Targeting Nitric Oxide and NMDA Receptor-Associated Pathways in Treatment of High Grade Glial Tumors

Nitric Oxide 79 (2018) 68–83

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Nitric Oxide

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Targeting nitric oxide and NMDA receptor-associated pathways in treatment of high grade glial tumors. Hypotheses for nitro-memantine and nitrones T

∗ Meric A. Altinoz , İlhan Elmaci

Neuroacademy Group, Department of Neurosurgery, Memorial Hospital, Istanbul, Turkey

ARTICLE INFO ABSTRACT

Keywords: Glioblastoma multiforme (GBM) is a devastating brain cancer with no curative treatment. Targeting Nitric Oxide Nitric oxide (NO) and glutamatergic pathways may help as adjunctive treatments in GBM. NO at low doses promotes tu- Nitrone morigenesis, while at higher levels (above 300 nM) triggers apoptosis. Gliomas actively secrete high amounts of Nitro-memantine glutamate which activates EGR signaling and mediates degradation of peritumoral tissues via excitotoxic injury. Glial tumor Memantine inhibits NMDA-subtype of glutamate receptors (NMDARs) and induces autophagic death of glioma Glioblastoma cells in vitro and blocks glioma growth in vivo. Nitro-memantines may exert further benefits by limiting NMDAR signaling and by delivery of NO to the areas of excessive NMDAR activity leading NO-accumulation at tumoricidal levels within gliomas. Due to the duality of NO in tumorigenesis, agents which attenuate NO levels may also act beneficial in treatment of GBM. Nitrone compounds including N-tert-Butyl-α-phenylnitrone (PBN) and its disulfonyl-phenyl derivative, OKN-007 suppress free radical formation in experimental cerebral ischemia. OKN-007 failed to show clinical efficacy in stroke, but trials demonstrated its high biosafety in humans including elderly subjects. PBN inhibits the signaling pathways of NF-κB, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX). In animal models of liver cancer and glioblastoma, OKN-007 seemed more efficient than PBN in suppression of cell proliferation, microvascular density and in induction of apoptosis. OKN-007 also inhibits SULF2 enzyme, which promotes tumor growth via versatile pathways. We assume that nitromemantines may be more beneficial concomitant with chemo-radiotherapy while nitrones alone may act useful in sup- pressing basal tumor growth and angiogenesis.

1. Introduction donors, such as nitromemantines may potentiate chemo-sensitivity selectively within glial tumor cells, as they may specifically accumulate in Glioblastoma (GBM) has the highest incidence among primary brain areas of NMDA-receptor (NMDAR) overreactivity, which is the case in tumors, second only to meningioma and exerts a very poor prognosis high grade glial tumors. At the first glance, it may seem paradoxical and [1,2]. In high-grade glial tumors, maximum surgical resection, radio- even discursive to propose that both NO synthesis inhibitors and NO therapy, and adjuvant temozolomide is the new standard of care. donors can be employed in the treatment of glioblastoma. However, However, even if these standards are employed at the optimal condi- substantial data provides evidence that constitutive low doses of NO tions, the median survival of patients with GBM is around 15 months propagates tumor growth, while higher doses (suggested to be above with a 5-year survival rate of < 4% from the time of diagnosis [1,2]. 300–500 nM in various resources) of NO acted tumoricidal. Hence, NO Hence, discovery of novel innovative approaches is an urgent need in inhibitors may block basal growth of glioblastoma while NO-donors treatment of these devastating tumors. Increasing recent evidence may potentiate chemo-radiotherapy efficacy of glioblastoma via en- suggest that nitric oxide (NO), NO-synthase (NOS) enzymes, glutamate hancing cell apoptosis and secondary necrosis. In sections below, we and its receptor (NMDA) play important roles in pathogenesis and will provide clues for our hypotheses after explaining general bio- treatment responses of glioma cells, which will be outlined below. As chemistry of NO and NO-derived molecules and their involvement in will be explained, we propose that drugs which block NO at basal tumor metabolism. conditions (such as nitrones) may block aggressive behaviour of high grade glial tumors by reducing growth, invasion and angiogenesis. As NO exerts dual and opposite functions on glioma metabolism, NO

∗ Corresponding author. E-mail address: [email protected] (M.A. Altinoz). http://dx.doi.org/10.1016/j.niox.2017.10.001 Received 10 August 2017; Received in revised form 26 September 2017; Accepted 7 October 2017 Available online 13 October 2017 1089-8603/ © 2017 Elsevier Inc. All rights reserved. İ M.A. Altinoz, . Elmaci Nitric Oxide 79 (2018) 68–83

formation of a mixed disulﬁde bridge between a cysteine residue and − the GSH thiol (Fig. 2). It can be induced by ONOO or formed after the reaction between a thiol and a nitrosothiol, but S-glutathionylation can also occur without NO [7]. Lipophilic compartments like the cell membrane will concentrate

NO and O2 and facilitate the reactions between them to trigger the production of N2O3 [10]. In aqueous systems, N2O3 in the presence of a nucleophile such as thiolate or an amine group will form a hybrid of + - covalent and ionic resonance structures NO –NO2 . Nitrosation reactions occur between NO+ and the thiolate or between NO+ and the amine form RSNOs (S-nitrosothiols) and nitrosamines, respectively [10]. Although RSNOs are widely considered as NO donors, they can also act through trans-nitrosylation, without release of NO [11]. Trans- nitrosylation is the exchange between a thiol group and a nitrosothiol group without release of NO [4]. Nitrosylating species of biological ff Fig. 1. cGMP dependent and independent pathways triggered by NO. Note that di ering relevance, such as S-nitroso-glutathione (GSNO) and S-nitrosocysteine, NO products may trigger cGMP-independent cascades. (Adapted from El-Sehemy et al. can nitrosylate/trans-nitrosylate signaling proteins [12]. Nitric oxide signaling in human ovarian cancer: A potential therapeutic target. Nitric Oxide. 2016; 54:30–7. http://dx.doi.org/10.1016/j.niox.2016.02.002). Protein S-nitrosylation (S-nitrosation) is the addition of a nitroso group moiety to a thiol group of a cysteine residue in peptides or proteins [4]. Protein S-nitrosylation is considered a mechanism for 2. Nitric oxide, S-nitrosylation and cancer signal transduction by NO and RSNOs [6] without requirement of enzymatic activities [4]. The nitrosylating agent's efficiency and the in- Nitric Oxide (NO) is a relatively stable free radical that neither di- tracellular redox environment regulated by the redox couples [oxidized merizes nor easily reacts with reducing or oxidizing biological sub- glutathione/reduced glutathione (GSSG/2GSH), oxidized thioredoxin/ stances. Rather, NO is selective, which weakly reacts with diamagnetic reduced thioredoxin (TrxSS/Trx)] determine S-nitrosylation [4,13] substances like the majority of organic molecules but strongly reacts (Fig. 2). Specificity of S-nitrosylation can be associated with the accu- with paramagnetic substances such as transition metals (iron and − mulation of high levels of nitrosylating species in the vicinity of specific copper), O % ,O, and nitrogen oxides [3]. NO regulates versatile 2 2 cysteine residues in subcellular compartments [14]. At least 1000 physiological pathways via activating two major signaling cascades: proteins modified by S-nitrosylation have been identified in mamma- The activation of the guanylyl cyclase to form cGMP and S-nitrosyla- lian cells [15]. tion, a covalent attachment of an NO moiety to a reactive cysteine re- The constitutive NOS isoforms have been detected in various can- sidue in peptides and proteins [4] (Fig. 1). Three NO synthases (NOS) cers. Activation of eNOS contributes to the initiation and progression of synthesize NO from the amino acid L-arginine and the NOS1, NOS2, and tumor growth in pancreatic cancer cell lines [16]. Tumor progression in NOS3 genes encode, respectively, neuronal NOS (nNOS), endothelial prostate cancer is associated with eNOS expression [17], while mela- NOS (eNOS), and inducible NOS (iNOS) [5]. The constitutively ex- noma progression is associated with nNOS expression [18]. The iNOS pressed eNOS and nNOS generally release low levels of NO that are isoform is ubiquitously distributed in malignant tumors [19]. The de- associated with cytoprotection and cell proliferation [4].Inflammatory composition of specific chemical compounds generally termed as NO cytokines induce iNOS in macrophages and production of NO under donors serves as exogenous sources for the generation of NO. Ni- these conditions is much higher compared to its synthesis by con- troglycerin (glyceryl trinitrate) is the oldest and the most well-known stitutive enzymes [6]. Higher NO fluxes such as those produced by iNOS NO donor [4]. Today, at least 16 different classes of nitrogen–ox- stimulated by inflammatory cytokines in macrophages or generated by ygen–bonded compounds were developed which decompose or to be millimolar concentrations of NO donors are cytotoxic and stimulate reduced/oxidized with the production of NO and other reactive ni- apoptosis [6]. trogen species. Clinical studies with glyceryl trinitrate to treat prostate NO-mediated nonclassical signaling includes modifications in tyr- cancer [20] and preclinical trials with NO-donating nonsteroidal anti- osine and cysteine residues of proteins [7]. Nitration of protein tyrosine − inflammatory drugs to treat colon cancer [21] indicate that NO donors residues happens by reaction with peroxynitrite (ONOO ), a powerful − potentially could act as antineoplastic. oxidant produced by the reaction of NO with O % and by NO gen- 2 2 It is generally assumed that low doses of NO stimulates cancer erated by neutrophil myeloperoxidase [8,9]. Both oxidants nitrate tyr- progression, while high amounts of NO helps in killing cancer cells osine at position 3 of the phenolic ring, producing 3-nitrotyrosine. [4,22,23] (Fig. 3). Cancer development involves S-nitrosylation of Tyrosine nitration may modify tyrosine phosphorylation/depho- specific enzymes and proteins, for instance: a) S-nitrosylation of specific sphorylation signaling cascades directly or indirectly [4]. S-glutathio- caspases which trigger apoptosis block apoptosis of cancer cells [24];b) nylation is the incorporation of a GSH thiol group into a protein and the S-nitrosylation of Bcl-2 hinders its proteosomal loss allowing it to

Fig. 2. Biochemical interactions of NO with glutathione (GSH) and thioredoxins (Trx). (Adapted from Lima et al. S-nitrosylation in cardiovascular signaling. Circ Res. 2010; 106(4):633–46. http://dx.doi.org/10.1161/CIRCRESAHA.109.207381).

