Cell survival was decreased by electron beam irradiation
To investigate possible effects of electron beam irradiation (EBI) on MCF-7 cells viability, MTT assay was performed 48h post-irradiation. MTT assay showed that irradiation caused significantly decrease in cell viability as compared to control group in a dose-dependent relationship (PControl vs.4 Gy group< 0.05; PControl vs.6 Gy and 8 Gy groups< 0.001; PControl vs. 10 Gy group< 0.0001; Fig. 1A). The same results were obtained when 2 Gy group compared to 6, 8, and10 Gy groups (P2Gy group vs.6 Gyand 8 Gy groups< 0.01; P2Gy group vs. 10 Gy group< 0.0001). Our finding indicated that EBI dose-dependently reduced viability of MCF-7 cells at the end point of 48 h post-radiation.
Electron beam irradiation enhanced apoptosis rate of MCF-7 cells
AnnexinV marker was used to monitor apoptosis rate of MCF-7 cells. Data showed a significant increase in apoptosis rate of MCF-7 cells subjected to electron beam in a dose-dependent manner (PControl vs.4 Gy group< 0.05; PControl vs.6 Gy group< 0.01; PControl vs. 8 Gy group< 0.001; PControl vs. 10 Gy group< 0.00001; Fig. 1B, C). In comparison to 2 Gy group, percentage of apoptotic cells elevated in other groups (P2 Gy group vs.6 Gy group< 0.05; P2 Gy group vs.8 Gy group< 0.01; P2 Gy group vs. 8 Gy group< 0.0001). Compared to the 4 Gy group, apoptosis was increased in 8 Gy and 10 Gy irradiated cells (P<0.05 and P<0.001, respectively). 10 Gy beam electron caused significantly increase in percentage of apoptotic cells as compared to 6 Gy (P<0.01), and 8 Gy irradiation (P<0.01) (Fig. 1B, C).
Electron beam irradiation increased ROS production in MCF-7 cells
Fluorometric method was used to measure ROS production in irradiated cell. Our finding indicated that EBI increased ROS production in MCF-7 cells in a dose-dependent relationship (PControl vs.2Gy and 4 Gy groups< 0.05; PControl vs.6 Gy group< 0.001; PControl vs. 8 Gy and10 Gy group< 0.000001; Fig. 2A). Compared to 2 Gy-CM, ROS production amplified in 6Gy, 8 Gy, and 10 Gy irradiated cells (P2 Gy group vs.6 Gy group< 0.05; P2 Gygroupvs.8Gygroup< 0.001; P2Gy groupl vs.10 Gy group<0.0001). We found an increased level of ROS production in 8 Gy and 10 Gy groups as compared to 4 Gy group (P4 Gy group vs. 8 Gy and10 Gy groups <0.001). In comparison to 6 Gy irradiation, 10 Gy irradiation caused a significant increase in ROS production in MCF-7 cells (P<0.01; Fig. 2A).
Electron beam irradiation increased the AChE activity in conditioned media of MCF-7 cells
AChE activity, an enzyme associated with extracellular vesicles, used to measure the extracellular vesicles content in CMs. According to data from Fig.2B, high intensity of EBI increased AChE activity as compared to control group (PControl vs.6 Gy group< 0.05; PControl vs.8 and 10 Gy groups< 0.01). Compared with both 2 Gy and 4 Gy groups, the AChE activity was elevated in 8 Gy and 10 Gy groups (P2 Gy and 4 Gy groups vs.8 Gy and 10 Gy groups< 0.05; Fig.2B).
Electron beam irradiation up-regulatedexpression of caspase-9, caspase-8, and caspase-3 genes
To further insight into apoptosis, we measured the mRNA level of apoptotic genes by real time-PCR. The application of higher doses of EBI (8 Gy and 10 Gy) showed an increase in the level of caspase-9 transcript (PControl and 2 Gy groups vs. 8 Gy group< 0.05; PControl and 2 Gy groups vs.10 Gy group< 0.01; Fig. 3). Compared to 4 Gy and 6 Gy groups, the mRNA level of caspase-9 augmented in10 Gy group (P4Gy and 6 Gy groups vs. 10 Gygroup< 0.05).
As shown in Fig. 3, compared to control group, 10 Gy and 8 GyEBI markedly induced expression of caspase-8 in MCF-7 cells (P<0.05 and P<0.01, respectively). In addition, 10 GyEBI caused a significant increase in mRNA level of caspase-8 as compared to other doses of EBI (P2 Gy and 4 Gy groups vs. 10 Gy group< 0.01; P6Gy groups vs.10 Gy group< 0.05; Fig. 3).
