Fractionated Radiation Exposure Enhances DNA Repair Capacity to Amplify Radioresistance of Cancer Cells

Fractionated radiotherapy is widely used in cancer therapy for its advantages in the preservation of normal tissues, but may amplify radioresistance of cancer cells. To understand whether and how fractionated radiation exposure amplies radioresistance, HCT-8 human colon cancer cells and MCF-7 human breast cancer cells were received a total dose of 5 Gy X-ray irradiation by a single exposure or fractionated exposures (1 Gy/day for 5 consecutive days), respectively. We then examined the radioresistance of cells. Underwent an additional exposing to 2 Gy, cells received fractionated exposures showed signicantly better cell proliferation and clonogenic ability than cells received a single exposure. Compared to the intact cells without radiation exposure, the expression of γ-H2AX, pATM and PARP was signicantly enhanced in only these cells received fractionated exposures. However, the expression of cyclin D1 and cyclin E1 was enhanced in only these HCT-8 cells received a single exposure. Otherwise, the expression of SOD1, SOD2 and caspase 3 was not signicantly changed in both cells received either a single exposure or fractionated exposures. Fractionated radiation exposure amplies radioresistance of cancer cells, predominantly by enhancing DNA repair capacity.


Introduction
Radiotherapy has proven to be an effective palliative and curative tool in cancer treatment, with approximately 50% of cancer patients receiving radiotherapy [1]. Usually, tumor can be controlled if a su ciently high dose radiotherapy be delivered. However, in clinical practice, the dose of radiation is unfortunately limited by the tolerance of patients [2], because therapeutic radiation also damages the surrounding normal tissue cells.
Radioresistance of cancer cells means the relatively low or poor susceptibility to damage from therapeutic radiation. Therefore, radioresistance of cancer cells is the major problem for radiotherapy.
Radioresistance of cancer cells can be either ''intrinsic'' or ''acquired''. Intrinsic radioresistance naturally exists in cancer cells even before the treatment has started. In contrast, acquired radioresistance is induced by radiation itself and is the process by which cancer cells adapt to changes induced by radiotherapy [3,4]. Acquired radioresistance to conventional radiotherapy is one of the main factors leading to failure of radiotherapy, leading to cancer recurrences, metastases and a poor prognosis of cancer patients [5]. It is already known from previous studies that complex mechanisms, including cell survival pathways, DNA repair systems, and hypoxia signaling pathways are involved in radioresistance of cancer cells [6][7][8][9]. However, it has not yet been completely understood the precise mechanism on radioresistance, especially the acquired radioresistance.
Fractionated radiotherapy is the most prominent technique to increase the dose of radiotherapy by selectively sparing of healthy tissue cells, but may induce radioresistance of cancer cells during repeated exposure progression [10]. In past decades, many clinical trials have tried to compare the outcomes of different regimens (hypo-vs. standard-vs. hyper-fractionated) of radiotherapy for cancer patients [11][12][13]. Therefore, it is critical to know whether and how fractionated radiotherapy ampli es the acquired radioresistance of cancer cells.
In this study, we treated HCT-8 human colon cancer cells and MCF-7 human breast cancer cells with a total of 5 Gy X-ray by fractionated exposures or a single exposure, and then compared the biological characteristics of cells, especially about the acquired radioresistance. Our data indicated that fractionated exposures ampli ed radioresistance of cancer cells, likely by enhancing DNA repair ability.

Materials And Methods
Cells culture HCT-8 cells (from ATCC) and MCF-7 cells (from JCRB) were used for the experiments [14]. Cells were maintained in RPMI-1640 medium (Wako Pure Chemical Industries Ltd, Osaka, Japan) and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco/Life Technologies, Grand Island, NY). For routine maintenance, cells were cultured at 37℃ in a humidi ed atmosphere of 5% carbon dioxide and 95% air.

Irradiation
All irradiations were performed using an ISOVOLT Titan-320 X-ray unit (200 kV, 15 mA, 5 mm aluminum ltration, GE Sensing and Inspection Technologies, Billerica, MA). The cells were irradiated with a total dose of 5 Gy at 1.0084 Gy/min by fractionated exposures or a single exposure. Fractionated exposures were done by daily exposure to 1 Gy X-ray for 5 consecutive days, but a single exposure was done by once exposure to 5 Gy at the rst day. Intact cells without irradiation exposure were used for control.

Cell counting
Cells were digested by 0.25% trypsin (Gibco) and collected as single cell suspension at day 5. The total number of collected cells was counted using the TC20 counter (Bio-Rad).

