BACKGROUND Non-targeting effects of radiotherapy have become as clinical concern due to secondary tumorigenesis in the patients receiving radiotherapy. Radiotherapy also affects non-tumoral cells present in the tumor microenvironment and surrounding tissues. As such, the irradiated cells are thought to communicate the signals that promote secondary tumorigenesis by affecting the function and fate of non-irradiated cells in the vicinity including endothelial cells. This may include up-regulation of genes in irradiated cells, secretion of paracrine factors and induction of gene expression in surrounding non-irradiated cells, which favor cell survival and secondary tumorigenesis. In the current study, we aimed to investigate whether the conditioned media from X-ray irradiated MCF-7 cells contribute to induction of gene expression in human umbilical vein endothelial cells (HUVECs) in vitro and modulate their angiogenic capability and migration. METHODS Following the co-culturing of X-ray irradiated MCF-7 media with HUVECs, the migration and wound healing rate of HUVECs was monitored using Transwell plate and scratch wound healing assay, respectively. The levels of angiogenic protein i.e. vascular endothelial growth factor (VEGF-A) in the conditioned media of MCF-7 cells was measured using ELISA. Additionally, we quantified mRNA levels of VEGFR-2, HSP-70, Ang-2, and Ang-1 genes in HUVECs by real time-PCR. Tubulogenesis capacity of endothelial cells was measured by growth factor reduced Matrigel matrix, whereas expression of CD34 (a marker of angiogenic tip cells) was detected by flow cytometry. RESULTS Data showed that VEGF-A protein content of conditioned media of irradiated MCF-7 cells was increased (P < 0.05) with increase in dose. Data showed that irradiated conditioned media from MCF-7 cells, when incubated with HUVECs, significantly enhanced the cell migration and wound healing rate of HUVECs in a dose-dependent manner (P < 0.05). The mRNA levels of VEGFR-2, HSP-70, Ang-2, and Ang-1 were dose-dependently enhanced in HUVECs incubated with irradiated conditioned media (P < 0.05). Importantly, HUVECs treated with irradiated conditioned media showed a marked increase in the tube formation capability as well as in expression of CD34 marker (P < 0.05). CONCLUSIONS Our findings indicate that conditioned media from irradiated MCF-7 cells induce angiogenic responses in endothelial cells in vitro, which could be due to transfer of overexpressed VEGF-A and possibly other factors secreted from irradiated MCF-7 cells to endothelial cells, and induction of intrinsic genes (VEGFR-2, HSP-70, Ang-2, and Ang-1) in endothelial cells. Video abstract.

中文翻译：

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电离辐射的旁观者效应：来自X射线辐射的MCF-7细胞的条件培养基增加了内皮细胞的血管生成能力。

背景技术由于接受放射治疗的患者的继发性肿瘤发生，放射治疗的非靶向作用已成为临床关注的问题。放射疗法还影响存在于肿瘤微环境和周围组织中的非肿瘤细胞。这样，被辐射的细胞被认为通过影响附近包括内皮细胞的未被辐射的细胞的功能和命运来传达促进继发性肿瘤发生的信号。这可能包括辐照细胞中基因的上调，旁分泌因子的分泌以及周围非辐照细胞中基因表达的诱导，这有利于细胞存活和继发性肿瘤的发生。在目前的研究中 我们旨在研究来自X射线照射的MCF-7细胞的条件培养基是否有助于体外诱导人脐静脉内皮细胞（HUVEC）中的基因表达，并调节其血管生成能力和迁移。方法在用X射线照射的MCF-7培养基与HUVEC共同培养后，分别用Transwell平板和刮擦伤口愈合试验监测HUVEC的迁移和伤口愈合速度。使用ELISA测量在MCF-7细胞的条件培养基中的血管生成蛋白，即血管内皮生长因子（VEGF-A）的水平。此外，我们通过实时PCR定量了HUVEC中VEGFR-2，HSP-70，Ang-2和Ang-1基因的mRNA水平。用生长因子还原的基质胶基质测量内皮细胞的肾小管生成能力，而通过流式细胞术检测CD34（血管生成尖端细胞的标志物）的表达。结果数据显示，随着剂量的增加，受辐照的MCF-7细胞条件培养基中的VEGF-A蛋白含量增加（P <0.05）。数据显示，与HUVEC一起孵育时，来自MCF-7细胞的辐照条件培养基以剂量依赖的方式显着增强了HUVEC的细胞迁移和伤口愈合率（P <0.05）。在用辐照条件培养基孵育的HUVEC中，VEGFR-2，HSP-70，Ang-2和Ang-1的mRNA水平呈剂量依赖性提高（P <0.05）。重要的是，用辐照条件培养基处理过的HUVEC在管的形成能力和CD34标记物的表达上均显着增加（P <0.05）。结论我们的研究结果表明，受辐照的MCF-7细胞的条件培养基可在体外诱导内皮细胞的血管生成反应，这可能是由于过表达的VEGF-A和辐照过的MCF-7细胞分泌的其他因子转移至内皮细胞以及诱导内皮细胞内在基因（VEGFR-2，HSP-70，Ang-2和Ang-1）的表达。录像摘要。