Rat C6 Glioma Model Research Articles

Abstract 556: Extracellular sulfatase 2; enzymatic inhibition by OKN-007 a disulfonylphenyl nitrone with anticancer activity and other sulfamate and sulfone compounds

Abstract Sulfatase 2 (Sulf2) is an extracellular enzyme which catalyzes the removal of 6-O-sulfate groups on glucosamine from subregions of the glycosaminoglycan heparin sulfate. Sulf2 action alters the binding of protein ligand growth factors such as VEGF, FGF-2, TGF and SDF-1 to heparin and cellular receptors. Sulf2 is enriched in several tumors including breast cancer, pancreatic cancer, lung cancer and liver cancer and studies in experimental models have shown its action is important in cancer development. We have shown that PBN (α-phenyl-tert-butylnitrone) nitrones have anti-cancer activity in experimental cancer models including the rat choline deficiency liver cancer model, the mouse APCmin model of colon cancer and the rat C6 glioma model. Studies of the PBN derivative 2,4-disulfonylphenyl-tert-butylnitrone in the C6 glioma model showed that it has more potent anti-cancer activity in this model than PBN. We considered that the sulfonyl groups on OKN-007 acting to inhibit the activity of Sulf2 could account for its anti-cancer activity and therefore set up experiments to test this concept. We have shown that OKN-007 does inhibit Sulf2 at biological meaningful concentrations. In addition we have partially completed an MCF-7 xenograph study that indicates OKN-007 administered orally does suppress cancer growth in this model. We have also shown that another compound suramin that has 6 sulfonyl groups and has been shown to have anti-cancer activity also suppressed the enzymatic activity of Sulf2. In addition we have also tested some compounds that have sulfamate and sulfone functional groups and have found that they also show some activity in suppressing the enzymatic action of Sulf2. Studies are continuing to elucidate the significance of the action of OKN-007 on Sulf2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 556. doi:10.1158/1538-7445.AM2011-556

Vascular Endothelial Growth Factor Increases Permeability of the Blood–Tumor Barrier via Caveolae-Mediated Transcellular Pathway

The first goal of this study was to determine the effect of vascular endothelial growth factor (VEGF) on permeability of the blood-tumor barrier (BTB). The second goal was to determine possible cellular mechanisms by which VEGF increases permeability of the BTB. In the rat C6 glioma model, the permeability of the BTB was significantly increased after VEGF injection at dose of 0.05ng/g and reached its peak at 45min. Meanwhile, we observed that the density of pinocytotic vesicles of brain microvascular endothelial cells (BMECs) in the BTB increased dramatically by transmission electron microscopy. The immunohistochemistry and western blot analysis revealed that the expression level of caveolae structure proteins caveolin-1 and caveolin-2 in BMECs was increased after VEGF injection, peaked at 45min, and then decreased to the untreated level. The time peak of expression level of caveolin-1 and caveolin-2 was identical with the peak time of permeability of the BTB and the density of pinocytotic vesicles. All of these results strongly indicated that VEGF increased permeability of the BTB caused by enhancement of the density of pinocytotic vesicles, and the molecular mechanism might be associated with upregulated expression of caveolin-1 and caveolin-2.

Role of ATP synthase alpha subunit in low-dose endothelial monocyte-activating polypeptide-II-induced opening of the blood–tumor barrier

This study was performed to determine whether the α subunit of ATP synthase (α-ATP synthase) on brain microvascular endothelial cells (BMECs) serves as the functional target for endothelial monocyte-activating polypeptide-II (EMAP-II)-induced increase in blood–tumor barrier (BTB) permeability. Using a rat C6 glioma model, we found that low-dose (80 ng/kg) EMAP-II significantly decreased the mRNA and protein expression levels of tight junction (TJ)-related proteins claudin-5, occludin, and ZO-1 on BMECs. Meantime, radioimmunity and Western blot assay showed a significant decrease in the expression levels of cAMP and catalytic subunit of protein kinase A (PKAcs) of tumor tissues. Also, pretreatment with specific α-ATP synthase antibody significantly blocked the effects of EMAP-II on TJ-related proteins, cAMP, and PKAcs. In addition, double immunofluorescence assay identified that EMAP-II was co-localized with α-ATP synthase on BMECs. This in vivo study demonstrated that α subunit of ATP synthase on BMECs serves as the functional target for EMAP-II selective opening of the BTB, and that cAMP/PKA signaling transduction pathway might be involved in the modulating process.