69 İ M.A. Altinoz, . Elmaci Nitric Oxide 79 (2018) 68–83

Fig. 3. Dose dependent dual roles of NO in stimulating and inhibiting apoptosis and modiﬁcation of chemosensitivity. (Adapted from El-Sehemy et al. Nitric oxide signaling in human ovarian cancer: A potential therapeutic target. Nitric Oxide. 2016; 54:30–7. http://dx.doi.org/10.1016/j.niox.2016.02. 002).

and protective responses via cGMP signaling, mTOR activation, phosphorylation of Akt, and stabilization of HIF-1α, and these signaling pathways were linked to the promotion of tumor proliferation, migration and angiogenesis [27]. NO-triggered PI3K/Akt activation at NO concentrations of 100–400 nM may occur via tyrosine nitration of TIMP-1 (tissue inhibitor matrix metalloproteinase-1), which activates pro-survival cascades through binding to the cell surface receptor CD63 [28]. NO at a level of 300 nM activates EGFR (epidermal growth factor receptor), Src kinase, and Ras by the mechanisms of nitrosylation which leads to the blockage of tumor suppressor protein phosphatase 2A activity and/or the activation of tumorigenic signaling cascades, including c-Myc, β-catenin, and Ets-1 [29,30]. Tyrosine phosphorylation and S-nitrosylation of β-catenin contributes to tumor progression [4]. Activation of Src in endothelial cells phosphorylates β-catenin on Tyr654, while eNOS-derived NO nitrosylates β-catenin on Cys619, causing dissociation of β-catenin from its partner VE-cadherin in adherent junctions [4]. Disassembled adherent junctions lead loss of cell- to-cell contacts in transformed cells. NO derived from β-estradiol stimulation of MCF-7 cells activates Src through Cys498 S-nitrosylation which increases cell invasion and metastasis [31]. S-nitrosylation of Cys118 is required for NO/RSNO-mediated activation of Ras. Low concentrations of RSNO/NO S-nitrosylate Ras, triggers downstream Raf/MEK/ERK1/2 MAP kinase cascades followed by cell proliferation [32]. Activation of the Ras signaling pathway by low levels of GSNO (S- nitrosoglutathione) in HeLa human cervical cancer cells enhances cell Fig. 4. NO activated pathways during stimulation of apoptosis. (Source: Yuste et al. proliferation [33]. S-nitroso-N-acetylpenicillamine (SNAP)-mediated Implications of glial nitric oxide in neurodegenerative diseases. Front Cell Neurosci. 2015; stimulation of ERK1/2 MAP kinases reduces the physiological inhibitor 9:322. http://dx.doi.org/10.3389/fncel.2015.00322). of Trx, Trx interacting protein (Txnip) and enhances cell survival [4]. Besides cell survival, permission of nuclear translocation of Trx is also accumulate and thereby blocking apoptosis [25];c)S-nitrosylation of associated with tumor progression and metastasis [34]. OGG1 (8-Oxoguanine glycosylase) DNA repair enzyme thus in- As NO levels increase above 300 nM, an increased phosphorylation activating it which leads the increase of 8-hydroxydeoxyguanine and of p53 occurs, which associates with anti-proliferative and/or apoptotic associated mutations [22];d)S-nitrosylation of PTEN tumor suppressor responses [27] (Fig. 4). Endoplasmic reticulum stress, disregulation of which then leads the increase of Akt-mediated signaling thus stimu- iron homeostasis, activation of p38 MAPK, blockage of mitochondrial lating tumorigenesis [26]. respiration/mitochondrial permeability transmission pore (MPT), re- Low to medium levels of NO (< 300 nM) trigger cell proliferative lease of cytochrome C from mitochondria to cytosol and subsequent

70 İ M.A. Altinoz, . Elmaci Nitric Oxide 79 (2018) 68–83 activation of intrinsic apoptotic cascades contribute to antitumor effects inhibiting p65-DNA binding activity and thus blocking NF-κB-depen- of high NO levels [27] (Fig. 4). The redox micro-environment also in- dent target gene expression (Fig. 5) [42]. Furthermore, nitroalkenes are fluences the actions of NO. Versatile biological responses elicited by NO robust endogenous ligands for peroxisome proliferator-activated re- can be attributed both to the concentrations of NO and the type of ROS ceptor-γ (PPARγ) although they also activate PPARα and PPARδ at which NO or NO derivatives interact with [27]. increasing concentrations (Figs. 5 and 6) [44]. Finally, nitroalkenes Intracellular nitrosative stress is caused by the combination of high exert other signaling features, such as releasing NO. Here it shall be NO concentrations with other reactive nitrogen species including underlined that inflammatory immune reactions may propagate tumor − ONOO [4]. Antitumor cytokines such as interferon-γ and tumor ne- growth, while selective acquired immune responses are necessary to crosis factor (TNF)-α stimulates iNOS in macrophages to produce high achieve a net inhibitory effect on cancer progression. PPARγ activation − levels of NO and of ONOO , leading tumoricidal efficacies [35,36]. is linked to anti-inflammatory and anti-glioblastoma effects and very Sustained overproduction of NO is pro-apoptotic, stimulating caspases noteworthy, usage of pioglitazone (an antidiabetic PPARγ agonist) was through the release of cytochrome-C from mitochondria into the cy- found to be lesser in anaplastic astrocytoma and glioblastoma patients, tosol, upregulation of p53, and downregulation of the antiapoptotic indicating a protective effect of PPARγ against high-grade gliomas [45]. protein Bcl-2 [37] (Fig. 4). NO donors have varying half-lives that ex- Therefore, PPARγ binding and anti-inflammatory features of nitrated tend from seconds to days. They potentially have the capacity to trigger fatty acids may indicate for their likely antitumor effects in gliomas. tumor cell apoptosis, block metastatic pathways, and inhibit angio- OA-NO2 and other fatty acid nitroalkenes may also signal via pleio- genesis. Classes of NO donors used in cancer include: Organic nitrates tropic mechanisms including Kelch-like erythroid cell-derived protein (glyceryl trinitrate), NONOates (Diethylene-triamine DETA/NO), hy- with CNC homology (EHC)-associated protein-1 (Keap1), and nuclear brid NO–drugs (NO–aspirin), metal–NO complexes (SNP), and RSNOs factor (erythroid derived)-like-2 (Nrf2)-regulated antioxidant response (GSNO) [37]. In the most studied SNP (sodium nitroprusside), the ni- genes (Fig. 5) [43]. trosyl moiety in the nitroprusside anion exerts NO+ character and is There exist many papers about nitrated nucleotides, which mediate very reactive against thiolate anions [38]. Upon addition of SNP to intracellular signaling events. Due to space constraints of this review tissues, there is production of nitrosyl-iron complexes with thiols, di- article, we mainly focused on 8-Nitroguanosine 3′,5′-cyclic monopho- nitrosyl-iron complexes, and RSNOs. RSNOs are decomposed by tran- sphate (8-nitro-cGMP), since it relates with cellular senescence and sition metal ions, Cu+ and Fe2+, and by ascorbate, generating NO [4]. mitochondrial heat shock proteins which associate with intrinsic In the presence of a thiol, RSNOs will react and form new RSNOs by the apoptosis pathways. 8-Nitro-cGMP is a unique derivative of guanosine trans-nitrosation reaction. The newly formed RSNOs might be more 3′,5′-cyclic monophosphate (cGMP) synthesized in mammalian and reactive for structural reasons [39]. The nonselective toxic systemic plant cells in response to formation of NO and ROS [46]. 8-Nitro-cGMP effects by most of the NO donors have limited their use. Selective tu- possesses signaling activity inherited from parental cGMP, including moricidal efficacy and reduced cytotoxicity in normal cells are desirable stimulation of vasorelaxation through activation of cGMP-dependent for a chemotherapeutic NO donor. A series of RSNOs derived from N- protein kinase. On the other hand, 8-nitro-cGMP activates cellular acetylpenicillamine and referred to as S-nitroso-aryl-butanamides were signaling pathways that are not seen with the native cGMP, e.g., it acts synthesized. The ortho- and meta-chloro derivatives of the S-nitroso- as an electrophile and reacts with protein sulfhydryls, which leads to aryl-butanamides acted potently cytotoxic on MCF-7 human breast cGMP adduction to protein sulfhydryls (protein S-guanylation) [46]. cancer cells, while sparing human fibroblasts from normal breast tissue Several proteins are targets for endogenous protein S-guanylation, in- [40]. cluding Kelch-like ECH-associated protein 1 (Keap1), H-Ras, and mitochondrial heat shock proteins. Keap1 is a negative regulator of nu- 3. Nitrated fatty acids and nitrated nucleotides as electrophilic clear factor erythroid 2-related factor 2 (Nrf2), a transcription factor secondary NO-metabolites involve in inflammation and cancer which controls the expression of phase II detoxifying and antioxidant enzymes for electrophiles and ROS (Fig. 5) [46]. Binding of Keap1 to Nitro-derivatives of unsaturated fatty acids are structurally defined Nrf2 maintains the cytosolic localization of Nrf2 and mediates rapid and measured in plasma, erythrocytes and urine of humans [41]. Al- degradation of Nrf2 by proteasomes. Chemical modifications of the though dietary sources may contribute to tissue nitro-fatty acids, these Keap1 sulfhydryls of thiol residues by electrophiles and ROS leads to molecules are formed by oxidative and nitrative reactions associated dissociation of Nrf2, which would lead to its nuclear translocation to with inflammation (Fig. 5). Mechanisms known to mediate fatty acid provide antioxidant defense [46]. On the other hand, cell culture stu- nitration to nitroalkene derivatives include nucleophilic (nitronium dies have shown that 8-nitro-cGMP strongly triggers cellular senescence group, NO2+) and/or radical (nitrogen dioxide, %NO2) addition reac- and growth arrest in a manner depending on H-Ras S-guanylation in tions with olefinic carbons [41]. Because they have a β-carbon adjacent addition to phosphorylation of p38 MAPK, ERK, p53 and Rb proteins to the nitro-bonded carbon, nitrated fatty acids exert electrophilic [46]. S-Guanylation proteomics revealed existence of S-guanylation on properties, and can facilitate nitroalkylating Michael addition reactions mitochondrial heat shock proteins including mitochondrial stress-70 with biological nucleophiles, such as cysteine, histidine and lysine re- protein (mortalin) and 60-kDa heat-shock protein (HSP60) in C6 glioma sidues of proteins (Fig. 6) [42]. Michael addition reactions overall link cells stimulated with LPS and cytokines [46]. HSP60 can directly as- cellular metabolic and redox homeostasis with the posttranslational sociate with various mitochondrial heat shock proteins to form multi- regulation of target proteins [43]. Nitrated free fatty acids (NO2-FA), chaperone complex, which involves in the maintenance of mitochon- mainly the nitroalkene derivatives of linoleic acid (nitrolinoleic acid; drial integrity and functions. In this context, 8-nitro-cGMP stimulates

