The mechanism of cell death induced by silver nanoparticles is distinct from silver cations.

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.

Low Doses of Silver Nanoparticles Selectively Induce Lipid Peroxidation and Proteotoxic Stress in Mesenchymal Subtypes of Triple-Negative Breast Cancer.

Molecular profiling of tumors shows that triple-negative breast cancer (TNBC) can be stratified into mesenchymal (claudin-low breast cancer; CLBC) and epithelial subtypes (basal-like breast cancer; BLBC). Subtypes differ in underlying genetics and in response to therapeutics. Several reports indicate that therapeutic strategies that induce lipid peroxidation or proteotoxicity may be particularly effective for various cancers with a mesenchymal phenotype such as CLBC, for which no specific treatment regimens exist and outcomes are poor. We hypothesized that silver nanoparticles (AgNPs) can induce proteotoxic stress and cause lipid peroxidation to a greater extent in CLBC than in BLBC. We found that AgNPs were lethal to CLBCs at doses that had little effect on BLBCs and were non-toxic to normal breast epithelial cells. Analysis of mRNA profiles indicated that sensitivity to AgNPs correlated with expression of multiple CLBC-associated genes. There was no correlation between sensitivity to AgNPs and sensitivity to silver cations, uptake of AgNPs, or proliferation rate, indicating that there are other molecular factors driving sensitivity to AgNPs. Mechanistically, we found that the differences in sensitivity of CLBC and BLBC cells to AgNPs were driven by peroxidation of lipids, protein oxidation and aggregation, and subsequent proteotoxic stress and apoptotic signaling, which were induced in AgNP-treated CLBC cells, but not in BLBC cells. This study shows AgNPs are a specific treatment for CLBC and indicates that stratification of TNBC subtypes may lead to improved outcomes for other therapeutics with similar mechanisms of action.

Combined Photothermal and Ionizing Radiation Sensitization of Triple-Negative Breast Cancer Using Triangular Silver Nanoparticles.

BACKGROUND: Ionizing radiation (IR) is commonly used in triple-negative breast cancer (TNBC) treatment regimens. However, off-target toxicity affecting normal tissue and grueling treatment regimens remain major limitations. Hyperthermia is one of the greatest IR sensitizers, but only if heat is administered simultaneously or immediately prior to ionizing radiation. Difficulty in co-localizing ionizing radiation (IR) in rapid succession with hyperthermia, and confining treatment to the tumor have hindered widespread clinical adoption of combined thermoradiation treatment. Metal nanoparticle-based approaches to IR sensitization and photothermal heat generation may aid in overcoming these issues and improve treatment specificity. METHODS: We assessed the potential to selectively treat MDA-MB-231 TNBC cells without affecting non-malignant MCF-10A breast cells using a multimodal approach based upon combined photothermal therapy, IR sensitization, and specific cytotoxicity using triangular silver nanoparticles (TAgNPs) with peak absorbance in the near-infrared light (NIR) spectrum. RESULTS: We found that TAgNP-mediated photothermal therapy and radiosensitization offer a high degree of specificity for treatment of TNBC without affecting non-malignant mammary epithelial cells. DISCUSSION: If given at a high enough dose, IR, heat, or TAgNPs alone could be sufficient for tumor treatment. However, when the dose of one or all of these modalities increases, off-target effects also increase. The challenge lies in identifying the minimal doses of each individual treatment such that when combined they provide maximum selectivity for treatment of TNBC cells with minimum off-target effects on non-malignant breast cells. Our results provide proof of concept that this combination is highly selective for TNBC cells while sparing non-malignant mammary epithelial cells. This treatment would be particularly important for patients undergoing breast conservation therapy and for treatment of invasive tumor margins near the periphery where each individual treatment might be at a sub-therapeutic level.