Lactoferrin-conjugated superparamagnetic iron oxide nanoparticles as a specific MRI contrast agent for detection of brain glioma in vivo

A specific contrast agent for magnetic resonance imaging (MRI) is crucial to brain tumor patients for the surgical operation or the postoperative radiology. This study explored lactoferrin-conjugated superparamagnetic iron oxide nanoparticles (Lf-SPIONs) as an MRI contrast agent for the detection of brain gliomas in vivo. The hydrodynamic diameter of about 75 nm, saturation magnetization of 51 emu/g Fe and T 2 relaxivity of 75.6 mM −1S −1 of the Lf-SPIONs suggested its applicability for MRI. Using a rat model of C6 glioma, Lf-SPIONs provided a better picture or more sensitivity to depict brain glioma on MR images than that of SPIONs. Significantly enhanced T 2 -weighted images of brain glioma were documented in vivo with Lf-SPIONs until 48 h after injection. Moreover, Lf-SPIONs were clearly observed around vascular region of the tumor slices after 48 h. High level expression of Lf receptors was confirmed in brain tumor tissues by RT-PCR and Western Blot compared to normal brain tissues. These findings suggested that Lf-SPIONs could be potentially employed as a sensitive and specific MRI contrast agent in the diagnosis of brain glioma.

Naringenin promote apoptosis in cerebrally implanted C6 glioma cells

Naringenin (NGEN), a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth. It has been also shown to access the brain and there is an increasing interest in its therapeutic applications. The up-regulated expression of Cx43 leads to the suppression of tumorigenicity with promoted apoptotic events. In this study, we investigated the in vivo effect of NGEN in fostering apoptosis in cerebrally implanted C6 glioma cells rat model. We analysed the expression of Cx43, caspase-3, caspase-9, Cyt C, Bcl-2 and Bax in vivo by immunoblot analysis and the ultra structure of brain cells by transmission electron microscopy. Supplementation of NGEN to experimental animals modulated Bcl-2/Bax ratio and up-regulation of caspase-3 and 9. NGEN was also found to up-regulate the expression of Cx43. These findings provide evidence that NGEN's apoptotic effect, modulation of Bcl-2/Bax ratio leads to release of Cyt C from mitochondria, thereby activation of caspase-3 and caspase-9 is mediated by enhanced expression of Cx43. These observations were well supported by the transmission electron microscopic results which showed the characteristic apoptotic features. In conclusion, our findings demonstrate that NGEN promotes apoptosis in rat C6 glioma model.

Additive effect of low-frequency ultrasound and endothelial monocyte-activating polypeptide II on blood–tumor barrier in rats with brain glioma

Brain glioma is a malignant tumor which needs surgery followed by chemotherapy. Low-frequency ultrasound (LFU) and Optison could open blood–tumor barrier (BTB) selectively and noninvasively and thus increase the permeability of BTB. Endothelial monocyte-activating polypeptide II (EMAP-II) induces cytoskeletal remodeling in endothelial cells. In this study, we asked whether LFU, Optison, and/or EMAP-II used in combination have additive effects on increasing the permeability of BTB by tight junction (TJ)-associated protein-dependent manner and thus help understand the possible mechanisms for TJ-based drug delivery to the central nervous system through BTB. Evans Blue assay was used to measure the permeability of BTB in rat model of C6 glioma. The mRNA and protein levels of TJ-associated proteins, claudin-5, occludin, and ZO-1, were determined. Results showed that Evans blue content significantly increased and the mRNA and protein levels of claudin-5, occludin, and ZO-1 significantly reduced after the treatment in groups treated with EMAP-II and LFU combined with or without Optison (LFU + EMAP-II and LFU + Optison + EMAP-II groups) and in the group treated with LFU and Optison (LFU + Optison group). In conclusion, LFU and EMAP-II used in combination have additive effects on increasing the permeability of BTB and remodeling of TJ-associated proteins.