Clonogenic assay
Clonogenic assay was used to evaluate the radiosensitivity of cells. We seeded cells into six-well plates at a density of 100 cells/well (HCT-8 cells) or 1000 cells/well (MCF-7 cells). After incubation overnight, the cells were exposed to radiation as indicated. Colony formation was quanti ed by counting the total number of colonies consisting of more than 50 cells in each well at day 10 (for HCT-8 cells) or day 14 (for MCF-7 cells).

MTT assay
Cell proliferation was performed using the Cell Proliferation Kit (Roche Life Science). Brie y, cells were seeded in 96-well culture plates (5 × 10 3 cells/well for HCT-8 cells and 1 × 10 4 cells/well for MCF-7 cells) and cultured overnight. The cells were exposed to radiation as indicated. We added 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) in medium and incubated for 4 h. The formation of formazan from MTT was stopped by adding solubilisation solution. The absorbance of formazan was measured at 570 nm using a microplate reader.

Western blot analysis
For Western blot analysis, total protein was puri ed from cells using RIPA buffer with the addition of protease & phosphatase inhibitor cocktail (Sigma, MO) at 4℃. Equal amounts of total protein (30 μg/lane) were electrophoretically separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h and incubated overnight with primary antibodies

Statistical analysis
All data were presented as the mean ± SD. Statistical signi cance of the differences was determined using one-way analysis of variance (ANOVA) followed by Tukey's test (Dr. SPSS II, Chicago, IL). A P value less than 0.05 was accepted as signi cant.

Fractionated exposures reserved the viability and clonogenic ability of cancer cells
To test the radiosensitivity, we exposed the HCT-8 cells and MCF-7 cells to X-ray, and then evaluated cell growth and clonogenic ability. As shown in the representative images ( Figure 1A), radiation exposure resulted in lower cell density than the control (0 Gy). Interestingly, compared to a single exposure to 5 Gy (5Gy x 1), fractionated exposures (1Gy x 5) was observed a better cell growth under microscopy and resulted in a signi cantly higher cell number by quantitative cell counting ( Figure 1A). We also tried to exposure the cells by daily exposure to 2 Gy for 5 days (a total dose of 10 Gy) and observed even higher cell density and cell number than that of cells giving a single exposure to 5 Gy ( Figure 1A). Similarly, clonogenic assay showed that a single exposure to 5 Gy formed signi cant a less number of colonies compared with fractionated exposures (P<0.05, Figure 1B), although the MCF-7 cells are relative radiosensitive because of the poorly colony formation following radiation exposures. These ndings suggests that fractionated exposures reserved the viability and clonogenic ability of cancer cells.

Fractionated exposures ampli ed radioresistance of cancer cells
To investigate the acquired radioresistance, irradiated cells with fractionated exposures or a single exposure were harvested at day 5, and then given an additional exposure to 2 Gy ( Figure 2A). As expected, clonogenic assay showed signi cantly higher number of colonies formed from cells that received fractionated exposures than that of a single exposure ( Figure 2B). MTT assay also showed that the proliferation of cells received fractionated exposures was signi cantly higher than those received a single exposure ( Figure 2C). These data con rmed that, comparing with a single exposure, fractionated exposures could amplify radioresistance of cancer cells.
Fractionated exposure to cancer cells signi cantly enhanced DNA repair capacity, but induced a limited changes on the expression of cell cycle regulators and antioxidant enzymes To further understand the underlying mechanism, we evaluated the expression of DNA repair molecules, cell cycle regulators, and antioxidant enzymes in the irradiated cells with fractionated exposures or a single exposure (Figure 2A).
Compared to the intact cells without radiation exposure, the expressions of γ-H2AX, PARP, and phospho-ATM were signi cantly enhanced in HCT-8 cells received fractionated exposures (p<0.05, Figure 3), but did not signi cantly change in HCT-8 cells received a single exposure to 5 Gy. Interestingly, for the radiosensitive MCF-7 cells that showing a relative poor PARP expression, only the expression of phospho-ATM was signi cantly enhanced by fractionated exposures (p<0.05, Figure 3).
About the cell cycle regulators, a single exposure but not fractionated exposures to 5Gy signi cantly inhibited the expression of Ki-67, an important proliferation marker in HCT-8 cells p < 0.05 vs. 0 Gy, Figure  4). The expression of cyclin D1 and cyclin E1, two of the most important cell cycle regulators was signi cantly enhanced in irradiated HCT-8 cells by a single exposure (p < 0.05 vs. 0 Gy, Figure 4), but not by fractionated exposures. Either a single exposure or fractionated exposures to 5Gy signi cantly inhibited the expression of Ki-67 in the MCF-7 cells (p < 0.05 vs. 0 Gy, Figure 4), but only a single exposure signi cantly enhanced the expression of cyclin E1 in the MCF-7 cells (p < 0.05 vs. 0 Gy, Figure 4).
Comparing to the intact cells without radiation exposure, the expressions of SOD1 and SOD2, two important antioxidant enzymes were only slightly enhanced in HCT-8 cells received 5 Gy by either fractionated exposures or a single exposure ( Figure 5). Although the expression of cleaved PARP was signi cantly increased in these irradiated HCT-8 cells by either fractionated exposures or a single exposure (p < 0.05 vs. 0 Gy, Figure 3), the expression of caspase 3, an important regulator of cell apoptosis did not signi cantly change in these irradiated HCT-8 cells ( Figure 5). For the MCF-7 cells, the expressions of SOD1 was not signi cantly changed after 5 Gy irradiation by either fractionated exposures or a single exposure ( Figure 5). The expression of SOD2 and caspase 3 were almost negatively detected in the MCF-7 cells ( Figure 5).

