MolecularAspects of Methylcadmium Toxicity: Effectson the H2O2 Reduction by Cysteine and SelenocysteineDisclosed In Silico

An effective steady-state redox balance is maintained in cancer cells, allowing for protection against oxidative stress and thereby enhancing cell proliferation and tumor growth. Disruption of this redox balance would increase the cellular content of reactive oxygen species (ROS) and potentiate oxidative stress-induced cell death in tumor cells, thus representing an effective strategy for cancer treatment. Glutathione (GSH) is a major reducing agent, and its cellular levels are determined at least partly by the availability of cysteine via xCT (SLC7A11)-mediated entry of cystine into cells. We developed a nanoplatform using ZnO nanoparticles (NPs) as a carrier, loaded with salicylazosulfapyridine (SASP), and stabilized with DSPE-PEG, to form ultra-small NPs (SASP/ZnO NPs). The goal of this NP strategy is to disrupt the redox balance in cells by two mechanisms: increased generation of ROS and decreased synthesis of GSH. Such an approach would be effective in killing tumor cells. As expected, the SASP/ZnO NPs enhanced ROS production because of ZnO and impaired GSH synthesis because of SASP-induced inhibition of xCT (SLC7A11) transport function. As a consequence, treatment of tumor cells with SASP/ZnO NPs in vitro and in vivo resulted in a synergistic disruptive effect on redox balance in tumor cells and induced cell death and decreased tumor growth. This ambidextrous approach has potential in cancer therapy by combining two complementary pathways to disrupt the redox balance in tumor cells.

In addition to the induction of cell proliferation and migration, bradykinin (BK) can increase c-fos mRNA expression, activate ERK 1/2 and generate reactive oxygen species (ROS) in vascular smooth muscle cells (VSMC). It is not known, however, whether BK can induce cellular proliferation and extracellular matrix production via redox-sensitive signaling pathways. We investigated the role(s) of ROS in proliferation, migration and collagen synthesis induced by BK in VSMC derived from Sprague Dawley rat aorta. BK (10 nM) increased VSMC proliferation by 30% (n=5); this proliferation was inhibited by the antioxidants N-acetylcysteine (20 mM) and alpha-lipoic acid (LA, 250 mM). In addition, BK induced an increase in cell migration and in collagen levels that were blocked by LA. ROS production induced by BK (n=10) was significantly inhibited by bisindolylmaleimide (4microM) and by PD98059 (40microM). These results suggest that: 1) ROS participate in the mechanism(s) used by bradykinin to induce cellular proliferation; 2) bradykinin induces ROS generation through a pathway that involves the kinases PKC and MEK; and 3) ROS participate in the pathways mediating cell migration and the production of collagen as a response to treatment with bradykinin. To our knowledge, this is the first report describing mechanisms to explain the participation of ROS in the cellular proliferation and extracellular matrix pathway regulated by BK.