Correspondingly, the transcript level of the caspase-3, an apoptotic executer, increased in irradiated cells as compared to control group (PControl vs. 4 Gy, 6 Gy and 8 Gy groups< 0.05; PControl vs.10 Gy group< 0.01; Fig.3). Compared to 2 Gy group (1.06 ± 0.14-fold change), the mRNA level of caspase-3 amplified in 8 Gy (1.57 ± 0.31fold change) and 10 Gy groups (2.17 ± 0.41-fold change) (P2 Gy group vs. 8Gy group < 0.05; P2 Gy group vs. 10 Gy group< 0.01). Furthermore, we showedthat10 Gy irradiation cased a significant upturn in expression of caspase-3 as compared to 4 Gy and 6 Gy irradiation (P<0.05; Fig. 3). Calculating caspase-9/caspase-8 ratio may signify extrinsic/extrinsic apoptosis pathway switch. We found that this ratio was rather than 1-fold change in all groups and upon increase in dose of irradiation, a slight increase but not significant was observed (P>0.05; Fig. 3). Data indicated that apoptotic genes increased in a dose-dependent manner.
Electron beam irradiation amplified expression of Sox-2 and Oct-4 genes
Comparative quantities of transcripts contributing in the stemness and self-renewal of MCF-7 cells were monitored by real-time PCR investigation. Results showed that 10 Gy irradiation elevated mRNA distribution of Sox-2 in MCF-7 cells (Pcontrol, 2 Gy, and 4Gy groups vs.10Gygroup< 0.05; Fig. 4). Additionally, the transcript level of Oct-4 increased in high doses of EBI (PControl vs.6 and 8 Gy groups< 0.05; PControl vs.10 Gy group< 0.01; Fig. 4). Compared to 2 Gy group, the expression level of Oct-4 increased in 6 Gy, 8 Gy, and 10 Gy groups (P2 Gy vs.6 Gy and 8 Gy groups< 0.05; P2 Gy vs.10 Gy group< 0.01). There was a significant difference between 4 Gy (1.88 ± 0.62 fold change) and 10 Gy (4.15 ± 0.41 fold change) groups (P<0.05) (Fig. 4). Data showed exposure to electron beam, up-regulated expression of stemness genes such as Sox-2 and Oct-4 inMCF-7 cells.
Wound healing rate of MCF-7 cells enhanced following exposure to electron beam
In vitro scratch assay was established to monitor effect of EBIon wound healing rate and migration potential of MCF-7 cells. Obtained results in this panel revealed that EBI dose-dependentlyenhanced wound healing rate of MCF-7 cells within 48 h (PControl vs.6 Gy group< 0.05; PControl vs.8 Gy and10 Gy groups< 0.00001; Fig. 5A, B). Compared to 2 Gy group, the wound healing rate significantly elevated in 6 Gy, 8 Gy, and 10 Gy groups (P2 Gy vs.6 Gy group< 0.05; P2 Gy vs 8 Gy and10 Gy groups< 0.0001). The same results obtained as either 4 Gy group or 6 Gy group compared with both 8 Gy and 10 Gy groups (P4 Gy group vs.8Gygroup< 0.001; P4 Gy group vs.10Gygroup< 0.0001; P6 Gy group vs. 8 Gy group< 0.01; P6 Gy group vs. 10 Gy group< 0.001; Fig. 5A, B).
Condition media from irradiated MCF-7 cells decreased survival rate of MCF-7cells, MSCs, and HUVECs
The cell survival rate of non-irradiated MCF-7 cells, HUVECs and MSCs co-cultured with CMs of MCF-7 cells investigated using MTT assay. As shown in Fig.6, there is a significant difference between control and control-CM groups (P<0.05). In addition, irradiated CMs decreased survival rate of MCF-7 cells (PControl and control-CM vs.2, 4, 6, 8, and10 Gy-CM groups< 0.00001; Fig. 6A). Incubation of HUVECs with CMs from MCF-7 cells represented a substantial reduction in cell viability as compared to control group (PControl vs. control-CM, 2, 4, 6, 8, and 10 Gy-CM groups< 0.00001; Fig. 6B). Compared with the Control-CM group, cell viability rate declined in 8 Gy-CM and 10 Gy-CM groups.
In the case of MSCs, irradiated CMs also profoundly decreased the viability of MSCs (PControl vs. control-CM, 2, 4, 6, 8, and 10 Gy-CM groups< 0.00001; Fig. 6C). Furthermore, the comparison of the viability of MCF-7 cells, HUVECs, and MSCs are presented in Fig. 6D.
Irradiated conditioned media from MCF-7 cells increased apoptosis rate of MCF-7 cells
We also monitored the apoptosis rate of MCF-7 cells following incubation with CMs from irradiated MCF-7 cells. Flow cytometric analysis showed that CMs increased the percentage of apoptotic cells in a dose-dependent manner (Pcontrol vs. control-cm group < 0.05; Pcontrol vs. 2Gy-CM group< 0.001; Pcontrol vs. 4, 6, and 10Gy-CM groups< 0.0001; Fig 6E, F).
Compared to the Control-CM, a significant increase in apoptosis rate of cells incubated with irradiated CMs was found (PCM-control vs. 2 and 4 Gy-cm groups< 0.05; PCM-control vs. 6, 8, and 10 Gy-cm groups< 0.01).
