Mitochondrial Peroxiredoxin Research Articles

182P Therapeutic activity of thiostrepton in patient-derived malignant pleural effusions

Malignant pleural effusion (MPE) is a common complication of many tumor types, arising at the onset of terminal cancer with a median survival of 3-12 months. Herein, we have characterized and evaluated the activity of thiostrepton (TS) on malignant and immune cell types present in MPE from patients diagnosed with primary lung, breast, prostate, pancreatic and ovarian cancers. TS targets mitochondrial peroxiredoxin 3 (PRX3) and disrupts redox homeostasis preferentially in malignant tissues, presenting a promising therapeutic approach currently under investigation in the MITOPE clinical trial.

Trypanosoma cruzi Mitochondrial Peroxiredoxin Promotes Infectivity in Macrophages and Attenuates Nifurtimox Toxicity.

Trypanosoma cruzi is the causative agent of Chagas disease which is currently treated by nifurtimox (NFX) and benznidazole (BZ). Nevertheless, the mechanism of action of NFX is not completely established. Herein, we show the protective effects of T. cruzi mitochondrial peroxiredoxin (MPX) in macrophage infections and in response to NFX toxicity. After a 3-day treatment of epimastigotes with NFX, MPX content increased (2.5-fold) with respect to control, and interestingly, an MPX-overexpressing strain was more resistant to the drug. The generation of mitochondrial reactive species and the redox status of the low molecular weight thiols of the parasite were not affected by NFX treatment indicating the absence of oxidative stress in this condition. Since MPX was shown to be protective and overexpressed in drug-challenged parasites, non-classical peroxiredoxin activity was studied. We found that recombinant MPX exhibits holdase activity independently of its redox state and that its overexpression was also observed in temperature-challenged parasites. Moreover, increased holdase activity (2-fold) together with an augmented protease activity (proteasome-related) and an enhancement in ubiquitinylated proteins was found in NFX-treated parasites. These results suggest a protective role of MPX holdase activity toward NFX toxicity. Trypanosoma cruzi has a complex life cycle, part of which involves the invasion of mammalian cells, where parasite replication inside the host occurs. In the early stages of the infection, macrophages recognize and engulf T. cruzi with the generation of reactive oxygen and nitrogen species toward the internalized parasite. Parasites overexpressing MPX produced higher macrophage infection yield compared with wild-type parasites. The relevance of peroxidase vs. holdase activity of MPX during macrophage infections was assessed using conoidin A (CA), a covalent, cell-permeable inhibitor of peroxiredoxin peroxidase activity. Covalent adducts of MPX were detected in CA-treated parasites, which proves its action in vivo. The pretreatment of parasites with CA led to a reduced infection index in macrophages revealing that the peroxidase activity of peroxiredoxin is crucial during this infection process. Our results confirm the importance of peroxidase activity during macrophage infection and provide insights for the relevance of MPX holdase activity in NFX resistance.

Altered Expression of Peroxiredoxins in Mouse Model of Progressive Myoclonus Epilepsy upon LPS-Induced Neuroinflammation.

Stefin B (cystatin B) is an inhibitor of endo-lysosomal cysteine cathepsin, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht–Lundborg disease (EPM1), a form of progressive myoclonus epilepsy. Stefin B-deficient mice, a mouse model of the disease, display key features of EPM1, including myoclonic seizures. Although the underlying mechanism is not yet completely clear, it was reported that the impaired redox homeostasis and inflammation in the brain contribute to the progression of the disease. In the present study, we investigated if lipopolysaccharide (LPS)-triggered neuroinflammation affected the protein levels of redox-sensitive proteins: thioredoxin (Trx1), thioredoxin reductase (TrxR), peroxiredoxins (Prxs) in brain and cerebella of stefin B-deficient mice. LPS challenge was found to result in a marked elevation of Trx1 and TrxR in the brain and cerebella of stefin B deficient mice, while Prx1 was upregulated only in cerebella after LPS challenge. Mitochondrial peroxiredoxin 3 (Prx3), was upregulated also in the cerebellar tissue lysates prepared from unchallenged stefin B deficient mice, while after LPS challenge Prx3 was upregulated in stefin B deficient brain and cerebella. Our results imply the role of oxidative stress in the progression of the disease.

Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton.

A central hallmark of tumorigenesis is metabolic alterations that increase mitochondrial reactive oxygen species (mROS). In response, cancer cells upregulate their antioxidant capacity and redox-responsive signaling pathways. A promising chemotherapeutic approach is to increase ROS to levels incompatible with tumor cell survival. Mitochondrial peroxiredoxin 3 (PRX3) plays a significant role in detoxifying hydrogen peroxide (H2O2). PRX3 is a molecular target of thiostrepton (TS), a natural product and FDA-approved antibiotic. TS inactivates PRX3 by covalently adducting its two catalytic cysteine residues and crosslinking the homodimer. Using cellular models of malignant mesothelioma, we show here that PRX3 expression and mROS levels in cells correlate with sensitivity to TS and that TS reacts selectively with PRX3 relative to other PRX isoforms. Using recombinant PRXs 1–5, we demonstrate that TS preferentially reacts with a reduced thiolate in the PRX3 dimer at mitochondrial pH. We also show that partially oxidized PRX3 fully dissociates to dimers, while partially oxidized PRX1 and PRX2 remain largely decameric. The ability of TS to react with engineered dimers of PRX1 and PRX2 at mitochondrial pH, but inefficiently with wild-type decameric protein at cytoplasmic pH, supports a novel mechanism of action and explains the specificity of TS for PRX3. Thus, the unique structure and propensity of PRX3 to form dimers contribute to its increased sensitivity to TS-mediated inactivation, making PRX3 a promising target for prooxidant cancer therapy.

Comparison of the protective effect of cytosolic and mitochondrial Peroxiredoxin 5 against glutamate-induced neuronal cell death

ABSTRACT Objectives: Although glutamate is an essential factor in the neuronal system, excess glutamate can produce excitotoxicity. We previously reported that Peroxiredoxin 5 (Prx5) protects neuronal cells from glutamate toxicity via its antioxidant effects. However, it is unclear whether cytosolic or mitochondrial Prx5 provides greater neuroprotection. Here, we investigated differences in the neuroprotective effects of cytosolic and mitochondrial Prx5. Methods: We analyzed patterns of cytosolic and mitochondrial H2O2 generation in glutamate toxicity using HyPer protein. And then, we confirmed the change of intracellular ROS level and apoptosis with respective methods. The mitochondrial dynamics was assessed with confocal microscope imaging and western blotting. Results: We found that the level of mitochondrial H2O2 greatly increased compared to cytosolic H2O2 and it affected cytosolic H2O2 generation after glutamate treatment. In addition, we confirmed that mitochondrial Prx5 provides more effective neuroprotection than cytosolic Prx5. Discussion: Overall, our study reveals the mechanisms of cytosolic and mitochondrial ROS in glutamate toxicity. Our findings suggest that mitochondrial ROS and Prx5 are attractive therapeutic targets and that controlling these factors be useful for the prevention of neurodegenerative diseases.

