Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet... more Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation.
The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the... more The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T 1 /T 2 -weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.
The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the... more The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T 1 /T 2 -weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.
Brain dysfunction is frequently observed in sepsis as a consequence of changes in cerebral struct... more Brain dysfunction is frequently observed in sepsis as a consequence of changes in cerebral structure and metabolism, resulting in worse outcome and reduced life-quality of surviving patients. However, the mechanisms of sepsis-associated encephalopathy development and a better characterization of this syndrome in vivo are lacking. Here, we used magnetic resonance imaging (MRI) techniques to assess brain morphology and metabolism in a murine sepsis model (cecal ligation and puncture, CLP). Sham-operated and CLP mice were subjected to a complete MRI session at baseline, 6 and 24 h after surgery. Accumulation of vasogenic edematic fluid at the base of the brain was observed in T 2 -weighted image at 6 and 24 h after CLP. Also, the water apparent diffusion coefficients in both hippocampus and cortex were decreased, suggesting a cytotoxic edema in brains of nonsurvival septic animals. Moreover, the N-acetylaspartate/choline ratio was reduced in brains of septic mice, indicating neuronal damage. In conclusion, noninvasive assessment by MRI allowed the identification of new aspects of brain damage in sepsis, including cytotoxic and vasogenic edema as well as neuronal damage. These findings highlight the potential applications of MRI techniques for the diagnostic and therapeutic studies in sepsis.
MRS Nitrone PBN Tumor metabolism Lipid Anti-glioma therapy Rodent glioma models (C6, RG2, GL261) ... more MRS Nitrone PBN Tumor metabolism Lipid Anti-glioma therapy Rodent glioma models (C6, RG2, GL261) Free radicals Gliomas, the most common primary brain tumors in adults, have a poor outcome. PBN (α-phenyl-tertbutylnitrone) and OKN007 (2,4-disulfophenyl-PBN) are nitrones that have demonstrated beneficial effects in many aging diseases. In this study, we evaluated the anti-tumor effects of PBN and OKN007 in several rodent glioma models (C6, RG2, and GL261) by assessing metabolite alterations with magnetic resonance spectroscopy (MRS). PBN or OKN007 was administered in drinking water before or after tumor formation. MR imaging and single-voxel point-resolved spectroscopy were done to assess tumor morphology and metabolites, after therapy. Major metabolite ratios (choline, N-acetylaspartate, and lipid (methylene or methyl), all compared to creatine), as well as quantification of individual metabolite concentrations, were assessed. Nitrones induced tumor metabolism changes that resulted in restoring major metabolite ratios close to their normal levels, in the glioma regression phase. Nitrone treatment decreased the lipid (methylene)-tocreatine ratio, as well as the estimated concentration of lipid (methylene) significantly. Alterations in lipids can be a useful marker for the evaluation of the efficacy associated with treatment and were found in this study to be related to the reduction of necrosis, but not apoptosis. OKN007 was more effective than PBN when administered after tumor formation in the C6 glioma model. In conclusion, 1 H MRS and conventional MRI are useful methods to assess and follow the response of varied glioma models to anti-tumor treatments.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2010
We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis ... more We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis model. Mice containing double mutations (DM) in c-myc and TGF-α (transforming growth factor-α), leading to liver neoplasia, were crossed with mice containing omega-3 desaturase. MRI analysis of triple mutant (TM) mice showed the absence of neoplasia at all time points for 92% of mice in the study. Pathological changes of TM (TGFα/c-myc/fat-1) mouse liver tissue was similar to control mouse liver tissue. Magnetic resonance spectroscopy (MRS) measurements of unsaturated fatty acids found a significant difference (p<0.005) between DM and TM transgenic (Tg) mice at 34 and 40 weeks of age. HPLC analysis of mouse liver tissue revealed markedly decreased levels of omega-6 fatty acids in TM mice when compared to DM (TGFα/c-myc) and control (CD1) mice. Mass spectrometry (MS) analysis indicated significantly decreased 16:0/20:4 and 18:1/20:4 and elevated 16:0/22:6 fatty acyl groups in both GPCho and GPEtn, and elevated 16:0/20:5, 18:0/18:2, 18:0/18:1 and 18:0/22:6 in GPCho, within TM mice compared to DM mice. Total fatty acid analysis indicated a significant decrease in 18:1n9 in TM mice compared to DM mice. Western blot analysis of liver tissue showed a significant (p<0.05) decrease in NF-κB (nuclear factor-κB) levels at 40 weeks of age in TM mice compared to DM mice. Microarray analysis of TM versus DM mice livers at 40 weeks revealed alterations in genes involved in cell cycle regulation, cell-to-cell signaling, p53 signaling, and arachidonic acid (20:4) metabolism. Endogenous omega-3 fatty acids were found to prevent HCC development in mice.
Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1... more Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.
We report a case of Parry-Romberg syndrome in a 32-year-old woman presenting with intermittent he... more We report a case of Parry-Romberg syndrome in a 32-year-old woman presenting with intermittent headache and mild sensory disturbance. MR imaging revealed minimal asymmetric atrophy of the right hemisphere with a few nonspecific white matter hyperintensities. Diffusion tensor imaging and fiber tractography, however, demonstrated clear fiber derangement, especially in the sensory tract of the right cerebral white matter.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2005
Liver cancer is one of the most common cancers worldwide. Altered lipid metabolism in the liver i... more Liver cancer is one of the most common cancers worldwide. Altered lipid metabolism in the liver is a key feature of developing liver nodules and tumors. Methods of analysis vary from the most sophisticated chromatography to the in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, we present a systematic method for the identification and quantitation of signature signals from lipid metabolites using 1D NMR proton spectroscopy. We assessed lipid metabolites in an epigenetic rat hepatocarcinogenesis model induced by treatment with a choline-deficient diet (CDAA, choline-deficient l-amino acid defined) over a period of 1 year, from the formation of steatosis, to the development of nodules and adenomas. A comparable choline-sufficient (CSAA) diet was used for the controls. The resonances of the methylene protons of the glycerol backbone in phospholipids were used to quantify the total concentration of such compounds. CDAA rat livers were found to have significantly higher levels of phospholipids, when compared to CSAA, throughout the entire carcinogenesis period. The tri-methyl protons of choline compounds serves to quantify total choline, and the vinyl and bis-allyl proton resonances can be used to not only quantify fatty acid concentrations but also to probe the number of double bonds in a fatty acid moiety. Early stages of carcinogenesis indicate a lower degree of double bonds in fatty acyl containing compounds in CDAA rat livers, when compared to CSAA. The results of this study are in agreement with those previously published in the literature on other rat hepatocarcinogenesis models.
Increased iNOS expression is often found in brain tumors, such as gliomas. The goal of this study... more Increased iNOS expression is often found in brain tumors, such as gliomas. The goal of this study was to develop and assess a novel molecular MRI (mMRI) probe for in vivo detection of iNOS in rodent models for gliomas (intracerebral implantation of rat C6 or RG2 cells or ethyl nitrosourea-induced glioma). The probe we used incorporated a Gd-DTPA (gadolinium(III) complex of diethylenetriamine-N,N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-pentaacetate) backbone with albumin and biotin moieties and covalent binding of an anti-iNOS antibody (Ab) to albumin (anti-iNOS probe). We used mMRI with the anti-iNOS probe to detect in vivo iNOS levels in gliomas. Nonimmune normal rat IgG coupled to albumin-Gd-DTPA-biotin was used as a control nonspecific contrast agent. By targeting the biotin component of the anti-iNOS probe with streptavidin Cy3, fluorescence imaging confirmed the specificity of the probe for iNOS in glioma tissue. iNOS levels in glioma tumors were also confirmed via Western blots and immunohistochemistry. The presence of plasma membrane-associated iNOS in glioma cells was established by transmission electron microscopy and gold-labeled anti-iNOS Ab. The more aggressive RG2 glioma was not found to have higher levels of iNOS compared to C6. Differences in glioma vascularization and blood-brain barrier permeability between the C6 and the RG2 gliomas are discussed. In vivo assessment of iNOS levels associated with tumor development is quite feasible in heterogeneous tissues with mMRI.
