Evaluating the Bystander Effects of Electron Beam Irradiation in Radiotherapy on MCF-7 Human Breast Cancer Cells

Background: The distinct direct and non-targeting effects of electron beam radiation on MCF-7 cells remain unclear. We aimed to investigate the effect of electron beam radiation and conditioned media of the irradiated MCF-7 cells on MCF-7 cells. Methods : The cell viability and apoptosis were assayed via MTT and flow cytometry analysis, respectively. Production of oxygen reactive species was evaluated by chemical fluorometric method, while the amount of extracellular vesicles was detected via acetylcholinesterase activity assay. Change in the expression of genes involved in apoptosis including caspase-3, caspase-8, and caspase9 and stemness such as Sox-2 and Oct-4 were calculated through q PCR. Wound healing rate of cells was monitored via in vitro scratch assy. Results: Result showed that compared to control group, electron beam radiation decreased cell viability and increased apoptosis and oxygen reactive species as well as acetylcholinesterase activity in a dose-dependent manner ( P <0.05). Concurrent with increasing dose of electron beam, the mRNA level of apoptotic genes (caspase-3, caspase-8, and caspase-9) and stemness related genes (Sox-2 and Oct-4) up-regulated following electron beam radiation. Wound healing rate of irradiated MCF-7 cells dose-dependently increased ( P <0.05). After treatment with conditioned media from irradiated MCF-7 cells, the similar results were observed. Conditioned media from irradiated MCF-7 cells decreased viability of MCF-7 cells, mesenchymal stem cells, and HUVECs ( P <0.05). Additionally, conditioned media from irradiated MCF-7 cells promoted apoptosis and wound healing rate as well as increased mRNA levels of apoptosis and stemness related genes ( P <0.05). Conclusion : Findings suggest electron beam radiation could affect apoptosis and stemness in MCF-7 cells in direct and bystander manners.

3 therapy, and radiation therapy have been exploited by physician to eliminated tumor cells [3,4]. One of the well-known treatments for breast cancer is radiation therapy, which ionizing radiation (IR) directly destroys cancer cells [5]. Intraoperative radiation therapy (IORT) is more effective method in reduction of local recurrence than the standard external beam radiotherapy of whole breast [6,7].
IORT is a method of directly irradiation of operated area during surgery which delivers increasing dose of radiation on operated area [8]. This method is an alternative to 6 weeks of external beam radiotherapy, which is equal to the deliverance of electron beam called intraoperative electron radiotherapy (IOERT) or 50-kv X-ray to tumor region during breast conserving surgery [9,10].
IOERT has been considered to improve treatment of breast cancer and prevent local recurrences of it after surgery in breast conserving therapy [8,9]. Indeed, electron beam therapy is a kind of radiation treatment that electrons are directed to a tumor site instead of photons. It is well established that electron beam damage bio-molecules and organelles of tumor cells, contributing in eliminating tumor cells [11,12]. Growing evidence reported that cells dwelled in tumor mass may indirectly were affected by IR, which previously known as non-targeting effects (NTEs) of radiation [13]. Bystander effects are a class of NTEs by which direct irradiated cells influence non-irradiated cells in an irradiated volume [14]. Of note, irradiated cells mediate the bystander effects of IR by releasing various soluble factors including cytokines, reactive oxygen species (ROS), and even exosomes into extracellular matrix that eventually affect fate and function of neighboring non-irradiated cells known as bystander cells [15]. In this regard, signals received from directly IR irradiated cells may induce mutations, DNA damage, apoptosis, chromosomal instability, migration, and resistance in nonirradiated cell [14,16]. In recent years, most studies have only focused to assess different responses of tumor cells to IR and to signals from directly irradiated cells [17][18][19].
Electron beam therapy is especially useful for treatment of superficial tumors. However, the biological and molecular effects of routine electron beam therapy and IOERT on tumor cells and its environment are not reported exactly up to now [10]. In line with these comments, the underling mechanisms involved in MCF-7 cells responses against directly and bystander effects of electron beam exposure need to be evaluated. Here, we aimed to highlight the direct and bystander effects of electron beam 4 treatment on MCF-7 human breast cancer cell line.