LNO2) and nitro-oleic acid (OA-NO2) exert several signaling features, the mitochondrial permeability transition pore (mPTP) opening, which which are formed via NO-dependent oxidative reactions (Fig. 5) [44]. is an important step in triggering the intrinsic apoptotic cascades [46].

Plasma concentrations are ∼0.5 μM for LNO2 and ∼0.6 μM for OA-NO2 in healthy human blood, and the combined blood levels of these two 4. Versatile effects of nitric oxide and S-nitrosylation on cellular fatty acid derivatives exceed 1 μM, suggesting their likelihood to act in energetic pathways physiological ranges. Nitroalkenes possess potent anti-inflammatory properties as they block human neutrophil superoxide generation, de- 4.1. Dual effects of NO on mitochondria: suppression of mitochondrial granulation, and integrin expression, and they also lower lipopoly- respiration and induction of mitochondrial biogenesis saccharide-induced secretion of inflammatory cytokines and NF-κB activity in macrophages [41,44]. Inhibition of NF-κB activity was NO enhances the energetic substances and oxygen availability to shown to occur via alkylation of the NF-κB p65 protein in macrophages, cells by increasing blood flow, yet NO also blocks mitochondrial

71 İ M.A. Altinoz, . Elmaci Nitric Oxide 79 (2018) 68–83

Fig. 5. Nitrated fatty acids and their involvement in cellular signaling. Nitrated oleic and linoleic acids were demonstrated as examples. Keap1: Kelch-like erythroid cell-derived protein with CNC homology (EHC)-associated protein-1. Nrf2: Nuclear factor (erythroid derived)-like-2. PPARγ: Peroxisome Proliferator Activating Receptor-γ. (Sources: Wang et al. Protection of nitro-fatty acid against kidney diseases. Am J Physiol Renal Physiol. 2016; 310(8):F697-F704. http://dx.doi.org/10. 1152/ajprenal.00321.2015; Web Site of Gustavo Bonacci, PhD from Pittsburgh University http://www.pharmacology.us/ Faculty/GustavoBonacci).

respiration in multiple pathways (Fig. 7a and b). CcO (Cytochrome c high levels of NO, likely via the disruption of iron-sulfur complex [55]. Oxidase, complex IV) is very vulnerable to NO inhibition compared to Complex III activity can be reversibly inhibited by high levels of NO in other mitochondrial complexes; nanomolar concentrations of NO at puriﬁed enzymes or submitochondrial particles. Tyrosine nitration in β physiological levels (1–200 nM) inhibit mitochondrial respiration at subunit of complex V inhibits the enzyme activity [56]. Overall, mi- CcO [47,48]. NO reversibly blocks mitochondrial oxygen consumption tochondrial respiration and ATP synthesis can be either reversibly or by competing with oxygen at the CcO heme iron:copper dinuclear irreversibly inhibited by NO and NO derivatives. center [27]. High levels of NO or prolonged incubation can also cause NO plays important roles in mitochondrial proliferation (Fig. 8). persistent inhibition of CcO via the S-nitrosylation of cysteine residues Mitochondrial biogenesis is mainly controlled at the level of tran- in subunit II [49] and by lowering of CcO protein level [50]; [51]. scription and can be stimulated by various factors [57]; [58]. Calorie- Mitochondrial complex I can be blocked by NO mainly through the restricted mice had increased eNOS expression, mitochondrial proteins indirect NO reactions of S-nitrosylation [52] and/or tyrosine nitration and respiration; in addition, lower mitochondrial density in tissues of [53]. NO-dependent inactivation of complex I is accelerated under eNOS-knockout mice indicates NO generated from eNOS involves in hypoxia [54]. Puriﬁed mitochondrial complex II can be inhibited by mitochondrial biogenesis [59]. In some cells the increase of

Fig. 6. Antiinﬂammatory roles of nitro-fatty acids and role of nitrated proteins in mitochondria-driven apoptosis. (Adapted from: Rubbo. Nitro-fatty acids: novel anti-in- ﬂammatory lipid mediators. Braz J Med Biol Res. 2013; 46(9):728–34. http://dx.doi.org/ 10.1590/1414-431X20133202).

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Fig. 7. NO blockage of mitochondrial respiration steps. (Adapted from: Brown. Nitric oxide and mitochondrial respiration. Biochim Biophys Acta. 1999; 1411(2–3):351–69; and from Carreras and Poderoso. Mitochondrial nitric oxide in the signaling of cell integrated responses. Am J Physiol Cell Physiol. 2007; 292(5):C1569-80). mitochondrial proteins and DNA (mtDNA) content by NO, from NO (GAPDH) [67] and aldolase [68]. donors or eNOS expression, were accompanied by an increase of cGMP and transcription factors responsible for mitochondrial biogenesis [27]. 5. Particular relevance of nitric oxide-associated pathways in These include PGC-1α (peroxisome proliferator-activated receptor treatment of glioblastoma gamma coactivator-1α), NRF-1 (nuclear respiratory factor-1), and mtTFA (mitochondrial transcription factor A) (Fig. 8) [59,60]. 5.1. Basal activity of NOS in gliomas and association with general features of malignancy 4.2. Nitric oxide effects on glycolysis Induction of iNOS was revealed in brain tumors and the extent of A biphasic effect of NO on glycolysis occurs, which is cell type-de- iNOS expression correlates with the degree of malignancy [69]. NO also pendent. In general, low/physiological levels of NO stimulate glycolysis changes vascular reactivity and triggers neoangiogenesis in favor of and high levels of NO inhibit it [27]. NO increases glycolysis in astro- tumor growth [69]. By using magnetic resonance imaging (MRI), in- cytes via mitochondrial inhibition, which results in glucose uptake creased NO secretion in experimental gliomas was demonstrated to [61,62]. This activation of glycolysis in astrocytes is cGMP-in- stimulate neovascularization in vivo [70] and chronic systemic NOS dependent, while NO failed to stimulate glycolysis in neurons sug- inhibition in rats bearing C6 glioma by Ng-nitro-D-arginine methyl ester gesting the NO-effect is context-dependent [61]. HIF-1α plays an im- treatment resulted in decreased tumor blood flow and lowered tumor portant role in NO-dependent activation of glycolysis [27]. Under volume [71]. In human gliomas, a positive correlation was found be- hypoxia, NO decreases HIF-1α stability by redistribution of oxygen tween in vivo nNOS and eNOS expression and tumor cell proliferation as from mitochondrial respiration to other uses, leading to HIF-1α-pro- measured by Ki-67 staining [70]. teasomal degradation [63]. In contrast, NO can create “pseudohypoxia” EGFRvIII is specifically implicated in glioblastoma pathogenesis, for HIF-1α activation under normoxia, which is an important pathway STAT3 triggers iNOS transcription specifically in EGFRvIII-expressing for tumor angiogenesis and progression [64,65]. NO also enhances HIF- astrocytes and selective iNOS inhibitors 1400W and S-MIU and the NO 1α levels in astrocytes via PI3K/AKT/mTOR signaling leading to the scavenger c-PTIO reduce the proliferation astrocytes and glioblastoma stimulation of glycolytic enzymes and glycolysis [66]. Nonetheless, cells that express EGFRvIII [5]. Noteworthy, inhibition of iNOS lowers excess levels of NO inhibit glycolysis. High flux of NO enhances the glioma invasiveness and iNOS knockdown in EGFRvIII-expressing as- production of NO derivatives which leads to the inhibition of glycolytic trocytes fail to produce tumors or lead to significantly smaller tumors enzymes, including glyceraldehyde-3-phosphate dehydrogenase [5]. Among the different NOS isoforms, iNOS appears to be the only

Fig. 8. NO involvement in mitochondrial injury versus biogenesis. (Source: Tengan et al. Nitric oxide in skeletal muscle: role on mitochondrial biogenesis and function. Int J Mol Sci. 2012; 13(12):17160–84. http://dx.doi.org/10.3390/ijms131217160).

