The different biological effects of single, fractionated and continuous low dose rate irradiation on CL187 colorectal cancer cells
© Wang et al.; licensee BioMed Central Ltd. 2013
Received: 28 March 2013
Accepted: 7 August 2013
Published: 9 August 2013
To determine the biological effectiveness of single, fractionated and continuous low dose rate irradiation on the human colorectal cancer cell line CL187 in vitro and explore the cellular mechanisms.
Materials and methods
The CL187 cells were exposed to radiation of 6 MV X-ray at a high dose rate of 4Gy/min and 125I seed at a low dose rate of 2.77 cGy/h. Three groups were employed: single dose radiation group (SDR), fractionated dose radiation group (FDR) by 2Gy/f and continuous low dose rate radiation group (CLDR). Four radiation doses 2, 4, 6 and 8Gy were chosen and cells without irradiation as the control. The responses of CL187 cells to distinct modes of radiation were evaluated by the colony-forming assay, cell cycle progression as well as apoptosis analysis. In addition, we detected the expression patterns of DNA-PKcs, Ku70 and Ku80 by Western blotting.
The relative biological effect for 125I seeds compared with 6 MV X-ray was 1.42. 48 hrs after 4Gy irradiation, the difference between proportions of cells at G2/M phase of SDR and CLDR groups were statistically significant (p = 0.026), so as the FDR and CLDR groups (p = 0.005). 48 hrs after 4Gy irradiation, the early apoptotic rate of CLDR group was remarkably higher than SDR and FDR groups (CLDR vs. SDR, p = 0.001; CLDR vs. FDR, p = 0.02), whereas the late apoptotic rate of CLDR group increased significantly compared with SDR and FDR group (CLDR vs. SDR, p = 0.004; CLDR vs. FDR, p = 0.007). Moreover, DNA-PKcs and Ku70 expression levels in CLDR-treated cells decreased compared with SDR and FDR groups.
Compared with the X-ray high dose rate irradiation, 125I seeds CLDR showed more effective induction of cell apoptosis and G2/M cell cycle arrest. Furthermore, 125I seeds CLDR could impair the DNA repair capability by down-regulating DNA-PKcs and Ku70 expression.
Colorectal cancer is one of the leading causes of death in the world, and more than 170,000 new patients are diagnosed in China each year. In general, rectal cancer has a relatively higher risk of recurrence. Once the rectal cancer recurred, regular treating strategies such as operation, chemotherapy and external beam radiotherapy failed to achieve appreciative outcomes [1, 2]. For those patients who could not receive an operation and re-irradiation, there were few strategies to control the disease progression, and their median survival time was between 3 and 6 months [2, 3]. Locally recurrent rectal cancer (LRRC) remained a challenging problem in clinics, due to poor survival and severe symptoms such as pain and bleeding. Image guided permanent implantation of radioactive 125I seeds into the tumor site shows major advantages of delivering a high dose of irradiation to the tumor with a very sharp fall-off outside the implanted volume. In this regard, 125I seed permanent implantation has been recommended by the National Comprehensive Cancer Network to treat patients with low and intermediate risk prostate cancer [4, 5]. 125I seed permanent implantation was also employed to treat pancreatic cancer and head and neck neoplasm because of its unique properties of locally limited irradiation [6, 7]. Several clinical studies confirmed that 125I seed implantation was feasible, effective and safe as a salvage or palliative treatment for those patients suffering recurrent and re-recurrent rectal cancer with minimally invasion [8, 9]. Although there are several reports on the biological effects of continuous low dose rate radiation, the different effectiveness of variant dose rate irradiation on the human colorectal cancer cell line and the relevant cellular mechanisms are still largely unidentified. The aim of the present study is to determine the direct biological effectiveness of single dose radiation (SDR), fractionated dose radiation (FDR) and continuous low dose rate radiation (CLDR) on human colorectal cancer cell line CL187 cultured in vitro, and we also aimed to unravel the underlying cellular and molecular mechanisms behind distinct modes of irradiation.