Irradiation
Following a dosimetry calibration, a clinical 5 MeV electron beam of a Siemens PRIMUS medical linear accelerator (Siemens AG, Germany) was used for irradiation. The output of machine is calibrated such that 1.0 monitor unit (MU) is equal to 1.0 cGy. MCF-7 cells were seeded in 96 and 6-well plates 24 h prior to irradiation in order to measure cell viability using MTT assay and other tests, respectively.
Medium level was adjusted to 11 mm over the cell monolayer to place the MCF-7 cells at the depth of maximum dose (d max ). A standard electron applicator (25 ×25 cm 2 ) was placed on the accelerator head for irradiation. The cells in the plates were positioned at 100 cm distance from the electron source, and irradiation was done at an output rate of 300 MU/min. The cell culture plates were placed at the center of the radiation field to ensure that all the cells irradiated with a uniform radiation dose. MCF-7 cells were divided into 5 groups and exposed to 2, 4, 6, 8, 10 Gy of electron beam. The nonirradiated cells were kept as control group and subjected to the same condition as the electron treated cells experienced. The MU values were calculated by taking into account that 1 cGy/MU. After irradiation, the cells were returned to the incubator and maintained under culture condition for 48 h post-irradiation, prior to evaluation. All biological experiments were repeated in three different series.

Conditioned medium (CM) preparation
48h after irradiation, CMs of all groups were collected and centrifuged (15,000g for 20 min at 4°C) and 0.2 mm syringe filters used to remove remained cells and debris. The filtrated cell conditioned medium was saved in -80 C freezer for next experiments.

5
To investigate bystander effects of irradiated MCF-7 cells, the non-irradiated MCF-7 cells, HUVECs, and MSCs co-cultured with CMs of irradiated and non-irradiated MCF-7 cells (control CM) for a period   of 48 h and kept as bystander cells. According to irradiation protocol, bystander cells are received the   CMs of relevant groups and named as: Control-CM, 2 Gy-CM, 4 Gy-CM, 6 Gy-CM, 8 Gy-CM, and 10 Gy-CM. Additionally, one group was kept as control group which cultured in DMEM/FBS media in the same condition.

Cell viability
Cell viability was determined 48h after irradiation using MTT assay. Prior to irradiation and CMs treatment, 5×10 3 cells seeded in per well of 96 well plates for 24 h. Next, MTT reagent was dissolved at a concentration of 5mg/ml in sterile phosphate-buffered saline (PBS) in the dark. 48h after irradiation, the cell media removed completely and replaced with 100 ml/well of MTT solution and then incubated for 4 h. After 4 h, wells were depleted and 100 ml of dimethyl sulfoxide (DMSO) added to each well for 20 min at room temperature. Absorbance was measured using a micro-plate reader at a wavelength of 540 nm. Analysis was accomplished by following formula: (optical density (OD) sample/OD control) ×100.

Flow cytometry analysisof apoptosis
We used Annexin-V (eBioscience) marker to monitor apoptosis rate in MCF-7 cells. For this purpose, 48 h after irradiation and incubation with CMs, Cells were subjected to Annexin-V kit protocol according to manufacture recommendation. In brief, cells were fixed with 100 ml of binding buffer at room temperature for 15 min. Next, Annexin-V (1ml/ ml) was added to each sample at room temperature over 15 min in a dark room. Following twice washing with PBS, cells were suspended in the 1ml final volume of binding buffer. Analysis was performed by a flowcytometric system (BioRad, Hercules, CA, USA) and FlowJo (version 7.6.1) software.

ROS production
Chemical fluorometric method was used for detection of ROS production using a ROS assay Kit (E-BC-K138, Elabscience). According to kit's protocol, 1 h after irradiation, 10 μM of dichlorofluorescein diacetate (DCFH-DA) reagent was added to cell culture medium and incubated at 37°Cover 30 min. 6 Then, cell suspensions were centrifuged at 1000 g for 5 min and washed with PBS twice. Finally, the fluorescence was measured using a micro-plate reader (Bio-Rad) at wavelength of 485 nm excitation and 525 nm emission.

Acetylcholinesterase activity assay
Acetylcholinesterase (AChE) activity was applied to evaluate extracellular vesicles in CMs of MCF-7 cells via cholinesterase kit (Cat No. BXC080; Iran).In brief, solution A (potassium hexacyanoferrateand pyrophosphate) was mixed to CMs and incubated for 5 min at room temperature. After that, solution B (2-butyrylthio-n,n,n-trimethylethanaminium iodide) was added and the absorbance was detected at 405 nm by three time points using a plate reader system (BioTek). AChE activity was analyzed by the recommended formula: Activity (U/l) = 65,800 × ΔAbs/min.

Real time-PCR analysis
In order to analyze expressions of apoptosis genes including caspase-3, 8, and 9 and; Sox-2 and Oct-4 genes dealing with stemness, real time-PCR analysis was used. Total RNA was isolated using RNA extraction kit (Cat.No: FABRK001, Iran). Next, RNA quality and quantity were evaluated using a Nanodrop system (BioTek). cDNA synthesis accomplished by a commercial cDNA synthesis kit (Cat:YT4500, Iran). Next, mRNA levels of genes were measured using SYBR Green dye-based PCR Master Mix (YT2551, Yekta Tajhiz Azma, Iran) and real time-PCR system (ABI 7500, Applied Biosystems). Data were normalized against GAPDH gene and analyzed using the 2 −ΔΔCt method.The sequences of primers are listed in Table 1.