This study focused on the detection of apoptosis related events in very early phases of choline-d... more This study focused on the detection of apoptosis related events in very early phases of choline-deficient (CD)-induced hepatocarcinogenesis (at 2-5 weeks). Flow cytometry of isolated intact primary hepatocytes from CD diet fed rats indicated increased expression of the apoptosis-associated protein Fas. Increased apoptosis in CD-treated livers was confirmed by Western blot analyses of caspases and cytochrome c. This study was also able to detect differences in apoptotic events following phenyl butyl nitrone (PBN) treatment. Fas expression was inhibited by PBN, indicating that PBN is anti-apoptotic. It is speculated that in the early stages of CD-induced hepatotoxicity, PBN is involved in inhibiting pro-inflammatory factor-driven apoptosis of normal hepatocytes, which protects against the initiation of carcinogenesis. The CD diet model is also considered as a model for non-alcoholic steatohepatitis (NASH) in humans and early expression of Fas could also be a good index of the progression of NASH.
Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet... more Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation.
Current detection methods (computed tomography, ultrasound, and MRI) for hepatocarcinogenesis in ... more Current detection methods (computed tomography, ultrasound, and MRI) for hepatocarcinogenesis in humans rely on visual confirmation of neoplastic formations. A more effective early detection method is needed. Using in vivo magnetic resonance spectroscopy (MRS), we show that alterations in the integral ratios of the bis-allyl to vinyl hydrogen protons in unsaturated lipid fatty acyl groups correlate with the development of neoplastic formations in vivo in a TGFalpha/c-myc mouse hepatocellular carcinoma (HCC) model. HPLC analysis of the TGFalpha/c-myc mice liver tissue revealed a significant increase in the amount of oleic acid, along with alterations in linoleic and gamma-linolenic acids, as compared with control CD1 mice. Electrospray ionization tandem mass spectrometry analysis indicated a significant increase in the abundance of specific glycerol phosphatidylcholine (GPCho) lipids containing palmitic and oleic acids between control CD1 and TGFalpha/c-myc mice liver tissue extracts. Western blot analysis of the mice liver tissue indicates alterations in the desaturase enzyme stearoyl CoA desaturase (SCD)1, responsible for palmitic and oleic acid formation. Microarray analysis detected alterations in several genes involved with fatty acid metabolism, particularly SCD2, in transgenic mouse liver tissue. In correlation with the HPLC, mass spectrometry, Western blot, and microarray analyses, we are able to confirm the ability of in vivo MRS to detect precancerous lesions in the mouse liver before visual neoplastic formations were detectable by MRI.
To apply fiber tractography to assess the effect of a possible antiglioma drug, phenyl N-tert-but... more To apply fiber tractography to assess the effect of a possible antiglioma drug, phenyl N-tert-butyl nitrone (PBN), on glioma-affected neuronal fibers. The fiber tractography method was able to differentiate between different tumor types, such as the C6 and F98 rat glioma models.
Angiogenesis is essential to tumor progression and a precise evaluation of angiogenesis is import... more Angiogenesis is essential to tumor progression and a precise evaluation of angiogenesis is important for tumor early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumor angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory system in angiogenesis and have been used as hot targets for radionuclide-based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti-VEGFR2 monoclonal antibody, shown by varied increases in T 1 signal intensity during a twohour period, demonstrated the heterogeneous expression of VEGFR2 in different tumor regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin-Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using MR angiography (MRA), which quantified tumor blood volume and provided a macroscopic view and a dynamic change of the correlation between tumor vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumor progression.
Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-spe... more Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-specific epsin deficiency abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting in dysfunctional tumor vasculature. Here, we designed a tumor endothelium–targeting chimeric peptide (UPI) for the purpose of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2. We determined that the UPI peptide specifically targets tumor endothelial VEGFR2 through an unconventional binding mechanism that is driven by unique residues present only in the epsin ubiquitin–interacting motif (UIM) and the VEGFR2 kinase domain. In murine models of neoangiogenesis, UPI peptide increased VEGF-driven angiogenesis and neovascularization but spared quiescent vascular beds. Further, in tumor-bearing mice, UPI peptide markedly impaired functional tumor angiogenesis, tumor growth, and metastasis, resulting in a notable increase in survival. Coadministration of UPI peptide with cytotoxic chemotherapeutics further sustained tumor inhibition. Equipped with localized tumor endothelium–specific targeting, our UPI peptide provides potential for an effective and alternative cancer therapy.
Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer ... more Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immuno-spin trapping (IST) methodologies. Free radicals are trapped with the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immuno-spin trapping by an antibody against DMPO-adducts. In this study, we combined mMRI with a biotin-Gd-DTPA-albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007 treated rat F98 gliomas. OKN-007 was found to significantly decrease (p<0.05) free radicals levels detected with an anti-DMPO probe in treated animals compared to untreated rats...
