Combined effects of C225 and 125-iodine seed radiation on colorectal cancer cells

To assess the radiation-enhancing effects of C225, the cells were exposed to CLDR from ¹²⁵I seeds with and without concurrent treatment with 100 nM of C225. Figure 1 shows the survival curves of in LS180 cells for treatment with radiation alone and in combination with C225. In our study, the survival fraction at 1 Gy, 2 Gy, 4 Gy in 125I-CLDR treated cells was 0.61 ± 0.09, 0.30 ± 0.04, 0.05 ± 0.003, and the corresponding rate in C225 + 125I-CLDR group was 0.51 ± 0.02, 0.18 ± 0.01, 0.02 ± 0.006, respectively. We found there were significant differences concerning clonogenic survival between 125I-CLDR and C225 + 125I-CLDR groups when we used two-way ANOVA for data analysis. The sensitizer enhancement ratio (SER) was approximately 1.4, indicating that C225 increased the radiosensitivity of LS180 cells to radiation from ¹²⁵I seeds. The radiobiological parameters of the LS180 cells are shown in Table 1.

We used the MTS proliferation assay to investigate the growth-inhibitory effects of radiation and C225. As shown in Figure 2A, C225 treatment and radiotherapy each significantly inhibited LS180 cell growth when used alone (72 h, Ctrl vs. C225, t = 25.5, P < 0.001; Ctrl vs. ¹²⁵I-CLDR, t = 53.1, P < 0.001), but their combined inhibitory effect was greater than that produced by either treatment alone (72 h, C225 + ¹²⁵I-CLDR vs. C225, t = 31.5, P < 0.001; C225 + ¹²⁵I-CLDR vs. ¹²⁵I-CLDR, t = 3.7, P < 0.01). To further clarify the inhibitory effects of this combination, we detected the cell cycle distribution at indicated times after treatment. G2/M cell cycle arrest only appeared in ¹²⁵I-CLDR and C225 + ¹²⁵I-CLDR treated group (0 h, Ctrl vs. C225, t = 2.4, P > 0.05; Ctrl vs. ¹²⁵I-CLDR, t = 3.5, P < 0.01; Ctrl vs. C225 + ¹²⁵I-CLDR, t = 4.8, P < 0.001), but disappeared at 24 h after treatment (24 h, Ctrl vs. ¹²⁵I-CLDR, t = 1.6, P > 0.05; Ctrl vs. C225 + ¹²⁵I-CLDR, t = 1.7, P > 0.05). G1 cell cycle arrest did not happen in all experiment groups (48 h, Ctrl vs. C225, t = 1.5, P > 0.05; Ctrl vs. ¹²⁵I-CLDR, t = 0.5, P > 0.05; Ctrl vs. C225 + ¹²⁵I-CLDR, t = 1.1, P > 0.05).S cell cycle decreased in three experiment groups within 48 hours after treatment (48 h, Ctrl vs. C225, t = 5.1, P < 0.001; Ctrl vs. ¹²⁵I-CLDR, t = 11.2, P < 0.001; Ctrl vs. C225 + ¹²⁵I-CLDR, t = 11.3, P < 0.001). The cell cycle distribution between C225 + ¹²⁵I-CLDR and ¹²⁵I-CLDR treated cells shew no significant differences within 48 hours after treatment (48 h, C225 + ¹²⁵I-CLDR vs. ¹²⁵I-CLDR, G1, t = 0.6, P > 0.05; S, t = 0.1, P > 0.05; G2/M, t = 0.6, P > 0.05) (Figure 2B, C, D).

We further detected the mitotic ratio at 48 h after treatment in the four groups and found that there were no significant differences (48 h, Ctrl vs. C225, t = 1.2, P = 0.4; Ctrl vs. ¹²⁵I-CLDR, t = 1.6, P = 0.3; Ctrl vs. C225 + ¹²⁵I-CLDR, t = 1.6, P = 0.3; unpaired t test) (Figure 2E, F).

