Chinese-German J Clin Oncol

April 2013, Vol. 12, No. 4, P171–P174

DOI 10.1007/s10330-012-1134-2

Effect of low-dose X-ray radiation on adaptive response in gastric cancer cell Shukai Wang, Gang Jiang, Hongsheng Yu, Xiangping Liu, Chang Xu Department of Oncology, The Affiliated Hospital of Medical College Qingdao University, Qingdao 266000, China Received: 27 December 2012 / Revised: 28 February 2013 / Accepted: 25 March 2013 © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2013 Abstract Objective: We aimed to study the effect and mechanism of low-dose radiation (LDR) on adaptive response of gastric cancer cell. Methods: SGC7901 cells were cultured in vitro, and divided into 4 groups: control group (D0 group), low-dose radiation group (D1 group, 75 mGy), high-dose radiation group (D2 group, 2 Gy), low-dose plus high-dose radiation group (D1 + D2 group, 75 mGy + 2 Gy, the interval of low and high-dose radiation being 8 h). Cell inhibition rate was detected by cytometry and CCK8 method; the proportion of cell cycle at different times after irradiation was determined by using a flow cytometry. The ATM mRNA levels were detected by using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Results: There was no significant different between groups D0 and D1, groups D2 and D1 + D2 cell inhibition rate (P > 0.05). There was a significant increase G2/M arrest in groups D2 and D1 + D2 than groups D0 and D1 after 6 h of radiation and did not recover at 48 h (P < 0.05). The ATM mRNA expression of group D2 and D1 + D2 increased highly than that of group D0 and D1 (P < 0.05). However, differences between group D2 and D1 + D2, group D0 and D1 were not statistical significant (P > 0.05). Conclusion: LDR cannot induce adaptive response in SGC7901 cells in vitro, which may be associated the regulation of cell cycle, and its ATM mRNA expression cannot be affected by 75 mGy X-ray radiation. Key words

low-dose radiation; SGC7901 cells; adaptive response; cell cycle

According to the report of United Nations Scientific Committee on the Effects of Atomic Radiation in 1986, in terms of human radiation, low levels radiation refers to high-LET radiation within 50 mGy or low-LET radiation within 200 mGy. In the actual study, low-dose radiation (LDR) refers to the radiation dose complied with the above conditions and the dose rate that is higher than 0.05 mGy/min irradiation [1]. In recent years, a large number of experimental results show that the response of the cells to LDR and high-dose radiation is obviously distinct, mainly reflected in: adaptive response (AR), low-dose hyper-radio sensitivity, bystander effect. AR, defined as the induction of radio resistance to subsequent high doses of radiation by pretreatment with LDR, is a hot research at home and abroad at present. It has been confirmed by related experiments that AR of immune system and some other normal tissues [1–3] can be induced by LDR; however, nearly no relevant research in tumor cells and its mechanism is still not yet clear. Ataxia-telangiectasia (A-T) is caused by the mutation of ATM (A-T mutated) gene, which is located on chromosome 11q22-23. ATM is the family members of phosphatidylinositol-3-kinase (PI3K), and it mainly plays a Correspondence to: Hongsheng Yu. Email: [email protected]

role in cell cycle arrest, DNA damage identification and repair and apoptosis. It is generally agreed that the ATM gene has a close relationship with tumor genesis and radio sensitivity. In the present study, SGC7901 cells were used to detect its cell growth, cell cycling changes and ATM mRNA expression after different doses irradiation.

Materials and methods Cell cultures Human gastric SGC7901 cells, provided by the Central Laboratory of our hospital, were cultured in RPMI 1640 medium (supplemented with 10% FBS, 100 kU/L penicillin and 100 mg/L streptomycin sulfate) in a humidified atmosphere of 5% CO2 at 37 ℃. The cells adherent growth, were passage about once every three days by 1 : 3 ratio. Grouping and radiation conditions Varian 23EX accelerator. D1 group: radiation field was 15 cm × 15 cm, source skin distance was 100 cm, lead weight was put between the source and the culture bottles, and 1 cm tissue compensator was put under the bottles. By dose verification dose rate = 3.438 cGy/min. D1 group: radiation field was 15 cm × 15 cm, source skin distance was 100 cm, 1 cm tissue compensator was put

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Fig. 1

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Cells counting at different times after X-ray irradiation

