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In sickle cell disease (SCD), adhesion of sickle red blood cells (SSRBCs) and activated leukocytes in inflamed venules affects blood rheology, causing vaso-occlusive manifestations and vital reduction in microvascular blood flow. Recently, we found that NADPH oxidases (NOXs) create a vicious feedback loop within SSRBCs. This positive feedback loop mediates SSRBC adhesion to the endothelium. We show for the first time the therapeutic effectiveness of the redox-active manganese (Mn) porphyrins MnTnBuOE-2-PyP5+(MnBuOE; BMX-001) and MnTE-2-PyP5+(MnE; BMX-010, AEOL10113) to treat established vaso-occlusion in a humanized sickle mouse model of an acute vaso-occlusive crisis using intravital microscopy. These Mn porphyrins can suppress SSRBC NOX activity. Subcutaneous administration of only 1 dose of MnBuOE or MnE at 0.1 to 2 mg/kg after the inflammatory trigger of vaso-occlusion, or simultaneously, reversed and reduced leukocyte and SSRBC adhesion, diminished leukocyte rolling, restored blood flow, and increased survival rate. Furthermore, MnBuOE and MnE administered to sickle mice subcutaneously at 0.1 to 1 mg/kg for 28 days (except on weekends) did not exacerbate anemia, which seemed to be due to downregulation of both SSRBC reactive oxygen species production and exposure of the eryptotic marker phosphatidylserine. In addition, Mn porphyrins ameliorated leukocytosis, venous blood gases, endothelial activation, and organ oxidative damage. Our data suggest that Mn porphyrins, likely by repressing NOX-mediated adhesive function of SSRBCs and activated leukocytes, could represent a novel, safe therapeutic intervention to treat or prevent the establishment of acute pain crises. These NOX-targeted antioxidants merit further assessment in SCD clinical trials.

In sickle cell disease (SCD), adhesion of sickle red blood cells (SSRBCs) and activated leukocytes in inflamed venules affects blood rheology, causing vaso-occlusive manifestations and vital reduction in microvascular blood flow. Recently, we found that NADPH oxidases (NOXs) create a vicious feedback loop within SSRBCs. This positive feedback loop mediates SSRBC adhesion to the endothelium. We show for the first time the therapeutic effectiveness of the redox-active manganese (Mn) porphyrins MnTnBuOE-2-PyP5+ (MnBuOE; BMX-001) and MnTE-2-PyP5+ (MnE; BMX-010, AEOL10113) to treat established vaso-occlusion in a humanized sickle mouse model of an acute vaso-occlusive crisis using intravital microscopy. These Mn porphyrins can suppress SSRBC NOX activity. Subcutaneous administration of only 1 dose of MnBuOE or MnE at 0.1 to 2 mg/kg after the inflammatory trigger of vaso-occlusion, or simultaneously, reversed and reduced leukocyte and SSRBC adhesion, diminished leukocyte rolling, restored blood flow, and increased survival rate. Furthermore, MnBuOE and MnE administered to sickle mice subcutaneously at 0.1 to 1 mg/kg for 28 days (except on weekends) did not exacerbate anemia, which seemed to be due to downregulation of both SSRBC reactive oxygen species production and exposure of the eryptotic marker phosphatidylserine. In addition, Mn porphyrins ameliorated leukocytosis, venous blood gases, endothelial activation, and organ oxidative damage. Our data suggest that Mn porphyrins, likely by repressing NOX-mediated adhesive function of SSRBCs and activated leukocytes, could represent a novel, safe therapeutic intervention to treat or prevent the establishment of acute pain crises. These NOX-targeted antioxidants merit further assessment in SCD clinical trials.

AbstractThis review aims to clarify (1) what is a true mimic of superoxide dismutase family of enzymes, SOD; and (2) whether such compound could act as SOD mimic in a complex biological milieu. Several groups of compounds (metalloporphyrins, metallocorroles, Mn biliverdins, Mn cyclic polyamines, Mn salens, and metal oxides and salts) have been described. Their ability to catalyze the dismutation of O2·–, [kcat(O2·–)], thermodynamic property that supports high catalytic ability (E1/2), kinetic factors that facilitate the catalysis, and the stability of compounds, which assures the integrity of metal coordination sphere where reactions of interest occur have been discussed. The other possible in vivoactions of those compounds, such as peroxynitrite and hypochlorite reduction, peroxidase-like activity, thiol oxidase activity etc., have been described as well. Based on in vivostudies it appears that kcat(O2·–) for Mn(III) N-substituted pyridylporphyrins parallels their therapeutic ability. The reason for that lies in their electrophilic nature which favors reactions with nucleophilic (anionic) reactive species (O2·–, ONOO–, ClO–, HO2–, CO3·–) and simple or protein thiolates. Their in vivomultiple rather than single modes of actions, would be determined by: (a) their redox properties; (b) localization at targeted cellular site; and (c) redox environment of diseased or mutated/cancer cell. Quality of any drug preparation and the knowledge of researchers on its properties are essential when its mechanistic aspects are explored.

