Effects of PBN and OKN007 in rodent glioma models assessed by 1H MR spectroscopy

Free Radical Biology & Medicine 51 (2011) 490–502

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Effects of PBN and OKN007 in rodent glioma models assessed by 1H MR spectroscopy Ting He a, Sabrina Doblas a, 1, Debra Saunders a, Rebba Casteel a, Megan Lerner b, Jerry W. Ritchey c, Tim Snider c, Robert A. Floyd d, Rheal A. Towner a,⁎ a

Advanced Magnetic Resonance Center, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA d Experimental Therapeutics Research Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA b c

a r t i c l e

i n f o

Article history: Received 23 August 2010 Revised 12 April 2011 Accepted 22 April 2011 Available online 30 April 2011 Keywords: MRS Nitrone PBN Tumor metabolism Lipid Anti-glioma therapy Rodent glioma models (C6, RG2, GL261) Free radicals

a b s t r a c t 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, 1H MRS and conventional MRI are useful methods to assess and follow the response of varied glioma models to anti-tumor treatments. © 2011 Elsevier Inc. All rights reserved.

Gliomas are the most frequent form of adult primary brain tumors. Less than 10% of patients with grade IV glioblastoma multiforme (GBM) survive after 2 years, despite efforts using chemotherapy, radiation therapy, and surgical removal of the tumor [1]. Precise grading of the tumor and evaluation of therapy efficiency would be very important to improve this poor outcome. Glioma grading based on histopathological analysis of tissues from biopsies was shown not to be highly accurate in some cases because of possible sampling errors. Conventional magnetic resonance imaging (MRI) has been used routinely in the clinic to assist in determining important elements of tumor grading and characterization, such as tumor size and position, hemorrhage level, and degree of necrosis [2,3]. However, conventional MRI could lead to false-positive diagnosis of low-grade astrocytoma when applied alone, because of

Abbreviations: Cho, choline; Cr, creatine; GBM, glioblastoma multiforme; HR-MAS, high-resolution magic angle spinning; Lip, lipid; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; NAA, N-acetylaspartate; PBN, α-phenyl-tertbutylnitrone; PCr, phosphocreatine; PRESS, point-resolved spectroscopy; TE, echo time; TR, repetition time; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Advanced Magnetic Resonance Center, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104 U.S.A. Fax: + 1 405 271 7254. E-mail address: [email protected] (R.A. Towner). 1 Current address: INSERM Unit 773, Beaujon Hospital, 92110 Clichy, France. 0891-5849/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2011.04.037

similar imaging features between low-grade astrocytomas and anaplastic astrocytomas or encephalitis [4]. Magnetic resonance spectroscopy (MRS) provides metabolite/ biochemical information about tissues in vivo. 1H MRS analyzes the signal from protons in various chemical environments. The signal intensity of an individual peak is proportional to the number of contributing protons. As a complement to MRI, MRS enables more specific tissue characterization, such as differentiating low-grade gliomas from infarction [5]. The x axis of the MR spectrum represents the differences in proton resonance frequencies of localized metabolites, referred to as the chemical shift, in parts per million (ppm), in reference to a standard compound (e.g., water arbitrarily set at 4.77 ppm). The y axis represents arbitrary units of signal intensity scaled relative to the highest signal intensity [6]. The major metabolites of the 1H spectrum seen in brain tumors are choline (Cho; 3.2 ppm), creatine (Cr; 3.0 ppm), N-acetylaspartate (NAA; 2.0 ppm), and lipids (Lip; 0.9–1.5 ppm). The choline peak is a combination of the total choline content of free choline, phospho-, glycerophospho-, and phospholipid-choline. Cho is involved in the synthesis and degradation of cell membranes, which may be altered in disease conditions, indicating tissue status. Cr consists of the creatine and phosphocreatine (PCr) pool and is associated with brain energy metabolism. NAA, which is generated by neurons, diffuses along

T. He et al. / Free Radical Biology & Medicine 51 (2011) 490–502

axons and is degraded in oligodendrocytes [7,8]. As a normal neuronal marker, NAA is reduced during any neuronal or axonal damage or loss. Membrane lipids are usually undetectable by 1H MRS, because of their decreased mobility and strong dipolar couplings. However, in pathological conditions, the release of fatty acids from membranes and/or lipid droplets increases the concentration of free lipids, which appears as sharp peaks at 1.3 (methylene group) and 0.9 ppm (methyl group) [9]. In brain tumors, the elevation of the lipid levels usually correlates with necrosis. α-Phenyl-tert-butylnitrone (PBN) was first used as a spin trapping agent to elucidate free radical chemistry and was later found to have potential therapeutic properties including antioxidant, neuroprotective, and anti-inflammatory activities in many pathologies such as stroke and Parkinson disease [10]. OKN007 (originally known as NXY059) is a derivative 2,4-disulfophenyl-N-tert-butylnitrone of PBN that was a candidate drug for the therapeutic treatment of acute ischemic stroke but failed to show a significant improvement in the outcome during phase III clinical trials [11–13]. Nakae et al. previously demonstrated the anti-tumor effect of PBN, which significantly decreased the formation of preneoplastic nodules in a rat hepatocellular carcinoma model [14]. Both PBN and OKN007 have been tested for glioma inhibition effects by our group in rodent glioma models. PBN resulted in rat C6 glioma regression, with inhibition of angiogenesis being one of the mechanisms of action [15]. Garteiser et al. assessed the anti-glioma effect of OKN007 in rat C6 gliomas, by assessing alterations in the apparent diffusion coefficient and perfusion rates [16]. In this study, we aimed at evaluating the anti-tumor effects of both PBN and OKN007 in several glioma models by assessing alterations in tumor metabolism with the use of MRS. Materials and methods Intracerebral orthotopic glioma implantation Rat C6 and RG2 glioma cell lines were obtained from the American Type Culture Collection. The murine GL261 cell line was provided by Dr. Safrany of the Frederic Joliot–Curie National Research Institute for Radiobiology and Radiohygiene. All cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The procedure of glioma cell implantation was similar to what was previously published by our group [15]. Briefly, 3-month-old male Fischer 344 rats (250–300 g; Harlan Laboratories, Indianapolis, IN, USA) were anesthetized with 2.5% isoflurane and 0.8 L/min oxygen and immobilized on a stereotaxic unit (Stoelting Co., USA). A hole was drilled through the skull 2 mm lateral and 2 mm anterior to bregma, on the right-hand side. Approximately 10,000 C6 cells in 10 μl cell culture medium and 1% ultralow-gelling-temperature agarose (Sigma) were injected into the cortex, at a 3-mm depth from the dura and at a rate of 2 μl/min. Bone wax was placed into the hole to prevent reflux of the cell suspension. After surgery, the rats were fed a choline-deficient diet to encourage tumor cell growth [17]. Two-month-old C57BL/6 male mice were injected with 2 × 10 4 GL261 cells in 4-μl volume, at 2 mm lateral and 1 mm anterior to bregma, at a 1.5-mm depth and at a rate of 0.6 μl/min. All animal studies followed the protocol approved by the Institutional Animal Care and Use Committee of our institution. The animals were kept under close observation for any sign of neurological or health problems. The animals were euthanized with CO2 at the end time points for MRI and MRS experiments. The animals were imaged at day 5 or 7 after cell implantation and then every 2–5 days for each glioma model. The end or last time points for the animals were defined as follows. Regarding untreated animals, the last time point was when tumors reached a maximum volume at days 17–31 for C6, days 14–26 for RG2, and day 23 for GL261; regarding nonrespondent treated animals, the last time point was when tumors reached a maximum