Temozolomide/PLGA microparticles: a new protocol for treatment of glioma in rats

Implantable and poly (d,l-lactide-co-glycolide) (PLGA) microparticles were developed to deliver temozolomide (TM) continuously in interstitial chemotherapy for glioma. The therapeutic effect of temozolomide/PLGA was evaluated in a rat C6 glioma model. C6 cells were implanted orthotopically into 100 rat brains in 5 groups (n=20 each): sham operation group, control group, local delivery of blank PLGA microspheres group, oral TM group, and local delivery of TM/PLGA group. Rats in oral TM group were orally administered temozolomide, and rats in TM/PLGA group were locally implanted with TM/PLGA microspheres. Ten rats were selected randomly from each group for observing the survival time, and the other 10 rats were killed on POD 14 to measure proliferation activity and apoptosis of the gliomas. Head MRI examination was performed before the rats were killed. The median survival time of sham operation group, control group, blank PLGA microspheres group, oral TM group, and TM/PLGA group was 19.5, 20, 19, 27, and 46.5 days, respectively. MRI demonstrated that the tumor volume was reduced in oral TM group and interstitial TM/PLGA group. PCNA-positive cell staining showed that proliferation activity of tumor cells treated with interstitial TM/PLGA therapy significantly decreased when compared with that of tumor cells treated with oral TM therapy. The apoptosis of C6 cells in interstitial TM/PLGA group significantly increased when compared with that in oral TM group. Interstitial TM/PLGA was effective in treating intracranial C6 rat gliomas and could prove to be a potential chemotherapy agent for human malignant gliomas.

Abstract 2267: Inhibition of extracellular sulfatase 2 as a possible mechanism to partially explain the anticancer activity of the nitrone OKN007

Abstract Sulfatase 2 (Sulf2) is an extracellular enzyme that has endosulfatase activity and catalyzes the removal of 6-O-sulfate groups on glucosamine from subregions of the glycosaminoglycans heparin sulfate. The action of Sulf2 has been shown to alter the binding of protein ligands to heparin which is involved in the binding of growth factors such as VEGF, FGF-2 and TGF to cellular receptors. Studies in experimental models have shown that fully active Sulf2 is important in the development of breast cancer and pancreatic cancer. Several tumors have been shown to be enriched in Sulf2 in comparison to the normal tissues where its level is either absent or present at very low levels. Sulf2 acts as a potentiator for the Wnt signaling and this is considered important in cancer stem cell growth. We have shown that PBN (alpha-phenyl-tert-butylnitrone) has anti-cancer activity in three experimental models, A) the choline deficiency induced liver cancer model, B) the APCmin model of colon cancer and C) the rat C6 glioma model. We have also shown that 2,4-disulfonyl-alpha-phenyl-tert-butylnitrone (OKN007) has potent anti-cancer activity in the rat C6 glioma model. We considered based on the presence of phenyl sulfonyl groups that the nitrone 2,4-disulfonyl-alpha-phenyl-tert-butylnitrone (OKN007) may act as a competitive inhibitor of Sulf2. We have found that Sulf2 is secreted into the media of Sulf2 transfected Hek-293 cells as well as several cancer cells, including from the highest to lower levels; MCF-7 breast cancer cells, Hek-293 cells, C6 glioma cells and BxPc-3 pancreatic cancer cells. Utilizing the concentrated media of these cells we have shown that Sulf2 has aryl sulfatase activity against the sulfate ester of 4-methylumbelliferone. We have shown that phenyl sulfonyl compounds suppress Sulf2 activity in an apparent competitive action. For instance, we found that the polysulfonated compound suramin potently suppressed the Sulf2 aryl sulfatase activity and that 2,4-disulfonylphenyl-tert-butylnitrone inhibited Sulf2 activity but less so than suramin. Utilizing concentrated extracellular media of several human cancer cell lines we have shown that Sulf2 activity is decreased by incubation with H2O2. Our results suggest that the anti-cancer activity of OKN007 may in part be explained by its inhibitory action against Sulf1. Research to test this concept is now underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2267.

