SOD Therapeutics: Latest Insights into Their Structure-Activity Relationships and Impact on the Cellular Redox-Based Signaling Pathways

Reactivity toward ONOO⁻

The log k_cat(O₂·⁻) is directly proportional to log k_red (ONOO⁻) (28, 84, 86). Thus, potent SOD mimics are potent peroxynitrite scavengers (Eqs. [15] and [16]). We have shown with MnPs that the reason lies in the fact that the most potent SOD mimics are electron-deficient and that such MnPs would favor reactions and/or binding of electron-rich anionic ligands such as ONOO⁻. The reactivity of pentacationic MnPs toward superoxide [k_cat (O₂·⁻)] is higher than toward peroxynitrite [k_red (ONOO⁻)]; pentacationic MnP would prefer O₂·⁻ compared with ONOO⁻ if it encounters both species under identical concentrations. The log k_cat (O₂·⁻)=7.76 for MnTE-2-PyP⁵⁺ at 25⁰C, while its log k_red(ONOO⁻)=7.53 but at 37°C (28). Once oxidized to O=Mn^IVP with ONOO⁻, MnP would regenerate itself as a catalyst with cellular reductants acting as electron pools and sparing biological targets from highly oxidizing Mn oxo species (86). \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}\quad&( { \rm H}_2{ \rm O} ) _2{ \rm Mn}^{ \rm III}{ \rm P}^{5 + } + { \rm ONOO}^ - \Leftarrow \Rightarrow ( { \rm H}_2{ \rm O} ) { \rm O} = { \rm Mn}^{ \rm IV}{ \rm P}^{4 +}\\ \quad + {\cdot}{ \rm NO}_2 + { \rm H}_2{ \rm O} \qquad [15]\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}({\rm H}_2{\rm O}) {\rm Mn}^{\rm II}{\rm P}^{\rm 4 + } + {\rm ONOO}^{-} \Leftarrow \Rightarrow ({\rm H}_2{\rm O}) {\rm O} = {\rm Mn}^{\rm IV}{\rm P}^{4 +} + {\rm NO}_{2}^{-} & \qquad \qquad [16]\end{align*} \end{document}

In the reaction with ONOO⁻, MnPs can cycle either one-electronically via O=Mn^IVP/Mn^IIIP redox, producing highly oxidizing radical, ·NO₂ (Eq. [15]), or two-electronically via O=Mn^IVP/Mn^IIP redox (Eq. [16]), producing benign nitrite NO₂ ⁻ (80, 196). The MnPs are in vivo maintained in reduced Mn +2 state by cellular reductants. Thus, the 2-electron- is more likely than the one-electron reaction.

The compounds with negative E_1/2 (such as Mn^IIITBAP³⁻, E_1/2=−194 mV vs. NHE) cannot participate in a 1st step of O₂·⁻ dismutation (Eq. [1]). However, Mn^IIITBAP³⁻ can be oxidized to O=Mn^IVP with ONOO⁻ (and perhaps other strong oxidants ClO⁻, H₂O₂, and lipid radicals), which may explain its reported in vivo efficacy (19). The log k_red (ONOO⁻)=5.02 for MnTBAP³⁻ is >100-fold lower than of MnTE-2-PyP⁵⁺ (log k_red=7.53) (28, 223). ONOO⁻ can also oxidize Mn texaphyrin with even lower k_red(ONOO⁻)=3×10⁴ M ⁻¹s⁻¹ (84, 196, 242) and some other MnPs, such as Mn(III) meso-tetracyclohexenylporphyrin as well as biscyclohexano-fused Mn(III) complex of bis(hydroxyphenyl)dipyrromethene (217, 218). The other compounds that cannot be oxidized by O₂·⁻ are MitoQH₂ (quinol), MitoQH (semiquinone), and nitroxides (177). Once it reaches mitochondria, the MitoQ readily gets reduced to MitoQH₂ by components of the electron transport chain (177). MitoQH₂can be oxidized by ONOO⁻ to MitoQH, which then dismutes (disproportionates) to MitoQ and MitoQH₂. Only MitoQ (quinone) and oxoammonium cation react with O₂·⁻ with high rate constants to yield MitoQH and nitroxide, respectively (177). Nitroxides get oxidized with the degradation product of ONOO⁻, CO₃·⁻ and ·NO₂, and protein-derived radicals, thiyl and peroxyl radicals giving rise to oxoammnium cation (28). Oxoammonium cation though reacts with O₂·⁻ (28). Mn(III) cyclic polyamines are often reported as specific to O₂·⁻ (59, 169, 171, 186, 228, 267). The reactivity of Mn cyclic polyamine M40403 toward ·NO has been reported (87). Mn salen derivatives show a wide range of reactivities toward ONOO⁻, H₂O₂, ClO⁻, and ·NO (74, 132, 234). The MCs are reactive toward O₂·⁻ and ONOO⁻ (28). The reactivity of cerium oxide (nanoceria) toward O₂·⁻, H₂O₂, and ·NO has been reported (28). Work in progress shows that potent SOD mimics are also very reactive toward ClO⁻ (109). One can easily envision that many other reactions are possible with each of those compounds within cells. Thus, we can only safely predict which reaction is possible but not which will occur.

MCs employ M^IVC/M^IIIC or O=M^VC/M^IIIC to cycle with O₂·⁻, H₂O₂, and ONOO⁻ (see earlier under SARs for diverse redox-active compounds—Mn(III) corroles). Similar to MnPs, two-electron oxidation with ONOO⁻ would lead to benign NO₂ ⁻ production (162). Reduction of strong oxidizing high-valent Mn will be achieved at the expense of reductants acting as electron pools.