Intracerebroventricular treatment with redox-regulating Mn(III) N-hexylpyridylporphyrin (MnPorphyrin) is remarkably efficacious in experimental central nervous system (CNS) injury. Clinical development has been arrested because of poor blood-brain barrier penetration. Mn(III) meso-tetrakis (N-hexylpyridinium-2-yl) porphyrin (MnTnHex-2-PyP) was synthesized to include four six-carbon (hexyl) side chains on the core MnPorphyrin structure. This has been shown to increase in vitro lipophilicity 13,500-fold relative to the hydrophilic ethyl analog Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP). In normal mice, we found brain MnTnHex-2-PyP accumulation to be ∼9-fold greater than MnTE-2-PyP 24 h after a single intraperitoneal dose. We then evaluated MnTnHex-2-PyP efficacy in outcome-oriented models of focal cerebral ischemia and subarachnoid hemorrhage. For focal ischemia, rats underwent 90-min middle cerebral artery occlusion. Parenteral MnTnHex-2-PyP treatment began 5 min or 6 h after reperfusion onset and continued for 7 days. Neurologic function was improved with both early (P = 0.002) and delayed (P = 0.002) treatment onset. Total infarct size was decreased with both early (P = 0.03) and delayed (P = 0.01) treatment. MnTnHex-2-PyP attenuated nuclear factor κB nuclear DNA binding activity and suppressed tumor necrosis factor-α and interleukin-6 expression. For subarachnoid hemorrhage, mice underwent perforation of the anterior cerebral artery and were treated with intraperitoneal MnTnHex-2-PyP or vehicle for 3 days. Neurologic function was improved (P = 0.02), and vasoconstriction of the anterior cerebral (P = 0.0005), middle cerebral (P = 0.003), and internal carotid (P = 0.015) arteries was decreased by MnTnHex-2-PyP. Side-chain elongation preserved MnPorphyrin redox activity, but improved CNS bioavailability sufficient to cause improved outcome from acute CNS injury, despite delay in parenteral treatment onset of up to 6 h. This advance now allows consideration of MnPorphyrins for treatment of cerebrovascular disease.

Cationic Mn(III) N-alkylpyridyl (MnTalkyl-2(or 3)-PyP5) and N, N-dialkylimidazolylporphyrins (MnTDalkyl-2-ImP5) have been regarded as the most powerful SOD mimics/peroxynitrite scavengers - i. e. antioxidants. The ethyl-, MnTE-2-PyP5 (AEOL10113), and hexylpyridyl-, MnTnHex-2-PyP5 and diethylimidazolylporphyrin, MnTDE-2-ImP5 (AEOL10150) have been mostly studied in vitro and in vivo. Given the in vivo abundance of cellular reductants, MnPs can couple with them in removing superoxide. Thus, they could be readily reduced from MnIIIP to MnIIP with ascorbate and glutathione, and in a subsequent step reduce either O2 .- (while acting as superoxide reductase) or oxygen (while exerting pro-oxidative action). Moreover, MnPs can catalyze ascorbate oxidation and in turn hydrogen peroxide production. The in vivo type of MnP action (anti- or pro-oxidative) will depend upon the cellular levels of reactive species, endogenous antioxidants, availability of oxygen, ratio of O2 .-- to peroxide-removing systems, redox ability of MnPs and their cellular localization/bioavailibility. To exemplify the switch from an anti- to pro-oxidative action we have explored a very simple and straightforward system - the superoxide-specific aerobic growth of SOD-deficient E. coli. In such a system, cationic MnPs, ortho and meta MnTE-2-(or 3)-PyP5 act as powerful SOD mimics. Yet, in the presence of exogenous ascorbate, the SOD mimics catalyze the H2O2 production, causing oxidative damage to both wild and SOD-deficient strains and inhibiting their growth. Catalase added to the medium reversed the effect indicating that H2O2 is a major damaging/signaling species involved in cell growth suppression. The experiments with oxyR- and soxRS-deficient E. coli were conducted to show that E. coli responds to increased oxidative stress exerted by MnP/ascorbate system by induction of oxyR regulon and thus upregulation of antioxidative defenses such as catalases and peroxidases. As anticipated, when catalase was added into medium to remove H2O2, E. coli did not respond with upregulation of its own antioxidant systems.

