Mitochondrial Superoxide Dismutase in Cisplatin-Induced Kidney Injury.

Cisplatin is a chemotherapy agent commonly used to treat a wide variety of cancers. Despite the potential for both severe acute and chronic side effects, it remains a preferred therapeutic option for many malignancies due to its potent anti-tumor activity. Common cisplatin-associated side-effects include acute kidney injury (AKI) and chronic kidney disease (CKD). These renal injuries may cause delays and potentially cessation of cisplatin therapy and have long-term effects on renal function reserve. Thus, developing mechanism-based interventional strategies that minimize cisplatin-associated kidney injury without reducing efficacy would be of great benefit. In addition to its action of cross-linking DNA, cisplatin has been shown to affect mitochondrial metabolism, resulting in mitochondrially derived reactive oxygen species (ROS). Increased ROS formation in renal proximal convoluted tubule cells is associated with cisplatin-induced AKI and CKD. We review the mechanisms by which cisplatin may induce AKI and CKD and discuss the potential of mitochondrial superoxide dismutase mimetics to prevent platinum-associated nephrotoxicity.

Avasopasem manganese synergizes with hypofractionated radiation to ablate tumors through the generation of hydrogen peroxide.

Avasopasem manganese (AVA or GC4419), a selective superoxide dismutase mimetic, is in a phase 3 clinical trial (NCT03689712) as a mitigator of radiation-induced mucositis in head and neck cancer based on its superoxide scavenging activity. We tested whether AVA synergized with radiation via the generation of hydrogen peroxide, the product of superoxide dismutation, to target tumor cells in preclinical xenograft models of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, and pancreatic ductal adenocarcinoma. Treatment synergy with AVA and high dose per fraction radiation occurred when mice were given AVA once before tumor irradiation and further increased when AVA was given before and for 4 days after radiation, supporting a role for oxidative metabolism. This synergy was abrogated by conditional overexpression of catalase in the tumors. In addition, in vitro NSCLC and mammary adenocarcinoma models showed that AVA increased intracellular hydrogen peroxide concentrations and buthionine sulfoximine- and auranofin-induced inhibition of glutathione- and thioredoxin-dependent hydrogen peroxide metabolism selectively enhanced AVA-induced killing of cancer cells compared to normal cells. Gene expression in irradiated tumors treated with AVA suggested that increased inflammatory, TNFα, and apoptosis signaling also contributed to treatment synergy. These results support the hypothesis that AVA, although reducing radiotherapy damage to normal tissues, acts synergistically only with high dose per fraction radiation regimens analogous to stereotactic ablative body radiotherapy against tumors by a hydrogen peroxide-dependent mechanism. This tumoricidal synergy is now being tested in a phase I-II clinical trial in humans (NCT03340974).

Persistent increase in mitochondrial superoxide mediates cisplatin-induced chronic kidney disease.

Severe and recurrent cisplatin-induced acute kidney injury (AKI) as part of standard cancer therapy is a known risk factor for development of chronic kidney disease (CKD). The specific role of superoxide (O2•-)-mediated disruption of mitochondrial oxidative metabolism in CKD after cisplatin treatment is unexplored. Cisplatin is typically administered in weekly or tri-weekly cycles as part of standard cancer therapy. To investigate the role of O2•- in predisposing patients to future renal injury and in CKD, mice were treated with cisplatin and a mitochondrial-specific, superoxide dismutase (SOD) mimetic, GC4419. Renal function, biomarkers of oxidative stress, mitochondrial oxidative metabolism, and kidney injury markers, as well as renal histology, were assessed to evaluate the cellular changes that occur one week and one month (CKD phase) after the cisplatin insult. Cisplatin treatment resulted in persistent upregulation of kidney injury markers, increased steady-state levels of O2•-, increased O2•--mediated renal tubules damage, and upregulation of mitochondrial electron transport chain (ETC) complex I activity both one week and one month following cisplatin treatment. Treatment with a novel, clinically relevant, small-molecule superoxide dismutase (SOD) mimetic, GC4419, restored mitochondrial ETC complex I activity to control levels without affecting complexes II-IV activity, as well as ameliorated cisplatin-induced kidney injury. These data support the hypothesis that increased mitochondrial O2•- following cisplatin administration, as a result of disruptions of mitochondrial metabolism, may be an important contributor to both AKI and CKD progression.

Superoxide Dismutase Mimetic GC4419 Enhances the Oxidation of Pharmacological Ascorbate and Its Anticancer Effects in an H2O2-Dependent Manner.