Reactivity toward ·NO

Cationic Mn(III) N-substituted pyridylporphyrins favor reactions with ·NO; at submicromolar concentrations and at 1:1 ratio, a fairly stable complex is formed, (NO)Mn^IIP, with Mn in +2 oxidation state (Eq. [18]). The reaction is very slow with t_1/2 ∼60 min; the same product is formed much faster if Mn^IIIP⁵⁺ is first reduced with cellular reductants such as thiol or ascorbate (HA⁻ and RS⁻, monodeprotonated species are major ones at pH 7.8) (Eq. [17]). The oxidation of cysteine (similar to the Eq. [17b] shown for glutathione) by Mn^IIIP resembles the reported thiol oxidase action of SOD enzyme (294). The rate constant for nitrosylation of MnP (Eq. [18]), estimated by stopped flow, is k ∼10⁶ M ⁻¹s⁻¹. The complex slowly undergoes the oxidation, whereby initial Mn^IIIP gets restored and nitrate is eventually formed (Eqs. [19] and [20]). Mn^IIP⁴⁺ can undergo oxidation with either oxygen or superoxide (the latter reaction being 2nd step of O₂·⁻ dismutation process), depending on their relative levels in cells. The reaction of Mn^IITE-2-PyP⁴⁺ with O₂ was estimated to occur with a rate constant of ∼8×10⁴ M ⁻¹s⁻¹ (248). Axially coordinated waters are omitted in the equations for simplicity, as no protonation equilibria are involved at the Mn site: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm Mn}^{{ \rm III}}{ \rm P}^{5 + } + { \rm HA}^- & \Leftarrow \Rightarrow { \rm Mn}^{{ \rm II}} { \rm P}^{4 + } +{ \rm HA}^{\cdot};\ 2 { \rm H}^ + + { \rm A}^{{\cdot}{-}} + { \rm O}_2^{{\cdot}{ -}} \\ \Leftarrow \Rightarrow { \rm A} + { \rm H}_2 { \rm O}_2 \qquad [17{\rm a}]\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm Mn}^{{ \rm III}}{ \rm P}^{5 + } + { \rm GS}^ - \Leftarrow \Rightarrow { \rm Mn}^{ \rm II} { \rm P}^{4 + }+ { \rm GS}^{\cdot} ; 2{ \rm GS}^{\cdot}\ \Leftarrow\Rightarrow { \rm GSSG} \qquad [17 {\rm b}]\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm Mn}^{{ \rm II}} { \rm P}^{4 + } + {\cdot} { \rm NO} \Leftarrow\Rightarrow ( { \rm NO} ) { \rm Mn}^{{ \rm II}} { \rm P}^{4 + } \qquad [18]\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}( { \rm NO} ) { \rm Mn}^{{ \rm II}}{ \rm P}^{4 + }+ { \rm O}_2 \Leftarrow\Rightarrow { \rm Mn}^{{ \rm III}} { \rm P}^{5 + } + { \rm NO}_3^ - \qquad [19]\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm Mn}^{{ \rm II}}{ \rm P}^{4 + } + { \rm O}_2 \Leftarrow\Rightarrow { \rm Mn}^{{ \rm III}}{ \rm P}^{5 + } +{ \rm O}_2^{{\cdot}{-}}; 2 { \rm O}_2^{{\cdot}{-}} + 2 { \rm H}^ + \\ \Leftarrow\Rightarrow { \rm H}_2 { \rm O}_2 + { \rm O}_2 & \qquad [20]\end{align*} \end{document}

The ·NO scavenging by MnP may affect cellular signaling pathways. The ·NO binding shifts the reduction potential for the Mn^IIIP/Mn^IIP redox couple by +600 mV (133, 184, 276), stabilizing Mn in +2 oxidation state. The reactivity of nitrosylated (NO)Mn^IIP⁴⁺ toward O₂·⁻ is presently under investigation.

Reactivity toward H₂O₂

Hydrogen peroxide (H₂O₂) is a strong oxidant; thus, very few organic ligand-containing compounds are stable enough in its presence to be efficient catalysts of its dismutation. In vivo H₂O₂ is produced by different oxidases, such as NADPH- and xanthine oxidases, and is also a product of O₂·⁻ dismutation during mitochondrial respiration. Under nM levels, it is a main signaling species due to its long life, lack of charge, and ability to cross cellular membranes (90). It is also a source of other highly oxidizing species, such as·OH radical, alkoxyl (RO·) and peroxyl (RO₂·) radicals, and protein thiyl radicals (89, 109).

The preliminary studies on the catalysis of H₂O₂ dismutation (producing H₂O and O₂) indicate that the log k_cat(H₂O₂) is in the range of 1–2 log units for several MnPs [MnTE-2(and 3)-PyP⁵⁺, MnTBAP³⁻, and MnTnHex-2(and 3)-PyP⁵⁺] (Weitner et al., unpublished). Data agree fairly well with those reported from Fridovich's group for MnTM-4-PyP⁵⁺ and MnTBAP³⁻ (66). Due to the excessive bleaching, Mahammed and Gross were not able to assess the k_cat(H₂O₂) for FeTSPP³⁻ (161). Starting with an FeP, containing three mesityl groups on porphyrin meso positions, Nocera's group added the fourth meso pendant group producing structure with k_cat(H₂O₂) increased ∼3000-fold relative to Fe(III) tetrakis-meso(2,4,6-methylphenyl)porphyrin (49). The rate constant for the reduction of H₂O₂ to H₂O with MnTDE-2-ImP⁵⁺ at pH ∼7 (128) is low, k_red(H₂O₂) ∼10² M ⁻¹s⁻¹, and is reportedly faster with less electron-rich MnTM-2-PyP⁵⁺ (30).

Corrole is a trianionic ligand, while porphyrin is dianionic; as a result, the metal site is more electron rich in MCs, giving rise to more stable complexes in reduced metal +3 oxidation state with regard to the loss of metal (Fig. 10). Further, after the reaction with H₂O₂, metal in MCs is stabilized much more in higher metal +5 oxidation state relative to MPs; consequently, Fe corroles are more efficient catalysts of H₂O₂ dismutation (161). The log k_cat(H₂O₂) of 3.8 was reported for Fe corrole-bearing pentafluorophenyl groups on three meso positions and two sulfonato groups at neighboring pyrrolic rings (161). Although possessing only ∼0.6% of enzyme activity [assuming log k_cat ∼6 for enzyme (109)], the Fe corrole may still have the highest k_cat(H₂O₂) among the synthetic redox-active compounds. Whether and how this affects its therapeutic efficacy is not clear. When an aqueous solution of a compound was given orally at 20 mg/kg/day for 7 weeks, it prevented cataract incidents, favorably affected kidney function, and decreased serum cholesterol and triglyceride levels. Such a study suggests sufficient stability of that compound toward the proton-dependent loss of metal. As much as 300 mg/kg caused only a mild adverse effect (103).

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FIG. 10.