American journal of nuclear medicine and molecular imaging, 2015
Angiogenesis is essential to tumor progression, and the precise imaging of the angiogenic marker ... more Angiogenesis is essential to tumor progression, and the precise imaging of the angiogenic marker vascular endothelial growth factor receptor 2 (VEGFR-2) may provide an accurate evaluation for angiogenesis during a therapeutic response. With the use of molecular magnetic resonance imaging (mMRI), an in vitro cell assay indicated significantly decreased T1 relaxation values when tumor endothelial cells (TEC), which positively expressed VEGFR-2 (Western blot), were in the presence of the VEGFR-2 probe compared to TEC alone (P < 0.001). For in vivo mMRI evaluations, we assessed VEGFR-2 levels in untreated and OKN-007-treated GL261 mouse gliomas. Regarding treatment response, OKN-007 was also able to significantly decrease tumor volumes (P < 0.01) and increase survival (P < 0.001) in treated animals. Regarding in vivo detection of VEGFR-2, OKN-007 was found to significantly decrease the amount of VEGFR-2 probe (P < 0.05) compared to an untreated control group. Fluorescence im...
Pediatric glioblastomas (pGBM), although rare, are one of the leading causes of cancer-related de... more Pediatric glioblastomas (pGBM), although rare, are one of the leading causes of cancer-related deaths in children, with tumors essentially refractory to existing treatments. Here, we describe the use of conventional and advanced in vivo magnetic resonance imaging (MRI) techniques to assess a novel orthotopic xenograft pGBM mouse (IC-3752GBM patient-derived culture) model, and to monitor the effects of the anti-cancer agent OKN-007 as an inhibitor of pGBM tumor growth. Immunohistochemistry support data is also presented for cell proliferation and tumor growth signaling. OKN-007 was found to significantly decrease tumor volumes (p<0.05) and increase animal survival (p<0.05) in all OKN-007-treated mice compared to untreated animals. In a responsive cohort of treated animals, OKN-007 was able to significantly decrease tumor volumes (p<0.0001), increase survival (p<0.001), and increase diffusion (p<0.01) and perfusion rates (p<0.05). OKN-007 also significantly reduced lipid tumor metabolism in responsive animals [(Lip1.3 and Lip0.9)-to-cre-atine ratio (p<0.05)], as well as significantly decrease tumor cell proliferation (p<0.05) and microvessel density (p<0.05). Furthermore, in relationship to the PDGFRα pathway, OKN-007 was able to significantly decrease SULF2 (p<0.05) and PDGFR-α (platelet-derived growth factor receptor-α) (p<0.05) immunoexpression, and significantly increase decorin expression (p<0.05) in responsive mice. This study indicates that OKN-007 may be an effective anti-cancer agent for some patients with pGBMs by inhibiting cell proliferation and angiogenesis, possibly via the PDGFRα pathway, and could be considered as an additional therapy for pediatric brain tumor patients., KMF) http://nih. gov/, Oklahoma State University (AE-1-50060; PCS) http://go.okstate.edu/, Musella Foundation (No. number; RAT) http://www.virtualtrials.com/musella. cfm, and Childhood Brain Tumor Foundation (No. number; RAT) http://www.childhoodbraintumor.org/. Competing Interests: The authors have declared that no competing interests exist.
Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet... more Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation.
The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the... more The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T 1 /T 2 -weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.
The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the... more The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T 1 /T 2 -weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.
Brain dysfunction is frequently observed in sepsis as a consequence of changes in cerebral struct... more Brain dysfunction is frequently observed in sepsis as a consequence of changes in cerebral structure and metabolism, resulting in worse outcome and reduced life-quality of surviving patients. However, the mechanisms of sepsis-associated encephalopathy development and a better characterization of this syndrome in vivo are lacking. Here, we used magnetic resonance imaging (MRI) techniques to assess brain morphology and metabolism in a murine sepsis model (cecal ligation and puncture, CLP). Sham-operated and CLP mice were subjected to a complete MRI session at baseline, 6 and 24 h after surgery. Accumulation of vasogenic edematic fluid at the base of the brain was observed in T 2 -weighted image at 6 and 24 h after CLP. Also, the water apparent diffusion coefficients in both hippocampus and cortex were decreased, suggesting a cytotoxic edema in brains of nonsurvival septic animals. Moreover, the N-acetylaspartate/choline ratio was reduced in brains of septic mice, indicating neuronal damage. In conclusion, noninvasive assessment by MRI allowed the identification of new aspects of brain damage in sepsis, including cytotoxic and vasogenic edema as well as neuronal damage. These findings highlight the potential applications of MRI techniques for the diagnostic and therapeutic studies in sepsis.