Discussion
Fractionated radiotherapy is widely used for cancer patients, but cancer cells may acquire radioresistance with repeated radiation exposures [15]. In the present study, we investigated in vitro whether and how fractionated exposures amplify radioresistance of cancer cells. We used HCT-8 human colorectal cancer cells and MCF-7 human breast cancer cells for experiments. Both of the HCT-8 cells and MCF-7 cells have wild type p53. Although radiotherapy is not commonly applied for colorectal cancer, HCT-8 cells are popularly used for experiments, including radiation studies. Comparing to the radiosensitive MCF-7 cells, HCT-8 cells are known to be relatively poor response to irradiation [16], and radioresistance can be easily and quickly induced in HCT-8 cells [16]. According to our data from MTT assay and colonogenic assay, fractionated exposures showed to amplify radioresistance in both of the HCT-8 cells and MCF-7 cells.
Complex mechanisms, such as DNA repair, cell cycle arrest, and redox regulation are known to be deeply involved in radioresistance of cancer cells. Previous studies have also demonstrated that fractionated exposures can induce radioresistance of cancer cells by various mechanisms [17][18][19]. However, data from this study indicated that fractionated exposures ampli ed radioresistance of cancer cells predominantly by enhancing DNA repair capacity.
Radioresistance could cause by all available DNA repair pathways, including those that go beyond the repair of DNA double-strand breaks [7]. Several fast-reacting proteins, including PARP, ATM, and γ-H2AX are known to quickly recruit to the damaged sites of DNA for repairing. PARP is inactivated by caspase cleavage. Cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis. Therefore, enhanced DNA repair ability can effectively prevent cells from apoptosis, which contributes to acquire radioresistance [20]. Cleaved PARP was enhanced in the irradiated cells received fractionated exposures and a single exposure in our study, fractionated exposures clearly enhanced the expressions of PARP, pATM, and γ-H2AX in HCT-8 cells, and only pATM in MCF-7 cells. The expression of γ-H2AX and pAMT is known to associate with both DNA damage and repair. In our study, the enhanced expression of γ-H2AX and pATM was more clearly found in cells received fractionated exposures. As these cells received fractionated exposures showed higher resistance to an additional radiation exposure to 2 Gy, the enhanced expression of γ-H2AX and pATM in cells received fractionated exposures should be closely associated with the increasing of DNA repair capacity rather than DNA damage. Therefore, fractionated exposures likely amplify radioresistance by increasing DNA repair capacity, especially the induction of pATM [21].
Radiosensitivity is known to be varied widely depending on the phase of cell cycle [22]. Cell cycle regulators have also well been demonstrated to contribute the acquired radioresistance after radiotherapy. Ki67 is highly expressed in cycling cells but strongly downregulated in resting G0 cells [23]. Cell proliferation always gets inhibited when cell cycle arrest happened. After the cell is exposed to ionizing radiation, the typical cell cycle is interrupted to allow enough time for DNA repair, or to prepare for cell death or senescence in the case of extreme or irreparable damage [24]. Cyclin D1 is known to be essential for cell progression from the G1 to S phase of cell cycle, and play important role in caner progression [25]. Cyclin D1 accumulation makes cell cycle progression uncontrolled [25,26]. It has been reported that cyclin D1 overexpression perturbs DNA replication and induces replication-associated DNA double-strand breaks in cells acquired radioresistance [27,28]. Targeting the cyclin D1/cyclin-dependent kinases 4 signaling pathway has been found to effectively eradicate tumor radioresistance followed by fractionated radiotherapy [15,28]. Cyclin E1 can regulate cell progression from G1 to S phase of the cell cycle [29]. The overexpression of cyclin E1 in cancer cells is well known, but there is not clear about the role of cyclin E1 in radioresistance. Very strangely, the expression of cyclin D1 and cyclin E1 was enhanced only in HCT-8 cells but poorly induced in the radiosensitive MCF-7 cells. Although cell cycle redistribution is the classic phenomenon of cell received fractionated irradiation, there has reported that cell cycle distribution seems to change marginally for the early days following radiation exposure [30]. As we puri ed the total protein from these irradiated cells for Western blot analysis 4 days after a single radiation to 5 Gy, but only 1 hr after the last exposure to 1 Gy in the fractionated exposures (Figure 2A), it is very di cult to interpret these ndings on the expression changes of cyclin D1 and cyclin E1 in this study.
Reactive oxygen species are a group of short-lived, highly reactive, oxygen-containing molecules that can induce DNA damage and affect the DNA damage response. As ionizing irradiation can produce reactive oxygen species to damage the cells, previous studies have demonstrated the relationship between the radioresistance and the expression of antioxidant enzymes in cancer cells [31,32]. Disagreed with previous studies, the expression of SOD1 and SOD2 was not signi cantly induced in irradiated HCT-8 cells and MCF-7 cells to 5 Gy X ray by either fractionated exposures or a single exposure. Actually, the expression of SOD2 was almost absent in MCF-7 cells. According to our data, redox regulation is poorly involved in the acquired radioresistance of HCT-8 cells and MCF-7 cells.
The reason on the different ndings among studies keeps unclear. The acquirement of radioresistance will be very complicated processes. It is still absence of consensus on the mechanism involving in radioresistance, especially the ampli cation of radioresistance by fractionated exposure. The redistribution state, as a phenomenon often observed in fractionated radiotherapy, has a decisive in uence on the prognosis of irradiated cells [33]. The dose and the interval between each fractionated exposure are also critical to the outcome of radiotherapy [34,35]. The same schedule of fractionated exposure for different cell lines may induce different prognosis. As the variation of intrinsic radioresistance and other biological properties among cells, it is di cult to optimize the best suitable dose and interval of fractionated exposures [36].