Wound healing assay
The MCF-7 cells were cultured in a 12-well plate until they reached 80% confluence. The cell monolayer was scratched using a yellow tip. Next, the supernatants were replaced by CMs for 48 h.
The cell-free scratched area was monitored at 0 h and 48 h of incubation and measured using an Image j software ver. 1.44p. Wound healing rate was reported by applying a formula :(( first surface area -second surface area)/ first surface area) × 100.

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 (P Control vs.4 Gy group < 0.05; P Control vs.6 Gy and 8 Gy groups < 0.001; P Control vs. 10 Gy group < 0.0001; Fig. 1A). The same results were obtained when 2 Gy group compared to 6, 8, and10 Gy groups (P 2Gy group vs.6 Gyand 8 Gy groups < 0.01; P 2Gy 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 (P Control vs.4 Gy group < 0.05; P Control vs.6 Gy group < 0.01; P Control vs. 8 Gy group < 0.001; P Control vs. 10 Gy group < 0.00001; Fig. 1B, C). In comparison to 2 Gy group, percentage of apoptotic cells elevated in other groups (P 2 Gy group vs.6 Gy group < 0.05; P 2 Gy group vs.8 Gy group < 0.01; P 2 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 (P Control vs.2Gy and 4 8 Gy groups < 0.05; P Control vs.6 Gy group < 0.001; P Control vs. 8 Gy and10 Gy group < 0.000001; 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 (P Control vs.6 Gy group < 0.05; P Control 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 (P 2 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 (P Control and 2 Gy groups vs. 8 Gy group < 0.05; P Control and 2 Gy groups vs.10 Gy group < 0.01; Fig. 3).
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 (P 2 Gy and 4 Gy groups vs. 10 Gy group < 0.01; P 6Gy 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 (P Control vs. 4 Gy, 6 Gy and 8 Gy groups < 0.05; P Control vs.10 Gy group < 9 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) (P 2 Gy group vs. 8Gy group < 0.05; P 2 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.

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 (P Control vs.6 Gy group < 0.05; P Control 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 (P 2 Gy vs.6 Gy group < 0.05; P 2 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 (P 4 Gy group vs.8Gygroup < 0.001; P 4 Gy group vs.10Gygroup < 0.0001; P 6 Gy group vs. 8 Gy group < 0.01; P 6 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

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 (P control vs. control-cm group < 0.05; P control vs. 2Gy-CM group < 0.001; P control 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 (P CM-control vs. 2 and 4 Gy-cm groups < 0.05; P CM-control vs. 6, 8, and 10 Gy-cm groups < 0.01).

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(P control-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 (P 8Gy-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.