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volume at days 16–18 for C6, days 11–19 for RG2, and days 23–26 for GL261; and regarding respondent treated animals, these animals were euthanized on days 35–40 for the PBN-C6 rats and days 35–55 for the OKN-C6 rats. PBN and OKN007 treatment PBN (purity of 99%) was synthesized by our group using the method described by Janzen et al. [18], and OKN007 was obtained from Ryss Laboratories (Union City, CA, USA). Both compounds were administered in the drinking water. PBN was given at a concentration of 0.06% w/v (approximately 75 mg/kg body wt/day for rats and 168 mg/kg/day for mice), whereas the concentration of OKN007 was 0.025% (14.3 ± 4.6 mg/kg body wt/day). The treatment was continuously given every day until the end of the study. For prophylactic treatment, PBN was administered 5 days before glioma cell implantation. For posttumor treatment, PBN was given at 14 days after glioma cell implantation when tumors reached volumes of 30– 60 mm 3. OKN007 was administered starting from 13 to 18 days after C6 glioma cell implantation (tumor volumes ranged from 5 to 46 mm 3). Rats receiving normal drinking water were used as controls. The amount of PBN or OKN007 consumed by rats was determined by weighing water bottles each day (rats were singly housed), with no significant deviations in the uptake of either compound observed. MRI and MRS MRI experiments were performed on a Bruker Biospec 7.0-T/30cm horizontal-bore magnet imaging system (Bruker Biospin, Ettlingen, Germany). Animals were restrained by using 1.5–2.5% isoflurane and 0.8 L/min O2 and placed in a 72-mm quadrature volume coil for signal transmission, and a surface coil was used for signal reception. Rat T2-weighted imaging was acquired by using a rapid acquisition with relaxation enhancement sequence with a repetition time (TR) of 3000 ms, an echo time (TE) of 63 ms, 20 transverse 1-mm-thick slices, a field of view of 3.5 × 3.5 cm 2, and an in-plane resolution of 137 × 137 μm 2. Mouse T2-weighted imaging was acquired with a slice thickness of 0.5 mm, a field of view of 2.0 × 2.0 cm 2, and an in-plane resolution of 78 × 78 μm 2. MR spectroscopy was acquired using a PRESS (pointresolved spectroscopy) sequence with a TE of 24 ms, a TR of 2500 ms, 256 averages, and a spectral width of 4006 Hz. Water was suppressed with a variable power RF pulse and optimized relaxation delay suppression scheme. A nonsuppressed MR spectrum was acquired beforehand to maximize signal intensity and decrease peak width. For all cases, the peak width (full width at half maximum) of the water peak was less than 30 Hz after localized shimming, which was conducted by using first- and second-order adjustments in Fastmap. A cubic voxel with sides of 3.0 mm (rat) or 2.0 mm (mouse) was positioned in either the tumor or the contralateral normal brain tissue, while maximizing the amount of tumor tissue present in the voxel at all times. Data processing Tumor volumes were obtained by compiling tumor areas from all slices that contain the tumors, using ImageJ software (NIH; version 1.40 g). The necrotic regions were assessed by calculating each necrotic lesion (determined as dark void regions) area and compiled as necrotic volume. The percentage necrosis was determined using the formula: % necrosis = necrotic volume/total tumor volume. MRS data were first processed using the built-in Bruker TopSpin tool in the Paravision software (PV4.0; Bruker Biospin). The free induction decay data were windowed using a line broadening of 10 Hz and Fourier transformed, and zero- and first-order phase corrections were applied with TopSpin. Using a Mathematica program written by our group (version 6.0; Wolfram Research, Champaign, IL, USA), the

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spectra were then scaled in ppm by calibrating on the water peak (4.78 ppm). The major brain metabolic peaks were identified as NAA at 2.02 ppm, choline and choline-containing compounds at 3.22 ppm, creatine and phosphocreatine (referred to as total Cr) at 3.02 ppm, and mobile lipids (absent from the normal brain tissue), which appeared sharply at 1.3 ppm for the methylene group (−CH2–) and 0.9 ppm for the methyl group (−CH3), in tumor tissue. The peak area, under the peak of interest, was obtained by curve-fitting the spectrum to a Gaussian model. The quantification of variations in metabolite levels were evaluated as peak area ratios: NAA/Cr, Cho/Cr, Lip (methylene, 1.3)/Cr, and Lip (methyl, 0.9)/Cr. The relative changes in metabolite ratios within the glioma tissue were normalized to the contralateral brain tissue spectral data. For estimation of absolute quantitation of 1H MRS metabolites, the totally automatic robust quantitation in NMR (TARQUIN) method (as more fully described by Reynolds et al. [19] and Wilson et al. [20]) was used to quantify metabolite signals of the in vivo 1H MRS data. The TARQUIN algorithm consists of (1) preprocessing (removal of postacquisition residual water by Hankel singular-value decomposition, automatic phasing), (2) basis set simulation (quantum mechanical simulation of 1H NMR signals from small molecules), and (3) the solution of a nonlinear least-squares fitting problem (modeling the experimental data as a linear combination of modified basis signals, and following the adjustment of all segments, the basis set is resimulated and synthesized at the new frequencies) [19,20]. The amplitudes of the metabolite signals are estimated to be expressed in absolute units, by scaling the fitted signal amplitudes to the amplitude of an unsuppressed water resonance (obtained from an additional experiment), in which the concentration of water is known for particular tissue types [19,20]. The TARQUIN program was obtained at http://tarquin.sourceforge.net/. 1H MRS sequence details (TE, pulse sequence, reference offset) and the spectrometer frequency (sampling and transmitter frequencies) are confirmed before peak simulation. Peak metabolite concentration estimates were further normalized to published rat brain total creatine (Cr + PCr) levels of 7.5 mmol/kg wet wt as reported by Cudalbu et al. [21]. Mathematical modeling of tumor growth and therapy response Mathematical modeling using Eq. (1) was used to characterize tumor growth and drug therapy response:
   −t t −t 2 V ðt Þ = Vmax exp − 0 : α β

ð1Þ

In this equation, Vmax is the maximum volume of the tumor (peak amplitude; mm 3), t0 is the time at which the maximum tumor volume occurs (days), t is the number of days monitored after cell implantation, β is a constant that determines the time (days) for reduction of tumor volume and is a measure of treatment efficacy, and α is the estimated time (days) from t0/Vmax until the tumor volume reaches 0. Eq. (1) was obtained by a modification of Eqs. (2) and (3), previously defined by Kirkby et al. [22] and Grushka [23], respectively:   p tage =

f ðt Þ =


   1 −1 tage;i −tage 2 N pffiffiffiffiffiffi ∙∑i = 1 exp ; 2 b Np b 2π

" #   A −ðt−tR −t ′ Þ2 −t ′ ∞ pffiffiffiffiffiffi ∙∫ exp exp dt ′ : 2 0 τ 2σ τσ 2π