under the bottles. Dose rate = 200 cGy/min. The cells of group D0 were treated similarly but were not irradiated. Cytometry and cells viability test After irradiation, 2 × 103 cells/well was seeded in a 96well microplate and 2 × 105 cells/well was inoculated in a 6-well microplate respectively. Each well was set 6 duplicate wells. Then the cells in microplates were cultured under the conditions as described previously. The cells in 6-well microplate were trypsinized and counted and the steps were repeated 6 days. CCK-8 solution was added per well in 96-well plates at a ratio of 1 : 10 for 1 h. Finally, absorbance at 540 nm was measured by using a microplate reader. Cells in each group counting and viability test continued for 6 days as described above. Cell proliferation inhibition rate (IR) was calculated as the following formula: IR = (ODControl group – ODBlank group) – (ODExperimental group – ODBlank group) / (ODControl group – ODBlank group). Cell cycle analysis The proportion of cell cycles-after 3, 6, 9, 12, 24 and 48 h radiation-was detected by using a flow cytometry and three samples were taken every time. The cells were harvested, fixed in 1ml of 70% cold ethanol at 4 ℃ overnight, centrifuged (1500 r/min, 5min), washed with PBS, filtered with 400 mesh nylon membrane, centrifuged again, added to 0.5 mL PI/RNase A solution. The mixed cells, after incubated in cell incubator for 30 min, were measured with flow cytometry. Detection of the ATM mRNA levels by RT-PCR The cells, after received irradiation up to 24 h, were washed with PBS. Total RNA was extracted using Trizol method and reverse-transcribed immediately. The ATM Table 1 Groups

mRNA expression levels were detected by RT-PCR. The primer sequences were designed by Primer Premier Software version 5.0 and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (China). The primers used for PCR amplification were as follows: ATM (forward primer: 5’-ACTGGCCTTAG CAAATGC-3’, reverse primer: 5’-TTGCAGCCTCTGTTC GAT-3’, amplified fragments: 128 bp); GAPDH (forward primer: 5’-TCACTGCCACCCAGAAGACT’, reverse primer: 5’-TTCTAGACGGCAGGTCAGGT-3’, amplified fragments: 128 bp). The cycling was performed at 95 ℃ for 30 s, the PCR reaction at 95 ℃ for 5 s, followed by 40 cycles of 60 ℃ for 20 s and 80 ℃ for 30 s to acquire fluorescent signal. Each dilution should be run in triplicate. The △△ct method [4] was used to calculate ATM gene expression levels. Statistical analysis The results were presented as means ± SD and analyzed with mono-agent analysis of variance of SPSS 17.0 statistical package. Data were analyzed by using Q-test between two groups and analysis of variance among multiple comparisons. Significance was considered at P < 0.05.

Results Effects of LDR on the growth of SGC7901 cells in vitro There was no significant difference between group D0 and group D1 for the cells counting and IR (P > 0.05). As compared with that in D1 group, the cells counting and IR were significantly different in the D2 and D1+D2 group (P < 0.05; Fig. 1 and Table 1). Although the cells IR decreased in D1 + D2 group compared with that in D2 group, the difference was no significant (P > 0.05). Cell cycle assay After SGC7901 cells were irradiated with X-rays at the dose of 75 mGy, the cell cycle almost had no changes as compared with D0 group (P > 0.05). Compared to D0 and D1 group, the percentage of G2 cells both in D2 and D1 + D2 group decreased significantly since 6 h after radiation (P < 0.05), and the difference still existed at 48 h after radiation. In comparison with D2 group, D1 + D2 group showed no statistical significance (P > 0.05; Fig. 2).

IR after irradiation at different times (n = 6, χ ± s, %) D1

D1 0.658 ± 0.06 D2 0.682 ± 0.05 D1 + D2 0.716 ± 0.02 Δ P﹤0.05 as compared with D1 group

D2

D3

D4

D5

D6

0.236 ± 0.07 2.236 ± 0.09Δ 2.107 ± 0.08Δ

0.358 ± 0.03 4.653 ± 0.14Δ 4.521 ± 0.17Δ

0.137 ± 0.08 15.264 ± 0.19Δ 14.984 ± 0.27Δ

0.416 ± 0.06 18.205 ± 0.28Δ 19.917 ± 0.31Δ

0.154 ± 0.04 18.716 ± 0.12Δ 18.493 ± 0.23Δ

Chinese-German J Clin Oncol, April 2013, Vol. 12, No. 4

Fig. 2 phase

Cell cycle distributions. (a) G1/G0 phase; (b) S phase; (c) G2/M

Expression of ATM Compared to D0 group, the ATM mRNA levels in D1, D2 and D1 + D2 group were 0.99, 1.93 and 1.85 times respectively. There was no significant statistic difference between D1 and D0 groups, D2 and D1 + D2 group (P > 0.05). Compared to D0 group, ATM in D2 and D1 + D2 group significantly increased (P < 0.05).

Discussion The biological effect of LDR is a hot topic in radiation biology research in recent years. Studies found that LDR could induce AR on normal cells, improve immunity and enhance radiation tolerance of normal tissues. However, it is unclear whether such stimulating effects induced by LDR can also occur in tumor cells. Most studies [5–8] showed that LDR could not induce AR on tumor cells, but it has been found that LDR can make HepG2

