RESUMEN
The precise mechanism of intercellular communication between cancer cells following radiation exposure is unclear. Exosomes are membraneenclosed small vesicles comprising lipid bilayers and are mediators of intercellular communication that transport a variety of intracellular components, including microRNAs (miRNAs or miRs). The present study aimed to identify novel roles of exosomes released from irradiated cells to neighboring cancer cells. In order to confirm the presence of exosomes in the human pancreatic cancer cell line MIAPaCa2, ultracentrifugation was performed followed by transmission electron microscopy and nanoparticle tracking analysis (NanoSight) using the exosomespecific surface markers CD9 and CD63. Subsequent endocytosis of exosomes was confirmed by fluorescent microscopy. Cell survival following irradiation and the addition of exosomes was evaluated by colony forming assay. Expression levels of miRNAs in exosomes were then quantified by microarray analysis, while protein expression levels of Cu/Zn and Mnsuperoxide dismutase (SOD1 and 2, respectively) enzymes in MIAPaCa2 cells were evaluated by western blotting. Results showed that the uptake of irradiated exosomes was significantly higher than that of nonirradiated exosomes. Notably, irradiated exosomes induced higher intracellular levels of reactive oxygen species (ROS) and a higher frequency of DNA damage in MIAPaCa2 cells, as determined by fluorescent microscopy and immunocytochemistry, respectively. Moreover, six up and five downregulated miRNAs were identified in 5 and 8 Gyirradiated cells using miRNA microarray analyses. Further analysis using miRNA mimics and reverse transcriptionquantitative PCR identified miR68235p as a potential candidate to inhibit SOD1, leading to increased intracellular ROS levels and DNA damage. To the best of our knowledge, the present study is the first to demonstrate that irradiated exosomes enhance the radiation effect via increasing intracellular ROS levels in cancer cells. This contributes to improved understanding of the bystander effect of neighboring cancer cells.
Asunto(s)
Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/radioterapia , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/metabolismo , Comunicación Celular/fisiología , Comunicación Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/fisiología , Daño del ADN , Exosomas/genética , Exosomas/metabolismo , Exosomas/efectos de la radiación , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Pancreáticas/genética , Tolerancia a Radiación , Superóxido Dismutasa-1/biosíntesis , Superóxido Dismutasa-1/metabolismoRESUMEN
The development of potentially safe radiosensitizing agents is essential to enhance the treatment outcomes of radioresistant cancers. The titanium peroxide nanoparticle (TiOxNP) was originally produced using the titanium dioxide nanoparticle, and it showed excellent reactive oxygen species (ROS) generation in response to ionizing radiation. Surface coating the TiOxNPs with polyacrylic acid (PAA) showed low toxicity to the living body and excellent radiosensitizing effect on cancer cells. Herein, we evaluated the mechanism of radiosensitization by PAA-TiOxNPs in comparison with gold nanoparticles (AuNPs) which represent high-atomic-number nanoparticles that show a radiosensitizing effect through the emission of secondary electrons. The anticancer effects of both nanoparticles were compared by induction of apoptosis, colony-forming assay, and the inhibition of tumor growth. PAA-TiOxNPs showed a significantly more radiosensitizing effect than that of AuNPs. A comparison of the types and amounts of ROS generated showed that hydrogen peroxide generation by PAA-TiOxNPs was the major factor that contributed to the nanoparticle radiosensitization. Importantly, PAA-TiOxNPs were generally nontoxic to healthy mice and caused no histological abnormalities in the liver, kidney, lung, and heart tissues.