Abstract 3574: Study of the anti-glioma properties of the nitrone OKN007 by magnetic resonance imaging

Abstract The nitrone compound OKN007, a disulfonated derivative of phenyl-tert-butylnitrone, is known to have anti-glioma properties in a rat C6 glioma model. In this study, the effect of OKN007 was studied using three orthotopically implanted glioma cell lines in the rat. In addition, some preliminary data investigating the involvement of the iNOS anti-inflammatory pathway in the anti-glioma activity of OKN007 is provided. The effect OKN007 on glioma was assessed by magnetic resonance imaging of rats orthotopically implanted with 10^6 C6, F98 or RG2 cells. Tumor volumes and growth rates were determined with T2-weighted imaging. The inflammatory edema and other properties of the glioma were assessed with diffusion-weighted imaging. Finally, tumor perfusion rates were measured by arterial spin labeling. The drug (25-30μmol/(kg·day)) was administered 15 days after tumor cell implantation in the drinking water or by a continuous intravenous perfusion, with appropriate saline controls. The expression levels of iNOS were measured using western blots. In addition, C6 cells stably transfected with an iNOS shRNA silencing plasmid were also implanted in an effort to delineate the involvement of this mediator in tumor growth. OKN007 was found to have an effect on the growth of C6 gliomas both when administered in the drinking water or intravenously. Doubling times were slower when OKN007 was administered orally (7.9±1.2 days, versus 2.7±0.3 days for the controls), but not when administered intravenously, although the latter route was tested on a smaller cohort. The drug affected the perfusion and diffusion profiles of the C6 tumors for both types of administration. In the C6 model, administration of the drug brought perfusion and diffusion values closer to those found in normal brain. Both administration routes had an effect on the tumor volume at day 35. In the RG2 model, OKN007 increased the doubling times of the tumors, although more controls are required to reach statistical significance. The effects of OKN007 on the F98 glioma cell line are currently under investigation. Western blot analyses on the C6 model revealed that in treated animals the iNOS levels, although higher than in the normal brain, were lower than in non treated tumors. Finally, preliminary evidence of the requirement of iNOS for tumor development in the context of orthotopic implantation of the tumor cells was obtained using the iNOS shRNA-transfected C6 cell line. In conclusion, OKN007 was found to have a suppressive effect on the growth of C6 rat glioma, with drinking water administration yielding the best results. The nitrone may also have a therapeutic potential for RG2 and F98 models. The precise mechanisms of action of OKN007 are still unknown, but may involve an interference with iNOS-mediated inflammation. Together with the established safety of this nitrone for humans, our results indicate that OKN007 may be a promising anti glioma agent for clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3574.

Multiparametric assessment of the anti‐glioma properties of OKN007 by magnetic resonance imaging

To demonstrate that OKN007, a disulfonyl derivative of phenyl-tert-butyl nitrone (PBN), has anti-glioma activity in the clinically relevant C6 rat glioma model using multi-parametric magnetic resonance imaging. Twenty-one rats were intracerebrally implanted with C6 cells and administered OKN007 or kept as controls. Animals were monitored with MRI at 7 Tesla (T), using morphologic, diffusion-weighted and perfusion imaging, followed by histology and Western blots of angiogenesis and inflammatory markers. OKN007 was found to decrease tumor volumes and increase survival. The glioma tissues of OKN007-treated rats were found to have longitudinal apparent diffusion coefficients (ADC(z)) of 0.76 +/- 0.06 x 10(-3) mm(2)/s, similar to the contralateral tissue and significantly smaller than untreated gliomas (0.97 +/- 0.13 x 10(-3) mm(2)/s). They had higher perfusion rates (66 +/- 4 mL/100 g.min) than untreated gliomas (26 +/- 7 mL/100 g.min). All examined molecular markers were decreased in OKN007-treated rat gliomas, compared with elevated levels in untreated rats. MRI assessment was successfully used to monitor a decrease in tumor growth, and corresponding alterations in ADC and perfusion rates in rat C6 gliomas treated with the anti-glioma agent, OKN007.

