Exosomes-another Underlying Mechanism Of Radiation-induced Bystander Effects (RIBEs)

Objective:First, the study was to demonstrate that ionizing radiation can induce medium-mediated bystander effects in H460non-small-cell lung cancer cells. Furthermore, itwas to explore a novel signal mediating mechanism of bystander effect besidesintercellular gap junctions and soluble factors. Based on the previous studies fromliterature and our preliminary experiment, the goal of this study was to investigatewhether exosomes can be a novel mechanism underlying RIBEs.Methods:Using DNA damage, micronucleus formation, clonogenic cell survival asendpoints, medium-mediated bystander effects in H460non-small-cell lung cancer cellsinduced by X-ray irradiation was studied adopting medium transfer method. Differentialcentrifugation method was used to isolate and purify exosomes from the media ofunirradiated and irradiated H460cells. The morphology of exosomes was observedusing transmission electron microscope, the size distribution of exosomes was measuredusing Zetasizer Nano ZS90size detector, and the expression of a marker of exosomes,hsp90β, was detected by Western Blot. Additionally, the size distribution of exosomes atdifferent dose and different time after irradiation were measured. Fluorescence probeswere applied to investigate the internalization of exosomes into recipient cells. Theeffects of exosomes on the proliferation of recipient cells were assessed using crystalviolet assay. At the same time, the effects of exosomes on the micronucleus formationand clonogenic cell survival ability of recipient cells were assessed. In order toinvestigate whether RNAs in exosomes played an important role in the induction ofbiological changes in recipient cells, exosomes were treated with RNase to inactivateRNAs in exosomes before they were added to recipient cells. The results show thatRNase-treated exosomes failed to induce an increase in micronucleus formation inrecipient cells. Origin8software was used to do data statistics, analysis and graphics. Differences between the different groups were analyzed using the Student’s t test. A pvalue of≤0.05between groups was considered significantly different. All data camefrom at least three independent experiment, represented as mean value±standarddeviation.Results:1. Medium-mediated radiation-induced bystander effects. Compared withconditioned medium from sham-irradiated (SCM) cells, conditioned medium fromirradiated H460cells (RCM) collected1hour after5Gy X-ray exposure induced an80%increase in micronucleus formation rate and a13%decrease in clonogenic cellsurvival in bystander cells. Compared with conditioned medium from sham-irradiated(SCM) cells, conditioned medium from irradiated H460cells (RCM) collected18hoursafter5Gy X-ray exposure induced a3.5-5-fold increase in micronucleus formation rate,a8.3%decrease in clonogenic cell survival and a18%increase in DNA damage inbystander cells. Moreover, no matter the time at which the conditioned medium wascollected,5Gy and10Gy X-rays induced similar changes in micronucleus formationrate in bystander cells.2. Exosomes isolation and identification. Exosomes could be isolated and purifiedeasily and rapidly using the differential centrifugation method. Typical “cup-shaped”and size of30-150nm characteristic could be observed under transmission electronmicroscope. To further confirm that the extracts were exosomes, the well-acceptedexosome marker Hsp90β was detected by Western Blot. Both unirradiated andirradiated H460cells secreted exosomes, but with different size distribution. Moreover,the size of secreted exosomes was dependent on the irradiation dose and the culture timeafter irradiation.3. The interaction of exosomes and H460recipient cells. The differentiallyfluorescent probe-labeled exsomes and H460recipient cells were cocultured for30minutes. The exosomes extracted from both conditioned medium from sham-irradiated(SCM) cells and conditioned medium from irradiated cells(RCM) can be internalizedquickly into recipient cells probably through membrane fusion.4. The effects of exosomes on H460recipient cells. Addition of exosomes isolatedfrom conditioned medium from sham-irradiated (SCM) cells did not change themicronucleus formation rate in H460recipient cells, however, exosomes isolated fromconditioned medium from irradiated cells(RCM) doubled the micronucleus formation rate in H460recipient cells, which could be abolished by the pretreatment of exosomeswith RNase. The exosomes secreted by both unirradiated and irradiated H460cellspromoted proliferation (increased11%) but had no effect on clonogenic cell survival inrecipient cells compared with untreated control.Conclusions:1. X-ray irradiation induced medium-mediated bystander effects in H460cells.2. No matter irradiation or not, H460cells secreted exosomes, but with differentsize distribution. Moreover, the size of secreted exosomes is dependent on theirradiation dose and culture time after irradiation.3. Exosomes extracted from both conditioned medium from sham-irradiated cells(SCM) and conditioned medium from irradiated cells (RCM) can be internalizedquickly into recipient cells probably through membrane fusion.4.Exosomes may be a novel mechanism mediate radiation induced bystandereffects and RNAs are one kind of signal transmitters.