The comparison of porphyrin versus corrole core. Porphyrin contains 2 while corrole contains 3 protonated pyrrolic nitrogens (encircled). Consequently, upon deprotonation porphyrin is a dianionic, while corrole is a trianionic ligand. Such a difference results in differential metal/ligand stability, and affects properties of metals. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Under physiological conditions, H₂O₂ is maintained at nM steady-state levels by abundant peroxide-removing enzymes. Under pathological conditions, particularly in cancer, some of the enzymes are reportedly down-regulated (8, 93, 191, 233, 235, 245). The reaction of MnP with oxygen (Eq. [21]), superoxide (Eq. [2]) or cycling with ascorbate (Eq. [17a]) or thiols (Eq. [17b]) would result in increased levels of H₂O_2. Subsequently, MnP can utilize H₂O₂ to glutathionylate thiols of subunits of critical anti-apoptotic transcription factor, NF-κB (124). Tome's group demonstrated that the glutathionylation occurs only in the presence of H₂O₂ and GSH and could be best presented with reactions [22] through [24], where MnP acts as glutathione peroxidase, GPx. This agrees with reported GPx-like activity of para isomer, MnTM-4-PyP⁵⁺ (9). A reaction given by equation [22] is proposed by Jin et al. for MnTDE-2-ImP⁵⁺, which is similar in reactivities to MnTE-2-PyP⁵⁺ (128). The reaction described by Eq. [24] presents the disulfide interchange reaction (109): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}( { \rm H}_2 { \rm O} ) { \rm Mn}^{{ \rm II}} { \rm P}^{4 + } + { \rm O}_2 + { \rm H}_2 { \rm O} \Leftarrow\Rightarrow ( { \rm H}_2 { \rm O} ) _2 { \rm Mn}^{{ \rm III}} { \rm P}^{5 + } + { \rm O}_2^{{\cdot}{-}} \qquad [21]\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}( { \rm H}_2 { \rm O} ) ( { \rm OH} ) { \rm Mn}^{{ \rm III}} { \rm P}^{4 + } + { \rm H}_2 { \rm O}_2 \Leftarrow\Rightarrow { \rm O} = { \rm Mn}^{ \rm V} { \rm P} = { \rm O}^{3 + } \\ + 2 { \rm H}_2{ \rm O} + { \rm H}^ + \qquad [22]\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm O} = { \rm Mn}^{ \rm V} { \rm P} = { \rm O}^{3 + } + 2 \ { \rm GS}^ - + 4{ \rm H}^ + \Leftarrow\Rightarrow ( { \rm H}_2{ \rm O} ) _2 { \rm Mn}^{{ \rm III}} { \rm P}^{5 + } \\ + 2 \ { \rm GS}^{\cdot} & \qquad [23{\rm a}]\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}2{ \rm GS}^{\cdot} \Leftarrow\Rightarrow { \rm GSSG} \qquad [23{\rm b}]\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{\rm GSSG} + {\rm Protein - SH} \Leftarrow \Rightarrow \hbox{Protein - S - S - G} + {\rm GSH} \qquad [24]\end{align*} \end{document}

The anticancer radiation or corticosteroid-based therapy gives rise to high levels of H₂O₂. When administered along with MnP, the metal complex would catalyze glutathionylation of p65 subunit of NF-κB by H₂O₂ and GSH. Use of the redox-active metal complex along with the source of H₂O₂ has been proposed by us and others (268, 299) as a prospective anticancer treatment.

The Mn salen derivatives have reportedly the advantage because of their dual SOD and catalase-like activities (74). While the first is fair [log k_cat(H₂O₂) ∼6, Table 1] (34, 74, 249), the catalase-like activity is, however, insignificant, representing only ∼7×10⁻⁵% of the enzyme activity (1, 73, 74, 98, 132, 202). Besides unfavorable thermodynamics, the low metal/ligand stability of Mn salens disfavors high catalase-like activity.

Reactivity toward cellular reductants

Due to the biocompatible E_1/2 and favorable electrostatics (26, 28, 177), the pentacationic electrophilic Mn(III) N-substituted pyridylporphyrins rapidly react with anionic deprotonated forms of cellular reductants: ascorbate, glutathione, cysteine, and protein thiols (29, 33, 299). In such reactions, the MnPs act as oxidants. When undergoing oxidation with strong oxidants (such as ONOO⁻ or H₂O₂), the highly oxidizing Mn in +4 and +5 oxidation state is formed. It gets reduced back to either Mn +3 or +2 oxidation states while oxidizing reductants (instead of biological targets) that serve as suppliers of electrons (Eq. [25]) (38, 84, 86, 277). \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} &( { \rm H}_2{ \rm O} ) { \rm O} = { \rm Mn}^{{ \rm IV}} { \rm P}^{4 + } + { \rm HA}^ - + 2 { \rm H}^ + \Leftarrow\Rightarrow ( { \rm H}_2{ \rm O} ) _2{ \rm Mn}^{{ \rm III}} { \rm P}^{5 + } \\ & + { \rm HA}^{\cdot};\ { \rm HA}^{\cdot}\ \Leftarrow\Rightarrow {\rm A}^{{\cdot}{-}} + { \rm H}^ + \qquad [25]\end{align*} \end{document}

These reductants also likely couple with MnP in removing O₂·⁻ in vivo; consequently, the MnP may act as superoxide reductase rather than as SOD (Eq. [17] as a first step and Eq. [2] as a second step). The antioxidative effect of MnP in removing O₂·⁻ is assured only with sufficient physiological levels of enzymes that are capable of removing the H₂O₂. Otherwise, similar to cancer cells in which such enzymes are frequently down-regulated, the redox cycling of MnP with ascorbate or glutathione or cysteine would result in the accumulation of peroxide and its involvement in the oxidation of biological targets.

Ascorbate

The antitumor potential of ascorbate was demonstrated in a number of tumor cell lines and in animal models only when given intravenously (iv) or intraperitoneally (ip) (44 –46, 82, 116, 151, 199). Supplementation of ascorbate to an immunodeficient mouse with rapidly spreading 9l glioblastoma reduced tumor growth and weight by 41%–53%; in 30% cases, cancer spreads to other organs, while no dissemination of cancer was seen in ascorbate-treated mice (46). Ascorbate antitumor action was assigned to its oxidation and subsequent cytoxic H₂O₂ formation, catalyzed by endogenous metalloproteins (116). A much higher yield of peroxide may be achieved if catalysts are optimized for ascorbate oxidation. Such are isomeric Mn(III) N-substituted pyridylporphyrins; their H₂O₂-producing potency has already been shown by us and others (82, 220, 273, 299) (Fig. 11).

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FIG. 11.

Differential effect of MnP/ascorbate-driven H₂O₂ production on tumor and normal cells. (A) Ascorbate oxidation produces H₂O₂ that normal cells remove readily. Depending on the type of cancer cell, peroxide may not be well taken care of, leading to higher oxidative stress than in normal cells (16) (B) Redox status of cancer and tumor cell differs significantly, which determines their differential sensibility to an additional increase in reactive species. Tumor cells already have high levels of reactive species, and any further increase could cause their death (42, 114). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Ascorbate distributes into cells via SVCT1 and SVCT2 transporters, whereas GLUT transporters are responsible for the dehydroascorbate uptake (55). Decreased tumor ascorbate levels in endometrial cancer have been associated with high hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and GLUT favoring tumor progression (141).

We were the first to show, using several tumor cell lines, that the bio-compatible pentacationic electron-deficient ortho isomeric MnPs catalyze ascorbate oxidation while producing high amounts of peroxide and exerting cytotoxic effects [Roberts et al., unpublished (299) (Fig. 12) (see also Reactivity toward signaling proteins section). With two normal cell lines, either none or lower cytotoxicity relative to tumor cell was demonstrated (Fig. 12). A normal cell is well equipped with multiple H₂O₂-removing systems such as catalases, glutathione peroxidases, glutathione transferases, peroxyredoxins, and thioredoxins. Tumor cells, however, with a lower buffering capacity, are much more sensitive to any further increase in oxidative stress (Fig. 12) (8, 35, 93, 142, 177, 191, 233, 235, 245). In a cell culture, the MnP/ascorbate showed promise as potential treatment of inflammatory breast cancer; the noncaspase-, but AIF- and NF-κB-based apoptosis was promoted (82). Even when the SOD enzyme was up-regulated, the suppression of tumorigenesis was demonstrated by Tome et al., which is likely due to the down-regulation of peroxide-removing enzymes (40). The rapamycin increased levels of reactive species in a cellular model of mantle cell lymphoma (Grant519 and NCEB1) via inhibition of mTORC2 signaling. These cells presumably have insufficient levels of MnSOD, which would have otherwise reduced tumor progression. The increased levels of reactive species consequently up-regulate the MnSOD enzyme. The up-regulation of MnSOD increases the levels of reactive species, which suppresses the tumor cell growth (41).