Endothelial-monocyte-activating polypeptide II increases blood–tumor barrier permeability by down-regulating the expression levels of tight junction associated proteins

This study was performed to determine whether endothelial-monocyte-activating polypeptide (EMAP) II increases the permeability of the blood–tumor barrier (BTB) in the rat model of C6 glioma, and whether EMAP II opens the BTB by affecting tight junction (TJ) associated proteins zonula occluden-1 (ZO-1), occludin and claudin-5. The rats were divided into eight groups randomly: control group, EMAPII 0 h group, EMAPII 0.5 h group, EMAPII 1 h group, EMAPII 2 h group, EMAPII 3 h group, EMAPII 6 h group and EMAPII 12 h group. The BTB permeability was assessed by Evans blue extravasation. The mRNA and protein expressions of ZO-1, occludin, and claudin-5 were determined by reverse transcriptase–polymerase chain reaction, western blot, and immunohistochemistry assays. The BTB permeability significantly increased after EMAP II injection in different doses (40 ng/kg, 80 ng/kg and 160 ng/kg). The BTB permeability started to increase from 0.5 h, reached a peak at 1 h, and finally returned to the level of EMAP II 0 h group after EMAP II injection at dose of 80 ng/kg. The mRNA and protein expression levels of ZO-1, occludin and claudin-5 were significantly decreased after EMAP II injection. This study demonstrates for the first time that EMAP II increases the permeability of BTB selectively, and the possible mechanism is associated with the down-regulation of ZO-1, occludin and claudin-5.

The effects of tibolone on the human primary glioblastoma multiforme cell culture and the rat C6 glioma model

Objectives: In the light of recent advances in tumor biology and genetics, we hypothesized that tibolone, an estrogen receptor agonist, may have antiproliferative effects on primary human glioblastoma cells and rat C6 malignant glioma cell lines. We thought that tibolone should exert its antiproliferative effects by augmenting glial cell differentiation through the naive, nonhypermethylated estrogen receptors in the glioma cells.Methods: Human primary glioblastoma multiforme (GBM) cells were acquired perioperatively from ten patients aged between 45 and 69 years, diagnosed clinically and radiologically with GBM. The diagnosis was confirmed using immunohistochemical assays. Human GBM and rat C6 malignant glioma cells were cultivated in vitro to obtain monolayer cell cultures. Tibolone was then applied to these cultures in wells, each containing 500,000 tumor cells.Results: Tibolone significantly decreased the number of human GBM cells at the concentrations of 10 and 100 mg/ml. For tibolone, a strong dose-dependent correlation in tumor inhibition was found (p=0.001). This antiproliferative effect of tibolone in human GBM cells was not observed in rat C6 malignant glioma cells. Tibolone demonstrated differential effects on human GBM and rat C6 glioma cells.Discussion: In vitro antiproliferative effects of tibolone on human GBM need to be investigated further in in vivo works.

Synergistic effect of low-frequency ultrasound and low-dose bradykinin on increasing permeability of the blood-tumor barrier by opening tight junction

Low-frequency ultrasound (LFU) and bradykinin (BK) have been shown separately to increase the permeability of the blood-tumor barrier (BTB) in the rat model of C6 glioma. This study examined the hypothesis that the combination of LFU and low-dose BK has a synergistic effect on increasing the permeability of BTB and explored the possible underlying mechanism including the involvement of tight junction (TJ). The rats were divided into six groups: control group, LFU group, BK group, 2/3LFU + 1/2BK group, 5/6LFU + 2/3BK group, and LFU + BK group. The BTB permeability was assessed by Evans blue extravasation. The mRNA and protein expressions of TJ-related proteins ZO-1, occludin, and claudin-5 were determined by reverse transcriptase-polymerase chain reaction, immunohistochemistry, immunolocalization, and Western blot test. BTB permeability increased in all the experimental groups, accompanied by opening of local TJ of the BTB, observed by transmission electron microscopy, and decreased mRNA and protein expressions of ZO-1, occludin, and claudin-5. In addition, there was a further increase in BTB permeability and a further reduction in the expressions of TJ-related proteins in 5/6LFU + 2/3BK and LFU + BK groups, compared with LFU or BK group. These results indicate that LFU and low-dose BK applied in combination act in a synergistic manner to increase BTB permeability. The down-regulation of TJ-related proteins ZO-1, occludin, and claudin-5 may be one of the underlying mechanisms of the increase in BTB permeability induced by LFU and BK.