ð2Þ

ð3Þ

In Eq. (2), p is the probability density function, b is the bandwidth for approximation of the distribution of tumor age at presentation (days), Np is number of patients in the virtual clinical trial (dimensionless), tage is the tumor age at presentation (days), and tage,i is the tumor age at presentation in the ith patient (days). In

Eq. (3), A is the peak amplitude, τ is the time constant of the exponential modifier, σ is the standard deviation of the Gaussian constituent, tR is the center of gravity of the Gaussian function, and t′ is the dummy variable of integration. Histology and immunohistochemistry (IHC) After the last MRI and MRS experiments, brain tissues were extracted and fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 5 μm for routine staining. Sections were deparaffinized and rehydrated through three changes of xylene and graded alcohol and stained with hematoxylin and eosin (H&E). Slides were coverslipped and images were acquired on a light-transmission microscope. The brain tissue sections were also examined for Ki-67 (cell proliferation marker) and the m30 antigen (apoptosis marker). Performance of the IHC stains was done according to the manufacturer's directions. The names and dilutions of antibodies are mouse anti-Ki-67 (1:25; Dako North America, Carpinteria, CA, USA) and mouse anti-m30 CytoDeath (1:50; Enzo Life Sciences, Plymouth Meeting, PA, USA). Visualization was accomplished using a stable DAB/Plus substrate (DBS, Pleasanton, CA, USA), and counterstaining was carried out with hematoxylin. Controls, incubated with mouse IgG (Biocare Medical, Concord, CA, USA), were obtained for each examined IHC antibody. Statistical analysis Statistical analyses were done using a Student two-tailed, unpaired t test. Data are represented as means ± SD and p values of b0.05, b0.01, and b0.001were considered statistically significant. Results The effects of nitrones (PBN and OKN007) on inhibition of glioma growth in various glioma models The structures of PBN and OKN007 are shown in Fig. 1. The basic structure of a nitrone in its simplest form can be represented as X– CH = NO–Y [10]. PBN or OKN007 was administered to rats or mice as a prophylactic or posttumor treatment. MR T2-weighted imaging was acquired to access tumor morphology, which included the evaluation of tumor heterogeneity and volumes. Fig. 2 depicts the use of the mathematical model fitting routine for assessing tumor growth and nitrone-treatment response from experimental tumor volumes using Eq. (1), and their respective correlations, which have R 2 ≥ 0.96. Figs. 2A and C indicate that most C6 rats responded to either PBN pretreatment or OKN007 posttreatment. Fig. 2B shows both responsive and nonresponsive examples for PBN posttreatment. As there is no tumor regression for either nontreatment (Fig. 2D) or nonrespondent cases, only the tumor growth phase was fitted. Inhibition of tumor growth is indicated by decreased tumor volumes at the end time point from treated gliomas, shown in Table 1, compared to untreated glioma volumes. For C6 gliomas, PBN was able to inhibit tumor formation almost completely by decreasing tumor volumes by 98.3% when applied as a prophylactic agent, but could only partially induce tumor regression when applied as a posttumor treatment [15]. PBN decreased the end tumor volume of RG2 gliomas but did not induce total tumor regression. PBN was found not to have a significant inhibition effect on the growth of GL261 mouse gliomas. OKN007 was studied only as a posttumor treatment here to imitate a clinical situation. OKN007 induced almost complete regression of C6 gliomas, with the final tumor volume decreased by 83%. (One of the OKN007treated C6 rats was euthanized for brain extraction and histology as soon as we found the tumor was undergoing regression. This resulted in an increase in the average tumor volume and the standard

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Fig. 1. Chemical structures of nitrone compounds. (A) PBN and (B) OKN007.

deviation.) Although OKN007 was administered at various time points (day 13 to day 18), when the tumor volumes ranged from 5 to 46 mm 3, all the tumors responded to OKN007 very well. Another previously reported study from our group had similar findings [16]. Effects of PBN as a prophylaxis on tumor metabolism For MRS, a similar region of interest of the same volume (voxel) was chosen in tumor or contralateral tissues. As shown in Fig. 3A, a representative spectrum from normal brain tissue is distinguished by

prominent Cr, NAA, and Cho peaks but no Lip peak. The C6 glioma (untreated) indicated a significant increase in Lip (− CH2–) and Lip (−CH3) peak, compared to decreased Cr and NAA peaks. Cho was found to usually be decreased in most of the cases, but was also enhanced in the RG2 case (e.g., see later in Tables 2–5). Morphological MR T2-weighted images indicated that C6 gliomas grew to only a small volume before regression (Fig. 4A, images iv, v, vi) when treated with PBN as a prophylaxis 5 days before C6 cell implantation. The tumor in the untreated group grew continuously to a larger volume with increased tumor heterogeneity until the animals

Fig. 2. Mathematical model fitting of C6 tumor volumes associated with tumor growth and nitrone-treatment response. Representative model fitting curves of therapy response to (A) pre-PBN treatment, (B) post-PBN treatment, and (C) post-OKN007 treatment. (D) Representative model fitting curves of tumor growth for untreated C6 gliomas. Each curve represents an individual animal.

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Table 1 Tumor volumes (mm3) at the last time point from C6-, RG2-, and GL261-gliomabearing rats treated with PBN or OKN007.

PBN

OKN

Nontreatment Pretreatment Posttreatment Nontreatment Posttreatment

C6

RG2

GL261

300.3 ± 103.5 5.0 ± 10.0*** 144.6 ± 107.0* 245.0 ± 47.0 41.6 ± 74.3***

161.6 ± 22.7 105.6 ± 50.9* — — —

111.6 ± 17.0 91.5 ± 42.7 — — —

The numbers of animals are n = 6 for PBN-C6 nontreatment, n = 4 for PBN-C6 pretreatment, n = 5 for PBN-C6 posttreatment; n = 7 for PBN-RG2 nontreatment, n = 5 for PBN-RG2 pretreatment; n = 5 for both PBN-GL261 nontreatment and pretreatment; n = 7 for OKN007-C6 nontreatment, and n = 5 for OKN007-C6 posttreatment. The values are represented as means ± SD. *p b 0.05 and ***p b 0.001, statistical significance comparing untreated to treated groups, using two-tailed unpaired Student's t test.

were euthanized (Fig. 4A, images i, ii, iii). The metabolite ratios (Cho/ Cr, NAA/Cr, Lip1.3/Cr, Lip0.9/Cr) were calculated for each time point over the course of tumor growth. Fig. 4B, graph i indicates that the metabolite ratios all increased as tumor volumes increased. The Lip1.3/Cr ratio was the most elevated in this representative C6 glioma (data in Fig. 4B, graph i) as well as in most of all the other untreated gliomas. When treated with PBN 5 days before glioma implantation, all the metabolite ratios were found to have minor fluctuations, with most metabolites remaining at similar levels throughout the assessment period, with a corresponding decrease in tumor volume (Fig. 4B, graph ii).

