EGFR inhibitor C225 increases the radiosensitivity of human lung squamous cancer cells

Background The purpose of the present study is to investigate the direct biological effects of the epidermal growth factor receptor (EGFR) inhibitor C225 on the radiosensitivity of human lung squamous cancer cell-H520. H520 cells were treated with different dosage of 60Co γ ray irradiation (1.953 Gy/min) in the presence or absence of C225. The cellular proliferation, colony forming capacity, apoptosis, the cell cycle distribution as well as caspase-3 were analyzed in vitro. Results We found that C225 treatment significantly increased radiosensitivity of H-520 cells to irradiation, and led to cell cycle arrest in G1 phase, whereas 60Co γ ray irradiation mainly caused G2 phase arrest. H-520 cells thus displayed both the G1 and G2 phase arrest upon treatment with C225 in combination with 60Co γ ray irradiation. Moreover, C225 treatment significantly increased the apoptosis percentage of H-520 cells (13.91% ± 1.88%) compared with the control group (5.75% ± 0.64%, P < 0.05). Conclusion In this regard, C225 treatment may make H-520 cells more sensitive to irradiation through the enhancement of caspase-3 mediated tumor cell apoptosis and cell cycle arrest.

In the in vitro studies, C225 combined with radiotherapy or chemotherapy inhibits the growth of head and neck squamous carcinoma cells dramatically. Furthermore, in the clinical treatment of head and neck squamous cell carcinoma [17,18], C225 combined with radiotherapy has also made a fantastic therapeutic effect [14,15]. At present, the clinical trials of C225 combined with radiotherapy and radiotherapy alone for patients with advanced NSCLC are under way [18], no clear conclusion has been drawn. In this experiment H-520 cell proliferation, apoptosis and cell cycle distribution were detected after treating with C225 combined with radiotherapy.

Cell lines and culture
The human non-small cell lung cancer cell line H-520 was purchased from Institute of Basic Medical Science, Peking Union Medical College (Beijing, China). H-520 cells were maintained in DMEM medium which was composed of 10% fetal calf serum, 1% penicillin and streptomycin (penicillin 100 U/ml, streptomycin 100 mg/ml), and 1% glutamine. Cells were cultured in 37°C incubator with 5% CO 2 .

Determination of EGFR expression in H-520 cells by flow cytometry
Exponentially growing H-520 cells were adjusted to 5 × 10 6 /ml and incubated with mouse-against human EGFR antibody for 30 min at 4°C. After washing with PBS twice, the cells were treated with FITC labeled goatanti-mouse IgG for 30 min. The EGFR expression was detected by flow cytometry.
Cell treatment with C225 and irradiation H520 cells were pre-cultured with 40 nM C225 for 12 h, these cells and control cells then received a single dose of γ ray irradiation from a 60 Co source (Peking University Health Science Center, China). The irradiation rate is 1.953 Gy/min. Cell growth analysis by methyl thiazolyl tetrazolium (MTT) assay Cell proliferation was determined by assessing the mitochondrial reduction of MTT. Cells were plated at 1 × 10 3 cells/well in 96-well plates containing 200 μl growth medium and allowed to attach for 24 h. The medium was removed. Blank control and C225 (0.008, 0.04, 0.2, 1, 5, 25, 125 and 625 nM) groups were prepared and cells were incubated for 0, 12, 24, 48, 72 and 120 h. At harvest, the medium was removed from the appropriate wells, replaced with 50 μl MTT solution (2.5 mg/ml), and incubated for 4 h at 37°C. After incubation, the MTT solution was carefully aspirated and replaced with 150 ul DMSO. Cell growth was analyzed on a plate reader by using Soft-Max program (Molecular Devices Corp., Menlo Park, CA). Experiments were performed in quadruplicate and repeated at least 3 times. Inhibition ratio (%) = (1-A190 of testing group/A190 of the control group) × 100%.