Effect of trans sodium crocetinate on brain tumor oxgenation

Glioblastoma multiforme tumors typically exhibit regions of hypoxia. Hypoxic regions within the tumor make cells less sensitive to radiosurgery and radiation therapy. Trans sodium crocetinate (TSC) has been shown to be a radiosensitizer. The goal of this research was to elucidate the underlying mechanism of TSC's radiosensitizing effect. A rat C6 glioma model was used. The C6 glioma cells were stereotactically injected into the rat brain to create a tumor. Two weeks later, MR imaging was used to confirm the presence of a glioma. Following demonstration on MR imaging of a brain tumor, animals were randomized into 1 of 2 groups: 1) TSC alone (100 microg/kg), or 2) saline control. Licox probes were inserted into the brain tumor and contralateral cerebral hemisphere. Tissue oxygenation measurements were recorded before and after intravenous infusion of either TSC or saline. Not surprisingly, tissue oxygenation measurements revealed that the brain tumor was hypoxic relative to the contralateral cerebral hemisphere brain tissue. Two to 8 minutes after TSC was infused, tissue oxygenation measurements in the brain tumor increased above baseline by as much as 60%. After this temporary elevation following TSC infusion, tumor oxygenation measurements returned to baseline. No significant elevations in tissue oxygenation were seen on the contralateral side. Similarly, the saline vehicle was not observed to increase tissue oxygenation in either the brain tumor or the contralateral brain tissue. Administration of TSC transiently improves tissue oxygenation in hypoxic gliomas. Such an effect is one potential mechanism for the radiosensitization previously observed after addition of TSC.

Mechanisms of the increase in the permeability of the blood–tumor barrier obtained by combining low-frequency ultrasound irradiation with small-dose bradykinin

The research was conducted to study the characteristics of the noninvasive, reversible, targeted opening of the blood-brain barrier (BBB) by use of low-frequency ultrasound (LFU) irradiation and the selective opening of the blood-tumor barrier (BTB) by intracarotid infusion of bradykinin (BK) in small-dose, with the objective of exploring maximum opening of the BTB by combining LFU irradiation with BK infusion. Thus, it provides new therapeutic strategies for targeted transport of macromolecular or granular drugs to the brain. By using the rat C6 glioma model it was shown that extravasation of Evans blue (EB) through the BTB was significantly increased by combining LFU irradiation (frequency = 1.0 MHz, power = 12 mW, duration = 20 s) with intracarotid small-dose BK infusion, compared with utilizing the two methods separately. By transmission electron microscopy (TEM) we observed that this combination significantly increased the number of pinocytotic vesicles of brain microvascular endothelial cells (BMECs) in the BTB. An even more significant increase was observed by using RT-PCR, western blot, immunohistochemistry, and immunofluorescence to detect mRNA and changes of expression of the caveolae structure proteins caveolin-1 and caveolin-2 of BMECs. In summary, this research concludes that LFU irradiation and small-dose BK together selectively enhance the permeability of the BTB and increase the number of pinocytic vesicles of BMECs to a maximum. Significant up-regulation of the level of expression of caveolae structure proteins caveolin-1 and caveolin-2 might be the molecular mechanism of the co-enhanced endocytotic transport by BMECs. Thus, this research provides new therapeutic strategies for targeted transport of macromolecular drugs and the design of drugs.