Materials and methods
RPMI 1640 media for cell culture was purchased from Gibco (Life Technologies Corporation, NY, USA). Propidium Iodide (PI) was purchased from Cell Signaling Company (Cell Signaling Technology, Beverly, MA, USA). Anti-DNA-PKcs, anti-Ku80 and anti-Ku70 antibodies were obtained from Cell Signaling Technology, Inc (Cell Signaling Technology, MA, USA).
Cell lines and cell culture
The human colorectal cancer cell line, CL187, was kindly gifted by the Beijing Institute for Cancer Research . In brief, CL187 cells are maintained in RPMI 1640 supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin, 4 mM glutamine, and 10% heat-inactivated fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Company, China) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The medium was replaced every two or three days.
125I seeds and X-ray irradiation
Cell death assay
Exponentially-growing CL187 cells was left untreated or exposed to 125I seeds and X-ray at the absorbed doses of 2, 4, 6 and 8 Gy, respectively. After irradiation, cells were cultured for another 24 or 48 hrs, followed by cell harvest and stained with trypan blue. The percentage of dead cells was counted for each delivered dose as: number of dead cells/total number of cells collected × 100%. The mean was calculated from three parallel samples for each dose point.
Clonogenic survival assay
Briefly, exponentially growing cells were exposed to different dosages (0, 2, 4, 6 and 8 Gy) with SDR, FDR or CLDR. After further culture for 14 days, colonies were fixed with methanol and stained with Giemsa. Colonies with more than 50 cells were manually counted. The plating efficiency (PE) and survival fraction (SF) were calculated as follows: PE = (colony number/inoculating cell number) × 100%. SF = PE (tested group)/PE (0-Gy group) × 100%. The SF2 of different groups were used to calculate the relative biological effect (RBE). RBE = SF2(CLDR)/ SF2(SDR/FDR).
Cell cycle and apoptosis analysis
Cells were harvested 24 and 48 hrs after SDR, FDR or CLDR. Cells were counted and washed twice with cold PBS. For the preparation of the cells, an apoptosis assay by Beyotime Biotech, Jiangsu, China, based on AnnexinV and PI staining was used. The cells were prepared according to the manufactures instructions, and the stained cells were manufactured with Beckman Coulter Epics XL as soon as possible. Cells used for cell-cycle analysis were fixed with cold 70% ethanol and stained with PI after RNase treatment and analyzed by fluorescence-activated cell sorting (FACS) using BD Calibur and ModFit software (Verity Software House, Topsham, MN). Three independent experiments were conducted for each group.
Immunoblotting and antibodies
Proteins were extracted from cells of different groups 30 minutes and 6 hours after radiation , cells without radiation served as the control. The protein concetration was determined by Bicinchoninic Acid (BCA) protein assay (Beyotime Biotech, China). Cell lysates containing 20 μg of protein were resolved on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Hybond-P, Amersham, Buckinghamshire, UK). The membranes were blocked with TBS containing 0.1% Tween-20 (TBST) containing 5% non-fat milk for 2 hrs at room temperature and then incubated overnight at 4°C with the primary antibodies. Membranes were washed three times with TBST, and then incubated with second antibody for 2 hrs at room temperature. After washing with TBST, proteins were detected by enhanced chemiluminescence (ECL) detection kit (Pierce Biotechnology). Antibodies for immunoblotting in this study were as follows: anti-DNA-PK, anti-Ku80, anti-Ku70 (Cell Signaling Biotechnology, Beverly, MA, USA), anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-rabbit IgG-HRP and anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).
The statistical analysis was performed with SPSS (version 13.0; SPSS Inc., Chicago, IL). Measurement data are presented as the mean ± standard deviation (x ± SD). The difference between the means of different groups was analyzed with a pair-designed t-test, a value of p < 0.05 was considered significant.
CLDR inhibited survival of CL187 cells more efficiently than SDR and FDR
Clonogenic survival of CL187 cells treated with different fraction model of irradiation
CLDR-treated CL187 cells displayed severe G2/M arrest
CLDR leads to increased apoptosis of CL187 cells
Impaired DNA repair capacity of CL187 cells upon CLDR
Management of local–regional recurrence of rectal cancer after primary radical resection and post-operative radiotherapy remains challenging. There are few effective options for those patients, and the prognosis remains poor. Majority of patients could not receive re-operation, and some of them even could not receive re-irradiation. They are suffering unbearable symptoms such as continuous pain and bleeding, and their survival time decrease dramatically. 125I radioactive seed interstitial implantation provides an alternative choice for those patients. The clinical results are inspiring, but the underlying mechanisms are not quite clear [17, 18]. Whether there are different radiobiological mechanisms between low and high dose rate irradiation is still undefined.