Medulloblastoma is the most common solid malignant pediatric brain tumor. These tumors arise in the cerebellum and can be molecularly subdivided into 4 consensus subgroups, one of which is marked by amplification and activation of Sonic hedgehog (Shh) pathway components and downstream targets. This subclass is proposed to arise from oncogenic transformation of cerebellar granule neuron precursors (CGNPs), whose expansion during post-natal brain development is driven by and requires activation of the Shh pathway. These tumors often demonstrate similarities with normal cerebellar development at the molecular level, thus allowing us to use primary CGNP cultures as a model system for the Sonic hedgehog (Shh) driven subclass of medulloblastoma. In addition to mitogens driving proliferation, it has been shown in the past that low levels of intracellular reactive oxygen species (ROS) are required for proliferation, through mechanisms as diverse as inhibition of receptor tyrosine phosphatases, stabilization of proliferation proteins, and modifications of metabolites, thus indicating additional roles for ROS in proliferation and perhaps tumor growth beyond their known capacity to cause DNA damage, thereby contributing to genomic instability and apoptosis. Although the Shh ligand does not bind to a receptor tyrosine kinase (RTK), it is known that Shh signaling cooperates with RTK- activated pathways such as the insulin-like growth factor pathway to drive proliferation. To determine whether intracellular ROS play a role in Shh-driven CGNP proliferation, we treated CGNPs with the ROS scavenger lipoic acid (LA) in the presence of Shh, and observed a significant decrease in proliferation. Conversely, addition of the ROS inducer tert-Butyl hydroperoxide to Shh treated CGNPs led to enhanced proliferation over Shh treatment alone. These results indicate that a certain level of ROS are required to support Shh-driven CGNP proliferation, and enhancing their levels can increase proliferation. To investigate whether Shh signaling may affect expression of ROS regulatory genes we carried out a qPCR analysis. We identified up-regulation of sod2, gstm1, and gsto1: genes known to respond to ROS and whose products neutralize ROS, over vehicle-treated CGNPs. Paradoxically, when we examined ROS regulatory enzyme expression in Shh-driven mouse medulloblastomas, we noted sharp drop in the expression of gstm1, gsto1, and sod2 compared to the adjacent cerebellum suggesting a reduced ability to inactivate ROS, which could be contributing to proliferation or DNA damage in this transgenic model. These findings suggest that normal Sonic hedgehog pathway activation contributes to the production and tight regulation of ROS via downstream effectors in addition to synergizing with ROS to drive proliferation, a highly regulated balance that may be lost in medulloblastoma. Citation Format: Chad R. Potts, Rachel D. Rotenberry, Anna M. Kenney. Reactive oxygen species in Sonic hedgehog-driven proliferation of cerebellar granule neuron precursors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3978. doi:10.1158/1538-7445.AM2014-3978

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. OS is associated with locally aggressive growth and high metastatic potential. The mechanisms that underlie these processes are currently elusive. Reactive oxygen species (ROS) and Na+/H+ exchanger 1 (NHE1) have been suggested to regulate proliferation and migration of tumor cells. However, the relationship between NHE1 and ROS in OS proliferation and migration has not been investigated before. To investigate the role of NHE1 and ROS in the proliferation and migration of OS. ROS levels and NHE1 expression were studied in cultured human OS cells and human OS xenografts in nude mice. In vitro, OS cells were treated with different doses of tert-butyl hydroperoxide (tBHP), a ROS inducer, and cariporide, an NHE1 inhibitor, to study the effect on cell proliferation and migration. In vivo, nude mice bearing OS cells were administrated with NHE1 inhibitor or antioxidant and the tumor weights were measured. This study reported for the first time that the expression of NHE1 and intracellular ROS level were both increased in OS tissues and cells. Exposure of OS cell to ROS derived from tBHP was able to accelerate cell proliferation and migration and also up-regulate NHE1 protein expression. Moreover, tBHP significantly increased intracellular pH (pHi), decreased extracellular pH (pHe) and induced upregulation of ERK, MMP2, and MMP9. Lowering of ROS levels with the anti-oxidant DMTU or inhibiting NHE1 activity via cariporide abolished the stimulatory effect of tBHP. However, there cariporide did not affect intracellular ROS levels. In vivo study we further confirmed that cariporide could inhibit tumor growth in the nude mouse xenografts of OS cells. The data demonstrate that up-regulation of NHE1 was induced by low concentrations of ROS contributes to the regulation of tumor proliferation and invasion of OS. There is potential application for cariporide as an effective antitumor agent during the development of human osteosarcoma. In addition, redox modulation on proton transport may represent a novel target of osteosarcoma prevention, and open a new avenues for future research.

The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.

Mammalian target of rapamycin controls glucose consumption and redox balance in human Sertoli cells

The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.

Heat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.

