Selective suppression of matrix metalloproteinase-9 in human glioblastoma cells by antisense gene transfer impairs glioblastoma cell invasion.

Increased expression of matrix metalloproteinases (MMPs) has been associated with human glioblastoma tumor progression. In this study, we sought to down-regulate MMP-9 expression by stably transfecting a high-grade glioblastoma cell line with a plasmid vector capable of expressing an antisense transcript complementary to a 528-bp segment at the 5' end of human MMP-9 cDNA. Stable transfectants were obtained through selection with G418. Of the clones transfected with vector, sense, and antisense constructs, Northern blotting, Western blotting, and gelatin zymography showed that MMP-9 expression was significantly reduced only in the antisense-transfected cells. A Matrigel invasion assay revealed marked reductions in invasiveness for the antisense clones relative to the parental, vector, and sense clones. Cocultures of tumor spheroids and fetal rat brain aggregates showed that the antisense-transfected stable clones showed no invasion of the rat brain aggregates; in contrast, 90% of the parental, vector, and sense clones invaded the rat brain aggregates. Intracerebral injection of antisense stable transfectants in nude mice produced no tumors or very small tumors, but intracerebral injection of parental or vector clones did produce tumors. These results suggest that MMP-9 expression is essential for the invasiveness of glioblastoma cells.

Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells.

Increases in abundance of cathepsin B transcript and protein correlate with increases in tumor grade and alterations in subcellular localization and activity of cathepsin B. The enzyme is able to degrade the components of the extracellular matrix (ECM) and activate other proteases capable of degrading ECM. To investigate the role played by this protease in the invasion of brain tumor cells, we transfected SNB19 human glioblastoma cells with a plasmid containing cathepsin B cDNA in antisense orientation. Control cells were transfected with vector alone. Clones expressing antisense cathepsin B cDNA exhibited significant reductions in cathepsin B mRNA, enzyme activity and protein compared to controls. Matrigel Invasion assay showed that the antisense-transfected cells had a markedly diminished invasiveness compared with controls. When tumor spheroids containing antisense transfected SNB19 cells expressing reduced cathepsin B were co-cultured with fetal rat brain aggregates, invasion of fetal rat brain aggregates was significantly reduced. Green Fluorescent Protein (GFP) expressing parental cells and antisense transfectants were generated for detection in mouse brain tissue without any post-chemical treatment. Intracerebral injection of SNB19 stable antisense transfectants resulted in reduced tumor formation in nude mice. These results strongly support a role for cathepsin B in the invasiveness of human glioblastoma cells and suggest cathepsin B antisense may prove useful in cancer therapy.

Stable transfection of urokinase-type plasminogen activator antisense construct modulates invasion of human glioblastoma cells.

The diffuse and extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modifications of the proteolysis of extracellular matrix components. A key molecule in regulating plasminogen-mediated extracellular proteolysis is the urokinase-type plasminogen activator (uPA). To investigate the role of uPA in the invasive process of brain tumors, we stably transfected a human glioblastoma cell line SNB19 with a vector capable of expressing an antisense transcript complementary to the 1020 bases at the 3' end of the uPA cDNA. Parental, vector-, and antisense construct-stably transfected cell lines were analyzed for uPA mRNA transcript by Northern blot analysis, for uPA enzyme activity by zymography, and for uPA protein levels by Western blotting. The levels of uPA mRNA, protein, and enzyme activities were significantly lower in antisense clones than in parental and vector controls. Radioreceptor binding studies demonstrated that uPA receptor levels remained the same in parental, vector-, and antisense-transfected cells. The antisense-transfected cells showed a markedly lower level of invasion in the Matrigel invasion assays, and their spheroids failed to invade the fetal rat brain aggregates in the coculture system. Green fluorescent protein (GFP) expressing parental and antisense transfectants was generated for detection in mouse brain tissue without any posttreatment. Intracerebral injection of antisense stable transfectants significantly reduced tumor formation compared with that in controls. Our results suggested that down-regulation of uPA expression may be a feasible approach to reducing the malignancy and invasiveness of glial tumors.

Regulation of MMP-9 (type IV collagenase) production and invasiveness in gliomas by the extracellular signal-regulated kinase and jun amino-terminal kinase signaling cascades.

Our previous studies have shown that MMP-9 levels are significantly elevated during the progression of human gliomas. In the current study, we examined the role of JNK- and ERK-dependent signaling modules in the regulation of MMP-9 production and the invasive behavior of the human glioblastoma cell line SNB19, in which JNK/ERK1 is constitutively activated. SNB19 cells that were transfected with dominant-negative JNK, MEKK, and ERK1 expression vectors showed reduced MMP-9 promoter activity. In addition, conditioned medium collected from SNB19 cells transfected with these expression vectors showed diminished MMP-9 activity in the presence of phorbol myristate acetate, as determined by gelatin zymography. The cotransfection of SNB19 cells with kinase-deficient c-raf also diminished MMP-9 promoter activity. Further, in the presence of a specific inhibitor of MEKK (PD098059), the Matrigel invasion assay showed the invasiveness of dominant-negative SNB19 cells transfected with dominant-negative JNK1 or ERK1 to be remarkably reduced. In conclusion, our studies showed for the first time that MMP-9 production and the invasive behavior of SNB 19 cells are regulated by JNK- and ERK-dependent signaling modules and that interfering with either of the pathways reduces invasiveness.