Colony formation
The cells in exponential growth were digested with trypsin into single cell suspension. Cells were seeded into 100 mm culture plates at various dilutions. Cells were distributed evenly in 10 ml medium and maintained in incubator at 37°C for 14 days. Then the cells were fixed with methanol and stained with Gimsa. The colonies with more than 50 cells were counted. The plating efficiency (PE) was calculated as the formula: PE = number of colonies/number of seeded cells × 100%. Results were shown as the mean value of quadruplicate samples in each dose.

Dose-survival curve and sensitization enhancement ratio (SER)
The H-520 cells were exposed to 60 Co γ ray irradiation, with the total dose of 100, 200, 400, 600, 800 and 1000 cGy. There are three samples in each dose group. After irradiation, cells were digested and seeded into 100 mm plates at different cell numbers. Then the cells were cultured in 35 mm plates for 14 days, and the formed colonies were counted. The survival fraction (SF) was calculated as the formula: SF = number of colonies/ number of seeded cells × PE. The dose-survival curve was fitted based on the one-hit multi-target formula Y-axis and absorbed dose as X-axis, in which D was the absorbed dose, D 0 was the mean lethal dose, and N was the extrapolation number. In addition, the N, D 0 , D q (quasi-threshold dose) and SERwere calculated based on the dose-survival curve of H-520 cells irradiated with 60 Co γ ray, SER is defined as following: SER = (D 10 of the group irradiated with 60 Co γ ray)/(D 10 of the group treated with combination of 60 Co and C225).
Cell apoptosis determined by Hoechst33258-staining H-520 cells were separated into three groups: control, C225-treated group and 8 Gy 60 Co γ ray irradiated group. After incubation for 72 h, the cells were fixed and stained with 100 μg/ml Hoechst 33258 (Beijing Lanbosite Biotech. Co. Beijing, China) for 30 min. Cells morphology were detected under fluorescence microscopy.

Cell cycle and apoptosis analysis by flow cytometry (FCM)
Cells from the control and C225 groups were exposed to different dose of irradiation (0, 2, 4 and 8 Gy). Cells were harvested at 72 h after irradiation for cell apoptosis analyses by FCM. Cells for cycle analysis (0 and 8 Gy) were harvested at 48 h after irradiation. Each test was performed 3 times. Cells used for cell-cycle tests were stained with propidium iodide. Cells used for apoptosis tests were stained with propidium iodide (PI) and annexin V-FITC and analyzed by fluorescence-activated cell sorting (FACS) using Coulter EPICS and ModFit software (Verity Software House, Topsham, MN).
The expression levels of Caspase-3 after treatment with C225 or/and irradiation Four groups were involved in this study: control group, C225 group (final concentration of 40 nM), irradiation group (8 Gy) and combination group (C225 of 40 nM combined with irradiation of 8 Gy). Cells were collected as before and 0.5 μL Caspase-3 specific antibody were added to each tube, and incubated for 15 min away from light after mixing the cell suspension. Cell suspension were adjusted to 300 μl with PBS, and analyzed with FACS.

Statistical analysis
GraphPad prism software 4.0 was adopted to produce dose-survival curve. The statistics was performed with SPSS11.5 version. Measurement data were presented as mean ± standard deviation (x ± SD). The difference between the means of two different groups was analyzed with t-test, while ANOVA was sued for comparing 4 groups of treatment. p < 0.05 was considered significant.

High expression of EGFR in H-520 cells
In order to determine the expression of EGFR in our cell line H520, we firstly performed indirect staining to detect EGFR expression with FACS. The percentage of EGFR expression in normal lung tissue cells is below 10%. However, as shown in Figure 1, 82% of the H-520 cells expressed EGFR.

Effects of C225 on H-520 cell growth
From our previous study, we found the 40 nM C225 treatment showed approximately 50% inhibition of H-520 cell growth. We thus used 40 nM C225 for further analysis. We incubated H-520 cells with 40 nM C225 for different time points, and found that the growth inhibition rate was about 50% 72 hours after 40 nM C225 treatment. Prolonged treatment did not lead to higher growth inhibition, as shown in Figure 2.
Enhanced radiosensitivity in EGFR inhibitor C225-treated H-520 cells The radiobiological parameters of C225-treated H-520 cells were D 0 = 1.32 Gy, Dq = 0.19 Gy, N = 1.352, while those of C225-untreated H-520 cells were D 0 = 1.82 Gy, Dq = 0.51 Gy, N = 1.81. In the present study, the SER was about 1.35, which indicated that treatment with C225 significantly improved the biological effect of irradiation. The results were shown in Figure 3.

