Exosome-mediated radiosensitizing effect on neighboring cancer cells via increase in intracellular levels of reactive oxygen species
Exosome-mediated radiosensitizing effect on neighboring cancer cells via increase in intracellular levels of reactive oxygen species
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DOI：
10.3892/or.2021.7964
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发表时间：
2021-02
期刊：
影响因子：
4.2
通讯作者：
A. Nakaoka;M. Nakahana;Sachiko Inubushi;H. Akasaka;M. Salah;Y. Fujita;H. Kubota;Mennaallah Hassan;R. Nishikawa;N. Mukumoto;T. Ishihara;D. Miyawaki;T. Sasayama;R. Sasaki
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作者：
A. Nakaoka;M. Nakahana;Sachiko Inubushi;H. Akasaka;M. Salah;Y. Fujita;H. Kubota;Mennaallah Hassan;R. Nishikawa;N. Mukumoto;T. Ishihara;D. Miyawaki;T. Sasayama;R. Sasaki
辐射暴露后癌细胞之间细胞间通讯的精确机制尚不清楚。外泌体是包含脂质双层的膜封闭的小囊泡，并且是细胞间通讯的介质，其转运多种细胞内组分，包括微小RNA（miRNA或miR）。本研究旨在确定从辐射细胞释放到邻近癌细胞的外泌体的新作用。为了确认人胰腺癌细胞系MIAPaCa-2中外泌体的存在，进行超离心，然后使用外泌体特异性表面标志物CD 9和CD 63进行透射电子显微镜和纳米颗粒跟踪分析（NanoSight）。通过荧光显微镜证实了外来体的后续内吞作用。通过集落形成测定评价照射和外泌体添加后的细胞存活。然后通过微阵列分析定量外泌体中miRNA的表达水平，同时通过蛋白质印迹法评估MIAPaCa-2细胞中Cu/Zn-和Mn-超氧化物歧化酶（分别为SOD 1和2）的蛋白质表达水平。结果表明，照射的外泌体的摄取显著高于未照射的外泌体。值得注意的是，照射的外来体诱导较高的细胞内活性氧（ROS）水平和较高的频率的DNA损伤MIAPaCa-2细胞，分别通过荧光显微镜和免疫细胞化学测定。此外，6个上调和5个下调的miRNA被确定在5和8 Gy照射的细胞使用miRNA微阵列分析。使用miRNA模拟物和逆转录-定量PCR的进一步分析将miR-6823- 5 p鉴定为抑制SOD 1的潜在候选物，导致细胞内ROS水平增加和DNA损伤。据我们所知，本研究是第一个证明辐射外泌体通过增加癌细胞中的细胞内ROS水平来增强辐射效应的研究。这有助于提高对邻近癌细胞的旁观者效应的理解。
The precise mechanism of intercellular communication between cancer cells following radiation exposure is unclear. Exosomes are membrane-enclosed 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 MIAPaCa-2, ultracentrifugation was performed followed by transmission electron microscopy and nanoparticle tracking analysis (NanoSight) using the exosome-specific 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 Mn-superoxide dismutase (SOD1 and 2, respectively) enzymes in MIAPaCa-2 cells were evaluated by western blotting. Results showed that the uptake of irradiated exosomes was significantly higher than that of non-irradiated exosomes. Notably, irradiated exosomes induced higher intracellular levels of reactive oxygen species (ROS) and a higher frequency of DNA damage in MIAPaCa-2 cells, as determined by fluorescent microscopy and immunocytochemistry, respectively. Moreover, six up- and five downregulated miRNAs were identified in 5 and 8 Gy-irradiated cells using miRNA microarray analyses. Further analysis using miRNA mimics and reverse transcription-quantitative PCR identified miR-6823-5p 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.