Lung cancer, together with head and neck cancer, accounts for more than one-fourth of cancer deaths worldwide. New, non-toxic therapeutic approaches are needed. High-dose IV vitamin C (aka, pharmacological ascorbate; P-AscH-) represents a promising adjuvant to radiochemotherapy that exerts its anti-cancer effects via metal-catalyzed oxidation to form H2O2. Mn(III)-porphyrins possessing superoxide dismutase (SOD) mimetic activity have been shown to increase the rate of oxidation of AscH-, enhancing the anti-tumor effects of AscH- in several cancer types. The current study demonstrates that the Mn(II)-containing pentaazamacrocyclic selective SOD mimetic GC4419 may serve as an AscH-/O2•- oxidoreductase as evidenced by the increased rate of oxygen consumption, steady-state concentrations of ascorbate radical, and H2O2 production in complete cell culture media. GC4419, but not CuZnSOD, was shown to significantly enhance the toxicity of AscH- in H1299, SCC25, SQ20B, and Cal27 cancer cell lines. This enhanced cancer cell killing was dependent upon the catalytic activity of the SOD mimetic and the generation of H2O2, as determined using conditional overexpression of catalase in H1299T cells. GC4419 combined with AscH- was also capable of enhancing radiation-induced cancer cell killing. Currently, AscH- and GC4419 are each being tested separately in clinical trials in combination with radiation therapy. Data presented here support the hypothesis that the combination of GC4419 and AscH- may provide an effective means by which to further enhance radiation therapy responses.

Mitochondrial Superoxide Increases Age-Associated Susceptibility of Human Dermal Fibroblasts to Radiation and Chemotherapy.

Elderly cancer patients treated with ionizing radiation (IR) or chemotherapy experience more frequent and greater normal tissue toxicity relative to younger patients. The current study demonstrates that exponentially growing fibroblasts from elderly (old) male donor subjects (70, 72, and 78 years) are significantly more sensitive to clonogenic killing mediated by platinum-based chemotherapy and IR (∼70%-80% killing) relative to young fibroblasts (5 months and 1 year; ∼10%-20% killing) and adult fibroblasts (20 years old; ∼10%-30% killing). Old fibroblasts also displayed significantly increased (2-4-fold) steady-state levels of O2•-, O2 consumption, and mitochondrial membrane potential as well as significantly decreased (40%-50%) electron transport chain (ETC) complex I, II, IV, V, and aconitase (70%) activities, decreased ATP levels, and significantly altered mitochondrial structure. Following adenoviral-mediated overexpression of SOD2 activity (5-7-fold), mitochondrial ETC activity and aconitase activity were restored, demonstrating a role for mitochondrial O2•- in these effects. Old fibroblasts also demonstrated elevated levels of endogenous DNA damage that were increased following treatment with IR and chemotherapy. Most importantly, treatment with the small-molecule, superoxide dismutase mimetic (GC4419; 0.25 µmol/L) significantly mitigated the increased sensitivity of old fibroblasts to IR and chemotherapy and partially restored mitochondrial function without affecting IR or chemotherapy-induced cancer cell killing. These results support the hypothesis that age-associated increased O2•- and resulting DNA damage mediate the increased susceptibility of old fibroblasts to IR and chemotherapy that can be mitigated by GC4419. Cancer Res; 77(18); 5054-67. ©2017 AACR.

O2⋠- and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate.

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2⋅- and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

Antioxidants in Translational Medicine.

SIGNIFICANCE: It is generally accepted that reactive oxygen species (ROS) scavenging molecules or antioxidants exert health-promoting effects and thus their consumption as food additives and nutraceuticals has been greatly encouraged. Antioxidants may be beneficial in situations of subclinical deficiency and increased demand or acutely upon high-dose infusion. However, to date, there is little clinical evidence for the long-term benefit of most antioxidants. Alarmingly, recent evidence points even to health risks, in particular for supplements of lipophilic antioxidants. RECENT ADVANCES: The biological impact of ROS depends not only on their quantities but also on their chemical nature, (sub)cellular and tissue location, and the rates of their formation and degradation. Moreover, ROS serve important physiological functions; thus, inappropriate removal of ROS may cause paradoxical reductive stress and thereby induce or promote disease. CRITICAL ISSUES: Any recommendation on antioxidants must be based on solid clinical evidence and patient-relevant outcomes rather than surrogate parameters. FUTURE DIRECTIONS: Such evidence-based use may include site-directed application, time-limited high dosing, (functional) pharmacological repair of oxidized biomolecules, and triggers of endogenous antioxidant response systems. Ideally, these approaches need guidance by patient stratification through predictive biomarkers and possibly imaging modalities.

Superoxide mediates acute liver injury in irradiated mice lacking sirtuin 3.