Significantly enhanced cell apoptosis in C225-treated H-520 cells after irradiation
Under fluorescent microscope, it appeared apparently that, compared with irradiation group, C225 treatment together with irradiation significantly induced morphological changes of H-520 cells, including the majority of cells' nuclei was wavy (rippled), cell membrane folded, chromatin condensed, some of which were cracking as the fragments of the nucleus, resulting in the appearance of apoptotic bodies (data not shown). The percentage of apoptosis cells was determined by flow cytometry. The apoptosis rate of H-520 cells under conventional culture conditions was about 5%, however C225 rose the percentage of cell apoptosis to 13.91 ± 1.88 (t = 3.59, P < 0.05, Figure 4). The apoptosis percentage of control group irradiated with 2, 4, or 8 Gy γ ray respectively was 13.88 ± 2.38, 24.67 ± 1.77, 30.36 ± 2.81, after treated with C225 coupled with irradiation the apoptosis percentage was 23.33 ± 3.18 (t = 2.16, P < 0.05), 39.01 ± 0.173 (t = 4.74, P < 0.05), and 42.46 ± 4.06 (t = 5.65, P < 0.05), respectively. There was significant difference between C225 and irradiation combination group and irradiation group. The results were summarized in Table 1.
C225 arrested the cell cycle of H-520 cells at G 0 /G 1 phase As shown in Figure 5, C225 arrested the cell cycle of H-520 at G 0 /G 1 phase from 43.64 ± 1.54% to 52.55 ± 2.67% determined by flow cytometry. Meanwhile, γ ray irradiation arrested the cell cycle at G 2 /M phase from 10.83 ± 1.52% to 29.95 ± 2.6%; when C225 coupled with irradiation the cell cycle arrested at both G 0 /G 1 and G 2 / M phase, meanwhile the proportion of S phase reduced from 45.46 ± 0.06% to 21.12 ± 6.84%.
Control C225 0Gy 2Gy 4Gy 8Gy Figure 4 Apoptosis of H520 cells induced by irradiation and C225. H520 cells were treated with C225 or/and different doses of irradiation. The apoptosis was determined by flow cytometry. The results were summarized in Table 1. One representative of three experiments with identical results was shown.

