Molecular Mechanisms Underlying Ionizing Radiation-induced Bystander Effects In H1299Non-small Cell Lung Cancer Cells

Radiation-induced bystander effects (RIBEs) refer to the responses detected in theunirradiated cells when the neighboring cells were irradiated. Along with the advance ofresearch techniques, many researchers have demonstrated the existence ofradiation-induced bystander effects both in vitro and in vivo using different detectiontechniques and methods. But the mechanisms of radiation-induced bystander effects arestill unclear. For example, transforming growth factor β1(TGF-β1) has been proposed tobe a candidate mediator of bystander effect. And it has been demonstrated to regulatebystander effects in some cell lines, yet the detailed mechanisms remained elucidated. Onthe other hand, microRNAome changes have been shown in bystander cells or tissues, butstudies on whether miRNAs have an important role in the regulation of radiation-inducedbystander effects are very limited. Therefor, in the present study, we first demonstrated thatX-irradiation could induce medium-mediated bystander effects in H1299non-small-celllung cancer cells and the biological changes in bystander cells depended on the timespost-radiation. We found that while the radiation-conditioned medium (RCM) harvestedfrom irradiated H1299cells1h post radiation (1h RCM) caused oxidative stress and DNAdamage in bystander cells, the RCM harvested18h after irradiation (18h RCM) inducedcell cycle delay and proliferation inhibition. Then we investigated the roles of TGF-β1signaling pathway in radiation-induced bystander effects in H1299cells. The results showthat the TGF-β1signaling pathway in both irradiated and bystander cells were critical tothe induction of RIBEs. More interestingly, we found that1h RCM and18h RCM led toup-regulation and down-regulation of miR-21expression in bystander cells, respectively. And the alterations of miR-21expression in bystander cells was dependent upon TGF-β1signaling pathway in both irradiated and bystander cells. Furthermore, transfection ofmiR-21inhibitor into bystander cells abolished the increase of intracellular ROS level andDNA damage caused by1h RCM, and transfection of miR-21mimics eliminatedproliferation inhibition caused by18h RCM. Taken all together, our data suggest animportant mediating role of TGF-β1-miR-21-ROS pathway in X-rays-induced bystandersignaling in H1299cells. In addition, we also observed an adaptive response inradiation-induced bystander H1299cells. We then carried on some studies on theunderlying mechanisms. The data show that there are some connections between bystandereffect and adaptive response. The conditioned medium from both unirradiated andirradiated cells induced an adaptive response in bystander cells when irradiated,manifesting as greater clonogenic cell survival of irradiated bystander cells compared withthe directly irradiated cells. And the TGF-β1signaling pathway in both irradiated andbystander cells played complex roles in the adaptive response. Furthermore, the expressionof SOD2of bystander cells decreased after cultured in radiation-conditioned medium for3h and5h compared to that after cultured in non-radiation-conditioned medium, suggestingthat SOD2may not be involved in the radiation adaptive response of bystander cells. Theelucidation of these phenomena and the underlying mechanisms enriches the molecularmechanisms of radiation-induced bystander effects, and provides the direct evidence for animportant mediating role of miR-21in RIBEs. And it may also suggest an implication ofRIBEs in the optimization of radiotherapy plan for lung cancer patients and prognosticassessment as well.