Irradiated CMs from MCF-7 cells induced expression of caspase-9, caspase-8, and caspase-3 genes in MCF-7 cells
As shown in Fig. 7, the mRNA level of casspase-9 dose-dependently amplified in 8 Gy-CM and 10 Gy-CM groups when compared with both control and control-CM groups (Pcontrol vs. 6 Gy-cm group < 0.05; Pcontrol and control-CM vs. 8Gy-CM group< 0.01; Pcontrol and control-CM vs. 10 Gy-CM group< 0.001). Compared fold changes in caspase-9 of both 2 Gy-CM (1.28 ± 0.15-fold change) and 4 Gy-CM (1.45 ± 0.09-fold change) groups, these values reached 1.80 ± 0.14 and 2.36 ± 0.32 fold changes in 8 Gy-CM and 10 Gy-CM groups respectively (P<0.01; Fig. 7). The similar result was achieved when 6 Gy-CM group compared with 10 Gy-CM group (P<0.01).
Similar to expression pattern of caspase-9, CMs from high dose-irradiated cells had potential to induce profoundly the expression of caspase-8 (Pcontrol vs. 8Gy-cm group< 0.05; Pcontrol vs. 10Gy-CM group< 0.01; Pcontrol-CM and 2 Gy-CM groups vs. 10 Gy-CM group< 0.01; Fig. 7). In comparison with 4 Gy-CM and 6 Gy-CM groups, the mRNA level of caspase-8 was significantly elevated in 10 Gy-CM group (1.40 ± 1.00 and 1.43 ± 0.13 vs. 2.12 ± 0.31-foldchange; P<0.01).
We also found that CMs from electron beam irradiated cells induced significantly expression of caspase-3 in MCF-7 cells in a dose-dependent manner (Pcontrol vs. 4 Gy-CM and 6 Gy-cm group < 0.05; Pcontrol vs. 8 Gy-CM group< 0.01; Pcontrol vs. 10 Gy-CM group< 0.001; Fig. 7). Data from Fig.7 also showed the same trend for control-CM group as compared with other groups (Pcontrol-CM vs. 6 Gy-CM < 0.05; Pcontrol-CM vs. 8 Gy-CM group< 0.01; Pcontrol-CM vs. 10 Gy-CM group< 0.001). Transcript level of caspase-3 increased in 8 Gy-CM and 10 Gy-CM groups when compared either with 2 Gy-CM group or 4 Gy-CM group (P<0.01). Furthermore, the mRNA form of caspase-3 significantly increased in 8 Gy-CM group and 10 Gy-CM group as compared with 6 Gy-CM group (P6 Gy-CM vs. 8 Gy-cm group < 0.05; P6 Gy-CM group vs. 10 Gy-CM group< 0.01; Fig.7).
Irradiated CMs from MCF-7 cells induced expression of Sox-2 and Oct-4 genes in MCF-7 cells
Gene expression assessment by real time-PCR showed that CMs from 8 Gy and 10 Gy irradiated MCF-7 cells induced expression of Sox-2 gene (Pcontrol, control-CM, 2 Gy-CM, and 4 Gy-Cm groups vs. 8Gy-CM < 0.05; Pcontrol, control-CM, 2 Gy-CM, and 4 Gy-Cm groups vs. 10Gy-CM <0.01; Fig. 8). The mRNA level of Sox-2 increased in 8 Gy-CM and 10 Gy-CM groups as compared to 6 Gy-CM group (P<0.05). Similar to Sox-2 gene, we also found that the other self-renewal gene, Oct-4, up-regulated in 8 Gy-CM and 10 Gy-CM groups as compared with other groups (Pcontrol, control-CM, 2 Gy-CM, and 4 Gy-Cm groups vs. 8 Gy-CM < 0.01; Pcontrol, control-CM, 2 Gy-CM, and 4 Gy-Cm groups vs. 10 Gy-CM <0.001; Fig. 8). Additionally, in comparison with 6 Gy-CM group (1.38 ± 0.26-fold change), the mRNA levels of Oct-4 were increased in 8 Gy-CM (2.48 ± 0.16) and 10 Gy-CM (3.35 ± 0.29-fold change) groups (P<0.05 and P<0.01; respectively). Analysis of caspase-9/caspase-8 ratio showed that this ratio reached slightly rather than 1-fold in all groups without any significant differences among groups (P>0.05; Fig. 8).
Conditioned media from MCF-7 cells increased wound healing rate of MCF-7 cells
To investigate the effect of irradiated CMs on wound healing rate and migration of MCF-7 cells, we established in vitro scratch assay. Our results showed that 8 Gy and 10 Gy irradiated CMs a long with control raised the wound healing rate of MCF-7 cells over 48 h incubation(Pcontrol-CM, 2 Gy-CM, 4 Gy-CM, 6 Gy-CM groups vs. 8 Gy-CM, 10 Gy-CM, and control groups< 0.00001; Fig. 9A,B). In addition, the wound healing rate increased in 10 Gy-CM and control groups as compared to 8 Gy-CM group (P8Gy-CM group vs. control and 10 Gy-CM groups<0.01). Interestingly, we found that the wound healing rate of control group increased; that supposedly due to incubation in fresh DMEM containing FBS.