Reactive oxygen species (ROS) are considered to be a main cause for cancer development, but they can also be used for cancer eradication. Because of this dual nature of ROS action, both antioxidant and prooxidant therapeutic agents have been developed and some have shown clinical promise. Selective uptake of porphyrins by malignant cells has for a long time been used for tumor imaging and for targeted delivery of ROS. Redox-active Mn porphyrins can act both as antioxidants and as prooxidants, and may thus be used in anticancer therapy. Porphyrins, which chelate redox inactive metals, for example Zn, demonstrate photo-sensitizing activity and thus can produce singlet oxygen and other reactive oxygen species in cancer cells on irradiation with visible light. Here we review the properties of Zn(II) N-alkylpyridylporphyrin-based photosensitizers, and their ability to damage selected cellular targets.

Despite intensive efforts to improve multimodal treatment of brain tumors, survival remains limited. Current therapy consists of a combination of surgery, irradiation and chemotherapy with predisposition to long-term complications. Identifying novel targeted therapies is therefore at the forefront of brain tumor research. This study explores the utility of a manganese porphyrin in a brain tumor model. The compound used is ortho isomer, manganese(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP5+. It is a powerful SOD mimic and peroxynitrite scavenger and a potent modulator of redox-based cellular transcriptional activity, able to suppress excessive immune and inflammatory responses and in turn proliferative pathways. It is further one of the most lipophilic compounds among cationic Mn(III) N-alkylpyridylporphyrins, and thus accumulates predominantly in mitochondria relative to cytosol. In mitochondria, MnTnHex-2-PyP5+ could mimic our key antioxidant system, mitochondrial superoxide dismutase, MnSOD, whose overexpression has been widely shown to suppress tumor growth. Importantly, MnTnHex-2-PyP5+ crosses blood brain barrier in sufficient amounts to demonstrate efficacy in treating CNS injuries. For those reasons we elected to test its effects in inhibiting brain tumor growth. This study is the first report of the antitumor properties of MnTnHex-2-PyP5+ as a single agent in adult and pediatric glioblastoma multiforme (D-54 MG, D-245 MG, D-256 MG, D-456 MG) and pediatric medulloblastoma (D-341 MED), and is the first case where a redox-able metal complex has been used in glioma therapy. When given subcutaneously to mice bearing subcutaneous and intracranial xenografts, MnTnHex-2-PyP5+ caused a significant (P ≤ 0.001) growth delay in D-245 MG, D-256 MG, D-341 MED, and D-456 MG tumors. Growth delay for mice bearing subcutaneous xenografts ranged from 3 days in D-54 MG to 34 days in D-341 MED. With mice bearing intracranial xenografts, MnTnHex-2-PyP5+ increases median survival by 33% in adult glioblastoma multiforme (D-256 MG; P ≤ 0.001) and 173% in pediatric medulloblastoma (D-341 MED, ≤ 0.001). The beneficial effects of MnTnHex-2-PyP5+ are presumably achieved either (1) indirectly via elimination of signaling reactive oxygen and nitrogen species (in particular superoxide and peroxynitrite) which in turn would prevent activation of transcription factors; or (2) directly by coupling with cellular reductants and redox-sensitive signaling proteins. The former action is antioxidative while the latter action is presumably pro-oxidative in nature. Our findings suggest that the use of Mn porphyrin-based SOD mimics, and in particular lipophilic analogues such as MnTnHex-2-PyP5+, is a promising approach for brain tumor therapy.

