|Year : 2016 | Volume
| Issue : 1 | Page : 25-28
Evaluation of gamma radiation-induced cytotoxicity of breast cancer cells: Is there a time-dependent dose with high efficiency?
M Fazel1, P Mehnati1, B Baradaran2, J Pirayesh1
1 Department of Medical Physics, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
2 Researches Center of Immunology, Tabriz University of Medical Sciences, Tabriz, Iran
|Date of Web Publication||28-Apr-2016|
Department of Medical Physics, School of Medicine, Tabriz University of Medical Sciences, Tabriz
Source of Support: Tabriz University of Medical Sciences., Conflict of Interest: None
Context: Radiotherapy is one of the important treatment modalities in the management of breast cancer. Aims: The aim of this study is to study the efficient treatment of breast cancer as related to the dose delivery. Materials and Methods: The human breast cancer cell lines (MCF-7) cells were cultured and exposed by 1, 2, 4, 6, 8, 10, and 20 Gy of γ-rays. Radiation-induced cell death was detected and evaluated, using three assay methods: Cell viability, clonogenic cell survival assay and induction of apoptosis. The cell viability was determined using trypan blue staining, 24 and 72 h post-irradiation. The survival fraction (SF) was determined by colony counting, 14 days after exposure and the apoptotic cell death was determined using the TUNEL assay. Statistical Analysis Used: One- or two-way analysis of variance was deemed as appropriate, followed by relevant post t-test to determine P values. Results: The difference of MCF-7 cell death through increasing post-radiation time from 24 to 72 h following the dose of 1, 6 and 10 Gy was found to be 2%, 9.6% and 7.14%, respectively. D0of MCF-7 was 220 cGy and the SF in the cells irradiated by 1 Gy and 10 Gy doses were 0.8 and 0.0001, respectively. The estimated variances were 2%, 11.1% and 8.4%, between 24 h and 72 h post-radiation apoptosis death for 1, 6, and 10 Gy, respectively. Conclusions: The dose and time dependence inducing apoptotic death was significant (P = 0.001). The delayed mortality and apoptosis was observed in MCF-7 cell, but the variance of total cell death and apoptosis in 24 and 72 h post-radiation with 6 Gy was obviously more than that with other doses.
Keywords: Apoptosis, dose, MCF-7 cell
|How to cite this article:|
Fazel M, Mehnati P, Baradaran B, Pirayesh J. Evaluation of gamma radiation-induced cytotoxicity of breast cancer cells: Is there a time-dependent dose with high efficiency?. Indian J Cancer 2016;53:25-8
|How to cite this URL:|
Fazel M, Mehnati P, Baradaran B, Pirayesh J. Evaluation of gamma radiation-induced cytotoxicity of breast cancer cells: Is there a time-dependent dose with high efficiency?. Indian J Cancer [serial online] 2016 [cited 2019 Aug 23];53:25-8. Available from: http://www.indianjcancer.com/text.asp?2016/53/1/25/180862
| » Introduction|| |
Currently, breast cancer, as a solid neoplasm, is the most frequent cancer type among women. Ionizing radiation (IR) causes damage and cell death, especially delayed cell death of breast cancer. Radiation therapy has been used as a method of inducing cell death and preventing the spreading of cancerous cells for quite a long time. Evaluation of breast cell response to IR is used to determine the clinical efficacy of radiation treatment., The Oxford overview data of radiation treatment-associated mortality indicated that the 17-30% of patients who are destined to die in the absence of radiotherapy will live as a result of radiation treatment preventing the systemic dissemination. The amount and type of cell death from IR in various cell lines are different. Breast cancer is a solid tumor introduced as a malignancy resistant to radiation. Breast cancer cells MCF-7 is classified as the most resistant breast cancer epithelial cell lines. In different studies, irradiation of MCF-7 cells with various doses has led to different results. For example, some studies have concluded that MCF-7 against high doses of radiation is less susceptible., Wang et al. exposed MCF-7 cells to 1 Gy X-rays and found a significant time-dependent decrease of cell viability compared with non-irradiated cells. Similar results were obtained with 4 Gy X-rays irradiation, but the rate of the cell viability decrease was slower than that of 1 Gy X-rays. Other studies showed that increasing of dose rate will increase radiation-induced apoptotic cell death. The mortality rate of apoptosis in MCF-7 cancer cells was time-dependent. A study showed that apoptosis observed over time will increase. Some studies have suggested that these differences may occur due to the differences in the type of cell death in irradiated MCF-7 cells, so the most common death observed in the MCF-7 is necrosis or mitotic death and isn't apoptosis.,,, There are different methods for evaluating radiation-induced cell death, but the level of clonogenic ability of the cell is a Gold standard., Various factors such as cell characteristics, type of stimulation and molecular composition are involved to create all types of cell death by radiation like apoptosis., This paper first gives a brief overview of the MCF-7 cell death exposed to various radiation doses and finally compares in detail, the dependence of cell death especially apoptosis death to radiation dose and the effect of time.