Nicotinamide, a glucose-6-phosphate dehydrogenase non-competitive mixed inhibitor, modifies redox balance and lipid accumulation in 3T3-L1 cells

Previous studies have shown that high glucose increases reactive oxygen species (ROS) in endothelial cells that contributes to vascular dysfunction and atherosclerosis. Accumulation of ROS is due to dysregulated redox balance between ROS-producing systems and antioxidant systems. Previous research from our laboratory has shown that high glucose decreases the principal cellular reductant, NADPH by impairing the activity of glucose 6-phosphate dehydrogenase (G6PD). We and others also have shown that the high glucose-induced decrease in G6PD activity is mediated, at least in part, by cAMP-dependent protein kinase A (PKA). As both the major antioxidant enzymes and NADPH oxidase, a major source of ROS, use NADPH as substrate, we explored whether G6PD activity was a critical mediator of redox balance. We found that overexpression of G6PD by pAD-G6PD infection restored redox balance. Moreover inhibition of PKA decreased ROS accumulation and increased redox enzymes, while not altering the protein expression level of redox enzymes. Interestingly, high glucose stimulated an increase in NADPH oxidase (NOX) and colocalization of G6PD with NOX, which was inhibited by the PKA inhibitor. Lastly, inhibition of PKA ameliorated high glucose mediated increase in cell death and inhibition of cell growth. These studies illustrate that increasing G6PD activity restores redox balance in endothelial cells exposed to high glucose, which is a potentially important therapeutic target to protect ECs from the deleterious effects of high glucose.

OBJECTIVES: Upregulated glutamine metabolism is an important metabolic signature for aggressive and treatment-resistant cancers, including many triple negative breast cancers (TNBCs). Glutamate (Glu) produced from glutamine (Gln) via mitochondrial glutaminase (GLS) feeds into the TCA cycle to provide energy and biosynthetic precursors for tumor growth. Recent studies from our lab and others have revealed that Glu also plays an important role in maintaining redox homeostasis which is essential for survival of cancer cells. Glu is a component amino acid of glutathione, the most abundant cellular antioxidant. Furthermore, via the xCT antiporter (SLC7A11), Glu is exported for import of cystine, a rate-limiting step for glutathione synthesis. We assessed the effect of metabolic inhibition of GLS and/or xCT pathways on cellular reactive oxygen species (ROS) level and induction of apoptosis. To track cytosolic Glu transport cross plasma membrane in vivo, we tested (4S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), a non-metabolized structural analog of Glu. We hypothesize that mitochondrial Glu supply is necessary for maintaining cellular redox homeostasis and [18F]FSPG PET can serve as a tool to track Glu transport in glutaminolytic TNBC and guide treatment targeting GLS and redox balance leading to unmitigated oxidative stress and cell death. METHODS: A human glutaminolytic TNBC cell line (HCC1806) and a non-glutaminolytic estrogen-receptor positive cell line (MCF-7) were used to generate xenografts in athymic mice. Cells were exposed to culture media containing GLS inhibitor CB839 (1 µM), xCT inhibitor Erastin (ERA, 3 µM) and/or chemotherapy doxorubicin (DOX, 0.2 µM) for 24 h. ROS were measured by DHE and CellROX Green assays; early apoptosis was estimated by flow cytometry analysis of cells double-stained with Annexin V-FITC and TOR-PR-3. In vivo dynamic [18F]FSPG PET imaging (1 h) was performed on HCC1806 and MCF-7 xenograft models. Lipid peroxidation induced by treatments was examined using C11-Bodipy, a fluorescent sensor. RESULTS: ROS level in HCC1806 cells increased robustly with CB839 (20% over untreated cells), ERA (30%), or ERA plus CB839 (50%), whereas ROS responses in MCF-7 cells were more blunted. Compared to untreated cells, DOX treatment increased ROS level in HCC1806 cells by 40% whereas DOX combined with CB839 plus ERA led to a remarkable increase of cellular ROS by more than 200%. Combined treatment also led to 31% of cells undergoing early apoptosis, compared to 3% in DOX only and 2% in untreated cells. Time-activity curves (TACs) from [18F]FSPG PET of these xenograft models were fit by a reversible one-compartment model in keeping with the known biology of the tracer. The tumor-to-blood ratio (T/B) averaged from the last 6 points (30 min) of the TACs reveal a large difference between HCC1806 vs. MCF-7 by a factor > 2. C11-Bodipy signal in HCC1806 cells was not changed after CB839 but increased substantially after ERA only (45%) or DOX only (68%) treatment and after combined treatment of CB839, ERA and DOX (111%). DISCUSSIONS & CONCLUSION: Our preliminary data support the efficacy of dual targeting of glutaminolysis and Glu transport for sensitizing glutaminolytic TNBC to cytotoxic chemotherapy. T/B of [18F]FSPG PET appears to be distinct for glutaminolytic versus non-glutaminolytic breast cancers. Future work will focus on developing [18F]FSPG PET, complementing our prior work with a Gln analog as a measure of Gln metabolism. The two PET tracers may guide treatment targeting both Gln and Glu usage to overcome chemoresistant TNBC. SUPPORT: R01CA211337 and Komen SAC130060. Reference: 1. Gross MI, et al. Mol Cancer Ther 2014;13(4):890-901. 2. Viswanath V, et al. J Nucl Med 2020. 3. Zhou R, et al. Cancer Res 2017;77(6):1476-84. Citation Format: Hoon Choi, Hsiaoju Lee, Austin Pantel, Christopher Hensley, David Mankoff, Rong Zhou. Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate transport and glutaminase [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-18.

