The rapid dissolution of copper oxide (CuO) nanoparticles (NPs) with release of ions is thought to be one of the main factors modulating their toxicity. Here we assessed the cytotoxicity of a panel of CuO NPs (12 nm ± 4 nm) with different surface modifications, i.e., anionic sodium citrate (CIT) and sodium ascorbate (ASC), neutral polyvinylpyrrolidone (PVP), and cationic polyethylenimine (PEI), versus the pristine (uncoated) NPs, using a murine macrophage cell line (RAW264.7). Cytotoxicity, reactive oxygen species (ROS) production, and cellular uptake were assessed. The cytotoxicity results were analyzed by the benchmark dose (BMD) method and the NPs were ranked based on BMD20values. The PEI-coated NPs were found to be the most cytotoxic. Despite the different properties of the coating agents, NP dissolution in cell medium was only marginally affected by surface modification. Furthermore, CuCl2(used as an ion control) elicited significantly less cytotoxicity when compared to the CuO NPs. We also observed that the antioxidant, N-acetylcysteine, failed to protect against the cytotoxicity of the uncoated CuO NPs. Indeed, the toxicity of the surface-modified CuO NPs was not directly linked to particle dissolution and subsequent Cu burden in cells, nor to cellular ROS production, although CuO-ASC NPs, which were found to be the least cytotoxic, yielded lower levels of ROS in comparison to pristine NPs. Hierarchical cluster analysis suggested, instead, that the toxicity in the current in vitro model could be explained by synergistic interactions between the NPs, their dissolution, and the toxicity of the coating agents.
- MeSH
- antioxidancia MeSH
- buněčná smrt účinky léků MeSH
- buněčné linie MeSH
- kovové nanočástice chemie toxicita MeSH
- makrofágy metabolismus MeSH
- měď chemie farmakokinetika toxicita MeSH
- myši MeSH
- povrchové vlastnosti MeSH
- reaktivní formy kyslíku metabolismus MeSH
- rozpustnost MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
To examine reciprocal or unilateral implications between two cell destruction processes, autophagy and apoptosis, in 5-Fluorouracil (5-FU)-treated tumor cells, a combination of chemical inhibitors, RNAi and genetic approaches were used. In contrast to cancer cells harboring obstructed apoptosis, either at the DISC or the mitochondrial level, p53-deficiency generated signs of autophagy deregulation upon chemotherapy. On the other, hand disruption of lysosomal function by chloroquine, caused a profound decrease in apoptotic markers appearing in response to 5-FU. DR5, which is essential for 5-FU-induced apoptosis, accumulated in lysosomes and autophagosomes upon chloroquine treatment. Since neither 3-MA, RNAi of critical autophagy regulators or inhibition of cathepsins reversed apoptosis in a similar manner, it is likely that not autophagy per se but rather correct receptor transport is an important factor for 5-FU cytotoxicity. We found that apoptosis generated by TRAIL, the cognate ligand for DR5, remained unchanged upon chloroquine lysosomal interference, indicating that 5-FU activates the receptor by a discrete mechanism. In support, depletion of membrane cholesterol or hampering cholesterol transport drastically reduced 5-FU cytotoxicity. We conclude that targeting of lysosomes by chloroquine deregulates DR5 trafficking and abrogates 5-FU- but not TRAIL-stimulated cell elimination, hence suggesting a novel mechanism for receptor activation.
- MeSH
- antimetabolity antitumorózní farmakologie MeSH
- apoptóza MeSH
- autofagie * MeSH
- buněčná membrána metabolismus MeSH
- chlorochin chemie MeSH
- cholesterol chemie MeSH
- fagozomy MeSH
- fluorouracil chemie MeSH
- HCT116 buňky MeSH
- lidé MeSH
- ligandy MeSH
- lyzozomy metabolismus MeSH
- makrolidy chemie MeSH
- mitochondrie metabolismus MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- protein TRAIL farmakologie MeSH
- RNA interference MeSH
- signální transdukce účinky léků MeSH
- TRAIL receptory metabolismus MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains a common clinical strategy for post-surgical treatment of solid tumors. Although it has been proposed that RNA metabolism is disturbed by 5-FU treatment, the key cytotoxic response is believed to be enzymatic inhibition of thymidylate synthase resulting in nucleotide pool disproportions. An operating p53 tumor suppressor signaling network is in many cases essential for the efficiency of chemotherapy, and malfunctions within this system remain a clinical obstacle. Since the fate of chemotherapy-insensitive tumor cells is rarely described, we performed a comparative analysis of 5-FU toxicity in p53-deficient cells and conclude that p53 acts as a facilitator rather than a gatekeeper of cell death. Although p53 can act as a regulator of several cellular stress responses, no rerouting of cell death mode was observed in absence of the tumor suppressor. Thus, the final death outcome of 5-FU-treated p53-/- cells is demonstrated to be caspase-dependent, but due to a slow pace, accumulation of mitochondrial reactive oxygen species contributes to necrotic characteristics. The oligomerization status of the p53 target gene DR5 is determined as a significant limiting factor for the initiation of caspase activity in an intracellular TRAIL-dependent manner. Using several experimental approaches, we further conclude that RNA-rather than DNA-related stress follows by caspase activation irrespectively of p53 status. A distinct 5-FU-induced stress mechanism is thereby functionally connected to a successive and discrete cell death signaling pathway. Finally, we provide evidence that silencing of PARP-1 function may be an approach to specifically target p53-deficient cells in 5-FU combinatorial treatment strategies. Together, our results disclose details of impaired cell death signaling engaged as a consequence of 5-FU chemotherapy. Obtained data will contribute to the comprehension of factors restraining 5-FU efficiency, and by excluding DNA as the main stress target in some cell types they propose alternatives to currently used and suggested synergistic treatment regimens.
- MeSH
- antimetabolity antitumorózní farmakologie MeSH
- apoptóza účinky léků MeSH
- fluorescenční protilátková technika MeSH
- fluorouracil farmakologie MeSH
- imunoblotting MeSH
- imunoprecipitace MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- polymerázová řetězová reakce MeSH
- protein TRAIL metabolismus MeSH
- RNA účinky léků MeSH
- signální transdukce účinky léků fyziologie MeSH
- transdukce genetická MeSH
- transmisní elektronová mikroskopie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