Miro1-mediated mitochondrial positioning supports subcellular redox status

Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular positioning of mitochondria supports subcellular levels of ATP, Ca2+ and reactive oxygen species (ROS, i.e. hydrogen peroxide, H2O2). Previous work from our group showed that deletion of the mitochondrial adapter protein Miro1 leads to perinuclear clustering of mitochondria, leaving the cell periphery devoid of mitochondria which compromises peripheral energy status. Herein, we report that deletion of Miro1 significantly restricts subcellular H2O2 levels to the perinuclear space which directly affects intracellular responses to elevated mitochondrial ROS. Using the genetically encoded H2O2-responsive fluorescent biosensor HyPer7, we show that the highest levels of subcellular H2O2 map to sites of increased mitochondrial density. Deletion of Miro1 or disruption of microtubule dynamics with Taxol significantly reduces peripheral H2O2 levels. Following inhibition of mitochondrial complex 1 with rotenone we observe elevated spikes of H2O2 in the cell periphery and complementary oxidation of mitochondrial peroxiredoxin 3 (PRX3) and cytosolic peroxiredoxin 2 (PRX2). Conversely, in cells lacking Miro1, rotenone did not increase peripheral H2O2 or PRX2 oxidation but rather lead to increased nuclear H2O2 and an elevated DNA-damage response. Lastly, local levels of HyPer7 oxidation correlate with the size and abundance of focal adhesions (FAs) in MEFs and cells lacking Miro1 have significantly smaller focal adhesions and reduced phosphorylation levels of vinculin and p130Cas compared to Miro1+/+ MEFs. Together, we present evidence that the intracellular distribution of mitochondria influences subcellular H2O2 levels and local cellular responses dependent on mitochondrial ROS.

The critical role of redox regulation of PTEN and peroxiredoxin III in alcoholic fatty liver

Hepatic steatosis and subsequent fatty liver disease are developed in response to alcohol consumption. Reactive oxygen species (ROS) are thought to play an important role in the alcoholic fatty liver disease (AFLD). However, the molecular targets of ROS and the underlying cellular mechanisms are unknown. Here, we investigate roles of peroxiredoxin III and redox regulation of phosphatase and tension homolog deleted on chromosome 10 (PTEN) in the alcoholic fatty liver. Alcohol-induced mitochondrial oxidative stress was found to contribute to reversible oxidation of PTEN, which results in Akt and MAPK hyperactivation with elevated levels of the lipogenesis regulators SREBP1c and PPARγ. Moreover, mitochondrial peroxiredoxin III was found to have antagonistic effects on lipogenesis via the redox regulation of PTEN by removing ROS, upon alcohol exposure. This study demonstrated that redox regulation of PTEN and peroxiredoxin III play crucial roles in the development of AFLD.

Structural and biochemical analysis of 1-Cys peroxiredoxin ScPrx1 from Saccharomyces cerevisiae mitochondria

BackgroundScPrx1 is a yeast mitochondrial 1-Cys peroxiredoxins (Prx), a type of Prx enzyme which require thiol-containing reducing agents to resolve its peroxidatic cysteine. ScPrx1 plays important role in protection against oxidative stress. Mitochondrial thioredoxin ScTrx3 and glutathione have been reported to be the physiological electron donor for ScPrx1. However, the mechanism underlying their actions, especially the substrate recognition of ScPrx1 requires additional elucidation. METHODS: The structure of ScPrx1 was obtained through crystallization experiments. The oligomeric state of ScPrx1 was monitored by Blue-Native PAGE. Mutations were generated by the QuikChange PCR-based method. The ScPrx1 activity assay was carried out by measuring the change of 340 nm absorption of the NADPH oxidation. ResultsScPrx1 exist as a homodimer in solution. The structure adopts a typical Prx-fold core which is preceded by an N-terminal β-hairpin and has a C-terminal extension. Mutations (Glu94Ala, Arg198Ala and Trp126) close to the active site could enhance the catalytic efficiency of ScPrx1 while His83Ala and mutations on α4-β6 region exhibited reduced activity. The biochemical data also show that the deletion or mutations on ScPrx1 C-terminal have 2–4.56 fold increased activity. ConclusionWe inferred that conformational changes of ScPrx1 C-terminal segment were important for its reaction, and the α4-β6 loop regions around the ScPrx1 active sites were important for the catalytic function of ScPrx1. Collectively, these structural features provides a basis for understanding the diverse reductant species usage in different 1-Cys Prxs.