Typical spectra were obtained from C6, RG2, and GL261 gliomas treated with PBN as a prophylaxis at time points when tumors were initialized (Fig. 5). Some tumors reached a maximum and then reduced if they were respondent to PBN treatment (Table 1). For PBNpretreated C6 gliomas, the metabolite peaks in the spectra were not substantially altered (Fig. 5A, spectra i, ii, iii) because of PBN treatment. Relative changes in Lip1.3/Cr were significantly decreased compared to untreated C6 gliomas (Fig. 5A, graph iv). Estimated metabolite concentrations (Table 2) that were altered were significantly increased (p b 0.01) for Cr and NAA and significantly decreased (p b 0.01) for Lip1.3, in PBN-pretreated gliomas compared to untreated gliomas. At the end time point for the PBN-treated RG2 gliomas, Cho and Lip1.3 peaks were still greatly elevated, whereas Cr and NAA peaks were decreased at this time point (Fig. 5C, spectra i, ii). Although PBN did not induce total regression of the RG2 gliomas (Table 1), the relative change in the Lip1.3/Cr ratio was decreased significantly, compared to that of untreated RG2 gliomas (Fig. 5B, graph iii). PBN did not seem to have an obvious effect regarding inhibition of GL261 mouse glioma growth, as there was no significant change in either final tumor volume or any of the metabolite ratios, compared to untreated GL261 gliomas (Fig. 5C, spectra i, ii, graph iii). Estimated concentrations of metabolites for the PBN-pretreated RG2 (Table 3) and GL261 (Table 4) rodent glioma model studies indicated no significant differences in any of the metabolites, comparing tumor spectral regions of PBN-pretreated animals versus untreated rats, although there were notable downward trends in the Lip1.3 and Lip0.9 peaks in the PBN-pretreated groups. Effects of OKN007 and PBN on tumor metabolism as a posttumor treatment

Fig. 3. Representative spectra from (A) normal rat brain tissue and (C) C6 glioma tissue at day 18 post-glioma cell implantation. (B) Square regions in the T2-weighted image indicate the positions from which the MRS data were obtained in the rat brain.

Fig. 6A, image i, shows another representative group of untreated C6 gliomas at initial, middle, and end time points for tumor growth. A representative C6 glioma was treated with OKN007 when the tumor volume was 15 mm 3 at day 15 after tumor cell implantation. The tumor continued to grow to a maximum size until day 27 (Fig. 6A, image v) and then was found to regress (the end tumor is shown in Fig. 6A, image vi). Changes in metabolite ratios correlated well with tumor growth curves over the course of tumor growth (Fig. 6B, graph i). The Lip1.3/Cr, NAA/Cr, and Cho/Cr ratios started to drop from their maximum values 2 days before the tumors started to regress. Of five C6 gliomas posttreated with PBN, 40% were respondent as shown in Fig. 6A, images vii–ix; the rest were nonrespondent as shown in Fig. 6A, images x–xii. Fig. 6B, graph ii, shows the changes in metabolite ratios for a respondent case, which follow a trend similar to that observed for OKN007-treated rats, except that all the ratios began to drop from the maximum 2 days after the tumor started to regress. As shown in Fig. 7A, spectrum ii, when a C6 glioma treated with OKN007 continued to grow to a certain volume (in this case, it was 128 mm 3) before regression, the spectral changes included absent Cr and Cho peaks, decreased NAA peak, and increased Lip1.3 and Lip0.9 peaks. When the tumor was almost gone, the spectrum reverted back to a normal appearance (Fig. 7A, spectrum iii). For Lip1.3/Cr and Lip0.9/Cr ratios, significant differences were found comparing maximum tumor to end tumor time points after OKN007 treatment (Fig. 7A, graph iv), suggesting that OKN007 had a significant effect on lipid metabolism. Significant differences were also observed between untreated and OKN007-treated maximum tumor time points, for NAA/Cr, Lip1.3/Cr, and Lip0.9/Cr ratios, as a result of the more decreased Cr levels in OKN007-treated maximum tumors (see also Table 5 for metabolite concentrations). Estimated metabolite concentrations (Table 5) indicated a significant increase (p b 0.01) in Cho and a decrease (p b 0.05) in Lip1.3, comparing untreated gliomas to OKNtreated gliomas. For C6 rats treated with PBN 14 days postimplantation, Fig. 7B depicts a respondent case, and Cho, Lip1.3, and Lip0.9 peaks were

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Table 2 Estimated concentrations (mM) of metabolites for C6-glioma-bearing rats with or without PBN treatment (including pre- and posttreatment). Metabolite

Untreated C6 Ctrl

Cr NAA Cho Lip1.3 Lip0.9

PBN-pretreated C6 Tum end

Ctrl a

7.5 ± 3.1 4.1 ± 2.5 2.4 ± 0.4 0.3 ± 0.5 0.1 ± 0.1

a,

10.4 ± 1.0 6.3 ± 1.1 2.3 ± 0.6 0±0 0±0

3.0 ± 2.2 1.8 ± 2.2a 2.6 ± 0.4 14.8 ± 6.1a 0.6 ± 0.5a

PBN-posttreated C6 Tum end 10.3 ± 2.0 ** 8.1 ± 2.3a,** 2.1 ± 0.4 0 ± 0a,** 0.1 ± 0.1

Ctrl

Tum end

10.4 ± 2.1 6.6 ± 2.8 4.2 ± 1.9 0±0 0.2 ± 0.3

3.6 ± 4.6 8.4 ± 9.8 2.4 ± 1.9 27.4 ± 11.3 1.7 ± 0.4a,**

The absolute concentrations of metabolites were obtained with TARQUIN. The data are presented as means ± SD. Ctrl, contralateral; Tum end, end phase of tumor progression. **p b 0.01, statistical difference comparing untreated (n = 5) to pretreated (n = 3), at Tum end, or to posttreated (n = 5), using two-tailed unpaired Student's t test. a Comparison between Untreated C6 at tumor (Tum) end phase to either PBN-pretreated or -posttreated C6 gliomas at Tum end phase.