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FIG. 12.

Differential cytotoxicity of MnP/ascorbate in tumor (A) and normal cell line (B). Two MnPs were tested, a hydrophilic MnTE-2-PyP⁵⁺ and a lipophilic MnTnHex-2-PyP⁵⁺ at 3 μM (shown) and 15 μM. The concentration of ascorbate was 1 mM. MnPs and ascorbate alone were not toxic. In the presence of ascorbate, both MnPs become toxic to cervical cancer cells, HeLa, but not to normal primary fibroblasts, NHDF cells. MTT assays were performed in a 96-well plate with an initial seeding density of 25,000 cells/well. Cells were incubated with MnP ±ascorbate for 48 h before the assay was performed (272, 299). The study was done in duplicate. Ascorbate was added soon after MnP. The effect of MnP/ascorbate on several other cancer cell lines has also been demonstrated (82, 299).

The para isomeric MnPs, such as MnTM-4-PyP⁵⁺, could be a better catalyst for ascorbate oxidation than their ortho analogs when judged solely on a thermodynamic basis. The E_1/2 of +60 mV versus NHE shows that, once reduced, the MnTM-4-PyP⁵⁺ more readily reoxidizes, thus producing H₂O₂. The ortho analog MnTE-2-PyP⁵⁺ with 162 mV more positive E_1/2 is more electron deficient and prefers residing in a reduced state. Further, the para compounds are planar and, thus, favor intercalation within groves of nucleic acids, which may prevent their action and/or cause toxicity to normal tissue (18, 220). Also, it would undergo oxidative degradation faster then ortho analog (30, 128). Rawal et al just reported the tumor suppression in sc nude mouse xenograft model of human pancreatic cell line (220).

It is important to note that the reduction of Mn^IIIP⁵⁺ with ascorbate reduces a total charge of MnP from penta- to tetracationic and, thus, enhances lipopilicity and cellular and tissue accumulation (251, 270). In cellular studies, the MnP/ascorbate was added exogenously into medium where it produced peroxide outside of the cell. Thus, under such conditions, no impact of the magnitude of MnP lipophilicity on its therapeutic efficacy was observed (299). In an E. coli model, however, the accumulation of ascorbate-reduced tetracationic MnP is greatly enhanced (251, 270). Thus, in addition to appropriate thermodynamics and kinetics of MnP/ascorbate redox system, the bioavailability of MnP would play a role in in vivo studies.

Welsh et al. just completed Phase I Clinical Trials in pancreatic cancer, where ascorbate was administered along with gemcitabine (291). Pancreatic cancer is the fourth leading cause of death in the United States, with 80% of mortality. The data obtained from Phase I are enthusiastic and warrant Phase II Clinical trials. As compared with an earlier report by Monti et al. (181), Welsh et al. infused ascorbate to achieve at least 20 mM levels in plasma, which would assure the antitumoral effect of ascorbate based on an earlier study of Du et al. (78). While Monti's study lasted only 8 weeks, to assure the safety and tolerability, the Welsh et al. study was several months long. In the absence of dose-limiting toxicity, the treatment was continued until progression (defined by Response Evaluation Criteria in Solid Tumors, RECIST). Even with only 8 weeks of ascorbate administration, the reduction in tumor volume was observed in 8 out of 9 patients in the Monti et al. study (181). The progression-free survival and overall survival was 12.7 weeks and 6 months with Monti et al. and 26 weeks and 12 months with Welsh et al. (181, 291). It is important to note that the high levels of ascorbate and peroxide did not induce systemic oxidative stress, as levels of F₂-isoprostanes remained the same or were reduced. The same was true for blood cells glutathione, which was unchanged as was the half-cell reduction potential. The ascorbate radicals were undetectable before treatment, while they were markedly increased in treated patients.

Buettner's group has also reported that ascorbate acts as a radiosensitizer in pancreatic cancer via production of reactive species (4, 290). The group is further exploring the role of transition metals and their porphyrin complexes on ascorbate-induced cytotoxicity in cancer (220).

Reactivity toward signaling proteins

The investigations of the effect of MnPs on the up-regulation of numerous cytokines (33) made it clear that either direct reactions with reactive species (which supposedly signal the start of the cellular transcription) or direct reactions with signaling proteins may be involved. The driving force for understanding such observations was the fact that MnPs were as efficient in reducing mouse or rat infarct volume when given at early (6 min) or at late time points (90 min or 6 h) after reperfusion (238 –240). Our knowledge at the time when the observation was made would predict that only immediate infusion of porphyrin into the brain at the time of reperfusion (the moment of peak production of reactive species) would ameliorate the primary oxidative damage. The protection demonstrated with delayed treatment, however, suggested that MnP must have suppressed the cellular transcription which would have otherwise perpetuated the oxidative stress (Fig. 13). Further exploration indicated that at least one major transcription factor, NF-κB, has been inactivated by the action of MnP (240). Similarly, the radioprotective effect of MnP was demonstrated when it was given for the duration of two weeks starting at any time postradiation from 2 h up to 8 weeks (96). HIF-1α and the genes it controls (VEGF, TGF-β) have been involved (95, 96). In cancer studies, the impact of MnP on activator protein-1 (AP-1) has been shown (304). In diabetes again, NF-κB and SP-1 have been identified as targets of MnPs (33, 278). Based on earlier and ongoing studies and our growing knowledge on MnPs, there is more to be identified.

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FIG. 13.

The impact of MnPs on transcription factors and kinases and phosphatases and, in turn, on the related genes. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

The mechanism of MnP-transcription factor interactions is not fully understood. We have initially assigned the effects of MnP almost exclusively to the removal of signaling reactive species (O₂·⁻, ONOO⁻), which resulted in the suppression of the cellular transcriptional activity. However, the most recent data suggest the direct and peroxide-driven oxidation of signaling proteins as a prevailing action of MnPs; depending on the cell type, the therapeutic outcome may be anti- or pro-oxidative (Fig. 13).