Cerebral Ischemia Promotes Rich Pseudopalisading Necrosis in the Rat C6 Glioblastoma Model

The effect of hypoxia on glioma growth including pathological changes was investigated in an experimental model of brain ischemia in the rat C6 glioma model. C6 glioma cells were inoculated into the subcortex of adult Wistar rats. Focal cerebral ischemia near the implanted glioma area was induced by permanent middle cerebral artery occlusion (PMCAO). Ten days later, the rats were sacrificed to compare tumor volume of C6 glioma without PMCAO (control group) versus C6 glioma with PMCAO (hypoxia group). The histological features were also observed. The mean tumor volume in the hypoxia group was significantly larger than that in the control group. The most prominent histological finding in the hypoxia group was abundant formation of pseudopalisading around the necrotic areas. Immunohistological examinations showed intensive staining for vascular endothelial growth factor and hypoxia-inducible factor in these pseudopalisading cells. These findings suggest that cerebral ischemia positively modulates glioma mass growth by the formation of pseudopalisading necrosis, a characteristic histological finding of glioblastoma.

In vivo 19F MRI and 19F MRS of 19F-labelled boronophenylalanine–fructose complex on a C6 rat glioma model to optimize boron neutron capture therapy (BNCT)

Boron neutron capture therapy (BNCT) is a promising binary modality used to treat malignant brain gliomas. To optimize BNCT effectiveness a non-invasive method is needed to monitor the spatial distribution of BNCT carriers in order to estimate the optimal timing for neutron irradiation. In this study, in vivo spatial distribution mapping and pharmacokinetics evaluation of the 19F-labelled boronophenylalanine (BPA) were performed using 19F magnetic resonance imaging (19F MRI) and 19F magnetic resonance spectroscopy (19F MRS). Characteristic uptake of 19F–BPA in C6 glioma showed a maximum at 2.5 h after compound infusion as confirmed by both 19F images and 19F spectra acquired on blood samples collected at different times after infusion. This study shows the ability of 19F MRI to selectively map the bio-distribution of 19F–BPA in a C6 rat glioma model, as well as providing a useful method to perform pharmacokinetics of BNCT carriers.

L-DOPA Preloading Increases the Uptake of Borophenylalanine in C6 Glioma Rat Model: A New Strategy to Improve BNCT Efficacy

Boron neutron capture therapy (BNCT) is a radiotherapeutic modality based on (10)B(n,alpha)(7)Li reaction, for the treatment of malignant gliomas. One of the main limitations for BNCT effectiveness is the insufficient intake of (10)B nuclei in the tumor cells. This work was aimed at investigating the use of L-DOPA as a putative enhancer for (10)B-drug 4-dihydroxy-borylphenylalanine (BPA) uptake in the C6-glioma model. The investigation was first performed in vitro and then extended to the animal model. BPA accumulation in C6-glioma cells was assessed using radiowave dielectric spectroscopy, with and without L-DOPA preloading. Two L-DOPA incubation times (2 and 4 hours) were investigated, and the corresponding effects on BPA accumulation were quantified. C6-glioma cells were also implanted in the brain of 32 rats, and tumor growth was monitored by magnetic resonance imaging. Rats were assigned to two experimental branches: (1) BPA administration; (2) BPA administration after pretreatment with L-DOPA. All animals were sacrificed, and assessments of BPA concentrations in tumor tissue, normal brain, and blood samples were performed using high-performance liquid chromatography. L-DOPA preloading induced a massive increase of BPA concentration in C6-glioma cells only after a 4-hour incubation. In the animal model, L-DOPA pretreatment produced a significantly higher accumulation of BPA in tumor tissue but not in normal brain and blood samples. This study suggests the potential use of L-DOPA as enhancer for BPA accumulation in malignant gliomas eligible for BNCT. L-DOPA preloading effect is discussed in terms of membrane transport mechanisms.

