Abstract:

Abstract:Objective
To explore the mechanism of radio-resistance of breast cancer stem cells by investigating the effects of ionzing  radiation on the reactive oxygen species (ROS),apoptosis and cycle distribution.Methods The breast cancer MCF-7 cells  were suspension cultured in serum-free medium containing a variety of  growth factors.There were MCF-7 (breast cancer cells),MCF-7-S (breast cancer stem cells),MCF-7+8 Gy and MCF-7-S+8 Gy groups in the experiment.4-24 h after 8 Gy irradiation,the ROS levels,percentages of apoptotic cells  and percentages of the cells at each cycle phage   were measured by FCM with 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA),AnnexinⅤ-FITC/PI and PI staining,respectively.Results The breast cancer stem cell microsphere accumulated hundreds of cells were obtained successfully at 7 d after suspension culture with serum-free medium containing a variety of growth factors;the FCM results showed that CD44+CD24－ phenotype breast stem cells were up to 75.20%.With the time prolongation,the ROS levels and apoptosis in MCF-7 group and MCF-7-S group showed increasing trendency,and reached for the maximum values at 12 and 24 h;the ROS levels in MCF-7-S group were significantly lower than those in MCF-7 group at 4,8,12 and 24 h (P<0.05 or P<0.01),and the   percentage of apoptotic cells in MCF-7-S group was significantly higher than that in MCF-7 group only at 8 h (P<0.05);the ROS levels (4,8,12 and 24 h) and   percentage  of apoptotic cells(12 h) were significantly increased in MCF-7+8 Gy group (P<0.05),and the  percentages of apoptotic cells (4,8,12 and 24 h) in MCF-7-S +8 Gy group were significantly decreased (P<0.05 or P<0.01),but the ROS levels had no obvious change in MCF-7-S+8 Gy.At 12 h,as compared with MCF-7 group,the  percentages of the cells at  G0/G1 phase and G2/M phase in MCF-7-S group were significantly decreased (P<0.05),and the  percentage of the cells at S phase was significantly increased (P<0.05 );the  percentage of the cells at G2/M phase in MCF-7+8 Gy group was significantly increased (P<0.05 ),but there were no significant changes in MCF-7-S+8 Gy group.Conclusion Ionizing irradiation can cause the increasing of ROS level and apoptosis and G2/M phase arrest in breast cancer cells,but has no obvious effects on the breast cancer stem cells;it indicates that radio-resistance might be related to ROS level,apoptosis and G2/M phase arrest.

Key words: ionizing radiation;breast neoplasms, stem cells;suspension culture;reactive oxygen species；apoptosis；cell cycle