73 İ M.A. Altinoz, . Elmaci Nitric Oxide 79 (2018) 68–83 isoform that exerts higher expression in brain tumor stem cells (BTSC), efficient agents for chemosensitization, while the reverse may be re- independent of whether CD133 or CD15 are used for their enrichment. levant for temozolomide where NO-donors may enhance its tumoricidal BTSCs produce higher levels of NO and depletion of NO impairs their activity. growth and sphere formation, without hindering neurosphere forma- Both oral L-arginine and hydroxyurea increased tumor permeability tion of healthy neural progenitors [5]. Lastly, and maybe most im- by increased production of NO from eNOS in tumor vessels in the rat 9L portantly, treatment with iNOS inhibitors in mice implanted with glioma model; increased vascular permeability resulted from the joint − human glioma xenografts reduces tumor volumes [5]. The authors have effects of NO and peroxynitrite (OONO ) [70]. This finding is espe- proposed several molecular cascades, which would associate with iNOS cially noteworthy, since very high dosages of hydroxyurea can be re- propagation of glial tumor growth. iNOS reduces Cell Division Auto- latively safely employed in oncological practice, which traverses the antigen-1 (CDA1) expression in BTSCs, which is a tumor-suppressive blood-brain barrier and was shown to slower growth of high grade- cell cycle inhibitor [5]. NO also increases expression of IL-8 which as- meningiomas [76]. In future, high dose hydroxyurea may also be em- sociates with higher invasion and poor prognosis of various cancers and ployed to enhance delivery of other anti-glioma drugs by increasing enhanced angiogenesis in gliomas [5]. eNOS and associated vascular permeability. Indeed, induction of en- Data obtained from the REMBRANDT (Repository of Molecular hanced vascular permeability by a NO-donor found to potentiate the Brain Neoplasia Data) database indicates that increased iNOS expres- effect of carboplatin on C6 glioma [70]. Nitromemantines may also sion correlates with decreased survival of patients with glioma [72]. enhance chemotherapeutic drug-permeability in gliomas via inducing iNOS stimulation of glioma growth was shown to associate with mi- selective accumulation of NO in the vicinity of NMDAR overreactive croglial activation and S-nitrosylation of mitochondrial caspase-3 [72]. tumor regions, as will be explained below. Until recent times, caspases are only appraised as apoptosis inducing Glutathione-S-transferases (GST) are stimulated in malignant “killers”, yet increasing evidence suggests that caspase-3 activation can gliomas and involve in their chemoresistance [77]. The diazeniumdio- occur in various brain cell types as a part of various essential cell late NO donor PABA/NO generates NO upon selective enzymatic acti- functions. In fact, controlled activation of caspase-3 contributes to the vation by GST-π [77]. PABA/NO monotherapy demonstrated a strong activation of microglia in the absence of cell death [72]. Glioma cells dose-dependent growth inhibitory effect in U87 glioma cells in vitro, recruit and presume on microglia for their growth and invasion as and a strong synergism was witnessed after combined treatment with microglia density in gliomas positively correlates with malignancy and temozolomide, but not with carboplatin [77]. Systemic and in- invasiveness. Tumor cells block the cytotoxic properties of microglia tratumoral PABA/NO administration also significantly decreased cell and transform them to act protumorigenic via release of extracellular proliferation of U87 gliomas implanted into nude rats, yet it was not matrix proteases and cytokines, which promote tumor invasiveness and sufficient to prolong survival alone [77]. A strong caspase 8 staining growth. Indeed, removal of microglia, in both brain organotypic slices was witnessed in U87 gliomas treated with PABA/NO compared to and genetic mouse models, reduces glioma invasiveness [72]. Glioma- normal controls. The authors have underlined that NO could suppress induced microglia transformation was linked to S-nitrosylation and a multidrug resistance genes in cancer and temozolomide resistance is decrease in the basal levels of microglial caspase-3 through inhibition of associated with NF-κB activity, which can be inhibited by S-nitrosyla- thioredoxin-2 (Trx2)’s denitrosylating activity [72]. Blockage of iNOS tion [77]. Furthermore, S-nitrosylation also occurs on GST and NO expression in vivo led to a decrease in both microglia recruitment and exerts inhibitory actions on DNA repair enzymes [77]. The authors tumor growth [72]. attributed the inefficacy of PABA/NO to enhance survival to the fact Finally, Ghosh compared the global gene expression of a glio- that this drug was poorly water-soluble and hypothesized that future blastoma cell line HTB15 in comparison to normal human astrocytes nanoparticle drug delivery systems may be harnessed to successfully using global gene expression microarray, coupled with the Ingenuity employ this NO-donor in glioblastoma treatment. ® ® Pathway Analysis (IPA ) [73]. IPA is a powerful analysis that illumi- Safdar et al. have transformed chlorotoxin (CTX) into a diaze- nates the significance of “omics” data and determines new targets or niumdiolate NO donor [78]. CTX is a 36 amino acid protein extracted candidate biomarkers within biological systems. They have established from the venom of the Deathstalker scorpion (Leiurus quinquestraitus), 5 main canonical pathways that distinguished molecular pathways of which exerts a high affinity for matrix metalloproteinase-2 (MMP-2) normal and malignant astocytes. Besides the inositol pathway, polo like receptor overexpressed in glioma cells but not present in the brain [78]. kinases and tetrapyrrole biosynthesis, the fourth important pathway CTX reacts with NO gas to form an NO-releasing compound, CTX–NO, was defined as nNOS pathway [73]. Here we propose, in therapy per- while sparing its ability to selectively target glioma cells. Using an NO- iods, where cytotoxic treatments were not applied, chronic treatments specific probe, NO release was observed from CTX–NO for over 72 h which suppress NOS activity, such as with nitrones may suppress basal and CTX–NO triggered tumor cell kill in a dose-dependent manner [78]. proliferation and angiogenesis of gliomas. The biochemistry of nitrones Moreover, CTX–NO in fluenced healthy cell viability only at high NO were explained below. concentrations. Preexposure of cells to CTX–NO followed by 48-h exposure to temozolomide resulted in enhanced efficacy of temozolomide 5.2. Nitric oxide and glioma chemosensitivity. Opposite interactions with [78]. They also demonstrated that this combination therapy lowered BCNU versus temozolomide active levels of MGMT and altered p53 activity, both of which are crucial in DNA repair. MGMT methylation and its subsequent silencing Before temozolomide, BCNU (carmustine in the PCV protocol – was shown to profoundly enhance clinical success of temozolomide procarbazine + carmustine + vincristine) was the most commonly chemotherapy due to diminished DNA-repair of glioma cells. S-ni- used adjunct chemotherapy for glioblastoma because of its lipophilic trosylation of the cysteine residue in the active site of MGMT causes the character [69]. Today, it is still being used for chemotherapy of high irreversible loss of DNA-repair activity and quickly targets this enzyme grade oligodendrogliomas. iNOS was shown to reduce cytotoxic activity for degradation [78]. A short graphical description of NO-mediated of BCNU, but not of temozolomide in C6 glioblastoma cells, and this chemo-radiotherapy sensitization is provided in Fig. 9. effect was found to occur via diminishing carbamoylating efficacy of BCNU without altering its alkylating actions [69]. A NOS-inhibitor L- 6. Mutual interactions between P53 and NO signaling with NAME (N(ω)-nitro-L-arginine methyl ester) restored tumoricidal ac- emphasis to glial tumors tivity of BCNU [69]. Recent studies also supported these findings, which demonstrated enhanced transcription of iNOS in C6 glioma cells, Tumor suppressor gene product, p53 is a nuclear phosphoprotein which gained resistance to BCNU [74]. In the context of BCNU, ni- with a molecular mass of 53 kDa [79]. Wild-type p53 protein is present trones, which selectively block iNOS transcription [75], may be in normal cells; but due to its very short half-life, p53 is present in too