The mitochondrial peroxiredoxin displays distinct roles in different developmental stages of African trypanosomes

Hydroperoxide reduction in African trypanosomes relies on 2-Cys-peroxiredoxins (Prxs) and glutathione peroxidase-type enzymes (Pxs) which both obtain their reducing equivalents from the trypanothione/tryparedoxin couple and thus act as tryparedoxin peroxidases. While the cytosolic forms of the peroxidases are essential, the mitochondrial mPrx and Px III appear dispensable in bloodstream Trypanosoma brucei. This led to the suggestion that in this developmental stage which is characterized by a mitochondrion that lacks an active respiratory chain, only one of the two peroxidases might be required. Here we show that bloodstream cells in which the Px III gene is deleted and mPrx is down-regulated by RNA interference, proliferate as the parental cells indicating that both mitochondrial peroxidases are dispensable. However, when we raised the culture temperature to 39 °C, mPrx-depleted cells died indicating that under conditions mimicking a fever situation in the mammalian host, the protein becomes essential. In contrast, depletion of mPrx in insect stage procyclic T. brucei causes a proliferation defect under standard conditions at 27 °C, in the absence of any stress. In the absence of mPrx, a tryparedoxin-coupled roGFP2 biosensor expressed in the mitochondrial matrix is unable to respond to antimycin A treatment. Thus mPrx reduces mitochondrial H2O2 with the generation of trypanothione disulfide and acts as peroxidase. However, mPrx-depleted procyclic cells neither display any alteration in the cytosolic or mitochondrial trypanothione redox state nor increased sensitivity towards exogenous oxidative stressors suggesting that the peroxidase activity is not the crucial physiological function. After prolonged mPrx-depletion, the cells almost stop proliferation and display a highly elongated shape and diminished MitoTracker Red staining. In contrast to the situation in the mammalian bloodstream T. brucei and Leishmania, mPrx appears to play a constitutive role for the morphology, mitochondrial function and proliferation of the insect stage of African trypanosomes.

Variable protein expression in marine-derived filamentous fungus Penicillium chrysogenum in response to varying copper concentrations and salinity.

Copper is one of the essential trace dietary minerals for all living organisms, but is potentially toxic at higher concentrations, mainly due to the redox reactions in its transition state. Tolerance of microbes towards copper is primarily attributed to chelation and biosorption. In this study, marine-derived filamentous fungi were evaluated for their ability to remove Cu(ii) from a culture medium. Further, the cellular response of a select isolate to salinity stress (0, 35 and 100 PSU) and Cu(ii) stress (0, 100, and 500 ppm) was studied using the peptide mass fingerprinting technique, which revealed expression of 919 proteins, of which 55 proteins were commonly expressed across all conditions. Housekeeping proteins such as citrate synthase, pyruvate carboxylase, ribosomal proteins, ATP synthases, and more were expressed across all conditions. Reactive oxygen species scavenging proteins such as glutaredoxin, mitochondrial peroxiredoxins and thioredoxins were expressed under Cu(ii) and salinity stresses individually as well as in combination. Up-regulation of glutaredoxin under Cu(ii) stress with fold change values of 18.3 and 13.9 under 100 ppm and 500 ppm of Cu(ii) indicated active scavenging of free radicals to combat oxidative damage. The common mechanisms reported were enzymatic scavenging of free radicals, activation of DNA damage and repair proteins and probable intracellular metal chelation. This indicated multiple stress mechanisms employed by the isolate to combat the singular and synergistic effects of Cu(ii) and salinity stress.

Overexpression of the mitochondrial antioxidant protein peroxiredoxin 3 reduces the severity of age-related osteoarthritis in mice

Purpose: Mounting evidence suggests that mitochondrial dysfunction that results in higher levels of mitochondrial reactive oxygen species (ROS) contributes to the development of OA. Excessive levels of ROS not only cause random damage to cellular proteins, DNA, and lipids but also disturb cell signaling promoting catabolic activity. Hydrogen peroxide (H2O2) is a key intracellular ROS responsible for the regulation of redox signaling. Levels of intracellular H2O2 are controlled by the peroxiredoxins (Prxs) with Prx3 controlling levels of mitochondrial H2O2.

Upregulation of Mitochondrial Redox Sensitive Proteins in LPS-Treated Stefin B-Deficient Macrophages.