MRS Nitrone PBN Tumor metabolism Lipid Anti-glioma therapy Rodent glioma models (C6, RG2, GL261) ... more MRS Nitrone PBN Tumor metabolism Lipid Anti-glioma therapy Rodent glioma models (C6, RG2, GL261) Free radicals Gliomas, the most common primary brain tumors in adults, have a poor outcome. PBN (α-phenyl-tertbutylnitrone) and OKN007 (2,4-disulfophenyl-PBN) are nitrones that have demonstrated beneficial effects in many aging diseases. In this study, we evaluated the anti-tumor effects of PBN and OKN007 in several rodent glioma models (C6, RG2, and GL261) by assessing metabolite alterations with magnetic resonance spectroscopy (MRS). PBN or OKN007 was administered in drinking water before or after tumor formation. MR imaging and single-voxel point-resolved spectroscopy were done to assess tumor morphology and metabolites, after therapy. Major metabolite ratios (choline, N-acetylaspartate, and lipid (methylene or methyl), all compared to creatine), as well as quantification of individual metabolite concentrations, were assessed. Nitrones induced tumor metabolism changes that resulted in restoring major metabolite ratios close to their normal levels, in the glioma regression phase. Nitrone treatment decreased the lipid (methylene)-tocreatine ratio, as well as the estimated concentration of lipid (methylene) significantly. Alterations in lipids can be a useful marker for the evaluation of the efficacy associated with treatment and were found in this study to be related to the reduction of necrosis, but not apoptosis. OKN007 was more effective than PBN when administered after tumor formation in the C6 glioma model. In conclusion, 1 H MRS and conventional MRI are useful methods to assess and follow the response of varied glioma models to anti-tumor treatments.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2010
We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis ... more We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis model. Mice containing double mutations (DM) in c-myc and TGF-α (transforming growth factor-α), leading to liver neoplasia, were crossed with mice containing omega-3 desaturase. MRI analysis of triple mutant (TM) mice showed the absence of neoplasia at all time points for 92% of mice in the study. Pathological changes of TM (TGFα/c-myc/fat-1) mouse liver tissue was similar to control mouse liver tissue. Magnetic resonance spectroscopy (MRS) measurements of unsaturated fatty acids found a significant difference (p<0.005) between DM and TM transgenic (Tg) mice at 34 and 40 weeks of age. HPLC analysis of mouse liver tissue revealed markedly decreased levels of omega-6 fatty acids in TM mice when compared to DM (TGFα/c-myc) and control (CD1) mice. Mass spectrometry (MS) analysis indicated significantly decreased 16:0/20:4 and 18:1/20:4 and elevated 16:0/22:6 fatty acyl groups in both GPCho and GPEtn, and elevated 16:0/20:5, 18:0/18:2, 18:0/18:1 and 18:0/22:6 in GPCho, within TM mice compared to DM mice. Total fatty acid analysis indicated a significant decrease in 18:1n9 in TM mice compared to DM mice. Western blot analysis of liver tissue showed a significant (p<0.05) decrease in NF-κB (nuclear factor-κB) levels at 40 weeks of age in TM mice compared to DM mice. Microarray analysis of TM versus DM mice livers at 40 weeks revealed alterations in genes involved in cell cycle regulation, cell-to-cell signaling, p53 signaling, and arachidonic acid (20:4) metabolism. Endogenous omega-3 fatty acids were found to prevent HCC development in mice.
Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1... more Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.