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Fig. 9. NO potentiation of chemotherapy and radiotherapy induced DNA damage and involvement in intrinsic apoptosis mechanisms driven by mitochondrial signals. (Adapted from: Turchi. Nitric oxide and cisplatin resistance: NO easy answers. Proc Natl Acad Sci U S A. 2006; 103(12):4337–8; and from Carreras and Poderoso. Mitochondrial nitric oxide in the signaling of cell integrated responses. Am J Physiol Cell Physiol. 2007; 292(5):C1569-80). few amounts in cells, generally below the detection level of im- diminished ability to trigger apoptosis but enhanced DNA repair ability, munohistochemical (IHC) methods [79]. Somatic p53 mutations are which results in increased chemoresistance, which can be blocked by common events in the development of human tumors. Because mutant NO-donors [78]. In this review, we propose to employ nitromemantines p53 proteins often are generally more stable than the wild-type p53 and nitrones in treatment of glioblastoma. Considering important in- protein, mutant p53 proteins accumulate and became detectable with teractions between NO and p53 signaling cascades, basic scientific re- IHC [79]. Wild-type p53 protein functions as a transcription factor, i.e. search which determine relations between the antitumor efficacy of as a modulator which can turn genes either on or off. P53 is a genomic these drugs and p53 status in glioma cells may pave to develop selective gatekeeper, it arrests cell cycle and triggers DNA repair following a and more efficient treatment strategies. DNA-damaging insult; however, when the DNA damage is irrepairable, it triggers apoptotic cell death [80]. Cytokines including interferon IFN-γ, tumor necrosis factor TNF-α 7. Particular relevance of glutamate and NMDA-receptor or interleukin IL-1β and lipopolysaccharide (LPS) stimulates NO associated pathways in treatment of glioblastoma synthesis in rat C6 and A172 human glioma cells, but not in LN-229, T98G or LN-18 human malignant glioma cells [81]. Stimulation of NO Based on the pharmacology of agonist sites, there exist three classes release involved increased expression of iNOS. iNOS gene is a target for of glutamate-gated ion (or ionotropic) channels, known as α-amino-3- negative transcriptional regulation by p53 [81]. In opposite to the hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate, and N-me- human p53 his175 mutant, the murine p53 val135 mutant in its pre- thyl-D-aspartate (NMDA) receptors [86]. Among these, classical NMDA sumptive mutant conformation had a similar inhibitory effect on iNOS receptors (NMDARs) are generally the most permeable to calcium gene activation and suppresses NO toxicity in a conformation-in- (Ca2+). As a consequence of Ca2+ accumulation, extravagant stimula- dependent manner. Forced expression of temperature-sensitive p53 tion of the NMDARs triggers production of noxious free radicals and val135 in C6 glioblastoma cells in either mutant or wild-type form other enzymatic processes leading cell death [86]. NMDARs include blocked cytokine/LPS-induced NO synthesis [81]. Moreover, accumu- different subunits: NR1 (whose existence is mandatory), NR2A-D, and, lation of p53 in both mutant or wild-type form saved glioma cells from in some cases, NR3A or B subunits. The subunit composition determines the toxicity of exogenous NO, consistent with a gain of p53 function the pharmacology of the receptor–ion channel complex [86]. Ex- associated with p53 accumulation [81]. Hence, it was proposed that the citotoxicity is the abnormally high exposure to glutamate or over- resistance to NO-dependent immune defense pathways may involve in stimulation of its membrane receptors, leading to neural cell death the malignant progression of human cancers with p53 alterations, [86]. First in 1992, it was shown that oocytes injected with mRNA from mostly those associated with the accumulation of mutant p53 protein an astrocytoma cell line (R-111) acquired acetylcholine and glutamate [81]. Nonetheless, reductionist approaches to define p53 and NO sig- receptors as well as a small number of NMDARs [87]. naling interactions shall be avoided since NO can cause a conforma- Malignant gliomas diffusely invade cerebral tissues and peritumoral tional change of p53 from wild-type to mutant [81]. It is also proposed brain tissue exerts various inflammatory changes, including activated that NO-dependent tumor cell toxicity could induce a selection process microglia, hypertrophic reactive astrocytes, vascular invasion and that would favor the emergence of cancer cell clones accumulating p53 edema [88]. In 1995, it was shown that NMDA treatment of T67 as- [81]. In glioblastoma cells exposed to irradiation, NO induces accu- trocytoma cells induces NO-mediated release of Prostaglandin E2, a mulation of p53, while wild-type p53 suppresses iNOS expression major inflammatory mediator [89]. Degenerated neurons are witnessed suggesting a feedback regulation following noxious insults [82]. It shall in the vicinity of glioma cells. Another feature of high grade gliomas is be also kept in mind that NO-induced p53 may lack transcriptional epileptia, and epileptic activity in glioblastoma approaches 50% of all activity as peroxynitrite treatment of glioblastoma cells induces tyr- cases, when glutamate may be a trigger and conventional antiepileptics osine nitration of p53 and loss of its DNA binding ability [83,84]. are often ineffective [88]. Glutamate is the primary excitatory neuro- Adding further complexity to p53 and NO interactions, NO toxicity in transmitter in the central nervous system, but it also regulates neu- benign astrocytes was shown to occur by a simultaneous increase in p53 roembryogenesis and neural and astrocytic migration [90]. These ef- serine 18 phosphorylation and p53 translocation from the cytoplasm to fects of glutamate are lost in the adult cerebral tissue but glioma cells the nucleus [85]. Noteworthy, p53-deficient astrocytes were much may regain their migratory response to glutamate [90]. Glutamate also more resistant than wild-type cells to the same NO treatment in this damages peritumoral neural tissues via glutamatergic neurotoxicity and setting. It is likely that differing redox status in benign versus malignant this excitotoxic remodeling of the tissue configuration may propagate cells or even between different types of cancer cells will determine the invasion of glioma cells by minimizing spatial constraints [90]. whether NO may activate or inhibit p53 dependent apoptosis. Indeed, Normal astrocytes serve as a glutamate “sump” contributing to T98G glioblastoma cells produce a mutated p53 that demonstrates a glutamate clearance from the extracellular milieu, which is reversed in gliomas, with decreased reuptake and enhanced secretion of glutamate

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[90]. Normally, glutamate is released from active neurons through can be inhibited with NMDAR antagonist dizocilpine [99]. calcium-dependent fusion of synaptic vesicles, but glioma cells release In an in vitro proliferation-inhibition assay, F98 and 9L cells were glutamate spontaneously; such that a monolayer of glioma cells main- treated with riluzole and memantine and mouse cerebellar organotypic tained in a culture flask for 12 h achieves glutamate concentrations cultures were implanted with F98 glioma cells and treated with radia- exceeding 500 μM [91]. Microdialysis in RG2 glioma implanted into the tion, radiation + riluzole, or vehicle and evaluated for tumor growth rat brain cortex revealed that the peritumoral region undergoes 4-fold [90]. Biosafety and antitumor efficacy of intracranially implanted ri- increase of the glutamate concentration compared with healthy-brain luzole and memantine biopolymer wafers were also studied in F344 rats tissue [91]. MR-spectroscopical studies in humans [92,93] and human [90]. The efficacies of these drugs were analyzed against the 9L model biopsy analyzes also revealed prominently higher glutamate levels in and riluzole was further tested with and without radiation therapy. In peritumoral brain tissues, at concentrations toxic to neurons and as- vitro assays demonstrated significant anti-proliferative effects of both trocytes in vitro [90]. Glutamate release occurs via system X(c), a cy- riluzole and memantine on F98 and 9L cell lines. F98 organotypic stine-glutamate exchanger that releases glutamate in exchange for cy- cultures revealed more prominent reduction of tumor growth treated stine being imported for the synthesis of glutathione, which protects with radiation and riluzole in comparison with cultures treated with tumor cells from reactive oxygen and nitrogen species and provides single radiation or riluzole [90]. Systemic riluzole monotherapy was resistance to radiation- and chemotherapy [94]. ineffective; however, riluzole concomitant with radiotherapy resulted First, it was shown that NMDA receptor antagonist dizolcipine re- in improved survival [90]. Furthermore, three separate experiments all duced cell division and increased cell death in astrocytoma cells [95]. revealed that local polymer-based delivery of riluzole or memantine Moreover, glutamate antagonists decreased motility and invasion of was effective to suppress 9L gliosarcoma tumor in vivo [90]. Over 80% tumor cells with reductions of membrane ruffling and pseudopodial of high-grade glioma recurrences occur within 2 cm of the resection protrusions [95]. These facts led further research, where aminoada- site, definitely due to living tumor cells invading the macroscopically mantanes and riluzole were tested in experimental models of glioma. normal brain [90]. Here, anti-glutamatergic treatment may act bene- Aminoadamantanes (aminotricyclo (3,3,1,13,7) decane) constitute a ficial in versatile mechanisms. At first, anti-glutamatergic agents ex- family of neuroprotective drugs showing non-competitive antagonism erted direct cytotoxic effects on glioma cell lines. Of equal importance, at the NMDA subtype of glutamate receptor [96]. In contrast to other is the novel concept of better outcome via neuroprotection in neuro- NMDA receptor antagonists such as phencyclidine and dizolcipine, oncology. Protection of the peritumoral functional, but possibly in- these molecules exert much higher clinical safety, which is correlated filtrated, brain may be beneficial for multiple reasons including re- with their rapid blocking and unblocking kinetic features [96]. Among duction of glioma cell invasion, seizure activity, peritumoral edema and these drugs, memantine (1-amino-3,5 dimethyladamantane) is em- maintaining 3-dimensional migratory constraints on glioma cell mi- ployed as a neuroprotective medicine in the treatment of dementia such gration [90]. Yoon et al. demonstrated antiproliferative effect of as Alzheimer's disease, whereas amantadine (1-aminoadamantane) is memantine on T98 glioblastoma cells, which expressed N-methyl-D-as- proposed in the management of Parkinson's disease [96]. Riluzole is a partate 1 receptor (NMDAR1), while NMDAR1-negative U251 glio- substituted benzothiazole that is approved by the FDA for the treatment blastoma cells were resistant to memantine [91]. Memantine enhanced of amyotrophic lateral sclerosis and blocks glutamate release [90]. the autophagic apoptosis proteins reflected by the ratio of light chain Implanted glutamate-secreting glioma cells stimulated neuronal protein 3-II (LC3-II)-/LC3-I and the expression of beclin-1 and increased degeneration in vivo and exerted a clear growth advantage in compar- formation of autophagic vacuoles observed under an electron micro- ison to their non-glutamate secreting counterparts. Furthermore, glu- scope [91]. Transfection of small interfering RNA (siRNA) to suppress tamate-secreting gliomas promoted an inflammatory response (CD68+ NMDAR1 in the T98 glioblastoma cells induced resistance to meman- microglia) within surrounding tissue, possibly due to debris of dying tine and lowered the LC3-II/LC3-I ratio in T-98 G cells confirming the neurons and/or glutamate [88]. Importantly, treatment with the NMDA NMDAR-specific antitumor mechanism of memantine [91]. receptor antagonists dizolcipine (1 mg/kg twice daily) or memantine (25 mg/kg twice daily) slowed the growth of glutamate-secreting 8. Nitro-memantines target NMDA-receptor and deliver NO. Could gliomas in vivo [88]. they be new players against glioblastoma? Early growth response-1 (EGR-1) is a central modifier of tumor cell proliferation, migration and angiogenesis [97]. In glioblastoma cell Because glutamate is the major excitatory neurotransmitter, a total lines, EGR-1 binds to GC-rich sites of the human transforming growth inhibition of a glutamate receptor subtype like the NMDAR causes factor-β1 promoter leading to inhibition of proliferation and transfor- untoward effects that limit the clinical utility of generalized inhibitors mation. At the same time, EGR-1 promotes glial tumor cell migration [86]. Open-channel block with uncompetitive antagonism is the most via activation of fibronectin [97]. Two EGR-1 mRNA variants were logical strategy for prevention of excessive NMDAR activation as this defined, one including an NMDAR responsive cytoplasmic poly- action of blockade necessitates prior activation of the receptor. Mem- adenylation element (CPE) [97]. After NMDA stimulation of im- antine, a low affinity NMDAR-blocker with a relatively rapid off-rate mortalized astrocytic cell line SV-FHAS and neoplastic astrocytes, RT- from the channel, is such a drug which exerts open-channel block with PCR analyses showed an increase of the CPE, containing EGR-1 splice uncompetitive antagonism leading its development as the first clinically variant only in astrocytoma cells [97]. The surface expression and tolerated yet effective agent against NMDAR neurotoxicity [86] functionality of NMDAR were shown by flow cytometric analysis and (Fig. 10). NitroMemantines acted highly neuroprotective in both in vitro measurement of increased intracellular Ca2+. Among the 5 astrocytoma and in vivo and exerted higher effectivity than memantine at lower cell line tested, 3 had NMDAR levels above 5%, (U373–5.02%; dosages [100,101] (Fig. 10). U158–6.16%; U251–15.93%). On the other hand, among 5 primary NitroMemantines are second-generation memantine derivatives that glioblastoma cells, only 2 expressed NMDAR above 3% (3.5% and 4,33, were designed to exert higher neuroprotective efficacy. A nitrosylation respectively). EGR-1 was limited to tumor cells expressing NMDAR and site, or sites, exists on the extracellular domain of the NMDA receptor, significantly increased in gliomas compared with normal brain [97]. and S-nitrosylation of this site reduces receptor activity. One of these, Later on, it was demonstrated that accelerating brain to-blood gluta- located at Cys399 on the NR2A subunit of the NMDAR, mediates about mate efflux by decreasing blood glutamate levels with systemic ad- 90% of the inhibitory effects of NO. Nitroglycerin, which generates NO, ministration of oxaloacetate reduces intracranially implanted gliomas can act at this site to limit excessive NMDAR activity and could prevent alone and in synergy with temozolomide [98]. In 2014, it was shown ischemia reperfusion injury in the brain, but its hypotensive effects may that NMDA stimulates growth and proinvasive MMP-2 (matrix me- limit its clinical utilization [86]. Above, we have indicated that ni- talloprotease-2) release from U251 human glioblastoma cells, which troglycerine was found to slower clinical progression of prostate cancer