Stefin B (cystatin B) is an intracellular inhibitor of cysteine cathepsins and mutations in the stefin B gene, resulting in the development of Unverricht–Lundborg disease, which is a form of myoclonic epilepsy. It was suggested that a key mechanism behind stefin B-mediated disease progression was impaired redox homeostasis. Stefin B-deficient mice were found more sensitive to lipopolysaccharide (LPS)-induced sepsis as a consequence of increased expression of caspase-11 and Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing (NLRP nflammasome activation and higher levels of mitochondrial reactive oxygen species (ROS). In the present study, we investigated if LPS-triggered oxidative stress affected the protein levels and redox status of redox sensitive proteins—thioredoxin, peroxiredoxins, and superoxide dismutases in macrophages and spleens of LPS-injected mice. LPS challenge was found to result in a marked elevation in mitochondrial peroxiredoxin 3 (Prx3), sulfiredoxin, and superoxide dismutase 2 (Sod2) in stefin B-deficient macrophages and spleens. We determined that sulfiredoxin is targeted to mitochondria after LPS challenge. In conclusion, the upregulation of mitochondrial redox-sensitive proteins Prx3 and Sod2 in stefin B-deficient cells implies a protective role of stefin B in mitochondrial function.

Phenotypic screen for oxygen consumption rate identifies an anti-cancer naphthoquinone that induces mitochondrial oxidative stress

A hallmark of cancer cells is their ability to reprogram nutrient metabolism. Thus, disruption to this phenotype is a potential avenue for anti-cancer therapy. Herein we used a phenotypic chemical library screening approach to identify molecules that disrupted nutrient metabolism (by increasing cellular oxygen consumption rate) and were toxic to cancer cells. From this screen we discovered a 1,4-Naphthoquinone (referred to as BH10) that is toxic to a broad range of cancer cell types. BH10 has improved cancer-selective toxicity compared to doxorubicin, 17-AAG, vitamin K3, and other known anti-cancer quinones. BH10 increases glucose oxidation via both mitochondrial and pentose phosphate pathways, decreases glycolysis, lowers GSH:GSSG and NAPDH/NAPD+ ratios exclusively in cancer cells, and induces necrosis. BH10 targets mitochondrial redox defence as evidenced by increased mitochondrial peroxiredoxin 3 oxidation and decreased mitochondrial aconitase activity, without changes in markers of cytosolic or nuclear damage. Over-expression of mitochondria-targeted catalase protects cells from BH10-mediated toxicity, while the thioredoxin reductase inhibitor auranofin synergistically enhances BH10-induced peroxiredoxin 3 oxidation and cytotoxicity. Overall, BH10 represents a 1,4-Naphthoquinone with an improved cancer-selective cytotoxicity profile via its mitochondrial specificity.

Methyl and Ethylmercury elicit oxidative stress and unbalance the antioxidant system in Saccharomyces cerevisiae

Methylmercury (MeHg) and Ethylmercury (EtHg) are toxic to the central nervous system. Human exposure to MeHg and EtHg results mainly from the consumption of contaminated fish and thimerosal-containing vaccines, respectively. The mechanisms underlying the toxicity of MeHg and EtHg are still elusive. Here, we compared the toxic effects of MeHg and EtHg in Saccharomyces cerevisiae (S. cerevisiae) emphasizing the involvement of oxidative stress and the identification of molecular targets from antioxidant pathways. Wild type and mutant strains with deleted genes for antioxidant defenses, namely: γ-glutamylcysteine synthetase, glutathione peroxidase, catalase, superoxide dismutase, mitochondrial peroxiredoxin, cytoplasmic thioredoxin, and redox transcription factor Yap1 were used to identify potential pathways and proteins from cell redox system targeted by MeHg and EtHg. MeHg and EtHg inhibited cell growth, decreased membrane integrity, and increased the granularity and production of reactive species (RS) in wild type yeast. The mutants were predominantly less tolerant of mercurial than wild type yeast. But, as the wild strain, mutants exhibited higher tolerance to MeHg than EtHg. Our results indicate the involvement of oxidative stress in the cytotoxicity of MeHg and EtHg and reinforce S. cerevisiae as a suitable model to explore the mechanisms of action of electrophilic toxicants.