Investigating effect of fusion gene therapy by MR diffusion-weighted imaging in a rat C6 glioma model

Objective To evaluate the use of diffusion-weighted imaging(DWI)for early detection of tumor response to Angiostatin-Endostatin(Statin-AE)fusion gene therapy in a rat C6 glioma model.Methods Fifty male wistar rats with C6 tumor cells implanted into the striatum were examined by a 3.0T MR scanner,then the rats beating tmors were divided into two groups,treatment group and control group.Rats in the treatment group received 107 plaque forming unit(pfu)recombinant herps simplex viral (R-HSV)mediated Statin-AE fusion gene therapy on day 7,and then the tumors were conformed on MRI.Conventional MR and DWI examination were acquired on 1,2,3 weeks after implantation with a 5-inch surface coil.Two(1 w),eight(2 w)and all the residual rats(3 w)of each group were sacrificed to perform the histopathological examination after each MBI examination.Pretreatment and post treatment tumor volulnes and apparent diffusion coefficient(ADC)values were calculated.Rank sum test and t test were employed for statistical analysis.Results On MRI,43 rats demonstrated tumors on day 7 with a successful rate of 86%,On week 2,the tumor volumes of the controh and treatment group were 90.6 and 91.64 mm3,with no significant difference(Z=-0.14,P＞0.05).On week 3,the tumor volumes of the controls and treatment group were 156.64 and 29.64 mm3,and a significant difference was observed(Z=-3.45,P＜0.01).On week 2.the ADC values of the tumor centers of the treatment group and the control group were (1.20±0.25)×10-3 and(0.99±0.08)×10-3 mm2/s,and the values of the tumor peripheral parts of the two groups were(1.00±0.25)×10-3 and(0.83±0.12)×10-3mm2/s,the ADC values of both tumor centers and peripheral parts of the treatment group were significantly higher than those of the control group (t=-0.82 and-0.46,P＜0.05).On week 3,the ADC values of the tumor centers of the treatment group and the control group were(0.92±0.21)× 10-3 and(0.99±0.09)×10-3mm2/s,and the values of the tumor peripheral parts of the two groups were(0.81±0.19)×10-3 and(0.78±0.11)×10-3 mm2/a,there were no statisfical difference between the two groups(t=0.82,and-0.46,P＜0.05).HE stained slices showed more prominent tumor interstifial edenla.swelling and death of tumor cells in the treated rats than the controls.Conclusions Combination of conventional MRI and DWI can be powerful to monitor tumor progression and therapy effecL Conventional MRI showed that the therapy slow the tumor progression in size while DWI demonstrated the tumor response even earlier than size change.DWI has potential use forthe detection of early response to antiangiogenic gene therapy. Key words: Rats; Glioma; Diffusion magnetic resonance imaging

Targeted Transport of 125I-Labeled Antibody to GFAP and AMVB1 in an Experimental Rat Model of C6 Glioma

Glioblastoma is the most common high-grade glioma characterized by strikingly poor therapeutic outcome with survival time of about a year. This makes a search for new therapeutic approaches to glioblastoma treatment an area of great clinical importance. The present study aims to explore the potential of targeted delivery of 125I-radiolabeled antibodies, specific to glial fibrillary acidic protein (GFAP) and AMVB1 (antigen of abluminal membrane of endotheliocytes predominantly expressed in glioblastoma microvessels) as a strategy for in vivo tumor targeting. Rat C6 glioma model was used to test this hypothesis. Tumor bearing animals, injected with radiolabeled monoclonal antibodies to GFAP or AMVB1, were compared to control group, which received nonspecific mouse IgG. Radioactivity of blood, brain hemispheres, and some other tissues was measured 6, 24, 48, 72, and 96 h posttreatment. Our results demonstrate accumulation of both types of antibodies in tumors. Concentrations of both antibodies were significantly increased in tumor-bearing hemisphere compared to intact hemisphere. Antibodies to GFAP specifically accumulated in brain and bound tumor tissue with the high affinity. In contrast, increased accumulation of anti-AMVB1 antibody was detected in antigen-expressing organs, such as spleen and kidney. Based on results presented, we propose that the monoclonal antibodies to GFAP can be used as vectors for the delivery of diagnostic and pharmacological agents to high-grade gliomas. Development of this strategy would open new clinical perspectives for glioblastoma diagnostics and therapy.