| » Materials and Methods|| |
Cell line and culture conditions
The human breast cancer cell lines MCF-7, originally obtained from the Pasteur Institute of Iran (Tehran), were grown as monolayer in 80 cm 2 flasks in Roswell park memorial institute 1640 medium (RPA, Australia) supplemented with 10% heat-inactivated fetal calf serum and an antibiotic formulation (100 units/ml penicillin and 100 μg/ml streptomycin), (RPA, Australia). MCF-7 is an estrogen and progesterone receptors positive and human epidermal growth factor receptor-2/neu protein over expression negative.,,
Cells in exponential growth condition were treated by single radiation with 1, 2, 4, 6, 8, 10 and 20 Gy doses of γ-rays of 60 Co by a radiotherapy unit (Theratron-1000, Canada) at a dose rate of 1.5 Gy/min. Control cells were in the same condition of treatment with radiation cells.
Determination of cell doubling time
MCF-7 cells (1.5 × 104/ml) were seeded and cultured in a 6 well plate for 12, 24, 29, 33, 36, and 48 h. Then, the cells were trypsinized and centrifuged and diluted with 1 ml culture medium and were counted in the hemocytometer under the light microscope (×40).
Cells were plated in triplicate in 6-cm dishes with densities varying from 300 to 1800 including control and classified cells depending on the radiation dose treatment. The cells were then cultured in a 37°C, 5% CO2 incubator for 14 days to allow forming colony. The colonies (> cells/colony) were fixed and stained with giemsa staining (code: 912309, ARJ) for 10 min and counted under the light microscope and survival fraction (SF) of MCF-7 cells was calculated.
Determination of cellular viability
Trypan blue exclusion assay was used to determine cell viability after IR. For these assays, MCF7 cells (1 × 104/ml) were plated in 6-well plates. 24 and 72 h after radiation treatment with 0, 1, 2, 4, 6, 8 and 10 Gy doses, cells floating in the supernatant were collected and pooled with adherent cells recovered from the plates by trypsinization. Total cell number and stained cells were counted in the hemocytometer and the percent of dead cells was calculated.
Death % = Dead cells/total cell × 100.
Apoptosis cell detection
Number of 105 cells per chamber slid well were cultured and treated with radiation doses of 0, 1, 2, 4, 6, 8 and 10 Gy. Study of apoptosis was performed 24 and 72 h after the exposure. Briefly, we washed slides twice with phosphate buffered saline (PBS) then added 0.5 ml fixing solution (4% paraformaldehyde in PBS, freshly prepared) and incubated at room temperature for 1 h. After removing fixing solution and washing the slides, 0.5 ml blocked solution was added (1 ml H2O2100% in 9 ml methanol) and incubated at the room temperature for 10-15 min. After removing and washing slides, permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) was added and incubated for 2 min on ice; the slides were washed twice with PBS. Then 50 ml TUNEL reaction mixture was added and placed in the dark at 37°C for 1 h in a humidified incubator and the slides were washed twice. Finally, the cells were exposed to peroxidase reaction and stained with 0.3 ml 3,3'-diaminobenzidine (one drop of each A, B and C solution), (code: Ltd., boster). Under light microscope, seven fields in different places of the chamber were selected and saved as images and soft web analysis was performed. Apoptotic cells were observed in brown color and counted in selected area; others were non-apoptotic cells that were counted for calculation.
Statistical evaluations of cell viability, survival and percentage apoptosis was carried out on experimental data of three experiments carried out in triplicates. Data are represented as mean ± standard deviation from the mean and compared by one- or two-way analysis of variance as appropriate, followed by relevant post t-test to determine the P values. A P <0.05 was considered to be significant.
| » Results|| |
Cell doubling time
Doubling time of MCF-7 cells with high efficiency (>%) viable cells was determined 28 ± 2.31 h.