Interplay Between Mitochondrial Peroxiredoxins and ROS in Cancer Development and Progression.

Mitochondria are multifunctional cellular organelles that are major producers of reactive oxygen species (ROS) in eukaryotes; to maintain the redox balance, they are supplemented with different ROS scavengers, including mitochondrial peroxiredoxins (Prdxs). Mitochondrial Prdxs have physiological and pathological significance and are associated with the initiation and progression of various cancer types. In this review, we have focused on signaling involving ROS and mitochondrial Prdxs that is associated with cancer development and progression. An upregulated expression of Prdx3 and Prdx5 has been reported in different cancer types, such as breast, ovarian, endometrial, and lung cancers, as well as in Hodgkin’s lymphoma and hepatocellular carcinoma. The expression of Prdx3 and Prdx5 in different types of malignancies involves their association with different factors, such as transcription factors, micro RNAs, tumor suppressors, response elements, and oncogenic genes. The microenvironment of mitochondrial Prdxs plays an important role in cancer development, as cancerous cells are equipped with a high level of antioxidants to overcome excessive ROS production. However, an increased production of Prdx3 and Prdx5 is associated with the development of chemoresistance in certain types of cancers and it leads to further complications in cancer treatment. Understanding the interplay between mitochondrial Prdxs and ROS in carcinogenesis can be useful in the development of anticancer drugs with better proficiency and decreased resistance. However, more targeted studies are required for exploring the tumor microenvironment in association with mitochondrial Prdxs to improve the existing cancer therapies and drug development.

Hyperoxidation of mitochondrial peroxiredoxin limits H2O2‐induced cell death in yeast

Hydrogen peroxide (H2O2) plays important roles in cellular signaling, yet nonetheless is toxic at higher concentrations. Surprisingly, the mechanism(s) of cellular H2O2 toxicity remain poorly understood. Here, we reveal an important role for mitochondrial 1‐Cys peroxiredoxin from budding yeast, Prx1, in regulating H2O2‐induced cell death. We show that Prx1 efficiently transfers oxidative equivalents from H2O2 to the mitochondrial glutathione pool. Deletion of PRX1 abrogates glutathione oxidation and leads to a cytosolic adaptive response involving upregulation of the catalase, Ctt1. Both of these effects contribute to improved cell viability following an acute H2O2 challenge. By replacing PRX1 with natural and engineered peroxiredoxin variants, we could predictably induce widely differing matrix glutathione responses to H2O2. Therefore, we demonstrated a key role for matrix glutathione oxidation in driving H2O2‐induced cell death. Finally, we reveal that hyperoxidation of Prx1 serves as a switch‐off mechanism to limit oxidation of matrix glutathione at high H2O2 concentrations. This enables yeast cells to strike a fine balance between H2O2 removal and limitation of matrix glutathione oxidation.

Molecular characterization of novel mitochondrial peroxiredoxins from the Antarctic emerald rockcod and their gene expression in response to environmental warming

In the present study we describe the molecular characterization of the two paralogous mitochondrial peroxiredoxins from Trematomus bernacchii, a teleost that plays a pivotal role in the Antarctic food chain. The two putative amino acid sequences were compared with orthologs from other fish, highlighting a high percentage of identity and similarity with the respective variant, in particular for the residues that are essential for the characteristic peroxidase activity of these enzymes. The temporal expression of Prdx3 and Prdx5 mRNAs in response to short-term thermal stress showed a general upregulation of prdx3, suggesting that this isoform is the most affected by temperature increase. These data, together with the peculiar differences between the molecular structures of the two mitochondrial Prdxs in T. bernacchii as well as in the tropical species Stegastes partitus, suggest an adaptation that allowed these poikilothermic aquatic vertebrates to colonize very different environments, characterized by different temperature ranges.