We report a case of Parry-Romberg syndrome in a 32-year-old woman presenting with intermittent he... more We report a case of Parry-Romberg syndrome in a 32-year-old woman presenting with intermittent headache and mild sensory disturbance. MR imaging revealed minimal asymmetric atrophy of the right hemisphere with a few nonspecific white matter hyperintensities. Diffusion tensor imaging and fiber tractography, however, demonstrated clear fiber derangement, especially in the sensory tract of the right cerebral white matter.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2005
Liver cancer is one of the most common cancers worldwide. Altered lipid metabolism in the liver i... more Liver cancer is one of the most common cancers worldwide. Altered lipid metabolism in the liver is a key feature of developing liver nodules and tumors. Methods of analysis vary from the most sophisticated chromatography to the in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, we present a systematic method for the identification and quantitation of signature signals from lipid metabolites using 1D NMR proton spectroscopy. We assessed lipid metabolites in an epigenetic rat hepatocarcinogenesis model induced by treatment with a choline-deficient diet (CDAA, choline-deficient l-amino acid defined) over a period of 1 year, from the formation of steatosis, to the development of nodules and adenomas. A comparable choline-sufficient (CSAA) diet was used for the controls. The resonances of the methylene protons of the glycerol backbone in phospholipids were used to quantify the total concentration of such compounds. CDAA rat livers were found to have significantly higher levels of phospholipids, when compared to CSAA, throughout the entire carcinogenesis period. The tri-methyl protons of choline compounds serves to quantify total choline, and the vinyl and bis-allyl proton resonances can be used to not only quantify fatty acid concentrations but also to probe the number of double bonds in a fatty acid moiety. Early stages of carcinogenesis indicate a lower degree of double bonds in fatty acyl containing compounds in CDAA rat livers, when compared to CSAA. The results of this study are in agreement with those previously published in the literature on other rat hepatocarcinogenesis models.
Increased iNOS expression is often found in brain tumors, such as gliomas. The goal of this study... more Increased iNOS expression is often found in brain tumors, such as gliomas. The goal of this study was to develop and assess a novel molecular MRI (mMRI) probe for in vivo detection of iNOS in rodent models for gliomas (intracerebral implantation of rat C6 or RG2 cells or ethyl nitrosourea-induced glioma). The probe we used incorporated a Gd-DTPA (gadolinium(III) complex of diethylenetriamine-N,N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-pentaacetate) backbone with albumin and biotin moieties and covalent binding of an anti-iNOS antibody (Ab) to albumin (anti-iNOS probe). We used mMRI with the anti-iNOS probe to detect in vivo iNOS levels in gliomas. Nonimmune normal rat IgG coupled to albumin-Gd-DTPA-biotin was used as a control nonspecific contrast agent. By targeting the biotin component of the anti-iNOS probe with streptavidin Cy3, fluorescence imaging confirmed the specificity of the probe for iNOS in glioma tissue. iNOS levels in glioma tumors were also confirmed via Western blots and immunohistochemistry. The presence of plasma membrane-associated iNOS in glioma cells was established by transmission electron microscopy and gold-labeled anti-iNOS Ab. The more aggressive RG2 glioma was not found to have higher levels of iNOS compared to C6. Differences in glioma vascularization and blood-brain barrier permeability between the C6 and the RG2 gliomas are discussed. In vivo assessment of iNOS levels associated with tumor development is quite feasible in heterogeneous tissues with mMRI.
This study focused on the detection of apoptosis related events in very early phases of choline-d... more This study focused on the detection of apoptosis related events in very early phases of choline-deficient (CD)-induced hepatocarcinogenesis (at 2-5 weeks). Flow cytometry of isolated intact primary hepatocytes from CD diet fed rats indicated increased expression of the apoptosis-associated protein Fas. Increased apoptosis in CD-treated livers was confirmed by Western blot analyses of caspases and cytochrome c. This study was also able to detect differences in apoptotic events following phenyl butyl nitrone (PBN) treatment. Fas expression was inhibited by PBN, indicating that PBN is anti-apoptotic. It is speculated that in the early stages of CD-induced hepatotoxicity, PBN is involved in inhibiting pro-inflammatory factor-driven apoptosis of normal hepatocytes, which protects against the initiation of carcinogenesis. The CD diet model is also considered as a model for non-alcoholic steatohepatitis (NASH) in humans and early expression of Fas could also be a good index of the progression of NASH.
Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet... more Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation.