Despite intensive efforts to improve multimodal treatment of brain tumors, survival remains limited. Current therapy consists of a combination of surgery, irradiation and chemotherapy with predisposition to long-term complications. Identifying novel targeted therapies is therefore at the forefront of brain tumor research. This study explores the utility of a manganese porphyrin in a brain tumor model. The compound used is ortho isomer, manganese(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin, MnTnHex-2-PyP5. It is a powerful SOD mimic and peroxynitrite scavenger and a potent modulator of redox-based cellular transcriptional activity, able to suppress excessive immune and inflammatory responses and in turn proliferative pathways. It is further one of the most lipophilic compounds among cationic Mn(III) N-alkylpyridylporphyrins, and thus accumulates predominantly in mitochondria relative to cytosol. In mitochondria, MnTnHex-2-PyP5 could mimic our key antioxidant system, mitochondrial superoxide dismutase, MnSOD, whose overexpression has been widely shown to suppress tumor growth. Importantly, MnTnHex-2-PyP5 crosses blood brain barrier in sufficient amounts to demonstrate efficacy in treating CNS injuries. For those reasons we elected to test its effects in inhibiting brain tumor growth. This study is the first report of the antitumor properties of MnTnHex-2-PyP5 as a single agent in adult and pediatric glioblastoma multiforme (D-54 MG, D-245 MG, D-256 MG, D-456 MG) and pediatric medulloblastoma (D-341 MED), and is the first case where a redox-able metal complex has been used in glioma therapy. When given subcutaneously to mice bearing subcutaneous and intracranial xenografts, MnTnHex-2-PyP5 caused a significant (P ≤ 0.001) growth delay in D-245 MG, D-256 MG, D-341 MED, and D-456 MG tumors. Growth delay for mice bearing subcutaneous xenografts ranged from 3 days in D-54 MG to 34 days in D-341 MED. With mice bearing intracranial xenografts, MnTnHex-2-PyP5 increases median survival by 33 in adult glioblastoma multiforme (D-256 MG; P ≤ 0.001) and 173 in pediatric medulloblastoma (D-341 MED, ≤ 0.001). The beneficial effects of MnTnHex-2-PyP5 are presumably achieved either (1) indirectly via elimination of signaling reactive oxygen and nitrogen species (in particular superoxide and peroxynitrite) which in turn would prevent activation of transcription factors; or (2) directly by coupling with cellular reductants and redox-sensitive signaling proteins. The former action is antioxidative while the latter action is presumably pro-oxidative in nature. Our findings suggest that the use of Mn porphyrin-based SOD mimics, and in particular lipophilic analogues such as MnTnHex-2-PyP5, is a promising approach for brain tumor therapy.

OBJECTIVE: Osteoarthritis is characterized by the degradation of articular cartilage. The catabolic activity of chondrocytes is partly regulated by nitric oxide (NO), which with superoxide (O2-) leads to the formation of peroxynitrite (OONO-), a potentially damaging reactive species. Cartilage is avascular and functions at reduced oxygen tension. We investigated whether oxygen tension influences the effects of interleukin 1 (IL-1) on peroxynitrite formation and cartilage matrix metabolism. METHODS: Porcine cartilage explants were incubated at either 1% O2 or 20% O2 with either 1 ng/ml IL-1a, 25 µM MnTE-2-PyP5+ [Mn porphyrin-based catalytic antioxidant, Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin], or 1 ng/ml IL-1 + 25 µM MnTE-2-PyP5+ to decrease peroxynitrite formation. Nitrotyrosine, formed by nitration of tyrosine by peroxynitrite, was measured by immunoblot. Proteoglycan and collagen synthesis and proteoglycan degradation were also determined. RESULTS: IL-1-induced peroxynitrite formation was decreased in 1% O2 as compared to 20% O2. MnTE-2-PyP5+ inhibited IL-1-induced peroxynitrite formation in either 1% O2 or 20% O2. In 1% O2 (but not in 20% O2), Mn porphyrin significantly inhibited IL-1-induced proteoglycan degradation. IL-1 decreased both proteoglycan and collagen II synthesis in cartilage explants in 1% O2 or 20% O2, but MnTE-2-PyP5+ did not prevent these anti-anabolic effects. MnTE-2-PyP5+ alone caused a significant decrease in collagen synthesis at 20% O2 but not at 1% O2. CONCLUSION: Our findings show that oxygen tension alters IL-1-induced peroxynitrite formation, which can influence proteoglycan degradation. Oxygen tension may influence the effects of reactive oxygen and nitrogen species on matrix homeostasis.

ABSTRACTManganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformansmanganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2? mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2? mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformanscells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 µM) at 30°C, but not at 37°C. Furthermore, sod2? mutant cells that were incubated for 24 h at 37°C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25°C in the presence of air. These data suggest that the C. neoformansSod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.

The purpose of this study was to develop a minimally invasive recovery model of spinal cord injury in the C57Bl/6J mouse. Without laminectomy, the epidural space was exposed by disruption of the T10-T11 interspinous ligament. Perpendicular to the rostral-caudal axis of the spine, a 1.5-mm silicone tube (O.D. 0.047 in.) was placed in the T11 epidural space. Prior to placement, a suture was passed through the tube allowing withdrawal of the tube after discontinuation of anesthesia. After 1, 30, 60, or 120 min (n = 5-8) of spinal cord compression (SCC), the tube was withdrawn. Neurological function was measured at 1, 3, 7, and 14 days after injury followed by histologic analysis. BBB locomotor score, rotarod latency, and screen grasping were worsened in a SCC duration-dependent manner (p < 0.0001). With increasing SCC duration, the number of histologically normal neurons in the ventral horns decreased (p < 0.0001) while the cross-sectional area of spinal cord with pancellular necrosis increased (p < 0.0001). Increased duration of SCC caused progressive rostral-caudal spread of histologic damage. The results indicate that this is a simple, reliable model with neurologic and histologic injury highly dependent on SCC duration. This model may be useful for study of spinal cord injury in genetically modified mice in the absence of anesthetic confounds while leaving the vertebral column intact.

The ortho, meta,and paraisomers of manganese(III) 5,10,15,20-tetrakis(N-methylpyridyl)porphyrin, MnTM-2-PyP5+, MnTM-3-PyP5+, and MnTM-4-PyP5+, respectively, were analyzed in terms of their superoxide dismutase (SOD) activity in vitroand in vivo.The impact of their interaction with DNA and RNA on the SOD activity in vivoand in vitrohas also been analyzed. Differences in their behavior are due to the combined steric and electrostatic factors. In vitrocatalytic activities are closely related to their redox potentials. The half-wave potentials (E½) are +0.220 mV, +0.052 mV, and +0.060 Vversusnormal hydrogen electrode, whereas the rates of dismutation (kcat) are 6.0 × 107, 4.1 × 106, and 3.8 × 106m−1s−1for theortho, meta,and paraisomers, respectively.

Detergents, such as Triton X-100, markedly increase the reduction of tetrazolium salts by xanthine oxidase plus xanthine, or by NADH. This effect of detergent, in the case of the xanthine oxidase catalyzed process, is seen aerobically but not anaerobically. Increasing the rate of accumulation of formazan, whether by increasing the concentration of the tetrazolium salt or by adding detergent, decreased susceptibility to inhibition by superoxide dismutase or by O2. These results are accommodated by a scheme of reactions the essence of which is the univalent reduction of the tetrazolium to an uncharged tetrazoinyl radical which can reduce O2to[formula]or which can partition into the detergent micelles and there dismute to generate the stable formazan.