MCF-7 cells colony forming after irradiation
The mean of plating efficiency for MCF-7 cells was obtained 93%. The SFs of the cells after 14 days with 0, 1, 2, 4, 6, 8 and 10 Gy were 1, 0.8, 0.69, 0.29, 0.04, 0.006 and 0.0001, respectively and for each dose cell death % is shown in [Table 1]. The survival curve of MCF-7 cells showed a large initial shoulder (n = 5.5) with the D0 equaling 220 cGy, which indicates that there have to be roughly four (n-1) damages to result in lethal effect. The doses required to reduce survival to 0.1 and 0.01 were 500 and 720 cGy, respectively [Figure 1].
|Table 1: The percentage of MCF-7 cell death; studied by colony forming assay. SD means±SD of surviving fraction|
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|Figure 1: Survival curve for MCF-7 cells after exposure to gamma radiation at doses of 1, 2, 4, 6, 8 and 10 Gy|
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MCF-7 cell death
Low and high percent of radiation induced death were obtained, for 1 Gy (24 h) and 10 Gy (72 h) irradiation; 11.61% and 69.83% respectively [Table 2]. The results indicated that the MCF-7 cells mortality by different doses and increasing of post-radiation times showed a statistically significant difference (P < 0.001).
|Table 2: Percentage of MCF-7 cell death post-exposure to gamma rays by different doses after 24 and 72 h post-radiation|
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Apoptosis death in MCF-7 cells
The results of studying the percentage of apoptotic cells following the 24 h post-irradiation with 1, 2, 4, 6, 8 and 10 Gy doses were 7.27%, 18.18%, 21.79%, 25.11%, 32.69% and 38.11%, respectively. And the same doses as 72 h post-radiation yielded the following results: 9.28%, 24.10%, 29.31%, 36.22%, 38.88% and 46.55%, respectively. Apoptosis death for 1 Gy (24 h) and 10 Gy (72 h) doses of radiation were as a low and high percent of radiation induced apoptotic doses. The elapsed time post-irradiation affects the apoptosis ratio significantly (P = 0.001). Furthermore, the effect of dose level in apoptosis induction ratio was statistically significant (P < 0.001).
When we used high dose, 20 Gy, the percent of apoptosis at 24 and 72 h after treatment increased to 58.82% and 72.82%, respectively. Control cells showed 4.8% and 5% apoptosis death at 24 and 72 h observing times. Apoptotic cells were observed in brown color and counted in selected area; others were non-apoptotic cells counted for calculation [Figure 2].
|Figure 2: Photos of apoptotic cells obtained under light microscopic × 20 magnifications from control and irradiated MCF-7 cells at 24 and 72 h post-radiation. Arrows refer to apoptosis death cells|
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The effect of time interval post-radiation on the cell death
The study of cell total death and apoptosis showed that the relation between dose and time variance post-radiation from 24 to 72 h is not linear. Delayed cell death in MCF-7 cell after radiation for each dose was observed. Furthermore, the interval difference between 24 h and 72 h post-radiation on the MCF-7 cell death by 1, 6 and 10 Gy were 2%, 9.6% and 7.14%, respectively. Delayed Apoptosis in MCF-7 cell after radiation for each dose was observed. Apoptotic death by doses 1, 6 and 10 Gy were 2%, 11.1% and 8.4%, respectively and was observed in interval studies between 24 h and 72 h post-irradiation [Figure 3].
|Figure 3: Difference of MCF-7 cell death by increasing post-radiation time from 24 to 72 h in different doses of γ-ray|
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| » Discussion|| |
The present study confirms that irradiation-induced cell death has different amounts in MCF-7 cells by dose and interval time. Despite the delayed total cell death and apoptosis, the difference of MCF-7 cell death was %9.6 and %11.1 in 24 h and 72 h post-radiation with a dose of 6 Gy, respectively. It is worth mentioning that the viability assay referred to the response of individual cells to IR, but colony forming assay shows the ability of the cells to proliferate and to form colonies. In a similar study, a radiation dose of 5 Gy reduced the viability of cells by 14% by 48 h post-radiation. In our study, the reduction of viability happened rapidly after 24 and 72 h in 4 and 6 Gy in comparison and also the viability of MCF-7 cells decreased with time and was dose dependent (P = 0.001). We measured the doubling time of MCF-7 precisely because according to Bergonie and Tribondeau law (1906), rapidly dividing cells have a more significant cell killing capacity as opposed to less effective in slowly growing cells such as MCF-7 breast cancer cell line. These phenomena have described and compared in chemotherapy or used in chemo sensitizer drugs, but have rarely been explained in radiotherapy.