Neurotoxicity evaluation of rats after subchronic inhalation exposure to isobutanol.

The subchronic neurotoxic effects of isobutanol were studied by exposing Sprague-Dawley rats to isobutanol vapor concentrations of 0, 250, 1000, and 2500 ppm for 6 hrs/day, 5 days/wk, for 3 months. A comprehensive set of neurotoxicity tests (functional observational battery, motor activity, perfusion fixation neuropathology, and schedule-controlled operant behavior) including an assessment of complex behavior dependent on learning and memory was conducted. In addition, full histopathology and blood chemistry evaluations were conducted in order to assess any potential functional/behavioral effects in the context of other possible systemic toxicities. There were no morphological or behavioral effects indicative of a specific, persistent or progressive effect of isobutanol on the nervous system at exposure concentrations up to 2500 ppm. A slight decrease in response to external stimuli was observed during exposures at all concentrations. These effects are likely transient effects of acute exposure to isobutanol.

Acute and subchronic neurotoxicological evaluation of tetrahydrofuran by inhalation in rats.

Groups of adult male and female rats received exposure to tetrahydrofuran (THF) vapor by inhalation in acute or subchronic exposure scenarios. Acute exposure concentrations were 0, 500, 2500, or 5000 ppm for 6 hr. Evaluations conducted immediately after exposure included clinical observations, motor activity assessments (MA), and a battery of functional tests (FOB) designed to reveal nervous system dysfunction. During exposure to 2500 and 5000 ppm, rats had a diminished or absent startle response to a punctate auditory alerting stimulus. Following exposure to 5000 ppm, male and female rats were lethargic, exhibited abnormal gait or mobility, and splayed rear feet. Lethargy and splayed rear feet were also observed in females exposed to 2500 ppm. During the subsequent FOB, males exposed to 5000 ppm had a lower incidence of palpebral closure, higher incidences of slow or absent righting reflex, and a biphasic pattern of reduced motor activity followed by increased motor activity. Females exposed to 5000 ppm had increased incidences of palpebral closure in the open field, increased incidences of slow or absent righting reflex, and decreased motor activity. During the 14-week subchronic exposure series, daily THF exposure concentrations were 0, 500, 1500, or 3000 ppm, and neurobehavioral evaluations occurred on non-exposure days at approximately monthly intervals. Diminished startle responses to an auditory alerting stimulus were observed during exposure to 1500 or 3000 ppm; however, repeated exposures did not cause additional neurobehavioral or pathological effects. This pattern of effects is suggestive of transient sedation. Despite daily reinstatement of acute sedative effects during repeated exposure with up to 3000 ppm, THF did not produce any persistent or cumulative effects on nervous system structure or function. The demonstrated no-observed-effect level of THF for both acute and subchronic exposure was 500 ppm.

Effects of radiation on the levels of MMP-2, MMP-9 and TIMP-1 during morphogenic glial-endothelial cell interactions.

Radiation-induced damage to the central nervous system (CNS) is believed to target glial or endothelial cells or both, although the pathophysiology of the process is poorly understood. We therefore used a coculture system, in which glioblastoma SNB19 cells induced bovine retinal endothelial (BRE) cells to form capillary-like structures, to examine the role of ionizing radiation in modulating the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase-1 (TIMP-1). In particular, we irradiated both BRE cells and cocultures of BRE and SNB19 cells with a single dose of X-rays and then estimated the levels of MMP-2, MMP-9 and TIMP-1. Gelatin zymography revealed a continuous increase in the levels of MMP-2 and MMP-9 during capillary-like structure formation. Of note, the levels of both MMP-2 and MMP-9 were markedly higher in irradiated cocultures at 72 hr after irradiation than in untreated cocultures. Northern blot analysis also demonstrated an increased expression of MMP-9 mRNA in the irradiated cocultures. In addition, TIMP-1 mRNA and protein levels increased up to 48 hr in both irradiated and nonirradiated BRE cells and in nonirradiated cocultures, but there was a significant decrease in the TIMP-1 mRNA and protein levels in irradiated cocultures. It takes about 72 hr for capillaries to form in nonirradiated cocultures, but these capillary networks fail to form in endothelial cells in irradiated cocultures. These findings establish that radiation differentially affects the production of MMP-2, MMP-9 and TIMP-1 during glial-endothelial morphogenesis and suggest mechanisms by which microvessels in the CNS respond to radiation.