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areas of the mouse implanted with human gliomas; and overexcitation of the eNMDARs in the peritumoral neurons were proposed to contribute to the development of peritumoral seizures [109]. Hence, Nitro- memantines may also provide very promising candidates to limit refractory epileptias associated with glial tumors. Excessive glutamate release by glial tumor cells may create a viscious cycle of neurotoxicity and tumor propagation due to the release of cytoplasmic glutamate from dying neurons and due to glutamate- stimulation of EGFR-dependent proliferative signals in glioma cells [29,30]. Very noteworthy, nitromemantine blockage of NMDARs was found to occur higher in hypoxic conditions [110], which would target hypoxic core of glial tumors and limit viscious paracrine pathways of self stimulation. Moreover, the higher lipophilic properties of the aminoadamantane nitrates should be beneficial clinically in that they increase their concentration traversing the blood-brain-barrier [110] and reaching therapeutic concentrations within gliomas. Indeed, the lead nitroMemantine candidate developed, designated YQW-036/NMI- 6979, has demonstrated a favorable pharmacokinetic profile, excellent brain penetration, and a good safety index in preclinical studies (Fig. 10) [111]. High levels of glycolysis within glial tumors may render them particularly vulnerable to the S-nitrosylation. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) catalyzes the sixth step of glycolysis and its S-nitrosylation enhances its ability to bind to the ubiquitin E3 ligase Siah. Because Siah possesses a nuclear localization Fig. 10. Binding site of memantine to the NMDA receptor and structures of memantine signal, the SNO-GAPDH/Siah complex translocates to the nucleus and and nitromemantine. (Adapted from Lipton. Pathologically activated therapeutics for activates apoptosis [111] . As suggested above, NO blocks multidrug neuroprotection. Nat Rev Neurosci. 2007; 8(10):803–8; and from Talantova et al. Aβ resistant genes, inactivates NF-κB via S-nitrosylation and enhances te- induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and sy- mozolomide sensitivity of glioblastoma cells [77]; and hence, ni- naptic loss. Proc Natl Acad Sci U S A. 2013; 110(27):E2518-27). tromemantines may be plausible candidates to augment chemotherapy efficacy in gliomas. Lastly, nitromemantine was hypothesized to act [20]. A drug, which traverses the blood brain barrier and releases NO beneficial in HAND (HIV-associated neurocognitive disorder), since it selectively on sites of NMDAR hyperactivation may provide dual ben- protected synapses in a HIV/gp120 transgenic (tg) mouse model [112]. fi e ts, the open-channel block mechanism of memantine would spare Cognitive disfunctioning is increasingly appreciated as important side normal brain tissues while acting as a homing signal for targeting NO to effects of anticancer chemotherapy [113] and radiotherapy [114]. hyperactivated open NMDA channels. In a microenvironment, such as Here, it is very tempting to question, whether nitro-memantines may ffi in the vicinity of a glial tumor with overt glutamate release, this e - spare normal brain tissues without sacrificing (or maybe even po- cacy may increase NO levels to untolerable levels inside glial tumors tentiating) antitumor efficacy of conventional cancer therapies. ff and may provide selective tumoricidal e ects. Lastly, we should admit that there exist no direct evidence that − Here, it shall be also underlined that superoxide anion (O2 ), reacts NMDAR is functionally expressed in glial tumor cell membranes. − fastly with free radical NO to form the very toxic product peroxyni- However, it is certain that glioma cells actively secrete glutamate and − trite (ONOO ) [100]. This would also induce selective toxicity of NO blocking agents of NMDAR signaling reduces glioma progression. within glial tumor cells, as glioblastoma cells have reduced glutathione, Hence it is not illogical to assume that memantine analogues may ac- glutathione reductase, glutathione peroxidase, superoxide dismutase cumulate within the “tumoral penumbra” which contains high levels of and ascorbate in accompaniment with high levels of TBARS (thio- glutamate and more invasive subclones of glioma cells. barbituric acid reactive substances, byproducts of lipid peroxidation) NitroMemantines may be very suitable candidates to target this zone to – [102 105], which may render them particularly vulnerable for addi- deliver cytotoxic NO levels. Here, one may propose that the healthy tional nitrosative/oxidant stress. neurons may also be damaged, but nitromemantines block NMDAR Extrasynaptic NMDAR (eNMDAR) (which contains predominantly activity stronger than memantine and exerted higher neuroprotective NR2B subunits) blocks neuroprotective pathways and triggers neuronal activity in various experimental models, which may balance risks and injury, whereas synaptic NMDAR (sNMDAR) induces neuroprotective benefits of these drugs against healthy neurons. Nitro-memantines were β β transcriptional and antioxidant cascades [106]. Amyloid- peptide (A ) not tested in glioma models as yet, but they highly deserve to be studied α engages 7 nicotinic acetylcholine receptors to induce release of as- in future animal models. We avoided to make exaggerated comments trocytic glutamate, which stimulates eNMDARs on neurons [106].In and underline that we do not recommend any clinical studies with Alzheimer Disease-transgenic mice, whole-cell recordings revealed ex- Nitro-memantines before their safety and efficacy are proven with cessive tonic eNMDAR activity. The Food and Drug Administration- various animal models of glioblastoma. approved drug memantine offered some beneficial effect in this model, but only NitroMemantine completely ameliorated Aβ-induced synaptic – ffi loss [106 108]. In theory, higher neuroprotective e cacies of ni- 9. Blocking basal NO activity in gliomas as a therapeutic strategy. fi tromemantines may add additional bene ts in management of glio- Nitrones as candidates blastoma. Above, a novel neuroprotective concept was suggested in neurooncology, where sparing peritumoral brain tissues may maintain At the sections below, we will define biochemistry and mechanisms 3-dimensional migratory constraints on glioma cell migration and even of action of nitrones and we will propose that they will be efficient protect against refractory seizures in patients with brain tumors. In- agents in treatment of glioblastoma during periods when cytotoxic deed, it was recently found eNMDAR containing NR2B is highly phos- treatments (temozolomide, radiotherapy) are not applied. phorylated at S1013 in the neurons located in the peritumoral brain