Current detection methods (computed tomography, ultrasound, and MRI) for hepatocarcinogenesis in ... more Current detection methods (computed tomography, ultrasound, and MRI) for hepatocarcinogenesis in humans rely on visual confirmation of neoplastic formations. A more effective early detection method is needed. Using in vivo magnetic resonance spectroscopy (MRS), we show that alterations in the integral ratios of the bis-allyl to vinyl hydrogen protons in unsaturated lipid fatty acyl groups correlate with the development of neoplastic formations in vivo in a TGFalpha/c-myc mouse hepatocellular carcinoma (HCC) model. HPLC analysis of the TGFalpha/c-myc mice liver tissue revealed a significant increase in the amount of oleic acid, along with alterations in linoleic and gamma-linolenic acids, as compared with control CD1 mice. Electrospray ionization tandem mass spectrometry analysis indicated a significant increase in the abundance of specific glycerol phosphatidylcholine (GPCho) lipids containing palmitic and oleic acids between control CD1 and TGFalpha/c-myc mice liver tissue extracts. Western blot analysis of the mice liver tissue indicates alterations in the desaturase enzyme stearoyl CoA desaturase (SCD)1, responsible for palmitic and oleic acid formation. Microarray analysis detected alterations in several genes involved with fatty acid metabolism, particularly SCD2, in transgenic mouse liver tissue. In correlation with the HPLC, mass spectrometry, Western blot, and microarray analyses, we are able to confirm the ability of in vivo MRS to detect precancerous lesions in the mouse liver before visual neoplastic formations were detectable by MRI.
To apply fiber tractography to assess the effect of a possible antiglioma drug, phenyl N-tert-but... more To apply fiber tractography to assess the effect of a possible antiglioma drug, phenyl N-tert-butyl nitrone (PBN), on glioma-affected neuronal fibers. The fiber tractography method was able to differentiate between different tumor types, such as the C6 and F98 rat glioma models.
Angiogenesis is essential to tumor progression and a precise evaluation of angiogenesis is import... more Angiogenesis is essential to tumor progression and a precise evaluation of angiogenesis is important for tumor early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumor angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory system in angiogenesis and have been used as hot targets for radionuclide-based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti-VEGFR2 monoclonal antibody, shown by varied increases in T 1 signal intensity during a twohour period, demonstrated the heterogeneous expression of VEGFR2 in different tumor regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin-Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using MR angiography (MRA), which quantified tumor blood volume and provided a macroscopic view and a dynamic change of the correlation between tumor vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumor progression.
Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-spe... more Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-specific epsin deficiency abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting in dysfunctional tumor vasculature. Here, we designed a tumor endothelium–targeting chimeric peptide (UPI) for the purpose of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2. We determined that the UPI peptide specifically targets tumor endothelial VEGFR2 through an unconventional binding mechanism that is driven by unique residues present only in the epsin ubiquitin–interacting motif (UIM) and the VEGFR2 kinase domain. In murine models of neoangiogenesis, UPI peptide increased VEGF-driven angiogenesis and neovascularization but spared quiescent vascular beds. Further, in tumor-bearing mice, UPI peptide markedly impaired functional tumor angiogenesis, tumor growth, and metastasis, resulting in a notable increase in survival. Coadministration of UPI peptide with cytotoxic chemotherapeutics further sustained tumor inhibition. Equipped with localized tumor endothelium–specific targeting, our UPI peptide provides potential for an effective and alternative cancer therapy.
Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer ... more Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immuno-spin trapping (IST) methodologies. Free radicals are trapped with the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immuno-spin trapping by an antibody against DMPO-adducts. In this study, we combined mMRI with a biotin-Gd-DTPA-albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007 treated rat F98 gliomas. OKN-007 was found to significantly decrease (p<0.05) free radicals levels detected with an anti-DMPO probe in treated animals compared to untreated rats...
American journal of nuclear medicine and molecular imaging, 2015
Angiogenesis is essential to tumor progression, and the precise imaging of the angiogenic marker ... more Angiogenesis is essential to tumor progression, and the precise imaging of the angiogenic marker vascular endothelial growth factor receptor 2 (VEGFR-2) may provide an accurate evaluation for angiogenesis during a therapeutic response. With the use of molecular magnetic resonance imaging (mMRI), an in vitro cell assay indicated significantly decreased T1 relaxation values when tumor endothelial cells (TEC), which positively expressed VEGFR-2 (Western blot), were in the presence of the VEGFR-2 probe compared to TEC alone (P < 0.001). For in vivo mMRI evaluations, we assessed VEGFR-2 levels in untreated and OKN-007-treated GL261 mouse gliomas. Regarding treatment response, OKN-007 was also able to significantly decrease tumor volumes (P < 0.01) and increase survival (P < 0.001) in treated animals. Regarding in vivo detection of VEGFR-2, OKN-007 was found to significantly decrease the amount of VEGFR-2 probe (P < 0.05) compared to an untreated control group. Fluorescence im...