NF-κB—diabetes-and stroke-related cellular and animal studies

MnTDE-2-ImP⁵⁺ and MnTE-2-PyP⁵⁺ inhibited NF-κB activation in nuclear extracts of LPS-treated bone marrow-derived macrophages, and, therefore, suppressed pro-inflammatory cytokine production INF-γ and TNF-α. These results were recapitulated in human pancreatic cells cultured for 30 min in medium containing proteolytic enzymes and byproducts generated during cell isolation. The addition of 34 μM MnTDE-2-ImP⁵⁺ to isolated islet cells increased their survival and reduced levels of pro-inflammatory cytokines IL-6 and IL-8. Monocyte chemoattractant protein 1, MCAP-1 as well as PARP (poly(ADP-ribose)[polymerase]) were greatly suppressed and islets gained the capacity to normalize diabetic recipient mice (39, 40, 209). MnTE-2-PyP⁵⁺ also significantly delayed or prevented the diabetes altogether upon the treatment of young nonobese diabetic-severe combined immunodeficient mice with diabetogenic T-cell clone, BDC-2.5. All other aspects of NF-kB pathways were not affected, such as IKKα/β phposphorylation, and IKβ-α phosphorylation/degradation and p50/p65 nuclear translocation. Two additional experiments provided key support that MnP acted as a pro-oxidant, oxidizing cysteines whereby the antioxidant effects (listed earlier) were observed: (i) in a cell-free experiment (in the absence of GSH), MnP oxidized Cys-62 of p50 subunit of NF-κB, whereby disulfide formed prevented NF-κB activation (33, 278); (ii) in aqueous potassium phosphate-buffered solution, when only MnP and either cysteine or N-acetylcysteine or glutathione were present, the spectrophotometric evidence supported one-electron reduction of MnP (33). In vivo studies suggest that such NF-κB modification happens in the nucleus (33); data are supported by the biodistribution study in which MnTE-2-PyP⁵⁺ accumulated in the nucleus of macrophages at a 3-fold higher level than in the cytosol (33). Further on, the inactivation of NF-κB seems to play a major role in suppressing stroke injury with 2 hydrophilic and one lipophilic MnP. While in earlier studies hydrophilic MnPs were given intracerebroventricularly, most recently for the first time, the effect was demonstrated when lipophilic MnTnHex-2-PyP⁵⁺ was given subcutaneously for 6 days after initial iv bolus dose given at 30 minutes postreperfusion (Fig. 14) (238, 240). Again, similar to diabetes, the pro-oxidative action of MnP in stroke accounts for the antioxidative therapeutic outcome.

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FIG. 14.

The effect of MnTnHex-2-PyP⁵⁺ (A) on suppression of stroke injury in a mouse middle cerebral artery occlusion stroke model (238). (B) Infarct volumes were measured 7 days after 90 min MCAO. Rats were treated with intravenous vehicle (0.3 ml phosphate-buffered saline) or MnTnHex-2-PyP⁵⁺ (225 μg/kg) 5 min after reperfusion onset. The doses were repeated twice daily as subcutaneous injections for 7 days, after which cerebral infarct volume was measured. Open circles indicate individual animal values. Horizontal lines indicate group median values. MnTnHex-2-PyP⁵⁺ reduced cerebral infarct volume in the cortex (p=0.05) and subcortex (p=0.01), which was reflected in a 32% reduction in total infarct volume (p=0.028). Open circles indicate individual animal values. Horizontal lines indicate group mean values. (C) In studies on cytokines, rats were subjected to 90 min of middle cerebral artery occlusion. Five minutes after onset of reperfusion, rats were randomly treated with vehicle (n=3) or 225 μg/kg IV MnTnHex-2-PyP (n=3) followed by subcutaneous vehicle or 225 μg/kg MnTnHex-2-PyP⁵⁺, respectively, at 12 and 18 h post-MCAO. Brains were harvested at 24 h post-MCAO and analyzed for TNF-α and IL-6 by fluorescent enzyme-linked immunosorbent assay. Values represent mean±s.d. Both TNF-α and IL-6 concentrations were decreased by MnTnHex-2-PyP⁵⁺ (*p<0.05). (D) In NF-κB studies, four rats were subjected to 90 min middle cerebral artery occlusion and then treated with vehicle or MnTnHex-2-PyP⁵⁺ (225 μg/kg IV). Six hours later, ischemic brain was harvested to obtain nuclear extracts (2.5 μg) for electromobility shift assay (EMSA). The intravenous MnTnHex-2-PyP⁵⁺ decreased postischemic NF-κB DNA binding to a κB consensus oligo. Data shown are from Upper gel (EMSA): A-C are control lanes (A=probe only, B=positive control [HeLa nuclear extract], C=cold competitor). D and E (without and with p65 antibody, respectively) relate to rat #1 (vehicle). F and G (without and with p65 antibody, respectively) relate to rat #2 (MnTnHex-2-PyP⁵⁺). H and I=vehicle rat #3 (with and without p65). J and K=rat #4 (MnTnHex-2-PyP⁵⁺) with and without p65. Two slower migrating DNA-binding complexes are observed (shift). The proteins in the slower migrating complexes were identified by supershift analysis with 1 μg of p65-specific antibody. A marked reduction in NF-κB binding is seen in rats #2 and #4 (lanes F, G, J, and K, both hexyl). MCAO, middle cerebral artery occlusion. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

AIF—inflammatory breast cancer study

The effect of MnTnBuOE-2-PyP⁵⁺ on AIF, ERK, and p38(MAPK) kinases and X-linked inhibitor of apoptosis protein (XIAP) has been demonstrated in cellular inflammatory breast cancer study when MnP was co-administered with ascorbate (82). The MnP/ascorbate system enhances the oxidative stress via production of H₂O₂. In such pro-oxidative scenario, the GSH levels were reduced and the nuclear translocation of apoptosis-inducible factor AIF was enhanced, and so was the cellular apoptosis. With normal cells, such as cardiomyocyte, the mitochondrially localized MnP prevented the doxorubicin-based AIF-NHE adduct formation and nuclear translocation, and, in turn, inhibited the apoptosis (see earlier AIF cardiomyocytes). In an inflammatory breast cancer cell study (82), similar to a lymphoma study, the NF-kB was also suppressed (125, 126); in the former study, MnP/ascorbate, while in the latter, MnP/dexamethasone was the source of H₂O₂ (see below NF-kB—lymphoma).

HIF-1α–mouse breast cancer model

In a 4T1 breast cancer study, the suppression of hypoxia-related proteins was observed as well as the decrease in the levels of 8-oxo-2′-deoxyguanosine (8-OHdG), protein 3-nitrotyrosine, and NADPH oxidase. This could be a consequence of the anti-oxidative action of MnP. However, an MnP-driven oxidation of signaling proteins may be also operative; such an action would suppress secondary oxidative stress and, in turn, the up-regulation of various genes such as VEGF which would have otherwise contributed to the tumor growth delay (Fig. 15) (213). The data from Kim et al. (134) as well as the most recent data on the inhibition of NF-κB by Tome's group (125, 126) provide the basis for such reasoning (see below). NF-κB is known to control HIF-1α and NADPH oxidases (279). Kim et al. report indicates that a mild pro-oxidative event may cause the up-regulation of endogenous antioxidative defenses which action may result in antioxidative effects. If such scenario is operative, the effects could be wrongly assigned to the anti-oxidative action of MnP (134).

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FIG. 15.

