modality for brain tumors. Vasculostatin, the fragment of brain-specific angiogenesis inhibitor-1, shows anti-angiogenic activity against malignant gliomas. Previously, a vasculostatin-expressing oncolytic herpes simplex virus-1, Rapid Antiangiogenesis Mediated By Oncolytic virus (RAMBO), was reported to have a potent antitumor effect. Here, we investigated the therapeutic efficacy of RAMBO and cilengitide, an integrin inhibitor, combination therapy for malignant glioma. In vitro, tube formation was significantly decreased in RAMBO and cilengitide combination treatment compared with RAMBO or cilengitide monotherapy. Moreover, combination treatment induced a synergistic suppressive effect on endothelial cell migration compared with the control virus. RAMBO, combined with cilengitide, induced synergistic cytotoxicity on glioma cells. In the caspase-8 and -9 assays, the relative absorption of U87ΔEGFR cell clusters treated with cilengitide and with RAMBO was significantly higher than that of those treated with control. In addition, the activity of caspase 3/7 was significantly increased with combination therapy. In vivo, there was a significant increase in the survival of mice treated with combination therapy compared with RAMBO or cilengitide monotherapy. These results indicate that cilengitide enhanced vasculostatin-expressing OV therapy for malignant glioma and provide a rationale for designing future clinical trials combining these two agents.

Annexin A2 regulates angiogenesis and invasion phenotypes of malignant glioma

We have established a pair of animal models (J3T-1 and J3T-2) with different invasive and angiogenic phenotypes, and demonstrated that annexin A2 is expressed at higher levels in J3T-1 than J3T-2 cells. The function of annexin A2 in relation to angiogenesis and invasion was investigated using these models. Stable silencing or overexpression of annexin A2 in J3T-1 and J3T-2 cells (J3T-1shA and J3T-2A cells) was established and used. Thirty human glioblastoma samples were evaluated for expression of annexin A2, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). Immunohistochemical and quantitative reverse-transcription polymerase chain reaction analyses revealed higher expression of annexin A2, VEGF and PDGF in J3T-1 and J3T-2A cells. Cultured J3T-1 and J3T-2A cells exhibited higher adhesive ability to endothelial cells. Histopathological analysis of animal brain tumors revealed that J3T-1 and J3T-2A tumors displayed marked angiogenesis and invasion along the neovasculature, whereas J3T-2 and J3T-1shA tumors exhibited diffuse, infiltrative invasion without angiogenesis. Positive expression of annexin A2 was observed in tumor cells surrounding dilated vessels in 25/30 human glioblastoma specimens. Our results reveal that the phenotype of glioma invasion is closely related to angiogenesis. We identify annexin A2 as a factor regulating angiogenesis and invasion of malignant gliomas.

The supposed intracavernous sinus arachnoid cyst with abducens neuropathy: a case report.

Intracavernous sinus arachnoid cysts are rare intracranial congenital lesions. When present, their anatomic location frequently results in cranial nerve palsy. A 15-year-old boy was admitted to our hospital with diplopia, which had gradually worsened over the previous several months. An arachnoid cyst was identified within the right cavernous sinus and fenestration surgery was performed. The patient recovered well and three months after the surgery, diplopia was disappeared. Surgical decompression of the intracavernous sinus arachnoid cyst is beneficial for symptomatic patients with this condition.