Ionizing Radiation Increases the Activity of Exosomal Secretory Pathway in MCF-7 Human Breast Cancer Cells: A Possible Way to Communicate Resistance against Radiotherapy
Ionizing Radiation Increases the Activity of Exosomal Secretory Pathway in MCF-7 Human Breast Cancer Cells: A Possible Way to Communicate Resistance against Radiotherapy
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DOI：
10.3390/ijms20153649
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发表时间：
2019-08-01
影响因子：
5.6
通讯作者：
Rezaie, Jafar
中科院分区：
文献类型：
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作者：
Jabbari, Nasrollah;Nawaz, Muhammad;Rezaie, Jafar
放射治疗是利用高能射线清除肿瘤细胞，被认为是乳腺癌患者的基本治疗方法。大多数肿瘤细胞分泌外切体，参与肿瘤组织中的细胞间通讯，并参与治疗抵抗和促进肿瘤侵袭。在这里，我们研究了临床适用的X射线照射剂量(2，4，6，8，10Gy)对MCF-7乳腺癌细胞外切体活性动态的影响。用四甲基偶氮唑盐比色法和流式细胞仪分别检测细胞对X射线剂量的存活率和凋亡率。荧光分光光度法检测X射线照射后细胞内的活性氧(ROS)水平。实时荧光定量聚合酶链式反应(Real-Time-PCR)检测与外切体生物发生和分泌有关的重要基因Alix、Rab11、Rab27a、Rab27b、TSPA8和CD63的mRNA水平。Western blotting检测CD63蛋白表达水平。此外，还通过监测乙酰胆碱酯酶活性、透射电子显微镜、大小测定和Zeta电位来表征外切体。结果表明，与对照组相比，照射组细胞存活率、细胞凋亡率和ROS含量分别呈剂量依赖性下降和升高(p<；0.05)。Alix、Rab27a、Rab27b、TSPA8、CD63等基因的表达水平以及CD63的蛋白水平随X射线剂量的增加而升高(p<；0.05)。我们发现，随着X射线剂量的增加，照射细胞的外切体的乙酰胆碱酯酶活性、大小和Zeta-电位值增加(p<；0.05)。数据表明，X射线可以剂量依赖的方式激活MCF-7细胞外切体的生物生成和分泌，提示细胞的治疗反应是通过ROS和外切体活性来实现的。
Radiation therapy, which applies high-energy rays, to eradicate tumor cells, is considered an essential therapy for the patients with breast cancer. Most tumor cells secrete exosomes, which are involved in cell-to-cell communication in tumor tissue and contribute therapeutic resistance and promote tumor aggressiveness. Here, we investigated the effect of clinically applicable doses of X-ray irradiation (2, 4, 6, 8, 10 Gy) on the dynamics of the exosomes' activity in MCF-7 breast cancer cells. Survival and apoptosis rate of cells against X-ray doses was examined using MTT and flow cytometry assays, respectively. Whereas, the levels of reactive oxygen species (ROS) in the X-ray-treated cells were detected by fluorometric method. The mRNA levels of vital genes involved in exosome biogenesis and secretion including Alix, Rab11, Rab27a, Rab27b, TSPA8, and CD63 were measured by real-time PCR. The protein level of CD63 was examined by Western blotting. Additionally, exosomes were characterized by monitoring acetylcholinesterase activity, transmission electron microscopy, size determination, and zeta potential. The result showed that in comparison with control group cell survival and the percentage of apoptotic cells as well as amount of ROS dose-dependently decreased and increased in irradiated cells respectively (p < 0.05). The expression level of genes including Alix, Rab27a, Rab27b, TSPA8, and CD63 as well as the protein level of CD63 upraised according to an increase in X-ray dose (p < 0.05). We found that concurrent with an increasing dose of X-ray, the acetylcholinesterase activity, size, and zeta-potential values of exosomes from irradiated cells increased (p < 0.05). Data suggest X-ray could activate exosome biogenesis and secretion in MCF-7 cells in a dose-dependent way, suggesting the therapeutic response of cells via ROS and exosome activity.