found to decrease and Cr remained relatively unchanged. As a group including both respondent and nonrespondent cases, there were no significant differences between the untreated and the treated groups, for any of the ratios. This indicates that PBN is not as effective as OKN007 for treatment regarding inhibiting C6 glioma growth when applied as a posttumor treatment. Estimated concentrations of metabolites for the PBN-posttumor-treated C6 (Table 2) rat glioma model study indicated no significant expected differences in any of the metabolites, comparing tumor spectral regions of untreated rats versus PBN-posttumor-treated animals. There was a significant increase (p b 0.01) in the Lip0.9 peak in PBN-treated rats compared to untreated animals and a notable upward trend in the Lip1.3 peak in the PBN-treated group. Fig. 8 presents the necrotic regions, depicted as dark spots in the glioma tissue in T2-weighted MR images (Fig. 8A). Fig. 8A, image I, depicts intense necrotic regions in a representative untreated C6 glioma, whereas a number of untreated C6 gliomas had less necrosis, resulting in the variation detected. At the end of the OKN007 treatment, some animals still had necrosis in the remaining glioma tissue (Fig. 8A, image iii), whereas in other animals necrosis and glioma tumors had completely disappeared (Fig. 8A, image iv). The percentage necrosis data indicated the same trend observed in Lip1.3/ Cr and Lip0.9/Cr metabolite ratios after OKN007 treatment. A significant difference was made comparing percentage necrosis at maximum tumor volume before drug response with minimum tumor volume at the end point after treatment. Effect of OKN007 on tumor histology, cell proliferation (Ki-67), and apoptosis (m30 antigen) For C6 gliomas without treatment, H&E images showed that the tumor is composed of a large irregular sheet of single nonpleomorphic cells, with one to five mitoses as detected in the high-power field. Extensive hemorrhagic necrosis is present in the midportion of the tumor (Fig. 9B, image i). In contrast, histology of a C6 glioma treated with OKN007, at the end period of tumor regression, is shown in Fig. 9B, image ii. A glioma is present only in a portion of the histological slide, with increased cellularity compared to normal

brain; however, there is an obvious reduction in the degree of glioma cells compared to untreated gliomas. There is no necrosis or mitoses seen in the whole tumor region. To establish the effect of OKN007 on cell proliferation and apoptosis, IHC was done for Ki-67, a cell proliferation marker, and the m30 CytoDeath antigen, a specific apoptotic marker. The IHC detection of Ki-67 indicated that untreated C6 gliomas had a medium degree (pathology score of 2+) of cell proliferation (Fig. 9C, image i), whereas there was no detectable expression of Ki-67 in the OKNtreated C6 gliomas (Fig. 9C, image ii). For assessment of apoptosis, C6 gliomas without treatment showed negative staining for m30 (Fig. 9D, image i), whereas treatment with OKN induced strong m30 expression in glioma cells within the tumor region, indicating the presence of apoptosis (Fig. 9D, image ii, pathological score: 2+). Discussion Our study indicated that the nitrones PBN and OKN007 were able to induce the inhibition of tumor growth in C6 gliomas when applied either as a prophylactic agent or after tumor formation, respectively.

Table 4 Estimated concentrations (mM) of brain metabolites in GL261-glioma-bearing rats with or without PBN pretreatment. Metabolite

Cr NAA Cho Lip1.3 Lip0.9

Metabolite

Cr NAA Cho Lip1.3 Lip0.9

Untreated RG2

PBN-pretreated RG2

Ctrl

Tum end

Ctrl

Tum end

7.5 ± 1.8 9.5 ± 4 1.7 ± 0.2 0±0 0.6 ± 0.8

3.2 ± 1.9 1.7 ± 1.9 3.0 ± 1.9 20.6 ± 15.0 0.9 ± 1.0

7.0 ± 1.1 12.0 ± 2.2 1.8 ± 0.1 0±0 0.5 ± 0.9

2.0 ± 1.3 2.0 ± 2.9 3.4 ± 3.2 7.9 ± 3.7 0.4 ± 0.7

The data are presented as means ± SD. There was no significance obtained, comparing untreated (n = 5) to treated groups (n = 5) at the end phase of tumor progression (Tum end), using two-tailed unpaired Student's t test. Ctrl, contralateral.

PBN-pretreated GL261

Ctrl

Tum end

Ctrl

Tum end

7.5 ± 2.4 7.2 ± 1.7 2.8 ± 0.6 1.8 ± 2.8 0.2 ± 0.3

2.2 ± 2.0 2.0 ± 0.8 2.2 ± 1.2 74.3 ± 31.8 6.2 ± 5.6

8.0 ± 2.2 7.7 ± 2.4 3.3 ± 1.2 3.5 ± 3.2 0.4 ± 0.7

2.0 ± 1.9 3.0 ± 1.1 2.7 ± 0.8 41.2 ± 28.1 2.6 ± 2.7

The data are presented as means ± SD. There was no significance obtained, comparing untreated (n = 5) to treated groups (n = 4) at the end phase of tumor progression (Tum end), using two-tailed unpaired Student's t test. Ctrl, contralateral.

Table 5 Estimated concentrations (mM) of brain metabolites in C6-glioma-bearing rats with or without OKN007 treatment (posttumor). Metabolite

Table 3 Estimated concentrations (mM) of brain metabolites in RG2-glioma bearing rats with or without PBN pretreatment.

Untreated GL261

Cr NAA Cho Lip1.3 Lip0.9

Untreated C6

OKN-treated C6

Ctrl

Tum end

Ctrl

Tum max

Tum end

7.8 ± 3.1 10.7 ± 2.9 2.7 ± 1.2 0.3 ± 0.6 0.7 ± 0.6

1.4 ± 1.2 3.3 ± 3.7 1.0 ± 0.4b,** 34.4 ± 32.8 2.0 ± 2.2

10.1 ± 2.3 11.4 ± 1.6 2.9 ± 0.7 0±0 0.9 ± 1.1

0.7 ± 0.7 2.3 ± 1.8 0.5 ± 0.8b,* 62.2 ± 18.5a,* 3.3 ± 1.3

3.6 ± 3.3 2.9 ± 2.3 2.5 ± 0.5b 6.1 ± 8.6a 0.5 ± 0.7

The data are presented as means ± SD. Ctrl, contralateral; Tum max, maximum tumor; Tum end, end phase of tumor. *p b 0.05 and **p b 0.01, statistical significance comparing Tum max to Tum end in OKNtreated C6 rats and untreated (n = 5) to treated (n = 3) groups at Tum end, using twotailed unpaired Student's t test. a Comparison between Untreated C6 at tumor (Tum) end phase to OKN-treated C6 gliomas at Tum end phase. b Comparison between OKN-treated C6 gliomas at Tum max and Tum end phases.

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PBN had less of an anti-tumor effect when applied to already established C6 gliomas in comparison to OKN007. PBN was found not to induce tumor regression in RG2 or GL261 mouse gliomas, which are more invasive than the C6 tumors. Our MRS results correlated with MRI in assessing changes in tumor volumes and the efficacy of PBN or OKN007. Previously, Doblas et al. used the Gompertz function to fit the first phase of C6 tumor growth, but this function did not fit the response phase to PBN treatment [15]. We improved the mathematical modeling by using a Gaussian function (Eq. (1)), which was found to fit both C6 tumor growth and response to PBN or OKN007 treatment with a high correlation index. This mathematical model fitting routine was found to accurately predict regressed tumor volumes in response to PBN or OKN007.