AIMS: This study determined whether acute radiation-induced liver injury seen in Sirtuin3(-/-) mice after exposure to Cs-137 γ-rays was mediated by superoxide anion (O2(•-)). RESULTS: Male wild-type (WT) and SIRT3(-/-) mice were given 2×2 Gy whole-body radiation doses separated by 24 h and livers were harvested 20 h after the second dose. Ex vivo measurements in fresh frozen liver sections demonstrated 50% increases in dihydroethidium oxidation from SIRT3(-/-) animals, relative to WT animals, before irradiation, but this increase was not detected 20 h after radiation exposure. In addition, irradiated livers from SIRT3(-/-) animals showed significant hydropic degeneration, loss of MitoTracker Green FM staining, increased immunohistochemical staining for 3-nitrotyrosine, loss of Ki67 staining, and increased mitochondrial localization of p53. These parameters of radiation-induced injury were significantly attenuated by an intraperitoneal injection of 2 mg/kg of the highly specific superoxide dismutase mimic, GC4401, 30 min before each fraction. INNOVATION: Sirtuin 3 (SIRT3) is believed to regulate mitochondrial oxidative metabolism and antioxidant defenses in response to acute radiation-induced liver injury. This work provides strong evidence for the causal role of O2(•-) in the liver injury process initiated by whole-body irradiation in SIRT3(-/-) mice. CONCLUSION: These results support the hypothesis that O2(•-) mediates acute liver injury in SIRT3(-/-) animals exposed to whole-body γ-radiation and suggest that GC4401 could be used as a radio-protective compound in vivo.

Peroxynitrite decomposition activity of iron porphyrin complexes.

Peroxynitrite (ONOO(-)/ONOOH), a putative cytotoxin formed by combination of nitric oxide (NO.) and superoxide (HO(2)(.)) radicals, is decomposed catalytically by micromolar concentrations of water-soluble Fe(III) porphyrin complexes, including 5,10,15,20-tetrakis(2',4',6'-trimethyl-3,5-disulfonatophenyl)porphyrinatoferrate(7-), Fe(TMPS); 5,10,15,20-tetrakis(4'-sulfonatophenyl)porphyrinatoiron(3-), Fe(TPPS); and 5,10,15,20-tetrakis(N-methyl-4'-pyridyl) porphyrinatoiron(5+), Fe(TMPyP). Spectroscopic (UV-visible), kinetic (stopped-flow), and product (ion chromatography) studies reveal that the catalyzed reaction is a net isomerization of peroxynitrite to nitrate (NO(3)(-)). One-electron catalyst oxidation forms an oxoFe(IV) intermediate and nitrogen dioxide, and recombination of these species is proposed to regenerate peroxynitrite or to yield nitrate. Michaelis-Menten kinetics are maintained accordingly over an initial peroxynitrite concentration range of 40-610 microM at 5.0 microM catalyst concentrations, with K(m) in the range 370-620 microM and limiting turnover rates in the range of 200-600 s(-1). Control experiments indicate that nitrite is not a kinetically competent reductant toward the oxidized intermediates, thus ruling out a significant role for NO(2)(.) hydrolysis in catalyst turnover. However, ascorbic acid can intercept the catalytic intermediates, thus directing product distributions toward nitrite and accelerating catalysis to the oxidation limit. Additional mechanistic details are proposed on the basis of these and various other kinetic observations, specifically including rate effects of catalyst and peroxynitrite concentrations, solution pH, and isotopic composition.

The contribution of oxidative stress in apoptosis of human-cultured astroglial cells induced by supernatants of HIV-1-infected macrophages.

Apoptosis of neurons and astrocytes has been found in patients undergoing AIDS dementia complex. We demonstrated that supernatants from human primary macrophages (M/M) infected by HIV-1 lead human astroglial cells to oxidative stress, as shown by elevated levels of malondialdehyde, and then to apoptosis. Electron microscopy of astrocytes shortly incubated with HIV-1-infected M/M supernatants showed apoptotic blebbing, cytoplasmic loss, and chromatin condensation. Apoptosis was antagonized by pretreating astrocytes with the nonpeptidic superoxide dismutase (SOD) mimetic M40401 but not with anti-HIV-1 compounds, thus showing that apoptosis of astrocytes driven by HIV-1-infected M/M supernatants is mainly mediated by abnormal production of superoxide anions without relationship to HIV-1 replication in such cells. Overall results support the role of oxidative stress mediated by HIV-1-infected M/M as one of the leading causes of neurodegeneration in patients with HIV-1 and suggest the use of nonpeptidic SOD mimetics to counteract HIV-1-related neurological disorders.