Pediatric glioblastomas (pGBM), although rare, are one of the leading causes of cancer-related de... more Pediatric glioblastomas (pGBM), although rare, are one of the leading causes of cancer-related deaths in children, with tumors essentially refractory to existing treatments. Here, we describe the use of conventional and advanced in vivo magnetic resonance imaging (MRI) techniques to assess a novel orthotopic xenograft pGBM mouse (IC-3752GBM patient-derived culture) model, and to monitor the effects of the anti-cancer agent OKN-007 as an inhibitor of pGBM tumor growth. Immunohistochemistry support data is also presented for cell proliferation and tumor growth signaling. OKN-007 was found to significantly decrease tumor volumes (p<0.05) and increase animal survival (p<0.05) in all OKN-007-treated mice compared to untreated animals. In a responsive cohort of treated animals, OKN-007 was able to significantly decrease tumor volumes (p<0.0001), increase survival (p<0.001), and increase diffusion (p<0.01) and perfusion rates (p<0.05). OKN-007 also significantly reduced lipid tumor metabolism in responsive animals [(Lip1.3 and Lip0.9)-to-cre-atine ratio (p<0.05)], as well as significantly decrease tumor cell proliferation (p<0.05) and microvessel density (p<0.05). Furthermore, in relationship to the PDGFRα pathway, OKN-007 was able to significantly decrease SULF2 (p<0.05) and PDGFR-α (platelet-derived growth factor receptor-α) (p<0.05) immunoexpression, and significantly increase decorin expression (p<0.05) in responsive mice. This study indicates that OKN-007 may be an effective anti-cancer agent for some patients with pGBMs by inhibiting cell proliferation and angiogenesis, possibly via the PDGFRα pathway, and could be considered as an additional therapy for pediatric brain tumor patients., KMF) http://nih. gov/, Oklahoma State University (AE-1-50060; PCS) http://go.okstate.edu/, Musella Foundation (No. number; RAT) http://www.virtualtrials.com/musella. cfm, and Childhood Brain Tumor Foundation (No. number; RAT) http://www.childhoodbraintumor.org/. Competing Interests: The authors have declared that no competing interests exist.
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Papers by Debra Saunders
abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting
in dysfunctional tumor vasculature. Here, we designed a tumor endothelium–targeting chimeric peptide (UPI) for the purpose
of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2. We determined that the
UPI peptide specifically targets tumor endothelial VEGFR2 through an unconventional binding mechanism that is driven
by unique residues present only in the epsin ubiquitin–interacting motif (UIM) and the VEGFR2 kinase domain. In murine
models of neoangiogenesis, UPI peptide increased VEGF-driven angiogenesis and neovascularization but spared quiescent
vascular beds. Further, in tumor-bearing mice, UPI peptide markedly impaired functional tumor angiogenesis, tumor growth,
and metastasis, resulting in a notable increase in survival. Coadministration of UPI peptide with cytotoxic chemotherapeutics
further sustained tumor inhibition. Equipped with localized tumor endothelium–specific targeting, our UPI peptide provides
potential for an effective and alternative cancer therapy.
abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting
in dysfunctional tumor vasculature. Here, we designed a tumor endothelium–targeting chimeric peptide (UPI) for the purpose
of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2. We determined that the
UPI peptide specifically targets tumor endothelial VEGFR2 through an unconventional binding mechanism that is driven
by unique residues present only in the epsin ubiquitin–interacting motif (UIM) and the VEGFR2 kinase domain. In murine
models of neoangiogenesis, UPI peptide increased VEGF-driven angiogenesis and neovascularization but spared quiescent
vascular beds. Further, in tumor-bearing mice, UPI peptide markedly impaired functional tumor angiogenesis, tumor growth,
and metastasis, resulting in a notable increase in survival. Coadministration of UPI peptide with cytotoxic chemotherapeutics
further sustained tumor inhibition. Equipped with localized tumor endothelium–specific targeting, our UPI peptide provides
potential for an effective and alternative cancer therapy.