From previous studies, various mechanisms could be hypothesized to be involved in the anti-tumor effects of PBN and OKN007. The inhibition of glioma formation by PBN was related with reduced tumor angiogenesis (translated by reduced blood volume ratios, vessel size, and numbers), observed by MR angiography and decreased expression of angiogenic factors, such as the vascular endothelial growth factor (VEGF) and the von Willebrand factor, as observed from immunostaining of glioma tissues [15]. PBN administration has been found to decrease inducible NO synthase (iNOS) activity and production, which may be one of the factors causing enhanced selective apoptosis of cells in preneoplastic lesions in liver cancer [24,25]. After OKN007 treatment, iNOS, c-Met, and VEGFR2 levels of expression were all reduced, as observed from Western blots of treated C6 glioma tissues compared to untreated tissue [16].

Fig. 4. (A) Tumor growth patterns (shown as T2-weighted MR images) in untreated (i–iii) and PBN-pretreated (5 days before cell implantation) (iv–vi) C6 gliomas. (i, iv) Gliomas at the initiating point (7 days after cell implantation). (ii, iii) Gliomas at days 16 and 21 (the end time point). (v, vi) Gliomas at days 18 and 27. (B) Metabolite ratios and tumor volume changes over the course of tumor growth from representative (i) untreated and (ii) pretreated C6-glioma-bearing rats.

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Fig. 5. Representative spectra from PBN-pretreated glioma tissues and relative changes in metabolite ratios from both treated and untreated glioma tissues. (A–C, i) Spectra obtained at tumor initiating time points. (A–C, ii) Spectra obtained when tumors reached maximum volumes. (A, iii) A spectrum obtained when the tumor decreased to its minimum volume. Prominent peaks are indicated in each spectrum as a, b, c, d, e, which are Cho, Cr, NAA, Lip (methylene), and Lip (methyl), respectively. (A, iv; B, iii; C, iii) Relative changes in metabolite ratios from treated and untreated glioma tissues at the end time points. (A), (B), and (C) represent C6, RG2, and GL261 gliomas, respectively, pretreated with PBN. The relative changes in metabolite ratios were calculated by peak area ratios in tumors (Tum) compared to contralateral regions (Ctrl) as follows: (Tum − Ctrl)/Ctrl. Values are represented as means ± SD, *p b 0.05 indicates statistical significance comparing untreated to treated groups. The numbers of animals were n = 5 for untreated C6 gliomas (C6 NTR), n = 3 for PBN 5-day-pretreated C6 gliomas (PBN d-5); n = 5 for both untreated RG2 gliomas (RG2 NTR) and PBN-5-day pretreated RG2 gliomas (RG2 PBN); n = 5 for untreated GL261 gliomas (GL261 NTR) and n = 4 for PBN 5-day-pretreated GL261 gliomas (GL261 PBN).

The results of this study demonstrate that OKN007 and PBN could affect tumor metabolism, which was denoted by significant changes in major metabolite ratios, particularly the Lip/Cr ratio, in different gliomas. Choline is commonly studied by MRS in human gliomas and shown to be a relative prognostic marker. Senft et al. showed that WHO grade II gliomas had significantly less maximum and mean choline levels, and also a decreased Cho/Cr ratio, compared to nonnecrotic grade III and IV gliomas [26]. Hlaihel et al. recommended that MRS should be used in the follow-up of low-grade gliomas because the choline/creatine ratio predicted anaplastic transformation earlier than MR perfusion abnormalities, with a high positive predictive value of 83% [27]. We found that the Cho/Cr ratio was generally elevated in the glioma models studied, compared to normal contralateral tissue and ipsilateral tissue at early post-cell-implantation time points, whereas this ratio tended to decrease, but not significantly, after treatment with OKN007 and PBN. For the OKNtreated group, Cho was found to significantly increase (p b 0.01) compared to the untreated tumors (Table 5), indicating in this case that metabolite ratios may not best represent alterations in some metabolites. The different contributions by choline, phosphocholine, and glycerophosphorylcholine to the total choline levels were

different for low- and high-grade gliomas [28]; thus a further investigation of the changes in the proportions of choline metabolites may better reflect the response to nitrones. Any process of neuronal injury induces decreased NAA concentration. The levels of NAA and Cr are inversely related with brain tumor lesion volumes [29]. In several studies of brain tumors, high levels of NAA were associated with a good prognosis [30]. This was consistent with our results. Both NAA and Cr decreased significantly in untreated C6, RG2, and GL261 gliomas, and as Cr was reduced more than NAA this resulted in an elevated NAA/Cr ratio as the tumor grew. With prophylactic PBN treatment, the NAA/Cr ratio was found not to significantly change; however, when NAA and Cr metabolite concentrations were determined, both of these metabolites were found to significantly increase in the PBN-pretreated group compared to untreated animals (Table 2). Again this is an example of where obtaining estimated metabolite concentrations provides information that is missed when measuring metabolite ratios. Although OKN007 was able to restore the NAA/Cr ratio to a relatively normal value when the tumors regressed, the estimated concentrations of NAA and Cr were not found to significantly increase in OKN-treated gliomas compared to untreated gliomas (Table 5). In OKN007-treated C6 gliomas, Cr was

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Fig. 6. (A) Tumor growth patterns (shown in T2-weighted MR images) from untreated or nitrone-posttreated C6 gliomas. (A) (i, ii, iii) Untreated glioma at days 7, 18, and 24 (last time point). (iv, v, vi) OKN007-posttreated glioma at days 7, 27, and 36 (OKN007 was given on day 15). (vii, viii, ix) A respondent PBN-treated glioma at days 7, 22, and 35 (PBN was given on day 14). (x, xi, xii) A nonrespondent PBN-treated glioma at days 7, 12, and 18. (B) Changes in metabolite ratios and tumor volumes over the course of tumor growth from representative (i) OKN007-treated and (ii) respondent PBN-treated gliomas. Changes in metabolite ratios from a nonrespondent PBN-treated glioma were similar to non-treated gliomas in Fig. 4B-i.

reduced to very low levels as the tumors grew to maximum volumes, and this resulted in somewhat exaggerated NAA/Cr, Lip1.3/Cr, and Lip0.9/Cr ratios, compared to those of untreated C6 gliomas. It is a limitation to use Cr as an internal reference for the ratio calculations because Cr is usually decreased in brain tumors [31–34] and, as observed in our study, some changes in individual metabolites can be missed. In a study involving the evaluation of glioma grade by multivoxel proton MRS, Yerli et al. used Cr from the contralateral normal tissue as an internal reference [35]. There are considerations to be made with single-voxel MRS as a result of different magnetic homogeneities and receiver gains between each MRS scan (when comparing disease tissue to normal tissue or when comparing longitudinal scans). Although estimated metabolite concentrations were also obtained in this study, assumptions are made that sample homogeneity is similar in tumor versus contralateral tissue, that therapeutic effects do not alter the tissue environment, and that water concentration in diseased versus normal tissue does not significantly change. Further studies are required to establish what effects a disease process has on tissue heterogeneity and water concentrations in relationship to 1H-MRS-measured metabolites. A series of longitudinal MRS scans does indicate a basic trend in metabolic changes from a disease process (untreated gliomas; Fig. 4B, graph i) and when evaluating therapeutic response to PBN (prophylactic treatment; Fig. 4B, graph ii) and OKN007 (posttumor treatment; Fig. 6B, graph i), by comparing spectra at various time points. Our results indicated that the lipid peaks were greatly increased in all the glioma models that we studied and that this metabolite responded more significantly to treatment than other metabolites such as Cho and NAA. For PBN pretreatment in C6 and RG2 models, and OKN007 posttreatment in C6 gliomas, the lipid peaks (1.3 and/or 0.9 ppm) were the main metabolites that significantly reflected and