Discussion
In the current study, the effect of different doses of EBI on dynamic of the MCF-7 cells was evaluated.
Furthermore, we examined the bystander effects of irradiated MCF-7 cells on the different cell lines, which are known play pivotal roles in cancer treatment. Consistent with previous studies (20,21), we found that the cell viability and apoptosis rate of irradiated MCF-7 cells were dose-dependently decreased and increased respectively (Fig. 1). Irradiation elicit cytotoxic effects on the dynamic of cells through damaging to biomolecules and organelles that induces apoptosis (22,23). According to increase in electron beam dose, the ROS production inside cells increased ( Fig. 2A), which may correlated to induction of toxicity in cells and elevated apoptosis rate (24). Different caspases contribute to flow apoptosis both in intrinsic and extrinsic pathways (25), which we examined the mRNA levels of caspase-3, -8, -9 in irradiated MCF-7 cells over a period of 48 h of post-irradiation.
Caspase-9 and caspase-8, the cysteine proteases, are implicated in intrinsic and extrinsic apoptosis respectively that activate apoptosis through interacting with caspase-3 (25). Our result showed an increase in the transcript levels of all caspase-3, -8, -9 genes (Fig 3). Increased expression levels of these genes were concomitantly seen with higher level of ROS production in MCF-7 cells that correlated with the result of apoptosis assay. We also calculated caspase-9/caspase-8 ratio to predict the relative superiority of intrinsic or extrinsic pathways in our study. This ratio was rather than 1 value in all irradiated groups, however, the significant difference between them was not observed (Fig   3). These data support an idea that the majority of apoptosis may occur through intrinsic pathway in electron beam received cells. Electron beam could harms cellular biomolecules and organelles (23) and produce ROS (26) that may activates apoptosis intrinsic pathway. In addition, we found that the which may explain the mechanism that intracellular cytotoxicity is expelled by these vesicles for maintain homeostasis and adaptation (29). Based on our knowledge this is first report of increased AChE activity of CMs of MCF-7 cells following exposure to electron beam. It seems that EBI influenced ROS/apoptosis/EVs secretion axis in MCF-7 cells. Beside these, we measured the effect of EBI on MCF-7 cells regarding stemness related genes. In this regard, data showed that the expression of Sox-2 and Oct-4 increased in irradiated cells (Fig. 4), supposing induction of a stem cell-like phenotype in MCF-7 cells (30,31).This was in accordance with a work by Ghisolfi et alwhich found that gamma irradiation of cancer cells increased mRNA level of Sox-2 and Oct3/4 genes (32). Similarly, it was demonstrated that IR induce the cancer stem like cells via dedifferentiation, and increasing the expression of stem cell markers such as SOX-2 in glioblastoma cells (33). Confirmed that, upregulation of Sox2 and Oct-4 genes may have a pivotal role in the supporting of self-renewal, reprogramming, and plasticity capacity in cancer stem cells (34,35). Our findings would seem to show the mRNA level of these genes following EBI may represent alternation in the MCF-7 cells population and enrichment of cancer stem cells (36).Concurs well with Imaizumi and colleagues (37), 14 we also found that EBI was capable of inducing migration response of MCF-7 cells by scratch wound healing assay (Fig. 5). Different authorities have shown that irradiation not only induce apoptosis in tumor cells but also encourage motility and invasion of tumor cells through several pathways (38,39).
Further data collection is required to uncover underling mechanisms involved in EBI mediated cell migration.
Furthermore, we aimed to investigate bystander effect of EBI on non-irradiated cells. In this scenario, we noted that treatment of MCF-7 cells, MSCs, and HUVECs with CMs from irradiated MCF-7 cells decreased viability of all cell lines (Fig. 6A-D). Jella and co-workers (40) (42). For further insight into apoptosis, we monitored expression of apoptotic genes including caspase-3, -8, and -9 in the CM-treated MCF-7 cells (Fig. 7). Consist with; our results confirmed the up-regulation of those genes in MCF-7 cells cultured with CMs from irradiated MCF-7 cells. Increased expression levels of these genes were correlated with increased percentage of Annexin V positive cells (Fig. 6E and F). It seems that both intrinsic and extrinsic pathways were activated in our experiment; however, the caspas-9/caspas-8 ratio was rather than 1 in all CM-groups (Fig. 7), indicating more activation of intrinsic pathway. To our knowledge, little is known about the impact of bystander effects of electron beam on cancer stem cell related genes. Interestingly, we found that CMs from irradiated cells induced expression of stemness and selfrenewal related genes; Sox-2 and Oct-4 in MCF-7 cells (Fig. 8). According to previous studies, these genes are involved in resistance and adaption to stress condition (43,44). Bystander effects may 15 mediate through different soluble factors, cytokines (10), and even EVs (12). It was well established that bystander effects of IR are consist of chromosomal inconsistence, DNA damage, alteration, resistance, proliferation, and apoptosis in non-irradiated cells (45). Xu and co-workers have reported that X-ray irradiation increase exosomes biogenesis and secretion (46). In addition, several laboratories demonstrated that EVs from irradiated tumor cells promoted genomic instability and resistance in non-irradiated cell (47,48). In our experiment, in part, increased EVs in CMs may explain In summary, through investigating irradiated MCF-7 cells with the different doses of electron beam affecting the viability, apoptosis, EVs release, expression of genes involved in apoptosis and stemness, and migration capacity, we shed light into cellular responses of MCF-7 against EBI (Fig. 10).
We showed that along with increase in doses of irradiation, the cellular responses increased. Although EBI showed cytotoxic effects, but facilitated EVs secretion, cell migration, and up-regulation of Sox2 and Oct-4 in MCF-7 cells, which may represent side effects of EBI. Additionally, by studying possibly bystander effects of EBI, we uncovered that irradiated MCF-7 cells sent signals through paracrine 16 manner that influence survival and apoptosis rate of non-irradiated cells (Fig. 10).These signals from high dose irradiated cells also induced stemness and resistance in tumor cell population; and even promoted wound healing rate indicating elevated migration potential of MCF-7 cells.

Conclusion
Although the current study analyzed in vitro response of MCF-7 cells against EBI, our results showed thatMCF-7 cells treated with electron beam and CMs from irradiated MCF-7 cells exhibit an upregulation in both genes involved in apoptosis pathway and stemness. Therefore, further studies are essential to elucidate the exact underlying mechanisms involved in EBI induced alternation in dynamic of tumor cells.  Real time-PCR analysis of caspase-9, caspase-8, and caspase-3 genes involved in apoptosis pathway.Data are presented as means ± SD; n = 3. One-way ANOVA with Tukey post hoc test. * P< 0.05 ** P< 0.01. Cas means caspase.

Figure 10
A representative diagram of study handling and cell treatment.