We then detected cell death by annexin V-FITC/PI assay. As shown in Figure 3, both C225 and radiation induced slight cellular apoptosis when administered alone (48 h, Ctrl vs. C225, t = 4.9, P = 0.008; Ctrl vs. ¹²⁵I-CLDR, t = 4.4, P = 0.012; unpaired t test), and in the combined treatment, C225 increased radiation-induced apoptosis (48 h, Ctrl vs. C225 + ¹²⁵I-CLDR, t = 24.9, P < 0.001; C225 + ¹²⁵I-CLDR vs. ¹²⁵I-CLDR, t = 6.6, P = 0.003; unpaired t test). Furthermore, the Bax/Bcl2 ratio was increased by C225 and radiation either (24 h, Ctrl vs. C225, t = 5.9, P = 0.03; Ctrl vs. ¹²⁵I-CLDR, t = 26.5, P = 0.0014; unpaired t test) and increased to highest level by the combined treatment (24 h, Ctrl vs. C225 + ¹²⁵I-CLDR, t = 107.4, P < 0.001; unpaired t test). Thus, the combined treatment produced antiproliferative effects by inducing cellular apoptosis as a result of imbalance in the ratio of the pro-apoptotic protein Bax and the pro-survival protein Bcl2.

Radiation plays a key role in cancer therapy due to its ability to directly induce DNA damage. In order to determine the cellular DNA damage and repair, immunofluorescence staining was used to determine the nuclear γ-H2AX foci 48 h after treatment. The results revealed a limited number of cells in the control group exhibiting γ-H2AX foci (6.5 ± 0.7%). However, cells receiving combined treatment (59.1 ± 2.2%)demonstrated a significant increase in the γ-H2AX focus-positive cells as compared to those treated with radiation (48.5 ± 0.1%) or C225 (4.5 ± 3.5%) alone. To determine whether DNA repair proteins were expressed, western blotting was performed using lysates from the cells that received the different treatment protocols. The expression levels of DNA-Pkcs (48 h, C225 + ¹²⁵I-CLDR vs. ¹²⁵I-CLDR, t = 5.7, P = 0.005; unpaired t test) and Ku70 (48 h, C225 + ¹²⁵I-CLDR vs. ¹²⁵I-CLDR, t = 6.6, P = 0.003; unpaired t test) proteins decreased with the combined treatment, suggesting that C225 reduced the cellular DNA repair capacity by reducing the DNA-PKcs and Ku70 protein levels.

When the cancer cells overexpressing EGFR were exposed to radiation, the survival and proliferation mechanisms were predominantly activated through signaling via PI3K-Akt and Ras-Erk. Western blot analysis was used to detect the activation of these two pathways. Our results revealed that the phosphorylation level of Akt was lower in the cells receiving the combined treatment (0 h, C225 + ¹²⁵I-CLDR vs. C225, t = 9.2, P < 0.001; C225 + ¹²⁵I-CLDR vs. ¹²⁵I-CLDR, t = 7.3, P = 0.0019; unpaired t test) than in those receiving either treatment alone (0 h, Ctrl vs. C225, t = 2.8, P = 0.051; Ctrl vs. ¹²⁵I-CLDR, t = 5.3, P = 0.006; unpaired t test). However, there were no significant differences in the activation level of Erk between the different treatment groups.

The human colon carcinoma cell line LS180 was kindly provided by the Beijing Institute for Cancer Research. Cells were grown in RPMI-1640 supplemented with 20 mM HEPES (pH 7.4), 100 IU/mL penicillin, 100 mg/mL streptomycin, 4 mM glutamine (Gibco, China), and 10% heat-inactivated fetal bovine serum (FBS; Hangzhou Sijiqing Biological Engineering Materials Company, China) in a humidified atmosphere of 5% CO₂ at 37°C.

We used an in vitro ¹²⁵I seed radiation model that had been developed in our laboratory [23, 24]. In this model, the exposure times for delivering doses of 1, 2, 4, 6, 8, and 10 Gy were 36, 73.7, 154.6, 245.8, 345.1, and 460.1 hours, respectively. 100 nM C225 was chosen to treat cells, and the C225 treatment was continuous and concurrent. For example, C225 + ¹²⁵I-CLDR treated cells needs 7 days’ continuous and concurrent C225 treatment when the exposure dose is 4 Gy.