Conclusion
In summary, data from our in vitro experiments demonstrated that fractionated exposures ampli ed radioresistance of cancer cells, predominantly through the enhancement of DNA repair capacity rather than the alternations of cell cycle and redox regulation. However, in vivo experiments are highly required to con rm our ndings. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests:
The authors declare that they have no competing interests Funding: This study was mainly supported by Japan Agency for Medical Research and Development, a Grant-in-Aid from the Ministry of Education, Science, Sports, Culture and Technology, Japan (grant no. 17H04265). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

Authors' contributions:
T-S L is the creator, directing experimental design, data analysis and manuscript revision. K H performed the irradiation and Western Blot analysis and was a major contributor in writing the manuscript. M O partly performed clonogenic assay data and MTT assay. L A participated in experimental design and data curation. X Z collected the samples and data curation. All authors read and approved the nal manuscript.     Western blot analysis on the expression of Ki-67, cyclin D1, and cyclin E1. Representative blot (left images) and semi-quantitative data (right bar graphs) show the expression of Ki-67, cyclin D1, and cyclin E1 in cells received 5 Gy X-ray by fractionated exposures (1 Gy x 5) or a single exposure (5 Gy x 1). Intact cells without radiation exposure (0 Gy) were used for control. Data represents as mean ± SD from three independent experiments. *P < 0.05 vs. 0 Gy, †P < 0.05 vs. 1 Gy x 5.

Figure 5
Western blot analysis on the expression of SOD1, SOD2, and caspase 3. Representative blot (left images) and semi-quantitative data (right bar graphs) show the expression of SOD1, SOD2, and caspase-3 in cells received 5 Gy X-ray by fractionated exposures (1 Gy x 5) or a single exposure (5 Gy x 1). Intact cells without radiation exposure (0 Gy) were used for control. Data represents as mean ± SD from three independent experiments. *P < 0.05 vs. 0 Gy, †P < 0.05 vs. 1 Gy x 5.