Discussion
The incidence and mortality of lung cancer are high and Lung cancer is the leading cause of cancer-related deaths [19]. Clinical diagnosis of 80-87% of patients with lung cancer is non-small cell lung cancer. Currently, radiotherapy and chemotherapy combined treatment for locally advanced NSCLC is the standard treatment program. One of the characteristics of chemotherapy is based on two kinds of platinum-based drugs in combination with radiation therapy to sequential or simultaneous delivery methods [20]. Though the new chemotherapy drugs and new drug combination has been introduced into the program, conventional radiotherapy has been substituted by three-dimensional stereotactic radiotherapy. Statistics show that over the past two decades, the survival time of patients with NSCLC remains no change.
We found that the clinical treatment of the same stages and even the same type of patients using the same treatment, the patient's prognosis are different, it prompt us to speculate that the tumor pathological type may have a great impact on therapy options and prognosis of patients. In recent years, bio-molecular targeting therapy increasingly receives attention. In non-Hodgkin's lymphoma, renal cell cancer, colorectal cancer, non-small cell lung cancer and head and neck squamous cells cancer treatment, bio-targeting drugs have made a good effect [18,21]. Clinically, anti-EGFR monoclonal antibody C225 combined with radiotherapy showed a significant effect on head and neck squamous cell carcinoma, the C225 combined with radiotherapy as the conventional treatment options on head and neck squamous cell carcinoma have been acceptable to the majority of doctors [21,22]. Many biological characteristics of advanced NSCLC are consistent with the head and neck cancer. Squamous cell carcinoma is the majority of NSCLC, so in this experiment we use H-520 cells as research materials to observe the effects of C225 and 60 Co γ treatment (alone/coupled) on the proliferation, apoptosis and changes in the cell cycle.
In the present study, we used H-520 cells as human non-small cell lung cancer cells. Though it is reported that H-520 cells do not express EGFR [20,23], we detected high level expression of EGFR as determined by flow cytometry. The inconsistency between our results and the published data may be due to the long term cell culture, the selected clone expansion and the other reasons in our labs, which should be determined in the future. The expression of EGFR may make our H-520 cells more sensitive to C225 treatment so that we can study the co-effect of radiation of C225 in this cell line. * * ** ** * ** ** Figure 5 C225 arrested the cell cycle of H-520 at G 0 /G 1 phase. C225 treatment caused H-520 cell cycle arrested at G 0 /G 1 phase, 52.55 ± 2.67% compared with control cells 43.64 ± 1.54%. In the meantime, γ-ray irradiation arrested the cell cycle at G 2 /M phase from 10.83 ± 1.52% to 29.95 ± 2.6%; C225 treatment together with irradiation led to cell cycle arrest at both G 0 /G 1 and G 2 /M phase, thus the proportion of S phase reduced from 45.46 ± 0.06% to 21.12 ± 6.84%.
Colony forming efficiency analysis is a classical method to evaluate the anti-tumor ability of γ ray irradiation. Through analysis of cell survival curves we found that the N value of C225 combined with radiotherapy is smaller, the curves had no apparent shoulder area, which indicate that the C225 increased the sensitivity of the H-520 cells to 60 Co γ ray irradiation. At the same time, the D 0 Value is relatively lower suggesting that the reasonable lower doses of 60 Co γ ray can also kill tumor cells when coupled with C225. The SER of C225 treatment is about 1.4 which suggested that C225 had radiosensitization effect on the H-520 cells. It has been studied that after irradiation the expression of EGFR and TGF-α increased which enhanced the ability of the tumor cells to repair DNA damage, and rapidly proliferate [24,25]. The C225 which can combine with EGFR not only blocked the secretion of TGF-α and signal transduction but also decreased the expression of EGFR on the cell membrane surface [16]. These impeded the cell survival and proliferation.
The cell-cycle phase is one of the determinative factors of cell radiosensitivity [26]. In the present study we observed C225 arrested the cell cycle at the G0/G1 phase, 60 Co γ ray irradiation resulted in G 2 /M phase arrest, C225 in combination with 60 Co γ ray irradiation at the same time led to the cell cycle arrested at G 0 /G 1 , G 2 /M phase. Under normal condition, cell cycle arrests at some point in order to repair damage, when the damage repairs successfully, cell cycle continues, otherwise cells undergo apoptosis. It needs the participation of many growth factors to repair the irradiation induced damage for the cells arrested at G 2 /M phase [27]. When the EGFR pathway is blocked by C225, the repair is difficult to proceed smoothly, the cells will undergo apoptosis. With this match, C225 in combination with irradiation significantly increased the rate of apoptosis.
Caspase-3 plays an important role in the apoptosis process which hydrolytic DNA led cells to apoptosis, caspase-3 positive cells will be apoptotic eventually [28]. We found that C225 increased the number of caspase-3-positive cells significantly, the mechanism may be that C225 against with EGFR blocked the PI3K pathway which reduced Akt phosphorylation (Akt is an important protein molecule in PI3K pathway, it regulates caspase-9, GSK-3β, NF-B, such as protein phosphorylation and ultimately controls cell survival and apoptosis) and then activated caspase-9 and process of apoptosis using caspase-3 to cut DNA to complete the task of apoptosis.
In summary, C225, which blocks EGFR pathway, inhibited H520 lung squamous carcinoma cell proliferation, and increased the sensitivity of lung squamous cancer cells to irradiation dramatically.