?(Fig.4C,4C, E, F). as a potential target of cancer therapy. values <0.05 were considered significant. Results Establishment of radioresistant breast cancer cell line Parental MDA-MB-231 cells were divided into two subsets. One received 20 times of fractioned irradiation with a total dose of 60Gy and designated as MDA-MB231-PR (MD-PR) cells. Another group was treated under the same condition but received no irradiation and named as MDA-MB231-PB (MD-PB) cells. The morphology of MD-PR cells was obviously different from that of MD-PB cells under microscopy (Fig. ?(Fig.1A).1A). MD-PR cells had a much more stretched and flatter appearance compared with the latter. We then BMS-777607 examined whether the abilities of migration and invasion were changed in MD-PR cells. Results showed that MD-PR cells had increased migration and invasion capacities compared with MD-PB cells (Fig. BMS-777607 S1A, B). Increased expression of mesenchymal markers (N-cadherin, Snail, Slug and beta-catenin) and decreased expression of epithelial marker E-cadherin in MD-PR were also detected (Fig. S1C). These results suggested that enhanced malignancy was induced in MD-PR cells 27. Open in a separate window Figure 1 MD-PR cells present enhanced radioresistance compared with MD-PB. (A) Morphology changes of MD-PB and MD-PR under the microscope of 100X magnification, with representative cells zoomed in on the right. (B-D) MD-PB and MD-PR cells were seeded in a 6-well plate in triplicate. 24-hours later, cells were subjected to 0-6 Gy of X-ray radiation (Elekta, 1.43 Gy/min). After 10-14 days of incubation, formed clones were fixed, stained and counted. Surviving fraction was calculated and fitted into the linear-quadratic model as described in the Materials and Methods (C). A representative image of three independent experiments was showed (B). Surviving fraction at certain doses as indicated in (D). (E) MD-PB and MD-PR cells were exposed to 0 or 10 Gy of X-ray. 48 hours later, cells were collected, stained with PI and Annexin V-FITC dye and subjected to flow cytometry analysis. Annexin-V-positive cells (Q3) were counted as pre-apoptotic cells and PI-positive, Annexin-V-positive cells (Q2) were apoptotic. Percentage MUC16 of apoptotic cells equals the sum of Q2 and Q3. Top panel: one representative result of apoptosis analysis. Bottom panel: the statistic results of 3 separate experiments. (* p= 0.04) (Fig. ?(Fig.1D)1D) along with significant changes in SF4. Enhanced radioresistance in MD-PR cells was furtuher evidenced by apoptotic assays. Results of apoptotic assays showed that, after a large dose of irradiation, the proportion of pro-apoptotic (Annexin V-FITC positive) and apoptotic (Annexin V-FITC positive, PI positive) cells was significantly reduced in MD-PR cells compared with MD-PB cells (13.222.17 vs 20.921.33,p=0.01, Fig. ?Fig.1E).1E). The above results showed that MB-PR cells were more radioresistant compared with MD-PB cells. At the same time, we tested whether repeated irradiation could change the cell proliferation rate and cell cycle distribution. The results showed that there was no significant difference between MD-PR cells and MD-PB cells in these aspects (Fig. S1D, E). Altered -H2AX kinetic in radioresistant MD-PR cells To find the possible mechanism explaining reduced apoptosis ratio in MD-PR cell line after irradiation, DSB repair efficiency was evaluated in MD-PB and MD-PR cells by detecting Phosphor-Histone H2A.X (Ser139) (-H2AX), which was a widely adopted marker for the detection of DSBs 11. Western blotting experiments BMS-777607 evidenced that kinetics of -H2AX assorted between MD-PR and M-PB cells (Fig. ?(Fig.2A,2A, B). -H2AX manifestation peaked at quarter-hour after irradiation and decreased to normal level at about 2-hour post-irradiation in MD-PR cells, while it gained a maximum at 1-hour and decreased to normal level at 2-hour in MD-PB cells (Fig. ?(Fig.2B).2B). The two patterns of -H2AX kinetics suggested that DSBs were repaired faster in MD-PR cells. Consistent with results of western blotting, the number of -H2AX foci per nuclei enumerated at 1-hour post-irradiation in MD-PR cells (307 foci per nuclei) was significantly lower than that in MD-PB (4411 foci per nuclei) (Fig. ?(Fig.2C,2C, D). Open in a separate window Number 2 Modified -H2AX kinetic in MD-PR.