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and HeLa cells grow up faster [9]. CCK-8 is better than MTT for analyzing cell proliferation, because it can be reduced to soluble formosan by dehydrogenates, therefore, it can reduce the error. By using cells counting and CCK-8 method to detect cells growth after different doses irradiation, we found that 75 mGy X-ray radiation could not induce proliferation of SGC7901 cells. It meant that LDR could not induce adaptive response of SGC7901 cells cultured in vitro. In recent years extensive research about the mechanism of AR could be roughly divided into the following 4 aspects: regulatory mechanism of cell cycle, DNA damage repair mechanism, anti-oxidative injury mechanism and stress proteins, and the cell cycle regulation is an important factor in determining the radiation resistance of cells. Eukaryotic cells have three cell-cycle checkpoint functions: G1 phase checkpoint can block damaged DNA into S phase, S phase checkpoint mainly mediates injuries in the reproduction phase and other endogenous damage, and the G2 checkpoint mainly prevents the damaged DNA without repair cells directly into the M phase [10]. The ATM, a DNA-damage sensor and receptor of cell cycle checkpoints, would activate G1/S checkpoint by activating p53 and p21 genes, and activate S and G2/M checkpoints by activating Chkl, Chk2, Cdc25 and Cdc2 genes when radiation-induced DNA double-strand breaks. Therefore, the damaged DNA would be blocked in specific checkpoints of cell cycle and be repaired [11, 12]. Now that G1 checkpoint activation and maintenance was dependent on p53 gene, while related studies showed that p53 gene was mutated in more than 50% of the malignant tumors, most of the tumor cells could not be repaired in G1. Therefore, the ATM was critical in activating G2 checkpoint as the damaged cells passed through G1 and S [13]. Our study showed that the proportion of cell cycle and ATM mRNA levels were no significant difference between D1 and D0 groups, while there was a significant increase in G2 arrest at 6 h post irradiation and significantly higher ATM expression levels in D2 and D1 + D2 groups. The results, as described above, were consistent with the study of Tao [14]. The distribution of cell cycle and expression levels of the ATM in D2 group were no significant difference compared to D1 + D2 group. Based on this study, we can conclude that LDR could not cause the cell cycle changes of SGC7901 cell lines, nor could change the cell cycle of cells subsequently exposed to high-dose radiation, which may be associated with ATMmediated cell cycle regulation. In the previous study, we found that LDR caused a G1-phase arrest of S180 sarcoma cells [5]; however, this phenomenon does not appear in this experiment. We speculate that it may be concerned with the mutation of p53 gene of SGC7901 cells. In summary, based on this and our previous studies, we may conclude that LDR most likely induces AR in normal

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cells but may not induce the same effect in tumor cells. This finding may have a great potential for clinical application of LDR, but detail mechanisms of AR remain to be further investigated.

References 1. Hei TK, Zhou H, Chai Y, et al. Radiation induced non-targeted response: mechanism and potential clinical implications. Curr Mol Pharmacol, 2011, 4: 96–105. 2. Bogdandi EN, Balogh A, Felgyinszki N, et al. Effects of low-dose radiation on the immune system of mice after total-body irradiation. Radiat Res, 2010, 174: 480–489. 3. Yu HS, Liu N, Ju B. Low dose radiation induced adaptive response of apoptosis in mouse spleen cells. Chinese-German J Clin Oncol, 2010, 9: 235–238. 4. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 2001, 25: 402–408． 5. Yu HS, Sun WH, Liu N. The effect of low-dose total body irradiation on tumor-inhibition and signal transduction in tumor tissues of mice bearing S180 sarcoma. Chinese-German J Clin Oncol, 2011, 10: 602–605. 6. Savickiene J, Treigyte G, Aleksandraviciene C, et al. Low-dose ionizing radiation effects on differentiation of HL-6o cells. Cent Eur J Biol, 2010, 5: 600–612.

7. Jiang HY, Li W, Li XY, et al. Low-dose radiation induces adaptive response in normal cells, but not in tumor cells: in vitro and in vivo studies. Radiat Res, 2008, 49: 219–230. 8. Sun WH, Yu HS, Shang QJ. The effect of pre-low-dose X-ray radiation on tumor inhibition of HepG2 cells in tumor-bearing nude mice. Chinese-German J Clin Oncol, 2012, 11: 340–343. 9. Xia JG, Li WJ, Wang JF, et al. Effects of Low Priming Dose of Radiation on Cell Cycle Arrest of Tumor Cells Caused by High Dose of Radiation. HEP & NP (Chinese), 2005, 29: 201–204. 10. Williams RS, Williams JS, Tainer JA. Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem Cell Biol, 2007, 85: 509–520. 11. Langerak P, Russell P. Regulatory networks integrating cell cycle control with DNA damage checkpoints and double-strand break repair. Philos Trans R Soc Lond B Biol Sci, 2011, 366: 3562–3571. 12. Mchinnon PJ. ATM and ataxia telangiectasia. EMBO Rep, 2004, 5: 772–776． 13. Mitchell J, Smith GC, Curtin NJ. Poly(ADP-Ribose) polymerase-1 and DNA-dependent protein kinase have equivalent roles in double strand break repair following ionizing radiation. Int J Radiat Oncol Biol Phys, 2009, 75: 1520–1527. 14. Tao D, Cheng J, Wu G, et al. Low dose hyper-radiosensitivity in human lung cancer cell line A549 and its possible mechanisms. Chin J Radiol Med Prot (Chinese), 2009, 29: 147–151.

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