The human-mouse chimeric anti-EGFR monoclonal antibody (mAb) cetuximab (C225, 5 mg/ml) was provided by ImClone Systems, Inc. (New York, NY, USA). The following primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): rabbit monoclonal anti-γ-H2AX, anti-EGFR, anti-Erk1/2, anti-phospho-Erk1/2, anti-Akt, anti-phospho-Ser473 Akt, anti-Bcl-2, anti-Bax, anti-Ku70, anti-Ku80, and anti-DNA-PKcs antibodies. The following secondary antibodies were obtained for use in this study: Alexa Fluor 546 goat anti-rabbit IgG (H + L) (Invitrogen, Carlsbad, CA, USA) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG.

After treatment, the cells were trypsinized to generate a single cell suspension, and a specific number of trypan blue-negative cells were seeded into each well of a six-well tissue culture plate. After the plates were incubated for 14–21 days, colonies were stained with Wright’s-Giemsa solution and manually counted. Colonies containing >50 cells were scored, and three replicate dishes containing 10–150 colonies per dish were counted for each treatment.

After treatment, the cells were trypsinized to generate a single cell suspension, plated at 5 × 10³ cells/well in 96-well plates containing 200 μL growth medium, and allowed to attach for 24 h. At harvest, the medium was removed from the appropriate wells, replaced with 100 μL MTS solution (2.5 mg/mL, Beijing PreGene Biotechnology Company, Ltd. Beijing, China), and incubated for 2 hours at 37°C. After incubation, the plate was analyzed on a plate reader by SoftMax program (Molecular Devices Corp., Menlo Park, CA, USA).

The cells were harvested by trypsinization at the indicated times after treatment, washed with ice-cold phosphate-buffered saline (PBS), fixed in 75% ethanol, and stored at −20°C. Prior to analysis, 1 μg/mL propidium iodide (PI) and 1 mg/mL RNAse A were used to label the DNA. Flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA) was used for cell cycle analysis.

Cells were harvested by trypsinization at 48 h after treatment, treated with ice-cold 0.56% KCl for 3 minutes, pelleted by centrifugation for 10 min at 400 × g, resuspended in Carnoy’s fluid (methanol:glacial acetic acid = 3:1), spread on a slide, air dried, and stained with Wright’s-Giemsa solution. The binuclear cells were observed under a light microscope.

The Annexin V-FITC/PI assay (Beijing Zoman Biotechnology Company, Ltd. Beijing, China) was performed according to the manufacturer’s instructions. Briefly, 5 × 10⁵ cells were added to 200 μL of staining solution containing 195 μL of apoptosis buffer and 5 μL Annexin V-FITC. Samples were incubated in the dark at room temperature or 4°C for 30 minutes, after which 300 μL of apoptosis buffer was added. Five minutes before analysis, 10 μL PI was added for chromatin staining. The samples were then analyzed using a Coulter Epics XL cytometer.

The cells were harvested by trypsinization 48 h after the treatment, treated with ice-cold 0.56% KCl for 3 minutes, and pelleted by centrifugation for 10 min at 800 rpm. The pellets were then resuspended in Carnoy’s fluid (methanol:glacial acetic acid = 3:1), spread on a slide, air dried, and rinsed with 95% ethanol and purified water. Cells were permeabilized in 0.3% Triton X-100 at room temperature for 15 min, and washed three times for 10 min each in PBS. The cells were then blocked with a solution of 1% BSA, 0.3% Triton X-100, and PBS (room temperature) for 2 h, and washed three times for 10 min each in PBS. The samples were then incubated with primary monoclonal anti-γ-H2AX antibody (1:400) in 1% BSA, 0.3% Triton X-100, and PBS overnight at 4°C, washed in PBS three times for 10 min each, and incubated with Alexa Fluor 546-conjugated secondary antibodies (1:200) for 1 h at room temperature. Thereafter, the antibodies were washed in PBS four times for 10 min, and counterstained with Hoechst 33342. The cells were examined on a Zeiss LSM510 confocal scanning microscope. In a single experiment, cells were counted by eyes in the stored images (original magnification, ×40) until at least 100 cells or 100 foci were registered.

The treated cells were lysed with Tween-20 lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 0.1% Tween-20, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF). Equal amounts of protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thereafter, the proteins were transferred to nitrocellulose membranes and analyzed by specific primary antibodies. Proteins were detected with enhanced chemiluminescence after incubation with horseradish peroxidase-conjugated secondary antibodies.