A therapeutic role for selenoprotein T in reducing ischaemia/reperfusion injury in the heart?

Iron is an essential trace mineral element in almost all living cells and organisms. However, cellular iron metabolism pathways are disturbed in most cancer cell types. Cancer cells have a high demand of iron. To maintain rapid growth and proliferation, cancer cells absorb large amounts of iron by altering expression of iron metabolism related proteins. However, iron can catalyze the production of reactive oxygen species (ROS) through Fenton reaction. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an important player in the resistance to oxidative damage by inducing the transcription of antioxidant genes. Aberrant activation of Nrf2 is observed in most cancer cell types. It has been revealed that the over-activation of Nrf2 promotes cell proliferation, suppresses cell apoptosis, enhances the self-renewal capability of cancer stem cells, and even increases the chemoresistance and radioresistance of cancer cells. Recently, several genes involving cellular iron homeostasis are identified under the control of Nrf2. Since cancer cells require amounts of iron and Nrf2 plays pivotal roles in oxidative defense and iron metabolism, it is highly probable that Nrf2 is a potential modulator orchestrating iron homeostasis and redox balance in cancer cells. In this hypothesis, we summarize the recent findings of the role of iron and Nrf2 in cancer cells and demonstrate how Nrf2 balances the oxidative stress induced by iron through regulating antioxidant enzymes and iron metabolism. This hypothesis provides new insights into the role of Nrf2 in cancer progression. Since ferroptosis is dependent on lipid peroxide and iron accumulation, Nrf2 inhibition may dramatically increase sensitivity to ferroptosis. The combination of Nrf2 inhibitors with ferroptosis inducers may exert greater efficacy on cancer therapy.

Mitochondrial dysfunction and oxidative stress are pivotal drivers of obesity-induced insulin resistance, posing significant challenges to therapeutic efficacy. Bezafibrate, a pan-peroxisome proliferator-activated receptor (PPAR) agonist, enhances mitochondrial metabolism and antioxidant defenses; however, its efficacy is hindered by poor solubility and bioavailability. In this study, we engineered biodegradable periodic mesoporous organosilica (BPMO) nanoparticles to improve bezafibrate delivery and intracellular efficacy. Spectroscopic, circular dichroism, and molecular modeling analyses confirmed that bezafibrate stably binds antioxidant enzymes catalase and superoxide dismutase (SOD), with minimal perturbation to their conformation. Molecular docking and dynamics simulations supported these findings by demonstrating stable binding and increased protein structural integrity. In insulin-resistant human adipose-derived cells (HPAd), BPMO-bezafibrate notably restored mitochondrial membrane potential, enhanced fatty acid oxidation, reduced intracellular reactive oxygen species (ROS), and upregulated endogenous gene expression of PPARγ and adiponectin. Compared to liposomal and free-drug delivery, BPMO-bezafibrate showed higher cellular uptake, reduced cytotoxicity, and improved metabolic rescue. Moreover, in vivo uptake and safety were validated using medaka embryos. Collectively, our findings establish BPMO-assisted bezafibrate delivery as a safe and potent strategy to restore mitochondrial function and redox balance in insulin-resistant cells, offering translational promise for treating metabolic disorders.

Antioxidant supplementation in critical illness: what do we know?