predicted tumor response to treatment, although, by measuring estimated metabolite concentrations, we were able to observe in the PBN-pretreated group that NAA and Cr levels were essentially restored to normal levels in the treated group (Table 2) and that Cho was significantly increased in the OKN-treated group compared to the untreated group (Table 5). Lipid levels have been previously demonstrated to be a marker of tumor malignancy and an indication of response to treatment [36,37]. A multicenter patient study showed that mobile lipids occurred in 41% of high-grade tumors, with higher amounts found in GBM [33]. In another study of grading for newly diagnosed gliomas, lipids were significantly increased in GBM [38]. The mobile lipid resonance that can be detected by 1H MRS mainly is attributed to cholesterol, triglycerides, and phosphatidylcholine in malignant gliomas, as detected by ex vivo diffusion edited high-resolution magic angle spinning (HR-MAS) MRS [39]. The lactate (1.33 ppm) signal often interferes with the lipids signal. However, lactate has a better chance of being present in a long TE PRESS, whereas mobile lipids are more prominent with short TE sequences (TE 30 ms or less) because of their very short relaxation times [40]. With a TE of 24 ms, most signals at 1.3 ppm in our spectra came from mobile lipids. In this study, the longitudinal MRS and MRI data reflected that the metabolite changes with treatment or without treatment match well with tumor volume changes over the whole course of tumor growth and/or course of tumor regression. However, the changes in metabolite ratios or estimated metabolite concentrations could also provide information on metabolic response to therapy. Possible mechanisms associated with the 1H MR spectroscopic findings that were involved in the therapeutic effects include cell proliferation due to tumor growth and lipid changes resulting from either necrosis or apoptosis. Prophylactic PBN treatment was able to restore Cr and NAA

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Fig. 7. Representative spectra from OKN007- or PBN-posttreated C6 glioma tissues (respondent case) and relative changes in metabolite ratios for both treated and untreated C6 gliomas. (A and B, i) Spectra obtained at the tumor initiating time point. (A and B, ii) Spectra obtained when the tumors reached maximum volumes. (A and B, iii) Spectra obtained when the tumors decreased to minimum volumes. Prominent peaks are indicated in each spectrum as a, b, c, d, and e, which are Cho, Cr, NAA, Lip (methylene), and Lip (methyl), respectively. (A, iv) Relative changes in metabolite ratios from untreated C6-glioma-bearing rats at the end time point (C6 NTR; n = 5), OKN007-treated C6-glioma-bearing rats when tumors reached maximum volume (C6 OKN Max; n = 3), and OKN007-treated C6-glioma-bearing rats at the end time point (C6 OKN End; n = 3). (B, iv) Relative changes in metabolite ratios from untreated C6-glioma bearing rats (C6 NTR; n = 5) and PBN-posttreated C6-glioma-bearing rats (C6 PBN d + 14; n = 5) at the end time point. The relative changes in metabolite ratios were calculated by peak area ratios in tumors (Tum) compared to contralateral regions (Ctrl) as follows: (Tum − Ctrl)/Ctrl. The values are represented as means ± SD, with *p b 0.05 and **p b 0.01 indicating statistical significance comparing C6 NTR to C6 OKN Max and C6 OKN Max to C6 OKN End groups.

metabolite concentrations to relatively normal levels, indicating that tissue energy metabolism (measured by Cr) and neuronal cell viability (measured by NAA) were restored. To demonstrate that nitrone treatment could have an effect on cell proliferation, we obtained IHC staining for Ki-67, a cell proliferation marker. A decrease in the levels of a cell proliferation marker, Ki-67, in glioma tissues after OKN treatment confirmed that OKN007 can inhibit cell proliferation (Fig. 9). It has been shown that a positive correlation between Cho and Ki-67 is found in tumor tissue when using single-voxel 1H MRS; however, the presence of necrosis has a negating effect on this correlation [41,42]. The degree of necrosis

(Fig. 8) could account for the decreased Cho levels detected in C6 gliomas (e.g., either untreated gliomas or OKN-treated gliomas at maximum tumor volumes, in Table 5). Spectroscopic evidence has shown that visible lipids correspond to lipid droplets in necrotic and perinecrotic tumor regions in animal studies [43]. Opstad et al. demonstrated that the lipid signals were cytoplasmic in origin and that lipid droplets were related with the 1.3ppm lipid peak, which forms before necrosis but is positively correlated with the degree of necrosis as observed with the use of HR-MAS [44]. In this study, with changes in lipids being the major metabolic change detected in response to treatment, we investigated

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Fig. 8. (A) Representative necrotic areas (dark voids) as shown in T2-weighted images from (i) untreated C6 gliomas, (ii) an OKN007-treated tumor when the volume reached a maximum value, (iii), an OKN007-treated tumor with necrosis when the volume reached a minimum value, and (iv) an OKN007-treated tumor without necrosis when the volume reached a minimum value. (B) Percentage necrosis in C6 gliomas with or without OKN007 treatment. The numbers of animals were n = 7 for untreated C6 gliomas (C6-NTR) and n = 5 for OKN007-treated C6 gliomas at maximum tumor (C6 OKN Max) and at end time point (C6 OKN End). Data are presented as means ± SD, with **p b 0.001 indicating significance comparing C6 OKN Max to C6 OKN End groups.

the relationship between lipids and necrosis in OKN007-treated and untreated C6 gliomas. We had previously identified that the dark void regions in gliomas from T2-weighted MR images were attributed to necrosis as observed by H&E staining [45]. The percentage necrosis in the OKN-treated group was found to significantly decrease after the treatment period, compared to maximum tumor volumes obtained at the initial treatment period (Fig. 8), and this may be related to the decreased levels of lipid observed in the 1H MR spectra. This finding of reduced necrosis in gliomas may partially explain the mechanisms involved in inhibiting tumor growth by OKN007. A previous study by Floyd et al. demonstrated that nitrones induce apoptosis in preneoplastic lesions in a choline-deficient rat liver tumor model [46], leading us to investigate the effect of OKN007 on apoptosis in gliomas. Caspases, are responsible for the morphological changes within cells during apoptosis, and caspase-3 cleaves most of the cellular substrates in the apoptotic process and is essential for certain processes associated with the formation of apoptotic bodies [47]. Therefore, measurement of caspase-3 is a reliable determinant of apoptosis [48]. Proteins degraded by caspase-3 are also used to detect apoptosis. The m30 antibody detects cytokeratin 18 (CK18), which is an epithelial-specific caspase-3 early cleavage product [49,50]. We found that the apoptotic marker, m30 antigen (CK18), was increased significantly in the glioma region of OKN-treated C6-glioma-bearing rats (Fig. 9). An untreated glioma and the contralateral normal brain tissue had negative staining for the m30 antigen. This result suggests that OKN007 induced apoptosis of the glioma cells in the C6 glioma model. In vitro 1H MRS has been used to show that there is an increase in triacylglycerol accumulation with increased apoptosis [51]. Although we do detect OKN007-induced apoptosis with IHC (Fig. 9), the lipid signals, detected by 1H MRS in our in vivo study (Fig. 7 and Table 5), indicate that Lip1.3 significantly decreases with OKN treatment (compared to untreated gliomas). It is possible that the overwhelming antinecrotic effect of OKN007 on gliomas, and the overall decrease in lipids as a result of the reduced necrosis, may be masking the lipidassociated apoptosis effect. In vitro it has been demonstrated that OKN007 (formerly known as NXY-059) is more effective in trapping hydroxyl and methanol

