The different biological effects of single, fractionated and continuous low dose rate irradiation on CL187 colorectal cancer cells

Hao Wang 0 Jinna Li 0 Ang Qu 0 Jingjia Liu 0 Yong Zhao 1 Junjie Wang 0 0 Department of Radiation Oncology, Peking University Third Hospital , North Road No. 49, Haidian District, 100191, Beijing , China 1 Transplantation Biology Research Division, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences , Beijing , China Purpose: 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. Results: 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. Conclusions: 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. - Introduction 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 reirradiation, 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 Reagents 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 [10]. 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 37C. The medium was replaced every two or three days. 125I seeds and X-ray irradiation An in-house model for in vitro iodine-125 seed irradiation (shown in Figure 1) was developed for this study [10-12]. The model consisted of a 3-mm-thick polystyrene panel, with a lower seed plate layer and an uppercell culture plate layer. In the seed plate, 14 seeds with the same activity were equally spaced within recesses (4.5 0.8 mm) around a 35-mm diameter (D) circumference. For the cell-culture plate, similar recesses were made around a 35-mm D circumference. The center of the cell-culture plate was vertically aligned above the seed plate, so that a 35-mm Petri dish could be placed on it during the experiment. The height (H) between the seed plate and the bottom of the Petri dish was 12 mm, with a D/H ratio of 2.9. The purpose of this design was to obtain a relatively homogeneous dose distribution at the bottom of the Petri dish. The polystyrene assembly was encased in a 3-mm-thick lead chamber with a vent hole, so that the whole model could be kept in the incubator during the study. The incubator played a protective role by maintaining constant cell-culture conditions. Model 6711 125I seeds were provided by the Ningbo Junan Pharmaceutical Technology Company (Ningbo, Zhe Jiang province, China). The activity of the single seed used in this study was 92.5 MBq (2.5 mCi), which translates to an initial dose rate of 2.77 cGy/h to model cells. The dose uniformity of the irradiation model in the cell plane was 1.34, which was in agreement with previous study [13]. This model was thus validated by Figure 1 Schematic representation of an in vitro model for iodine-125 seed irradiation. The model was consi (...truncated)