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κB DNA binding activity with a lower concentration than that for the non-steroidal anti-inflammatory drug (NSAID), salicylate [75]. NF-κBis a transcription factor that can swiftly trigger the expression of genes involved in inflammatory, immune and acute phase responses. The binding of NF-κB to iNOS gene is critical for iNOS gene expression, and the promoter region of COX2 gene also contains an NF-κB consensus sequence [75]. Blockage of iNOS, COX-2 [118] and NF-κB [119] activities were all shown to reduce tumor growth and indeed, it was found that nitrone compounds have promising effects in inhibiting cancer cell Fig. 11. General structure of a nitrone molecule (Source: http://www.wikiwand.com/en/ proliferation and angiogenesis [115]. First, PBN was shown to inhibit Nitrone). hepatocarcinogenesis [120] and the angiogenic, ischemic, and growth features in F98 and C6 models of rat glioma [121,122]. In hepatocytes 10. Chemical structure of nitrones and their abilities to block free isolated from livers of rats treated with the choline deficient diet (to radical reactions stimulate liver carcinogenesis), it was revealed that a significant amount of these cells produced NO and that PBN lowered the produc- A nitrone is a functional group in organic chemistry, and is an N- tion of NO, while isolated hepatocytes from rats receiving the normal + − oxide of an imine. The general structure is R1R2C=NR3 O where R3 diet did not produce NO [22]. PBN decreased iNOS expression in the is not H. A nitrone is a 1,3-dipole and is used in 1,3-dipolar cycload- liver of rats fed the choline deficient diet. Therefore, the researchers ditions. Other reactions of nitrones include formal [3 + 3] cycloaddi- have proposed that NO enhanced cancer development and that PBN tions to form 6-membered rings, as well as formal [5 + 2] cycloaddi- mediated-suppression of NO production was associated with apoptosis tions to form 7-membered rings. The general chemical structure of a of pre-neoplastic cells and prevention of tumorigenesis [22]. nitrone can also be described as X-C=N(O)Y; and in the case of PBN (N- tert-butyl-α-phenylnitrone) X is a phenyl group and Y is a tert-butyl 13. Bioactivity of nitrones in experimental glial tumor models group [22] (Fig. 11). PBN and related nitrones were first employed in experimental studies mainly due to their ability to “spin-trap” free ra- Initial studies to evaluate the anti-cancer activity of nitrones in dicals. The spin trapping is the reaction of a spin trap compound (e.g. a gliomas were performed by prophylactically treatment with the parent nitrone) with reactive free radicals (e.g. carbon centered radicals, such nitrone, PBN, in drinking water (0.065% w/v, 75 mg/kg/day) [122]. as lipid radicals; or oxygen-centered radicals, such as the hydroxyl (% Rats were given PBN starting at 5 days prior to intracerebral im- OH) or lipid alkoxyl (%OL), or lipid hydroperoxyl (%OOL) radicals) to plantation of C6 rat glioma cells, and continuously for a period of 45 develop more stable radicals that can be quantified with electron spin days. MRI was employed to determine tumor volumes and doubling resonance (ESR) or electron paramagnetic resonance (EPR) spectro- times, and MR angiography (MRA) was used to monitor angiogenesis scopy [22]. Nitrones became popular spin traps in biological system [122]. PBN was able to prevent and/or suppress tumor volumes (by studies due to their relative hydrophilicity, low toxicity, solubility and ∼60-fold, with significance, p < 0.001), enhance animal survival stability [22]. (> 90%), and lower total tumor blood volumes (by ∼20%) [122]. Another cohort of rats were intracerebrally implanted with C6 glioma 11. High biosafety of nitrones cells, monitored for tumor growth, and when tumors reached a volume of ∼50 mm3 (approximately at 15 days postintracerebral implanta- Important chemical features differentiate PBN from its disulfonyl- tion), PBN was administered (drinking water, 0.065% w/v) for a period phenyl derivative, OKN-007. OKN-007 is an ionic and highly water of 25 days. In the post-tumor treatment group, PBN increased survival soluble molecule with relatively little uptake into cells unless they are (40% of the treated rats, p < 0.05), and lowered tumor volumes by anaerobic [22]. OKN-007 is first synthesized as NXY-059 in the mid ∼2-fold [122]. 1990s and taken through clinical studies of ischemic stroke. Although Garteiser et al. employed the T2-, diffusion-, and perfusion- phase III human clinical trials failed to demonstrate its efficacy in stroke weighted MR-imaging to assess the anticancer efficacies of the nitrone [115], OKN-007 was demonstrated to be very safe when given i.v. at compound OKN007 in a C6 rat glioma model [115]. In this study, rats high levels in healthy individuals as well as older subjects and in those in the treated group were given OKN007 at a dose of 9.42 ± 0.66 mg/ that had a stroke [22]. OKN-007 has undergone intensive human safety kg body weight/day starting at day 14 after cell implantation [115]. testing and has been demonstrated to be safe, even at high micromolar The implantation of C6 cells in the brain resulted in the death of all concentrations (300 μM) at which it is active [116]. In the clinical trials animals, with a median survival time of 21 days. Animals in the for acute stroke (SAINT I and II), a serum concentration of 260 μM for treatment group had a higher survival rate (p < 0.0001), as only 8.3% OKN-007 was maintained, which was proven to be safe. OKN-007 is of the cohort died during the study. The volume difference between more effective in trapping hydroxyl radicals compared to the parent groups was marked at day 25 (untreated: 151.6 ± 36.1 mm3, treated: nitrone PBN; and OKN-007-adduct that was formed also resulted in 50.3 ± 12.3 mm3). The initial growth phase observed in treated tu- more rapid degradation, possibly due to acidic conditions [22]. mors exerted a slower growth rate than the fastly proliferating untreated tumors. During this phase, the treated animals had normal 12. Pharmacological mechanisms of nitrones and their general diffusion and perfusion values at the tumor site, whereas the tumors in anticancer activity untreated animals were hyperdiffusive and hypoperfused [115]. Later, the treated animals exerted a phase of tumor volume decrease, where in Besides neuroprotection, pharmacological activities of PBN in an- some instances, characterized by the replacement of tumor areas by imal models include anti-aging and anti-diabetic effects, indicating that tissue that was histologically indistinguishable from healthy brain. In the action of PBN is largely anti-inflammatory. The abilities of PBN to this recession phase, tumors were characterized by a markedly high inhibit the signaling pathways of NF-κB, inducible nitric oxide synthase ADCz and by perfusion values returning to normal levels [115]. (iNOS) and cyclooxygenase (COX) were proposed as possible mechan- OKN007 efficacies on the early stage perfusion and diffusion prop- isms for its pharmacological properties [75,117]. PBN lowers steady erties of C6 tumors indicates some promising effects on the tumor state COX-2 mRNA level and COX-2 catalytic activity in macrophages vasculature. The normalization of tumor vasculature is a beneficial [75]. While PBN decreases iNOS mRNA, it does not block iNOS catalytic feature in cancer therapies by increasing tumor oxygenization to sen- activity. PBN inhibits lipopolysaccharide-mediated enhancement of NF- sitize against chemo-therapy and to reduce the morbidity of