Fig. 9. Effects of OKN007 on glioma histology and IHC levels of Ki-67 and the m30 antigen in C6 glioma-bearing rats. (A) T2-weighted MR images of (i) untreated C6 glioma (day 20) and (ii) OKN-treated (day 55) C6 glioma at last day of study. (B) H&E-stained images of tumors from (i) untreated and (ii) OKN-treated C6 gliomas. (C) Detection of Ki-67 in brain tissues of (i) untreated and (ii) OKN-treated C6 gliomas. (D) Detection of the apoptosis marker m30 antigen in brain tissues of (i) untreated and (ii) OKN-treated C6 gliomas. The histology and IHC images were taken at an original magnification of 200×.

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radicals compared to the parent nitrone PBN [52]. It may be possible that the increased spin trapping or free radical scavenging capability of OKN007, and its aqueous solubility compared to PBN, may in part contribute to its increased anti-cancer activity observed in rat glioma models. In conclusion, 1H MRS, along with conventional MRI, is a useful method to assess and follow the response of varied glioma models to either OKN007 or PBN treatment. The Lip1.3/Cr ratio or Lip1.3 peak could be a diagnostic metabolic marker for anti-glioma therapies. Based on the effect of OKN007 treatment on posttumor formation in C6 gliomas, and its tested nontoxic property [11,53], further studies should be done to investigate the anti-glioma effect of OKN007 clinically. Acknowledgments The authors thank Dr. Nataliya Smith, Jessica Hoyle, and Charity Njoku for their help in maintaining and preparing the glioma cells for intracerebral implantation. We thank Dr. Yasvir Tesiram for assistance in the mathematical modeling. We also thank Dr. Geza Safrany for kindly donating the GL261 cells. We also thank Dr. Daniel Brackett (Department of Surgery, University of Oklahoma Health Sciences Center) for the use of his histology facilities. Funding was provided by the Oklahoma Center for the Advancement of Sciences and Technology, Grant AR092-049, and the Oklahoma Medical Research Foundation. References [1] Law, M.; Yang, S.; Wang, H.; Babb, J. S.; Johnson, G.; Cha, S.; Knopp, E. A.; Zagzag, D. Glioma grading: sensitivity, specificity, and predictive values of perfusion MR imaging and proton MR spectroscopic imaging compared with conventional MR imaging. Am. J. Neuroradiol. 24:1989–1998; 2003. [2] Dean, B. L.; Drayer, B. P.; Bird, C. R.; Flom, R. A.; Hodak, J. A.; Coons, S. W.; Carey, R. G. Gliomas: classification with MR imaging. Radiology 174:411–415; 1990. [3] Felix, R.; Schorner, W.; Laniado, M.; Niendorf, H. P.; Claussen, C.; Fiegler, W.; Speck, U. Brain tumors: MR imaging with gadolinium-DTPA. Radiology 156: 681–688; 1985. [4] Kondziolka, D.; Lunsford, L. D.; Martinez, A. J. Unreliability of contemporary neurodiagnostic imaging in evaluating suspected adult supratentorial (lowgrade) astrocytoma. J. Neurosurg. 79:533–536; 1993. [5] Soares, D. P.; Law, M. Magnetic resonance spectroscopy of the brain: review of metabolites and clinical applications. Clin. Radiol. 64:12–21; 2009. [6] Marshall, I.; Wardlaw, J.; Cannon, J.; Slattery, J.; Sellar, R. J. Reproducibility of metabolite peak areas in 1H MRS of brain. Magn. Reson. Imaging 14:281–292; 1996. [7] Panigrahy, A.; Bluml, S. Neuroimaging of pediatric brain tumors: from basic to advanced magnetic resonance imaging (MRI). J. Child Neurol. 24:1343–1365; 2009. [8] Young, G. S. Advanced MRI of adult brain tumors. Neurol. Clin. 25:947–973 viii; 2007. [9] Griffin, J. L.; Lehtimaki, K. K.; Valonen, P. K.; Grohn, O. H.; Kettunen, M. I.; YlaHerttuala, S.; Pitkanen, A.; Nicholson, J. K.; Kauppinen, R. A. Assignment of 1H nuclear magnetic resonance visible polyunsaturated fatty acids in BT4C gliomas undergoing ganciclovir–thymidine kinase gene therapy-induced programmed cell death. Cancer Res. 63:3195–3201; 2003. [10] Floyd, R. A.; Kopke, R. D.; Choi, C. H.; Foster, S. B.; Doblas, S.; Towner, R. A. Nitrones as therapeutics. Free Radic. Biol. Med. 45:1361–1374; 2008. [11] Green, A. R.; Ashwood, T.; Odergren, T.; Jackson, D. M. Nitrones as neuroprotective agents in cerebral ischemia, with particular reference to NXY-059. Pharmacol. Ther. 100:195–214; 2003. [12] Lees, K. R.; Zivin, J. A.; Ashwood, T.; Davalos, A.; Davis, S. M.; Diener, H. C.; Grotta, J.; Lyden, P.; Shuaib, A.; Hardemark, H. G.; Wasiewski, W. W. NXY-059 for acute ischemic stroke. N. Engl. J. Med. 354:588–600; 2006. [13] Diener, H. C.; Lees, K. R.; Lyden, P.; Grotta, J.; Davalos, A.; Davis, S. M.; Shuaib, A.; Ashwood, T.; Wasiewski, W.; Alderfer, V.; Hardemark, H. G.; Rodichok, L. NXY-059 for the treatment of acute stroke: pooled analysis of the SAINT I and II trials. Stroke 39:1751–1758; 2008. [14] Nakae, D.; Kishida, H.; Enami, T.; Konishi, Y.; Hensley, K. L.; Floyd, R. A.; Kotake, Y. Effects of phenyl N-tert-butyl nitrone and its derivatives on the early phase of hepatocarcinogenesis in rats fed a choline-deficient, L-amino acid-defined diet. Cancer Sci. 94:26–31; 2003. [15] Doblas, S.; Saunders, D.; Kshirsagar, P.; Pye, Q.; Oblander, J.; Gordon, B.; Kosanke, S.; Floyd, R. A.; Towner, R. A. Phenyl-tert-butylnitrone induces tumor regression and decreases angiogenesis in a C6 rat glioma model. Free Radic. Biol. Med. 44: 63–72; 2008.

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