Manganese superoxide dismutase (MnSOD/SOD2) is a mitochondria-resident enzyme that governs the types of reactive oxygen species egressing from the organelle to affect cellular signaling. Here, we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting MnSOD expression or inhibiting AMPK suppress the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical in support cancer cell bioenergetics. Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes. Taken together, our results indicate that MnSOD serves as a biomarker of cancer progression and acts as critical regulator of tumor cell metabolism.

The inositol pyrophosphates 5-InsP (diphosphoinositol pentakisphosphate) and 1,5-InsP (bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular signals interconverted by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks). Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP and raises 5-InsP levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP] provides direct support for an energy-sensing attribute (i.e., 1 mM for ATP) of the 5-InsP-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP and 5-InsP as drivers of the hypermetabolic and p53-elevated phenotypes, we used RNAi and the pan-IP6K inhibitor,2-(-trifluorobenzyl), 6-(-nitrobenzyl) purine (TNP), to return 5-InsP levels in cells to those of WT cells without rescuing 1,5-InsP levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP regulates p53 expression, whereas 1,5-InsP regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP to bioenergetic homeostasis.

Background and Aims Progression from steatosis to steatohepatitic lesions is hypothesized to require a second hit. These lesions have been associated with increased oxidative stress, often ascribed to high levels of leptin and other proinflammatory mediators. Here we have examined the role of leptin in inducing oxidative stress and Kupffer cell activation in CCl4-mediated steatohepatitic lesions of obese mice. Methods Male C57BL/6 mice fed with a high fat diet (60%kcal) at 16 weeks were administered CCl4 to induce steatohepatitic lesions. Approaches included use of immuno-spin trapping for measuring free radical stress, gene-deficient mice for leptin, p47 phox, iNOS and adoptive transfer of leptin primed macrophages in vivo. Results Diet-induced obese (DIO) mice, treated with CCl4 increased serum leptin levels. Oxidative stress was significantly elevated in DIO mice liver but not in OB/OB mice, or in DIO mice treated with leptin antibody. In OB/OB mice, leptin supplementation restored markers of free radical generation. Markers of free radical formation were significantly decreased by the peroxynitrite decomposition catalyst FeTPPS, the iNOS inhibitor 1400W, the NADPH oxidase inhibitor apocynin, or in iNOS or p47 phox-deficient mice. These results correlated with the decreased expression of TNF-alpha and MCP-1. Kupffer cell depletion eliminated oxidative stress and inflammation, whereas in macrophage-depleted mice, the adoptive transfer of leptin-primed macrophages significantly restored inflammation. Conclusions These results, for the first time, suggest that leptin action in macrophages of steatotic liver through induction of iNOS and NADPH oxidase caused peroxynitrite-mediated oxidative stress thus activating Kupffer cells.

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1], [2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H2O2) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H2O2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O2•–) and H2O2 in the presence of NADH. Generation of the free radical O2•– and H2O2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies.

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of “stemness” genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.

Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H2O2 outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism and apoptosis thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase (GPx) activity along with increases in H2O2 further increase cancer risk in the face MnSOD overexpression. These facts led us to hypothesize that, like the Cu, Zn-counterpart, MnSOD may work as a peroxidase, utilizing H2O2 to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed.

Superoxide radical anion (O) and other reactive oxygen species are constantly produced during respiration. In mitochondria, the dismutation of O is accelerated by the mitochondrial superoxide dismutase 2 (SOD2), an enzyme that has been traditionally associated with antioxidant protection. However, increases in SOD2 expression promote oxidative stress, indicating that there may be a prooxidant role for SOD2. Here we show that SOD2, which normally binds manganese, can incorporate iron and generate an alternative isoform with peroxidase activity. The switch from manganese to iron allows FeSOD2 to utilize HO to promote oxidative stress. We found that FeSOD2 is formed in cultured cells and in vivo. FeSOD2 causes mitochondrial dysfunction and higher levels of oxidative stress in cultured cells and in vivo. We show that formation of FeSOD2 converts an antioxidant defense into a prooxidant peroxidase that leads to cellular changes seen in multiple human diseases.

Astaxanthin (ASTA) is a pinkish-orange carotenoid commonly found in marine organisms, especially salmon. ASTA is a powerful antioxidant and suggested to provide benefits for human health, including the inhibition of LDL oxidation, UV-photoprotection, and prophylaxis of bacterial stomach ulcers. Exercise is associated to overproduction of free radicals in muscles and plasma, with pivotal participation of iron ions and glutathione (GSH). Thus, ASTA was studied here as an auxiliary supplement to improve antioxidant defenses in soleus muscles and plasma against oxidative damage induced by exhaustive exercise. Long-term 1 mg ASTA/kg body weight (BW) supplementation in Wistar rats (for 45 days) significantly delayed time to exhaustion by 29% in a swimming test. ASTA supplementation increased scavenging/iron-chelating capacities (TEAC/FRAP) and limited exercise-induced iron overload and its related pro-oxidant effects in plasma of exercising animals. On the other hand, ASTA induced significant mitochondrial Mn-dependent superoxide dismutase and cytosolic glutathione peroxidase antioxidant responses in soleus muscles that, in turn, increased GSH content during exercise, limited oxidative stress, and delayed exhaustion. We also provided significant discussion about a putative “mitochondrial-targeted” action of ASTA based on previous publications and on the positive results found in the highly mitochondrial populated (oxidative-type) soleus muscles here.