The anticancer action of MnP as a sole agent. Anticancer effects of MnTE-2-PyP⁵⁺ (A) in a mouse sc 4T1 xenograft breast cancer model. 4T1 murine breast tumors were grown in Balb/C mice, allowed to reach ≥200 mm³ in size, and randomized to one of the three treatment groups: PBS; 2×1 mg/kg/day MnTE-2-PyP⁵⁺ (low) and 2×7.5 mg/kg/day MnTE-2-PyP⁵⁺ (high). MnP was given sc throughout the duration of the study. Immunohistochemistry of HIF-1α (C), carbonic anhydrase CAIX (D), and pimonidazole (E) and representative Western blot for HIF-1α is shown (B). Densitometric readings (HIF-1α/α-tubulin) of the Western blot are expressed as percentage control. While significant changes in different signaling proteins that impact the tumor growth were demonstrated, the effect on tumor growth delay was significant but not large, even at such a high dose as 15 mg/kg/day (213). HIF-1α, hypoxia inducible factor-1α. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

AP-1–a mouse skin carcinogenesis study

In skin carcinogenesis model, St. Clair's group has shown the inhibition of AP-1 by MnTE-2-PyP⁵⁺ and the reduction in the cell proliferation accompanied by the reduction in markers of oxidative stress (8-OHdG and protein carbonyls), all of which led to the remarkable suppression of skin papillomas (304). An antioxidative mechanism was proposed (304).

NF-κB—a lymphoma study

In a comprehensive study, Tome's group showed the pro-oxidative action of MnTE-2-PyP⁵⁺ on the enhancement of corticosteroid (dexamethasone)-induced lymphoma cell apoptosis (Fig. 16). The effect was ascribed to S-glutathionylation of p65 in cytosol, which prevented NF-κB DNA nuclear binding and, in turn, inhibited the up-regulation of anti-apoptotic genes (125, 126). Such modification required H₂O₂ and GSH. In turn, the cells were deprived of GSH—a major contributor to the physiological redox status. Tome's group also demonstrated that H₂O₂ was produced primarily in mitochondria by the action of dexamethasone (269)]. In addition, glucocorticoid treatment inactivates MnSOD. Were MnP a mimic of MnSOD, it would suppress (and not enhance) glucocorticoid-induced cell death.

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FIG. 16.

The chemosensitizing effect of MnE-2-PyP⁵⁺ in cellular lymphoma study (125). Dexamethasone alone (1 μM) and MnTE-2-PyP⁵⁺ (50 nM) each increased levels of reactive species, which resulted in glutathionylation of p65 and subsequent suppression of NF-κB DNA binding. The effects are largely enhanced with their co-administration to murine thymic lymphoma WEHI7.2 cells maintained in suspension in DMEM+10% calf serum. DMEM, Dulbecco's Modified Eagle Medium-low glucose. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

The aqueous-based studies have confirmed that MnP readily gets reduced one electronically by thiols (glutathione, cysteine, N-acteylcysteine, and dithionite), thereby producing thiyl radical. Thiyl radical production is a necessary step in S-glutathionylation. The glutathione peroxidase-like or cysteine oxidase-like activity of MnPs seems to be operative in S-glutathionylation. It is only natural that with similar thermodynamic features (reduction potentials), SOD enzymes and MnPs would undergo same reactions; however, the rates of such reactions would be limited by steric and electrostatic factors. While the low-molecular-weight MPs react readily with amino acids and proteins, the SOD enzymes have high specificity to small O₂·⁻ molecules, and will react with other large molecules that are orders of magnitude slower. For example, MnSOD reacts with ONOO⁻ at log k_red(ONOO⁻)=5, while MnPs reacts at log k_red(ONOO⁻)=7.34 (28, 84, 86). Cu,ZnSOD was shown to have Px-like activity, and, most recently, Bonini's group showed that MnSOD has Px-like activity also (7). The cysteine oxidase activity of Cu,ZnSOD was reported to be log k ∼5 (294). Our preliminary studies in simple aqueous systems have demonstrated such thiol oxidase activity for cationic Mn(III) N-alkylpyridylporphyrins (272). Araujo-Chaves et al. have indicated GPx activity of MnTM-4-PyP⁵⁺ (9). The scheme in Figure 17 proposes the GPx-like and thiol oxidase activity of MnP. We have shown that MnP/ascorbate system kills cancer cells, while it may be nontoxic or mildly toxic to normal cells (Fig. 12) (220, 272, 299). Please see the anticancer mechanism elucidated by Tome's group originating from the impact of MnP/dexamethasone on cellular bioenergetics (124). In the presence of dexamethasone, the mitochondrially located MnP glutathionylates, and in turn, inactivates critical proteins in electron transport chain, complexes I and III of mitochondrial respiration. Tome's group suggested to further combine MnP/dexamethasone therapy with 2-deoxyglucose inhibitor of glycolysis to deprive cancer cells from both energy sources and to down-regulate the anti-apoptotic pathways (124). Their study has vast therapeutic implications. Somewhat similar studies were reported by Kalyanaraman's group, where breast cancer MiaPaCa-2 cells were co-treated with redox-active mitochondrially targeted nitroxide, Mito-carboxypropyl (Mito-CP), and 2-deoxy-D-glucose (2-DG), which resulted in suppression of energy resources (48).

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FIG. 17.

The glutathione peroxidase (GPx) and thiol oxidase (TO) activities may contribute to the biological actions of MnPs. The proposed GPx-like (A) and thiol oxidase (B) activities of MnPs. The scheme is proposed based on the aqueous chemistry and experimental data in cellular and animal studies (272).

NF-kB- brain tumor

In an MnP/radiation study of nude/nude Balb/c sc xenograft mouse study of pediatric D-341-MED medulloblastoma, the preliminary data show the effect on NF-κB pathways (see under Therapeutic effects section; cancer studies).

Will pro-oxidative action of MnP promote oxidation or prevent oxidation of biological targets?

Our data imply that, while the reactions of MnP are likely identical, the magnitude and the end results of such reactions may be profoundly different in normal versus cancer cells, as much as in one versus another cancer cell type. Based on our present understanding, the resulting effects of electron shuttling by MnPs may be pro-oxidative rather than antioxidative, and particularly so in cancer cells that are already under the conditions of increased oxidative stress (16). The modes of action, other than antioxidative, have also been speculated for Mn salens (229) and polyphenols, and discussed by Forman et al. for natural compunds commonly viewed as antioxidants (16, 107, 108, 260). It may, thus, be concluded that the redox environment of the cell, including the levels and activities of the endogenous antioxidative enzymes, are main factors determinining how the cell will respond to MnP that is, the outcome of the therapy.

Pulmonary studies

In a rat pulmonary radioprotection study, 6 mg/kg MnTE-2-PyP⁵⁺ normalized breathing rate frequencies, as well as suppressed HIF-1α pathways and its gene VEGF when the two-week-long sc administration started at different time points between 2 h and 8 weeks after radiation. MnTnHex-2-PyP⁵⁺ was radioprotective also and at as low a dose as 0.05 mg/kg/day; the sc dosing started at 2 h after radiation and lasted for 2 weeks (95, 96). A significant decrease in HIF-1α, TGF-β, and VEGF A, as well as an overall reduction in lung damage (histopathology), was observed in animals in which MnP treatment started at the time of fully developed lung injury 8 weeks post-IR (96).