The Special Issue titled “SOD enzymes and their mimics in cancer: pro- vs anti-oxidative mode of action” aimed at challenging our thoughts on what is the true mechanism behind the beneficial effects we observe with redox-active compounds, both endogenous and exogenous, in different in vitro and animal models of diseases, which could help us in treating human diseases more successfully. SOD enzymes were proved essential for the functioning of our body in numerous publications since their mechanism of action had been established nearly half a century ago by McCord and Fridovich in a seminal J Biol Chem paper on Cu,ZnSOD [1]. Moreover, the mitochondrial Mn form of superoxide dismutase, MnSOD, is absolutely essential to our lives; the MnSOD-knockout mice survive only days after birth. It is therefore no wonder that therapeutic strategies targeting mitochondria have been sought. The review on MnSOD in this Issue was prepared by Daret St. Clair who has dedicated herself to understanding the critical role of this enzyme under physiological and pathological conditions and most specifically to the role of mitochondria in cancer [2]. Through the years a substantial body of evidence has emerged from a number of groups for an anticancer role of MnSOD. While this is true for many cancers Melendez and coworkers, in their chapter, discuss the strong connection between high level MnSOD expression and redox-signaling in metastatic disease. The effects were ascribed to the increased levels of H2O2, that was thought to be a direct consequence of overexpression of MnSOD. However the possibility is allowed, and elaborated by Melendez group in this Issue, that MnSOD overexpression could modify the cellular redox environment to indirectly enhance the production of O2- and H2O2 [3]. Several possible mechanisms of peroxide increase as a consequence of MnSOD overexpression, other than that resulting directly from MnSOD action, were proposed by Irwin Fridovich [4]. Further, Lee Ann MacMillan-Crow and John Crow reported that in their hands overexpression of MnSOD did not lead to increased levels of peroxide [5]. MacMillan-Crow and John Crow also noted that both peroxynitrite and peroxide behave similarly with respect to the effects that they produce in assays which we routinely use for measurements of reactive species. Hardly an assay exists that is specific to a particular reactive species. Thus, what we attribute to the increased levels of peroxide might indeed relate to the increased peroxynitrite levels. Based on their experiments, the authors illustrate how MnSOD overexpression could lead to peroxynitrite formation. Recently it became obvious that both the activity of redox-active proteins and their expression must be studied in order to fully comprehend their in vivo actions and their role in cellular signaling processes. For example as shown by Terry Oberley, the thioredoxin levels may be upregulated, while its activity suppressed due to the excessive oxidation of thioredoxin SH groups [6]. The references listed in the contribution from the Melendez group in this Issue have shown that such scenario may be operative for MnSOD also [3]. Understanding the impact of SOD enzymes on our health has resulted in a desire to mimic their activity. A number of compounds have been developed and used in different models of diseases with varying success over the last three decades: metalloporphyrins, Mn(III) salens, Mn(II) cyclic polyamines, nitroxides, different metal oxides [reviewed in 7], quinones and others. A number of naturally occurring antioxidants have been tested also. For a long time, all of those compounds were viewed as antioxidants. To be as successful SOD mimic as the enzyme itself, the compound must reduce and oxidize O2- with equal rate constants. For that to happen, the metal-centered reduction potential must be positioned at the midway between the potential for the oxidation and reduction of O2- (∼300 mV vs NHE); that fact immediately tells us that powerful SOD mimics are equally good pro-oxidants as they are antioxidants. Years ago, Barry Halliwell wrote an article questioning the actions of polyphenols [8]. The role of ascorbate was questioned too [9], particularly in the presence of metals and metal complexes [10]. Are such compounds pro- or antioxidants? The same question holds true for the metalloporphyrins also. The Mn(III) cationic N-substituted pyridylporphyrins are very active in the dismution of O2- [11]. Further, it has been shown that under certain circumstances in the presence of ascorbate, the pro-oxidative action of Mn porphyrins is enhanced. In such a scenario metal complexes (or other redox-active compounds) catalyze the ascorbate-driven peroxide production, as evidenced by us and others [12, 13], and proposed by Chen et al., to result in anticancer effects [14, 15]. An ascorbate/menadione system has been approved by FDA for the treatment of metastatic, locally advanced, inoperable transitional cell carcinoma of the urothelium (stage III and IV bladder cancer). The ascorbate/ menadione system is discussed by Pedro Buc Calderon group in this Issue [16]. Ines Batinic-Haberle and Ludmil Benov groups (Part II) have recently shown that Mn porphyrins clearly act as SOD mimics in allowing SOD-deficient E. coli to grow aerobically, but become cytotoxic in the presence of ascorbate. Depending upon the circumstances, such action provoked an adaptive response and we observed the upregulation of endogenous antioxidative defenses [12]. Under stringent conditions, at high concentrations and longer exposure to proxidants, the cells were not able to adapt and died [12]. With redundant peroxide-removing systems (catalases, glutathione peroxidases, glutathione reductases, glutathione transferases, thioredoxins, peroxyredoxins) such pro-oxidative action of Mn porphyrins would not create a problem to a normal cell. Tumors are frequently under oxidative stress and also have compromised reactive species-removing systems, particularly an inadequate ratio of superoxide- to peroxide-removing systems. Thus they may not be able to tolerate the additional oxidative burden and would perish. Therefore, under particular circumstances and in the presence of cellular reductants such as ascorbate and glutathione, and at inadequate ratio of superoxide- to peroxide removing systems, metalloporphyrins could act as powerful cytotoxic agents and kill tumor via excessive production of reactive species.....

