The current study determined if depletion of glutathione (GSH) and inhibition of thioredoxin reductase (TR) activity could enhance radiation responses in human breast cancer stem cells by a mechanism involving thiol dependent oxidative stress. Buthionine sulfoximine (BSO), a GSH synthesis inhibitor; sulfasalazine (SSZ), an inhibitor of xc- cysteine/glutamate antiporter; auranofin (Au), a thioredoxin reductase inhibitor; or 2AAPA, a GSH-reductase inhibitor, were used to inhibit GSH- and thioredoxin (Trx)-metabolism. Clonogenic survival, Matrigel assays, flow cytometry cancer stem cell assays (CD44+CD24-ESA+ or ALDH1), and human tumor xenograft models were used to determine the antitumor activity of drug and radiation combinations. Combined inhibition of GSH and Trx-metabolism enhanced cancer cell clonogenic killing and radiation responses in human breast and pancreatic cancer cells via a mechanism that could be inhibited by N-acetylcysteine (NAC). Au, BSO, and radiation also significantly decreased breast cancer cell migration and invasion in a thiol dependent fashion that could be inhibited by NAC. In addition pre-treating cells with Au sensitized breast cancer stem cell populations to radiation in vitro as determined by CD44+CD24-ESA+ or ALDH1. Combined administration of Au and BSO, given prior to radiation significantly increased the survival of mice with human breast cancer xenografts as well as decreasing the number of ALDH1 positive cancer stem cells. These results indicate that combined inhibition of GSH- and Trx-dependent thiol metabolism using pharmacologically relevant agents can enhance responses of human breast cancer stem cells to radiation both in vitro and in vivo.

SUMMARYHepatocellular carcinoma (HCC) is a devastating cancer increasingly caused by non-alcoholic fatty liver disease (NAFLD). Disrupting the liver Mitochondrial Pyruvate Carrier (MPC) in mice attenuates NAFLD. Thus, we considered whether liver MPC disruption also prevents HCC. Here, we use the N-nitrosodiethylamine plus carbon tetrachloride model of HCC development to test how liver-specific MPC knock out affects hepatocellular tumorigenesis. Our data show that liver MPC ablation markedly decreases tumorigenesis and that MPC-deficient tumors transcriptomically downregulate glutathione metabolism. We observe that MPC disruption and glutathione depletion in cultured hepatomas are synthetically lethal. Stable isotope tracing shows that hepatocyte MPC disruption reroutes glutamine from glutathione synthesis into the tricarboxylic acid (TCA) cycle. These results support a model where inducing metabolic competition for glutamine by MPC disruption impairs hepatocellular tumorigenesis by limiting glutathione synthesis. These findings raise the possibility that combining MPC disruption and glutathione stress may be therapeutically useful in HCC and additional cancers.

Background Precisely how silver nanoparticles (AgNPs) kill mammalian cells still is not fully understood. It is not clear if AgNP-induced damage differs from silver cation (Ag+), nor is it known how AgNP damage is transmitted from cell membranes, including endosomes, to other organelles. Cells can differ in relative sensitivity to AgNPs or Ag+, which adds another layer of complexity to identifying specific mechanisms of action. Therefore, we determined if there were specific effects of AgNPs that differed from Ag+ in cells with high or low sensitivity to either toxicant. Methods Cells were exposed to intact AgNPs, Ag+, or defined mixtures of AgNPs with Ag+, and viability was assessed. The level of dissolved Ag+ in AgNP suspensions was determined using inductively coupled plasma mass spectrometry. Changes in reactive oxygen species following AgNP or Ag+ exposure were quantified, and treatment with catalase, an enzyme that catalyzes the decomposition of H2O2 to water and oxygen, was used to determine selectively the contribution of H2O2 to AgNP and Ag+ induced cell death. Lipid peroxides, formation of 4-hydroxynonenol protein adducts, protein thiol oxidation, protein aggregation, and activation of the integrated stress response after AgNP or Ag+ exposure were quantified. Lastly, cell membrane integrity and indications of apoptosis or necrosis in AgNP and Ag+ treated cells were examined by flow cytometry. Results We identified AgNPs with negligible Ag+ contamination. We found that SUM159 cells, which are a triple-negative breast cancer cell line, were more sensitive to AgNP exposure less sensitive to Ag+ compared to iMECs, an immortalized, breast epithelial cell line. This indicates that high sensitivity to AgNPs was not predictive of similar sensitivity to Ag+. Exposure to AgNPs increased protein thiol oxidation, misfolded proteins, and activation of the integrated stress response in AgNP sensitive SUM159 cells but not in iMEC cells. In contrast, Ag+ cause similar damage in Ag+ sensitive iMEC cells but not in SUM159 cells. Both Ag+ and AgNP exposure increased H2O2 levels; however, treatment with catalase rescued cells from Ag+ cytotoxicity but not from AgNPs. Instead, our data support a mechanism by which damage from AgNP exposure propagates through cells by generation of lipid peroxides, subsequent lipid peroxide mediated oxidation of proteins, and via generation of 4-hydroxynonenal (4-HNE) protein adducts. Conclusions There are distinct differences in the responses of cells to AgNPs and Ag+. Specifically, AgNPs drive cell death through lipid peroxidation leading to proteotoxicity and necrotic cell death, whereas Ag+ increases H2O2, which drives oxidative stress and apoptotic cell death. This work identifies a previously unknown mechanism by which AgNPs kill mammalian cells that is not dependent upon the contribution of Ag+ released in extracellular media. Understanding precisely which factors drive the toxicity of AgNPs is essential for biomedical applications such as cancer therapy, and of importance to identifying consequences of unintended exposures.

