Search

OBJECT High-grade gliomas are the most common form of adult brain cancer, and patients have a dismal survival rate despite aggressive therapeutic measures. Intratumoral hypoxia is thought to be a main contributor to tumorigenesis and angiogenesis of these tumors. Because hypoxia-inducible factor 1α (HIF-1α) is the major mediator of hypoxia-regulated cellular control, inhibition of this transcription factor may reduce glioblastoma growth. METHODS Using an orthotopic mouse model with U87-LucNeo cells, the authors used RNA interference to knock down HIF-1α in vivo. The small interfering RNA (siRNA) was packaged using a novel multifunctional surfactant, 1-(aminoethyl) iminobis[N-(oleicylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO), a nucleic acid carrier that facilitates cellular uptake and intracellular release of siRNA. Stereotactic injection was used to deliver siRNA locally through a guide-screw system, and delivery/uptake was verified by imaging of fluorescently labeled siRNA. Osmotic pumps were used for extended siRNA delivery to model a commonly used human intracranial drug-delivery technique, convection-enhanced delivery. RESULTS Mice receiving daily siRNA injections targeting HIF-1α had a 79% lower tumor volume after 50 days of treatment than the controls. Levels of the HIF-1 transcriptional targets vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT-1), c-MET, and carbonic anhydrase-IX (CA-IX) and markers for cell growth (MIB-1 and microvascular density) were also significantly lower. Altering the carrier EHCO by adding polyethylene glycol significantly increased the efficacy of drug delivery and subsequent survival. CONCLUSIONS Treating glioblastoma with siRNA targeting HIF-1α in vivo can significantly reduce tumor growth and increase survival in an intracranial mouse model, a finding that has direct clinical implications.

Object Meningioma research has been hindered by the inability to sequentially measure intracranial tumor growth in a cost-effective, efficient manner. Recently, the luciferase gene has been transfected into cancer lines to obtain cells that express the luciferase enzyme, which oxidizes luciferin in a reaction that releases photon energy that can be measured noninvasively by bioluminescence imaging (BLI) systems. The authors describe a mouse model of intracranial meningioma that uses this novel BLI system. Methods The immortal meningioma cell lines CH-157-MN and IOMM-Lee were transfected with luciferase and neomycin phosphotransferase (LucNeo) and selected with G418. These cells were stereotactically implanted at skull base and cerebral convexity locations in nude mice. Animals were imaged for bioluminescence biweekly, and 5 mice underwent magnetic resonance (MR) imaging. Tumors were harvested for immunohistochemical and ultrastructural analysis. Results The CH-157-MN-LucNeo and IOMM-Lee-LucNeo cell lines were successfully implanted intracranially in mice. The tumor induction rate for CH-157-MN-LucNeo skull base tumors was 90% (36 of 40 procedures). Statistical analysis of CH-157-MN-LucNeo skull base tumor volume measured on MR imaging correlated with the results of BLI showed an R value of 0.900. The tumors exhibited characteristics of aggressive meningiomas by insinuating along arachnoid planes and invading brain. Conclusions Noninvasive BLI was successfully used to image intracranial meningiomas in mice. The tumors grew in a fashion similar to that of aggressive meningiomas in humans, and exhibited the microscopic, immunohistochemical, and ultrastructural features characteristic of meningiomas. This animal model overcomes the main obstacle in studying intracranial meningiomas by enabling sequential noninvasive tumor measurement in a cost-effective manner.

Object. Meningiomas are the second most common symptomatic primary central nervous system tumor in adults. Findings of epidemiological studies link meningiomas with a history of head trauma, indicating a causal relationship between the inflammatory response and meningioma tumorigenesis. Cyclooxygenase-2 (COX-2), an inducible inflammatory enzyme, converts arachidonic acid to prostaglandins, which have angiogenic, cell-proliferative, and antiapoptotic effects. The authors investigated COX-2 expression in meningiomas and the effects of celecoxib, a COX-2 inhibitor, on meningioma cell growth in vitro. Methods. Four meningioma surgical specimens were immunohistochemically stained and graded (0 to 4) for COX-2. In addition, a Western blot analysis was performed to detect the presence of COX-2. Human meningioma cells grown in cell culture were treated with vehicle or celecoxib (0.25‐1 mM). An immunohistochemical analysis of COX-2, a methylthiotetrazole cell proliferation assay, a TUNEL apoptosis assay, and a Western blot analysis for the proapoptotic protein BAX were performed in vitro. One hundred eleven (87%) of 128 benign meningiomas and six (86%) of seven atypical meningiomas displayed a high COX-2 immunoreactivity (Grade 4 staining). In the Western blot analysis all four surgical specimens (100%) stained positive for a 70-kD band consistent with COX-2. Celecoxib inhibited cell growth in a dose-dependent fashion and induced apoptosis by Day 2, with no change noted in the expression of the BAX protein. Conclusions. The COX-2 enzyme is universally expressed in meningiomas. Celecoxib inhibits meningioma growth in vitro in a dose-dependent fashion, with evidence of apoptosis. Inhibitors of COX-2 may have a role in the treatment of recurrent meningiomas.

Object. Endolymphatic sac (ELS) tumors are low-grade malignancies of the temporal bone that are associated with von Hippel—Lindau (VHL) disease but can also occur sporadically. The VHL gene product VHL protein is important in the regulation of hypoxia inducible factor (HIF)-1α, which controls expression of molecules that are important in angiogenesis and cell metabolism. In this study the authors examine the role of VHL and HIF-1 in ELS tumors. Methods. The ELS tumors from three patients were examined using the following method: DNA from tumor tissue was isolated, amplified by polymerase chain reaction and the VHL gene sequence was compared with the known wild-type sequence. Loss of heterozygosity (LOH) studies were performed to confirm the sequencing data. Immunohistochemical evaluation for VHL, HIF-1α, vascular endothelial growth factor (VEGF), and carbonic anhydrase IX (CA IX) was performed. Snap-frozen tumor tissue was examined using Western blot and HIF-1 immunoassays for HIF-1α and VHL expression. Two patients had sporadic ELS tumors and the other one suffered from VHL disease. Results of VHL gene sequencing were normal in the tissue derived from the sporadic ELS tumors. The ELS tumor, pheochromocytoma, and spinal hemangioblastoma were heterozygous for the same C-to-A transversion found in the germline carried by the patient with VHL disease. No LOH was detected in the tumor tissue obtained in the patient with VHL disease. Expression of HIF-1α, VEGF, and CA IX evaluated using immunohistochemical studies was elevated in the VHL-associated tumors. Nevertheless, Western blots and immunoassays for HIF-1α did not show elevated expression in these tumors. Conclusions. The sporadic and VHL disease—associated ELS tumors in this study had normal VHL-mediated HIF-1 regulation. This is a result of normal VHL gene expression in the case of the sporadic ELS tumor. In the VHL-associated ELS tumor, this is due to one normal copy of the VHL gene and adequate VHL gene expression.