Abstract 3589: Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy

Abstract Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells. Citation Format: Gang Cheng, Jacek Zielonka, Micael Hardy, Michael B. Dwinell, Balaraman Kalyanaraman. Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3589.

Abstract 3589: Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy

Invasive melanoma is an aggressive form of skin cancer with poor clinical outcomes. Nearly 50% of melanoma patients exhibit oncogenic BRAFV600E mutations. Existing BRAF kinase inhibitor drugs provide a short-term benefit prior to the rapid onset of drug resistance. Emerging results suggest that BRAF and other kinase inhibitors induce metabolic reprogramming from glycolytic energy production to oxidative phosphorylation (OXPHOS) in a subset of malignant melanomas. Metabolic reprogramming to OXPHOS is emerging as an important mechanism of resistance to BRAF kinase inhibitors and is associated with worse prognosis. Consequently, there is a significant opportunity for multimodal therapeutic approaches that selectively target the metabolic reprogramming associated with resistance to BRAF kinase inhibitors and promote immune-targeted therapy. We have previously developed mitochondria-targeted drugs by exploiting the enhanced negative mitochondrial membrane potential of cancer cells to selectively target and disrupt tumor growth. We hypothesized that metabolic reprogramming from glycolysis to OXPHOS in BRAF inhibitor-resistant melanoma cells would make them susceptible to OXPHOS inhibitors. In this study, we have modified the structure of a naturally occurring polyphenol, magnolol into a mitochondria-targeted-magnolol (Mito-MGN). We tested the effects of Mito-MGN on mitochondrial respiration and bioenergetic function, cellular redox status, and mitophagy in wild type (WT) and BRAF-resistant (metabolically reprogrammed) melanoma cells. Our data show that Mito-MGN inhibits mitochondrial respiration, melanoma cell proliferation and invasion at concentrations more than 200-fold lower than that of magnolol. Furthermore, Mito-MGN increases ROS formation and oxidation of mitochondrial peroxiredoxin. Preliminary results also indicate that BRAF inhibitor resistant melanoma cells exhibit enhanced OXPHOS as a consequence of metabolic reprogramming and that Mito-MGN effectively inhibits proliferation and mitochondrial respiration in the resistant cells. Mito-MGN inhibited mitochondrial complex I activity in both WT and resistant cells. The IC 50 values to inhibit complex I-mediated respiration was consistent with the values calculated for inhibition of cell respiration. We conclude that inhibition of complex I is a key initial event that is responsible for the anti-proliferative effects observed in the presence of Mito-MGN against wild-type as well as drug-resistant melanoma cells. Citation Format: Gang Cheng, Jacek Zielonka, Micael Hardy, Michael B. Dwinell, Balaraman Kalyanaraman. Targeting metabolic reprogramming and OXPHOS as a viable anti-melanoma strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3589.

Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis

Lung cancer often has a poor prognosis, with brain metastases a major reason for mortality. We modified lonidamine (LND), an antiglycolytic drug with limited efficacy, to mitochondria-targeted mito-lonidamine (Mito-LND) which is 100-fold more potent. Mito-LND, a tumor-selective inhibitor of oxidative phosphorylation, inhibits mitochondrial bioenergetics in lung cancer cells and mitigates lung cancer cell viability, growth, progression, and metastasis of lung cancer xenografts in mice. Mito-LND blocks lung tumor development and brain metastasis by inhibiting mitochondrial bioenergetics, stimulating the formation of reactive oxygen species, oxidizing mitochondrial peroxiredoxin, inactivating AKT/mTOR/p70S6K signaling, and inducing autophagic cell death in lung cancer cells. Mito-LND causes no toxicity in mice even when administered for eight weeks at 50 times the effective cancer inhibitory dose. Collectively, these findings show that mitochondrial targeting of LND is a promising therapeutic approach for investigating the role of autophagy in mitigating lung cancer development and brain metastasis.