In another mouse pulmonary radioprotection study, the forty-four genes associated with metabolism, cell growth, apoptosis, inflammation, oxidative stress, and extracellular matrix synthesis were up-regulated throughout 6 months postradiation (122). The messenger RNA expression of adrenomedulin (Adm), 1-acylglycerol-3-phosphate O-acyltransferase 2 (Agpat2), N-acetyltransferase ARD 1 homolog (Ard1), connective tissue growth factor (CTGF), Enolase 1, α noneuron (Eno1), HIF-2α, guanine nucleotide binding protein, α11 (Gna11), protein kinase, AMP-activated, α catalytic subunit (Prkaa1), sorbitol dehydrogenase (Sdh1), and tubulin α3 (Tub α 3) were elevated in irradiated animals relative to control. The expression of DNA damage repair (Dr1), fatty acid-binding protein 4 (Fabp4), formin-binding protein 3 (Fnbp3), and solute carrier family 2, member 1 (Slc2a1) was elevated only at 6 month postradiation. The animals were treated with imidazolium derivative, MnTDE-2-ImP⁵⁺ (AEOL10150) (Fig. 1) starting at 24 h after 15 Gy whole thorax radiation with sc loading dose of 40 mg/kg, followed by 20 mg/kg (sc) every other day for 4 weeks. The impact of MnTDE-2-ImP⁵⁺ was evaluated at 6 weeks after radiation. The elevated expression of 31 of these genes was attenuated in animals treated with MnTDE-2-ImP⁵⁺, suggesting that expression of a number of hypoxia-associated genes is regulated by early development of oxidative stress after radiation. Such genes are: IL-1 β, TGF- β1, PPAR-α (peroxisome proliferator-activated receptor α), HIF-2β, carbonic anhydrase 12 (Car12), neuroblastoma myc-related oncogene (Nmyc1), cell division cycle 42 homolog (Cdc42), denactin 2 (Dctn2), death-associated kinase 3 (dapk3), matrix metalloproteinase 14 (MMP14), CTGF, Sdh1, and so on (122). Data substantiated the earlier observation (96) that MnP radioprotects even when its injections started at 24 h after injury.

The impact of MnTDE-2-ImP⁵⁺ on apoptotic pathways as a consequence of mouse pulmonary irradiation was also explored (Fig. 22) (302). The involvement of oxidative stress was verified via observed up-regulation of NOX4 and 8-OHdG, which were increased at 6 weeks after radiation. The apoptosis was observed primarily in type I and II pneumocytes and endothelium. Apoptosis correlated well with increased phosphoinositide 3-phosphatase (PTEN) and transforming growth factor TGF-β1 (excreted by many cells, including macrophages and involved in apoptosis via Sma- and Mad-related proteins), SMAD [SMAD proteins are homologs of both the Drosophila protein, mothers against decapentaplegic (MAD), and the Caenorhabditis elegans protein SMA (from gene sma for small body size)] and death-associated protein 6 (DAXX transcriptional activities), as well as inhibition of downstream PI3K/AK signaling, and increased p53 and Bax protein levels. MnTDE-2-ImP⁵⁺ suppressed oxidative stress also and, thus, the pro-apoptotic signaling, which consequently reduced the number of apoptotic cells (Fig. 22) (302).

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FIG. 22.

The effect of MnTDE-2-ImP⁵⁺ on oxidative stress and, in turn, on PTEN signaling pathways involved in a mouse pulmonary radioprotection. Shown are changes in PTEN signaling as measured by PTEN expression and protein levels obtained by immunostaining; the same finding was supported with Western blotting. Also shown are protein levels of Bax and p53 (western blotting). PI3K-AKT is a key signaling pathway that is negatively regulated by PTEN. Finally, the changes in transforming growth factor expression (TGF)-β1 (by immunostaining), NOX4 expression and protein levels (by immunostaining, and Western blotting), and 8-OHdG levels (by immunostaining) are also presented (302). 8-OHdG, 8-oxo-2′-deoxyguanosine; PTEN, phosphoinositide 3-phosphatase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

One study of pulonary radioprotection of nonhuman primates has been completed, and the other is in progress. Both studies administer low doses of MnTnHex-2-PyP⁵⁺. The treatment with MnP (sc twice daily at 0.05 mg/kg) delayed onset of radiation-induced lung lesions, prevented elevation of respiratory rate, and reduced lung weight, inflammation, edema, and epithelial hyperplasia (50).

Head and neck cancer studies

Significant radioprotection of mouse salivary glands were observed in a C57BL/6 study with MnTnBuOE-2-PyP⁵⁺ injected sc twice daily at 1.5 mg/kg for the duration of the study, starting one week before radiation (13). The study is a part of head and neck cancer studies (Ashcraft et al. in preparation). In preliminary studies, no protection of cancer was observed (Park et al., unpublished).

Hematopoietic studies

MnTE-2-PyP⁵⁺ protected bone marrow-derived hematopoietic stem cells (HSCs) against sublethal 6.5 Gy total body radiation (TBI) (152, 205). Mice were treated with 6 mg/kg MnTE-2-PyP⁵⁺ sc at 6 h after radiation and then, every day for 30 days. After treatment, the irradiated mice showed a significant recovery in the frequency and number of HSCs and a dramatic improvement in HSC and function of hematopoietic progenitor cells, HPC. The clonogenic function of HSCs from irradiated mice after MnP treatment was comparable to that of HSCs from normal controls, suggesting that MnP treatment inhibited the induction of HSC senescence by TBI. The effects were attributed to the inhibition of senescence p16 pathways, which is supported by the finding that MnP treatment also reduced the expression of p16Ink4a (p16) mRNA in HSCs induced by TBI and improved the long-term and multilineage engraftment of irradiated HSCs after transplantation (205). The authors reported that the reactive species produced by NADPH oxidases played a critical role.

Protection of erectile dysfunction during prostate radiation

The role of oxidative stress in prostate radiation-caused erectile dysfunction was demonstrated by Koontz group in a rat model (Fig. 23D–F) (136). Irradiated animals (eight to nine per group) received prostate-confined radiation in a single 20 Gy fraction. Radiation caused a significant decrease in intracavernous pressure and increased expression of NADPH oxidase isoaform 4 (NOX4), DNA damage (8-OHdG), and lipid peroxidation (measured by 4-hydroxynonenal) in prostate tissue and corpora cavernosa accompanied by a trend toward an increase in endogenous antioxidant defense, nuclear factor-erythroid-derived 2-like 2 (Nrf-2) (136).

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FIG. 23.