As a continuation of Part I, herein Garry Buettner [1] addressed the critical role of manganese superoxide dismutase, MnSOD - the enzyme that we cannot live without. MnSOD is a central player in the redox biology of cells and tissues [1]. It is critical for establishing the appropriate balance in redox circuitry of the mitochondria. Thus, there has been an increasing demand to develop powerful SOD mimics. In the studies where MnSOD was overexpressed, the increased levels of H2O2 were found, which suggests that H2O2 has a major role in metastases. The loss of MnSOD is likely an early event in tumor progression allowing for further propagation of the tumorigenic phenotype resulting from the steady state increases in free radical production [2]. Garry Buettner contribution, and manuscripts by Irwin Fridovich [3] and Lee Ann MacMillan Crow and John Crow [4], as well as from Melendez [2] and St. Clair [5] groups addressed the possible, and still controversial origin of the increased peroxide as a direct or indirect consequence of MnSOD overexpression. More work is needed to gain a profound insight into the dichotomous role of MnSOD as a tumor suppressor or oncogene [2]. Similarly, much is to be learned about redoxbased compounds developed originally as SOD mimics. Major classes of such compounds are addressed in this Issue: Mn porphyrins, metallotexaphyrins, Mn salens, metallocorroles, nitroxides, nitrones, and quinones. Many were shown to exert anticancer effects, acting as tumor suppressors as exemplified with Mn porphyrins in contribution from Keir et al., [6], corroles by Zeev Gross group [7], texaphyrins by Jonathan Sessler group [8], nitrones by Robert Floyd et al., [9], and quinones by Pedro Buc Calderon group [10]. However, the mechanism of action of those compounds is not yet fully understood. Cationic Mn(III) N-substituted pyridylporphyrins are potent SOD mimics and protect SOD-deficient E. coli when it grows aerobically. Ines Batinic-Haberle and Ludmil Benov groups showed that in the presence of cellular reductant ascorbate, which is abundant in vivo, Mn porphyrins suppressed E. coli growth via cytotoxic H2O2 production. Under milder conditions and in a rich growing medium, over time the adaptive response of E. coli was observed, whereby oxyR regulon was induced and endogenous antioxidants - peroxide-removing enzymes, peroxidases and catalases upregulated [11]. The data exemplify how a pro-oxidative event could exert antioxidative effects [11]. Such data caution us to differentiate between the nature of the actions of synthetic antioxidants and the type of the effects observed. A manuscript by Robert Floyd et al., [9] discusses the potent anticancer effects of nitrones observed in three experimental cancer models: (1) the rat choline-deficiency liver cancer model; (2) the rat C6 glioma model; and (3) the mouse APCMin/ colon cancer model. Originally, nitrones were developed as spin traps for free radicals. The two mostly studied nitrones are -phenyl-tert-butylnitrone (PBN) and its derivative, 2,4-disulfophenyl-tert-butylnitrone (OKN-007, formerly known as NXY-059 and developed earlier for stroke therapy [9]). The ionic PBN derivative was shown to cause shrinkage of fully formed tumors in the rat C6 glioma model. The extensive human safety studies, showing that it is a safe compound to use, combined with its demonstrated potency to decrease the size of tumors, makes it an ideal candidate for clinical trials, which Robert Floyd is currently pursuing. Again, the mechanism of action has not yet been fully understood. The decreased .NO production due to the PBN-mediated suppression of iNOS expression, S-nitrosylation of critical proteins such as caspases and Bcl-2, as well as free radical scavenging, may play a role. Nitrones were previously shown to exert general anti-inflammatory effects. The exacerbated inflammatory cellular signaling processes are suppressed by their administration [9]. The inhibition of NF-B master transcription factor was reported with PBN, and needs to be tested with OKN-007 [9]. Mn porphyrin, MnTnHex-2-PyP5 has been shown in this Issue [6] to exert anticancer effect in mouse glioma study. Both Mn porphyrin and nitrones have been shown to affect cellular transcriptional activity as they inhibit NF-B activation [6,9]. Both also scavenge reactive species [6,9]. It may be challenging, from therapeutic and mechanistic perspective, to compare Mn porphyrin action to the anticancer effect of nitrones in the same glioma cell line. The other papers in Part II of this Issue relate to three different classes of redox-active compounds developed originally as SOD mimics. The metal-containing compounds are Mn(III) corroles developed by Zeev Gross group [7], and Mn(III) salen derivatives developed by Susan Doctrow group [12]. The third group comprises the nitroxides, described by Ryan Davies et al., [13]......