78 İ M.A. Altinoz, . Elmaci Nitric Oxide 79 (2018) 68–83 peritumoral edema. Anti-inflammatory properties of nitrone com- NAA may associate with a good prognosis (reflecting the reduced pounds including OKN-007 may have positively effected the integrity neuronal degradation directly induced by tumor cells or their meta- and function of the tumoral microvascular bed [115]. In turn, this early bolites); and OKN007 was found to increase NAA concentrations [123]. stage normalization may have provided the necrosis of the tumor tissue In OKN007-treated gliomas, Ki67-indices were found to be lower, in- at later phases, which was characterized by tumor volume-regression, a dicating suppression of tumor cell proliferation. The findings of this dramatically increased ADCz and a normal perfusion for the tumor. The study also indicated that apoptotic pathways may mediate antitumor expressions of iNOS, VEGF-R2, and c-Met in treated tumors were also actions of OKN007. Caspase-3 is essential to ensue apoptosis by much lower which were also are consistent with the effects of the drug cleaving the cellular substrates including cytokeratin 18 (CK18), which on the microvasculature [115]. The decrease of VEGF-R2 and c-Met, is an early cleavage product of caspase-3. CK18 was significantly in- both of which are often overexpressed in tumors and correlated with creased in the OKN007-treated C6-glioma, while an untreated glioma malignancy and angiogenesis [115], is also an important indicator for and the contralateral normal brain tissue had negative staining [123]. the prominent potential of OKN007 in treatment of cancers and GBM. Towner et al. evaluated efficacy of OKN-007 in more aggressive He et al. analyzed the antitumor efficacies of PBN and OKN007 in glioma models, such as the F98 model, and in human derived U87 several rodent glioma models (C6, RG2, and GL261) by measuring glioma cell xenografts [125]. The F98 rat glioma exerts an infiltrative metabolite alterations with MR-spectroscopy (MRS) [123]. PBN or pattern of growth, has features similar to human GBM gliomas, and is OKN007 was administered in drinking water before or after tumor classified as an anaplastic tumor. Similarly, U87 human glioma tumors formation [123]. MRI and single-voxel point-resolved spectroscopy in immunocompromised rodents are highly cellular with atypia, in- were employed to determine tumor morphology and metabolites. In- cludes mitotic figures and irregular nucleoli, and intense neovascular- dividual metabolite concentrations and major metabolite ratios (cho- ization [125]. Animal survival was significantly enhanced (p < 0.001 line, N acetylaspartate, and lipid (methylene or methyl), all compared for F98, p < 0.01 for U87) in the group that was treated with OKN-007 to creatine) were determined [123]. The major metabolites of the 1H [125]. After MRI detection of F98 gliomas, OKN-007, administered spectrum seen in brain tumors are choline (Cho; 3.2 ppm), creatine (Cr; orally, reduced tumor growth (p < 0.05). U87 glioma volumes sig- 3.0 ppm), N-acetylaspartate (NAA; 2.0 ppm), and lipids (Lip; nificantly decreased (p < 0.05) after OKN-007 treatment. OKN-007 0.9–1.5 ppm) [123]. The Cho peak is a combination of the total choline treatment significantly decreased tumor hypoxia (HIF-1α (p < 0.05) content of free choline, phospho-, glycerophospho-, and phospholipid- in both F98 and U87), angiogenesis (Microvessel Density – MVD choline [123]. An increase in total Cho in tumors arises primarily from (p < 0.05) in F98 or U87), and cell proliferation (Glut-1 (p < 0.05) in the synthesis and accumulation of phosphocholine, a metabolite pro- F98, (p < 0.01) in U87; and MIB-1 (p < 0.01) in F98) and caused a duced by the rapid uptake and phosphorylation of choline that is ne- significant enhancement of apoptosis (cleaved caspase 3 (p < 0.001) cessary for the synthesis of phosphatidyl-choline, a major membrane in F98, (p < 0.05) in U87), compared with untreated animals [125]. phospholipid constituting around 25% of mammalian cellular lipids OKN-007 is also an inhibitor of human sulfatase 2 (SULF2) which [124]. As enhanced membrane synthesis associates with enhanced acts as an oncoprotein in hepatocellular carcinoma (HCC) by provoking proliferation, decreases in Cho/Cr ratio may reflect decrease in tumor tumor growth and metastasis via increasing fi broblast growth factor-2 cell proliferation [124]. Cr peak includes the creatine and phospho- (FGF-2)/extracellular signal-regulated kinase (Erk) and WNT/β-catenin creatine (PCr) pool and is a mediator of energy metabolism [123]. NAA cascades [116]. SULF2 also activates the TGF-β and Hedgehog/GLI1 is generated by neurons and reduced during neuronal or axonal da- pathways in HCC. SULF2 is an extracellular enzyme which removes 6- mage. O-sulfate groups from heparan sulfate proteoglycans (HSPGs), which Membrane lipids are usually below the detection limits of 1H MRS, present on the cell surface and in the extracellular matrix. HSPGs act as because of their decreased mobility and strong dipolar couplings [123]. sequestration sites for growth factors and as coreceptors in cell sig- On the other hand, in disease conditions, fatty acid-release from naling pathways [116]. Desulfation of HSPGs by SULF2 release growth membranes and/or lipid droplets increases the free lipids, which appear factors from sequestration sites and making them available to activate as sharp peaks at 1.3 (methylene group) and 0.9 ppm (methyl group) cell growth and migration. SULF2 is upregulated in breast cancer and [123]. In brain tumors, the elevation of the lipid levels usually corre- exerts protumorigenic effects in lung and pancreas cancer [116]. OKN- lates with necrosis [124]. Necrosis is a hallmark of GBMs, with some 007 triggered apoptosis of HCC cells with accompanying increases in studies indicating it exists in over 85% of cases, which is caused by caspases-3 and -7 and significantly blocked tumor cell proliferation, tumor hypoxia as a result of increased cell proliferation, as well as in- viability, and migration. All of the anticancer effects occurred dose- adequate tissue perfusion [124]. Necrotic cascades begin with acute dependently, but the effects on apoptosis and migration occurred at cellular ATP decrease as a result of electron transport chain collapse lower doses than the effects on cell proliferation and viability [116].In and subsequent lowered oxidative phosphorylation. This lack of ATP HCC cells, the anticancer activity of OKN-007 was more prominent in causes a failure in the ATP-dependent ion channels, which triggers a cells expressing SULF2, which was also verified with in vivo experi- massive cell volume increase through Na+ influx. As the cell membrane ments. OKN-007 also suppressed both TGF-β1/SMAD and Hedgehog/ ruptures, the contents of the cells are released into the extracellular GLI1 signaling cascades [116]. space, which will lead to the presence of high lipid peaks that are de- OKN-007 was found to significantly decrease (p < 0.05) free ra- tected in 1H-MRS [124]. PBN and OKN007 suppressed tumor growth in dical levels in treated and F98-glioblastoma implanted rats compared to C6 gliomas when applied either as a prophylactic or after tumor for- untreated rats [126]. OKN-007 also lowered NFE2L2 (nuclear factor mation, respectively; and OKN007 was more effective than PBN when erythroid 2-related factor 2), iNOS, 3-nitrotyrosine, and mal- administered after tumor formation in the C6 glioma model [123]. ondialdehyde in ex vivo F98 glioma tissues via IHC, as well as lowered Nitrones changed tumor metabolism and shifted major metabolite ra- 3-nitrotyrosine and malondialdehyde adducts in vitro in F98 cells tios close to their normal levels including decreases in the lipid (me- measured via ELISA [126]. One of the strongest proangiogenic mole- thylene)-to creatine ratio, as well as the estimated concentration of lipid cules is the vascular endothelial growth factor (VEGF), which acts on (methylene) significantly [123]. Alterations in lipids reflected the vascular endothelial cells through compatible VEGF receptors (VEGFR)- therapeutic efficacy associated with treatment and were found to be 1, -2, and -3 [127]. VEGF also triggers reactive oxygen and nitrogen related to the reduction of necrosis, but not apoptosis [123]. species (RONS) production via Rac1-mediated NADPH oxidase and also

PBN had less anti-tumor eﬀect when applied to established C6 elevates levels of mitochondria-derived hydrogen peroxide (H2O2) gliomas in comparison to OKN007. PBN was also found not to suppress [127]. RONS, in turn, potentiate VEGFR phosphorylation and upregu- the growth of more invasive RG2 or GL261 mouse gliomas. The level of late VEGF secretion and VEGFR expression through induction of HIF-1α NAA is inversely related with brain tumor volumes and high levels of inducing a viscious cycle of tumor angiogenesis. VEGFR-1 and VEGFR-2

79 İ M.A. Altinoz, . Elmaci Nitric Oxide 79 (2018) 68–83 are highly expressed in different types of cancers such as gliomas [127]. 14. Future prospects and conclusions Glioblastoma is one of the most vascular tumors and intra-tumoral VEGF and VEGFRs correlate with the histological grade of gliomas. In this review, we tried to collect data which suggest that agents With a molecular MRI (mMRI) method employed in a mouse GL261 targeting NO and glutamate/NMDAR-associated pathways may exert glioma model, it was shown that OKN-007 could enhance survival and antitumor activity, with special emphasis for high grade glial tumors. suppress level of VEGFR-2, which correlates with the endothelial Enhanced basal activity of NOS associates with enhanced growth, in- marker CD31 [127]. vasion, angiogenesis and protumorigenic inflammation in high grade De Souza et al. analyzed an F98 rat glioma model by 1H-MRS, dif- gliomas. Therefore, nitrones, which demonstrate a very high biosafety fusion-weighted imaging (DWI), morphological T2-weighted imaging and clinical applicability in humans, may be chronically applied as (T2W) at 7 T (30 cm-bore MRI), as well as IHC and microarray studies, single agents in management of glial tumor patients. Gliomas actively at maximum tumor volumes (15–23 days following cell implantation in release glutamate at high amounts and NMDAR-blocking agents such as untreated (UT) tumors, and 18–35 days in OKN-007-treated tumors) riluzole and memantine suppress glioma growth in vitro and in vivo. [124]. 1H-MRS data revealed that Lip0.9/Cho, Lip0.9/Cr, Lip1.3/Cho, Nitromemantines may exert superior benefits in comparison to classical and Lip1.3/Cr ratios were significantly decreased (all P < 0.05) in the NMDAR-antagonists, as they would both block tumorigenic activity of OKN-007-treated group in comparison to UT F98 gliomas, which was NMDAR and to increase NO levels to intolerable levels specifically associated with reduced tumor necrosis [124]. Reduction of tumor within glioma tissues due to their overt glutamate content. In conclu- necrosis reflect that the intensity of tumor cell proliferation is lowered. sion, we believe that nitromemantines and nitrones strongly deserve to The Cho/Cr ratio is also significantly decreased in the OKN-007-treated be investigated in future animal models of glioblastoma. Only if their group compared with UT gliomas. As suggested above, Cho is involved efficacy and safety would be proven in various models, they could be in membrane synthesis; and hence, decrease in Cho levels suggested tested in Phase-1 trials in the treatment of a grave malignancy, glio- decreases in active cell membrane synthesis, which indirectly indicates blastoma. lowered cell proliferation. OKN-007-treated group exerted significantly lower apparent diffusion coefficient (ADC) values in the necrotic tumor References core and the nonnecrotic tumor parenchyma (both p < 0.05) [124]. Both intra- and extracellular fluid spaces contribute to the measured [1] M.A. Altinoz, S. Guloksuz, I. Elmaci, Rabies virus vaccine as an immune adjuvant ADC. As cellular density enhances, the increased tortuosity to extra- against cancers and glioblastoma: new studies may resurrect a neglected potential, Clin. Transl. Oncol. 19 (7) (2017) 785–792. cellular mobility paths reduces water mobility and, subsequently, the [2] M.A. Altinoz, I. Elmaci, B. 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