D-penicillamine (DPEN), a copper chelator, has been used in the treatment of Wilson's disease, cystinuria, and rheumatoid arthritis. Recent evidence suggests that DPEN in combination with biologically relevant copper (Cu) concentrations generates H2O2 in cancer cell cultures, but the effects of this on cancer cell responses to ionizing radiation and chemotherapy are unknown. Increased steady-state levels of H2O2 were detected in MB231 breast and H1299 lung cancer cells following treatment with DPEN (100 μM) and copper sulfate (15 μM). Clonogenic survival demonstrated that DPEN-induced cancer cell toxicity was dependent on Cu and was significantly enhanced by depletion of glutathione [using buthionine sulfoximine (BSO)] as well as inhibition of thioredoxin reductase [using Auranofin (Au)] prior to exposure. Treatment with catalase inhibited DPEN toxicity confirming H2O2 as the toxic species. Furthermore, pretreating cancer cells with iron sucrose enhanced DPEN toxicity while treating with deferoxamine, an Fe chelator that inhibits redox cycling, inhibited DPEN toxicity. Importantly, DPEN also demonstrated selective toxicity in human breast and lung cancer cells, relative to normal untransformed human lung or mammary epithelial cells and enhanced cancer cell killing when combined with ionizing radiation or carboplatin. Consistent with the selective cancer cell toxicity, normal untransformed human lung epithelial cells had significantly lower labile iron pools than lung cancer cells. These results support the hypothesis that DPEN mediates selective cancer cell killing as well as radio-chemo-sensitization by a mechanism involving metal ion catalyzed H2O2-mediated oxidative stress and suggest that DPEN could be repurposed as an adjuvant in conventional cancer therapy.

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To determine if mitochondrial pyruvate transport could represent a therapeutic target for sensitizing cancer cells to oxidative stress, lung and breast cancer cells were treated with 5 µM UK5099 to inhibit the mitochondrial pyruvate carrier (MPC). Treatment with UK5099 selectively sensitized lung and breast cancer cells to clonogenic cell killing when combined with depletion of glutathione using 1 mM buthionine sulfoximine (BSO; a glutathione synthesis inhibitor) for 48 h and 72 h, relative to normal lung and breast epithelial cells. Furthermore, cancer cell killing mediated by UK5099 combined with BSO was inhibited by the thiol antioxidant, N-acetylcysteine (NAC; 20 mM), independent of GSH levels, indicating a mechanism of toxicity involving reduced thiols and metabolic oxidative stress. In addition, treatment with UK5099 alone for 48 h also decreased levels of total glutathione in cancer cells that could be reversed by NAC. Using oxidation-sensitive fluorescent dyes (CDCFH2 and MitoSOX), treatment of lung and breast cancer cells for 24 h and 48 h with UK5099 induced increases in steady state levels of pro-oxidants (presumably hydroperoxides and mitochondrial superoxide), which were further increased with BSO. Finally, treatment of breast cancer cells with UK5099 for 24 and 48 hours significantly sensitized breast cancer cells to clonogenic cell killing mediated by paclitaxel. These data support the hypothesis that inhibition of the MPC selectively causes an impairment of antioxidant capability in cancer cells that is enhanced by depletion of glutathione. Furthermore, these results also support the hypothesis that inhibition of the MPC represents a significant target for sensitizing human breast cancer cells to chemotherapy agents thought to induce oxidative stress. Supported by R01CA182804. Citation Format: Shane R. Solst, Samuel N. Rodman, Melissa A. Fath, Eric B. Taylor, Douglas R. Spitz. Inhibition of mitochondrial pyruvate transport selectively sensitizes cancer cells to metabolic oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3527.

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Thioredoxin Reductase (TrxR) is a key enzyme in reactive oxygen species (ROS) detoxification and in redox regulation. Because cancer cells produce increased steady-state levels of ROS (i.e., superoxide and hydrogen peroxide), TrxR is viable target in clinical trials using the anti-rheumatic drug, auranofin (AF). To extend these observations to small cell lung cancer (SCLC), AF-mediated TrxR inhibition as well as tolerability and tumor growth inhibition was determined in a xenograft model. AF was administered intraperitoneal, daily or twice daily for 1 to 5 days in mice bearing DMS273 xenografts. AF uptake was determined by mass spectrometry of gold and inhibition of TrxR in the tumor was determined. The optimal dose was 4 mg/kg once daily resulting in 18 μM gold in the plasma and 50% inhibition of TrxR activity in DMS273 SCLC tumors. This regimen given for 14 days provided a trend for prolonged median survival from 17.5 to 22 days (p=0.058, N=20 controls, 19 AF) without causing changes in bodyweight, bone marrow toxicity, blood urea nitrogen or creatinine. These results support the hypothesis that AF is an effective inhibitor of TrxR and suggest that AF could be used as an adjuvant in radio-chemotherapy protocols to enhance therapeutic efficacy.