Percentage of MCF-7 proliferation death by colony forming assay in different studies has been presented by different results. We concluded from Coco Martin et al. that the percentage of proliferation death by 2, 4 and 6 Gy were 55%, 93% and 99.4%, respectively, 10 days following incubation. In another study, with the same doses, proliferation deaths were 75%, 94% and 99.5% 5 days after incubation. According to these results, it is concluded that the delayed death and increased death percentage occurred when the time of incubation increased. In another research, percentage of cell death following 1, 2 and 3 Gy were 10%, 34% and 60%, respectively 12 days after incubation, which confirmed the importance of doubling time determination before treatment. Perhaps, increasing incubation time for colony formation studies is necessary for getting brief results in MCF-7 cells, due to long dividing time. In the present research, a delay of 14 days was selected and we determined percentage of proliferation cell death in MCF-7 cells for different doses higher than viability and apoptosis results. Thus, the results of counted colonies could show the main effect of radiation by increasing doses and it shows their inability for colony forming in MCF-7 cells and their tendency to proliferation death. Kumar et al. showed that apoptosis in the radiation-treated groups with 2 and 5 Gy radiation was 6% and 10%, respectively. They showed that radiation-induced apoptosis is time and dose dependent. In another study, apoptosis increase in MCF-7 cells with 5 Gy in 24 and 50 h post-radiation were about 18% and 28%, respectively. The two researches mentioned above showed a lower cell death percentage than that observed in our study. In our study, the apoptosis percentage, 24 h and 72 h post-radiation by different doses showed a dose and time dependent relation (P = 0.001). Furthermore, for a higher dose (20 Gy) our results showed time and dose dependency but Jänicke et al. showed that apoptosis death is not dose dependent. Their study by MCF-7 cells showed that, although the IR-induced deoxyribonucleic acid double-strand breaks did not induce the high intrinsic death pathway in MCF-7 cell the possibility remained that these cells die through the necrotic pathway. Wang et al. showed that the exposure of MCF-7 cells to 1 Gy X-rays resulted in a significant time-dependent decrease of cell viability compared with non-irradiated cells. Similar results were obtained with 4 Gy X-rays irradiation, but the rate of this decrease was slower than that of 1 Gy X-rays. Moreover, this cell growth inhibition was observed at 1 h post-irradiation for 1 Gy, but at 8 h post-irradiation for 4 Gy. The studies showed that the breast cancer cells have different sensitivity to cytotoxic material, for example, SKBR-3 cell is more sensitive and BT-474 cell is more resistant than MCF-7 to apoptosis death because of different BCL-2 level.,
Our study showed that the exposure of MCF-7 cells to γ-rays from 1 to 10 Gy resulted in a significant time-dependent decrease of cell viability compared with non-irradiated cells. Results indicated delayed cell death and apoptosis after exposure to all radiation doses. The effect of interval time after irradiation and cell survival showed that there is a time-dependent response for the apoptosis frequency. The difference between 6 Gy, 8 and 10 Gy is related to cell analysis time after irradiation at 24 and 72 h intervals. [Figure 3] shows mortality and apoptosis in MCF-7 cells with 8 and 10 Gy γ-rays were slower than 6 Gy after 72 h. The results obtained in the studies of others in the articles referenced as 4, 9 by 1 and 4 Gy were controversial and these controversies served as the motivation for our study.
Post-radiation time efficiency on cell death showed variation of cell death in different time 24 h and 72 h with 6 Gy. It was obviously more than other doses for total and apoptosis cell death of %9.6 and %11.1, respectively, but this variance in 10 Gy was less for total and apoptosis cell death of %7.14 and %8.4, respectively.
Although, other researchers used high or low doses of radiation, the present study evaluated the amount of cell death by different doses and the post-irradiation time and has compared the results for cell death. In conclusion, dose and time dependent induced apoptotic death was significant for MCF-7 cells (P = 0.001). The percentage of mortality and apoptosis observed in MCF-7 cell was moderate, but the variation of total cell death and apoptosis in 24 h and 72 h post-radiation with 6 Gy was obviously more than that with other doses.
| » Acknowledgment|| |
The authors would like to thank to laboratory members of researches center of immunology and radiobiology laboratory members of medical physics.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]