MnP protects against prostate radiation-induced injury to male reproductive system. The reversal of erectile dysfunction and the reduction in testes shrinkage by MnTE-2-PyP⁵⁺, induced after rat prostate radiation (A–C) as a consequence of radiation-induced oxidative stress (D–F) (192). Intracavernous pressure (ICP) was obtained after cavernous nerve stimulation as a measurement of erectile function 12 weeks postirradiation. Irradiation caused a significant decrease in ICP (RAD group) (B) as compared with the nonirradiated group (PBS). MnTE-2-PyP⁵⁺ protected from the irradiation-induced loss in ICP (MnTE-2-PyP RAD). n=8 rats/group, “*” denotes a significant difference from PBS group, p=0.05, and “#” denotes a significant difference from RAD group, p=0.05. The MnP also prevented testes shrinkage (C), hair loss (not shown), and the damage to prostate tissue (not shown) (192). The oxidative stress is demonstrated as the increase in NAPDH oxidase expression (F), macrophage infiltration (ED-1) (E), and Nrf-2 (the primary cellular defense against the cytotoxic effects of oxidative stress) up-regulation (D) (136) Nrf-2, nuclear factor-erythroid-derived 2-like 2. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

The MnTE-2-PyP⁵⁺ fully reversed the erectile dysfunction as a consequence of prostate tumor radiation (Fig. 23) (192). Mice were irradiated for five sequential days at 7.5 Gy/day in a lower pelvic region, which mimics the irradiation scheme of prostate tumor patients. MnP was injected at 5 mg/kg 24 h before starting with radiation and then, 2.5 mg/kg every other day for the next two weeks and 5 mg/kg once a week for 12 weeks postradiation. MnP reduced DNA damage as seen by the decrease in 8-OHdG levels in prostate epithelial cells. It also prevented the ∼60% of shrinkage of rat testes due to radiation, and prevented damage of penile tissue. Intracavernous pressure in was obtained after cavernous nerve stimulation as a measurement of erectile function 12 weeks postirradiation. A significant decrease in erectile function was demonstrated as compared with the nonirradiated mice and was greatly attenuated with MnP. The reduction in tissue damage and hair loss by MnP was demonstrated as well.

Prostate cancer

A study on the prostate cells evaluated the antitumor potential of MnTE-2-PyP⁵⁺. MnP was used in a dose-dependent fashion (1–30 μM). It significantly inhibited the growth of three prostate cancer cells: PC3 (98.2%±0.9%), Du145 (94.3%±0.7%), and LnCAP (100%±0%). MnTE-2-PyP⁵⁺ further reduced the growth of tumor cells when co-administered with radiation (5 Gy), but did not inhibit the proliferation of the prostate tumor cells. However, MnP significantly inhibited the invasiveness of PC3 cells on matrigel (67%±3.1%) and significantly inhibited Du145 cells (56.7%±5.6%) ability to migrate across a filter. MnTE-2-PyP⁵⁺ reduced NF-κB activity, which may inhibit tumor colony formation. It further reduced the phosphorylation of the focal adhesion kinase (involved in breaking down the extracellular matrix to allow cells to migrate), and VEGF-A in hypoxic prostate cells. VEGF-A is another protein of VEGF family that is involved in the growth migration of tumor cells (287).

Breast cancer

Significant radiosensitization via HIF-1α suppression and, in turn, vastly reduced vascular density exhibited by MnTE-2-PyP⁵⁺ was demonstrated in a 4T1 mouse breast cancer sc xenograft study (180). The MnTE-2-PyP⁵⁺exerted an anticancer effect as a single agent at a high (but not at low dose of 2×1 mg/kg/day) dose of 2×7.5 mg/kg/day for the duration of the study. Strong suppression of oxidative stress and angiogenesis (reduced NADPH oxidase expression, HIF-1α, VEGFR, macrophage infiltration, and protein nitration) was demonstrated (Fig. 15) (213).

Brain tumor—MnP-driven radiosensitization

Recently, a brain tumor study was conducted on sc xenografts of glioblastoma multiforme D-245 MG (eight mice per group). Similarly, lipophilic and redox-active drugs, MnTnHex-2-PyP⁵⁺ (shown in Fig. 2) and MnTnBuOE-2-PyP⁵⁺ exert similar radio- and temozolomide-sensitizing effects (21, 22). Tumor growth was delayed for ∼12 days as a result of MnP impact on radiation and temozolomide and in both of these treatments combined (Fig. 24). The significance in tumor growth delay with MnP/radiation therapy of pediatric medulloblastoma D-341 MED cell line (in a sc xenograft Balb/c nude/nude mouse was not reached under single conditions tested. However, the metastatic pathways (ctss, catepsin L, becn1, and beclin1) were largely down-regulated, as were antiapoptotic and NF-κB pathways (Nfkb1, Bcl211, and Bcl2) and PI3kinase and mTOR (Rsp6kb1). The protein translation changes were also implicated (EIF5b and Rsp6kb1)(22). The data are in agreement with the data on NF-κB inhibition by MnP obtained with a number of other models (see also under Reactivity towards signaling proteins section).

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FIG. 24.

The radiosensitizing effect of a lipophilic MnTnHex-2-PyP⁵⁺ in a sc xenograft nu/nu mouse Balb/c D 245-MG glioblastoma multiform model (21, 22). The mice (8/group) were treated twice daily via sc injections of 1.6 mg/kg of MnP (starting at 24 h before radiation and continued during the duration of the study), or 1 Gy radiation (3 days 1 Gy per day) and their combination. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Lymphoma

As discussed under Reactivity toward signaling proteins section, MnTE-2-PyP⁵⁺ enhanced corticosteroid (dexamethasone) therapy in a lymphoma cellular study via NF-κB pathways (Fig. 11) (125). The most recent study indicates that MnP additionally inactivates complexes I and III of mitochondrial electron transport chain, suppressing largely mitochondrial ATP production (123). MnP also suppresses glycolysis in its own right and when combined with glycolysis inhibitor 2-deoxyglucose (124); in turn, energy production of the cell seems to be greatly suppressed by the action of MnP (123).

Head and neck cancer

Preliminary data indicate the radiosensitizing effect of MnP, MnTnBuOE-2-PyP⁵⁺ in head and neck mouse sc xenograft study, which along with its ability to protect salivary glands and oral mucosis indicates its high therapeutic potential (13) (Ashcraft et al., in preparation).

Skin cancer

The anticancer effects of MnP as a single anticancer agent were previously reported in skin carcinogenesis model (304). See also under Reactivity towards signaling proteins section; Cancer studies. Please see Holley et al. contribution in this Forum, which suggests that the pharmacological intervention by an MnSOD mimic, MnTnBuOE-2-PyP⁵⁺, may play an important role in prevention of aberrant cell signaling that may contribute to carcinogenesis (117).

As discussed under Reactivity towards cellular reactants, the MnP is a promising anticancer agent in combination with ascorbate. Such a system, very similar to radiation therapy, is a source of cytotoxic H₂O₂ that MnP employs to oxidize a variety of biological targets (Tovmasyan et al., unpublished). Under certain conditions, such as in pediatric tumors, radiation should be avoided and could be potentially replaced with MnP/ascorbate. Related studies are still at cellular stage, and exploration of optimal ratio MnP/ascorbate is required as well as a thorough insight into the redox status of the each targeted cancer cell line. Another cycling system employing ascorbate is ascorbate/menadione combination, which is already in clinics for the treatment of prostate cancer (262, 284).