In the present study, we first investigated the killing effects of SDR, FDR and CLDR on CL187 cells in vitro. Compared with SDR and FDR, CLDR caused severe total cell number decrease, due to enhanced cell death. The reported RBE of 125I seed continuous low dose rate irradiation of different models and cell lines were among 1.2 and 2.1, and most of which were ranged from 1.3 to 1.5 [11, 12, 19–21]. Lehnert et al showed that 125I irradiation caused increased RIF-1 cell death, and the RBE for 125I compared with 60Co gamma rays was 1.5 . Wang et al reported that cell apoptosis of PANC-1 cell line increased after CLDR, with RBE for 125I compared with 60Co gamma rays being 1.39 . Liao et al demonstrated that CLDR resulted in increased PC-3 cell apoptosis, with RBE for 125I compared with 60Co gamma rays being 1.41 . In the present study, we have shown that the RBE for 125I compared with 6 MV X- rays was 1.42, which is consistent with the previous data reported by our colleagues and others.
Impairment of cell cycle checkpoint control and disrupted balance of proliferation and apoptosis are very important attributes leading to cancer development and progression. G2/M arrest is one of the major characteristics of cancer cells after irradiation, which forces to slow down cell-cycle progression to repair DNA damage, otherwise damaged cell will be programmed to undergo apoptosis. Previous studies have suggested that CLDR mediated cancer inhibition mainly via induction of cell cycle arrest and apoptosis [22–25]. We found that CLDR triggered more G2/M phase arrest of CL187 cells and also induced increased level of early and late apoptosis.
The DNA damages caused by irradiation are positively correlated to the G2/M phase arrest. In this regard, we next decided to determine the expression levels of several important regulators of DNA damage. DNA DSBs caused by irradiation tend to impair the integrity of the genome and cause cell death, and agents regulating DNA repair progress were tend to affect radiosensitivity of CLDR [17, 18]. We have known that the Ku70 and Ku80 heterodimer bind to free DNA ends at the DSB and thus recruit DNA-PKcs, to activate its kinase activity [26–28]. It has been well known that functional defects or absence of Ku70, Ku80 or DNA-PKcs subunit result in remarked deficiencies in DNA-DSB repair and thus are hypersensitive to ionizing radiation. Korabiowska et al found that loss of Ku70 and Ku80 expression occurs frequently in hereditary and sporadic colorectal tumors . Mazzarelli et al demonstrated that Ku70 and Ku80 heterodimer expression associated with DNA end binding activity were significantly increased in human colorectal tumor tissue than normal tissue . After studying 101 colorectal cancer patients, Komuro et al found that 52 patients had elevated Ku70 expression, which were related with depth of tumor invasion and prognosis of survival in the multivariate analysis . It has been shown that the capacity of repairing damaged DNA varied according to the radiation dose rate. The increased frequency of cell apoptosis upon CLDR was, at least in part, due to the decreased expression level of DNA-PKcs and Ku proteins, which efficiently mediate the damaged DNA repair program caused by ionizing irradiation. However, the link connecting DNA repair program and cell cycle arrest as well as cell apoptosis is still missing. It will be interesting to investigate how the down-regulation of DNA-PKcs and Ku proteins occur in the context of CLDR.
In summary, our study provides a beneficial exploration of biological effects of CLDR. The induced G2/M arrest, increased cell death and the decreased DNA repair ability caused by CLDR may contribute to the enhanced inhibitory effects of CLDR on tumor cells, compared with SDR and FDR. Though several questions remained to be addressed, we believe that with development of fundamental research and accumulation of clinical experiences, 125I radioactive seed permanent implantation can be further transformed and properly used in malignant tumor therapy.
We would like to thank Jianxia Peng, Lianjun Zhang, Yuzhu Hou, Bo Ma and Tao Yang for helpful discussions and their technical assistance. This study was supported by the National Science Foundation of China, item NO. 81071834.
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