Chinese-German Journal of Clinical Oncology
November 2012, Vol. 11, No. 11, P638–P643
DOI 10.1007/s10330-012-1082-x
Study on effect of BSO on esophageal cancer cell line TE-1 Guogui Sun1, Wanning Hu1, Jun Zhang2, Shaowu Jing2 1 2
Department of Chemoradiology, People’s Hospital Affiliated to Hebei United University, Tangshan 063000, China Department of Radiology, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050017, China Received: 7 September 2012 / Revised: 20 September 2012 / Accepted: 15 October 2012 © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012 Abstract Objective: The aim of the study was to investigate the sensitizing effect of buthionine sulfoximine (BSO) and radiation on esophageal cancer cell line TE-1. Methods: Methyl thiazolyl tetrazolium (MTT) assay was used to observe the inhibition of BSO and radiation on cell proliferation, and to investigate the sensitizing effect of BSO on esophageal cancer cell line TE-1. Flow cytometry (FCM) was used to observe the effect of BSO and radiation on cell apoptosis and cycle. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to observe the effect of BSO on manganese superoxide dismutase (MnSOD) mRNA and protein expression. Results: BSO could inhibit the proliferation of TE-1 esophageal cancer cells, and had significant dose- and time-dependent radiosensitizing effects on TE-1 esophageal cancer cells. After the combined effects of BSO and radiation on TE-1 cells, the rate of apoptosis and G2/M phase proportion increased significantly, and MnSOD mRNA and protein expression decreased. Conclusion: BSO may reduce MnSOD mRNA and protein expression by affecting TE-1 cell cycle, thus inhibiting and inducing the apoptosis of esophageal cancer cells and enhancing the killing effect of the radiation on esophageal cancer cells. Key words buthionine sulfoximine (BSO); manganese superoxide dismutase (MnSOD); esophageal cancer cells; radiosensitization enhancement
Esophageal cancer is one of the digestive malignancies with poor prognosis. Although the conventional levels of cancer treatment have been continuously improved, the early diagnosis and long-term effect of esophageal cancer are still not ideal. Therefore, the exploration and development of new therapies for esophageal cancer has become the clinical problems to be solved. Buthionine sulfoximine (BSO) is a new radiosensitizing substance discovered in recent years that reduces intracellular antioxidant capacity and improves the killing effect of the radiation on tumor cells by reducing the levels of glutathione (GSH) in tumor cells [1]. It has been found that BSO is correlated with the radiosensitivity of malignant melanoma and other malignancies [2], but its relationship with radiosensitization of esophageal cancer was still rarely reported. Thus, methyl thiazolyl tetrazolium (MTT) assay, flow cytometry (FCM), reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used in this study to investigate the effect of BSO and radiation on esophageal cancer cell line TE-1 proliferation inhibition as well as cell cycle and apoptosis, and to elucidate its sensitizing mechanism, providing reference and theoretiCorrespondence to: Wanning Hu. Email:
[email protected]
cal basis for clinical treatment.
Materials and methods Materials Human TE-1 esophageal cancer cells (the Research Center of the Fourth Clinical Medical College of Hebei Medical University, China); Manganese superoxide dismutase (MnSOD) rabbit anti-human monoclonal antibody (Epitomics, Burlingame, CA, USA); GAPDH mouse anti-human monoclonal antibody (bought from Santa Cruz, Santa Cruz, CA, USA); IRDye 700 labeled goat antirabbit IgG antibody (LICOR bioscience, Lincoln, NE); RNATrizol Extraction Kit (Solarbio, Beijing, China); Superscript III Reverse Transcription Kit (Invitrogen, Grand Island, NY, USA); TaqDNA polymerase, OligodT, dNTPs, reverse transcriptase and RNases Inhibitor (Fermentas, Glen Burnie, MD, USA); Annexin V-FITC/PI Apoptosis Detection Kit (Beijing 4A Biotech Co., Ltd, China). Cell growth inhibition and radiosensitivity experiments The cells were irradiated with linear accelerator with a 6 MV X-ray beam over an area of 20 cm × 20 cm with a
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source target distance of 100 cm and gantry angle of 180°, and placed in 5 cm solid water under the 96-well culture plate or flask. Human TE-1 esophageal cancer cells were adherent monolayer growth cells, and cultured in high glucose medium, DMEM containing 10% fetal calf serum in a 37 ℃, 5% CO2 incubator. The cells were harvested, counted and inoculated with a 96-well culture plate, 200 μL each well, 5 × 103 counts/μL. After 24 h of culture, the cells were intervened with different concentration of BSO (0.1, 0.5, 0.75, 1, 1.5, 2 and 2.5 mg/mL) and radiation (1, 2, 4, 6 and 8 Gy) with 1 mg/mL BSO + 2 Gy radiation respectively, and cultured for 48 h. Four hours before the end of the culture, 20 μL MTT (5 μg/mL) was added to each well. The cells were cultured for 4 h, then 150 μL of 10% DMSO was added to each well, and each well was oscillated for 10 minutes. The value of the absorbance (A) or optical density (OD) of each well were detected with an automatic microplate reader. The detection wavelength and reference wavelength were 570 nm and 620 nm respectively. Tumor cell inhibition rate (%) = [1 – (experimental OD value/control OD value)] × 100%. Tumor cell survival rate (%) = experimental OD value/control OD value × 100%. The cell curves were fitted to a multi-target single-hit model. In the formula SF = 1 – (1 – e-D/D0)N, D represents the radiation dose, D0 represents the mean lethal radiation dose, Dq represents the quasi-field dose (the shoulder width of the survival), and N represents the extrapolated value. Sensitization enhancement ratio (SER) = D0 of single radiation group / D0 of sensitization enhancement group.
Determination of MnSOD mRNA with RT-PCR Upon the instructions of Trizol and reverse transcription reagents, the total RNA of TE-1 esophageal cancer cells treated with BSO, radiation and BSO + radiation for 24 hours was extracted, and reversely transcripted into cDNA. cDNA was used as template for PCR amplification. The upstream and downstream primer of MnSOD gene were 5’-AAGGTCGGAGTCAACGGATT-3’ and 5’-GCTCCTGGAAGATGGTGAT-3’ respectively, and the length of the amplified fragment was 158 bp. When GAPDH was used as the internal reference, the upstream and downstream primer were 5’-CCGACCTGCCCTACGACTA-3’ and 5’-CTGGGCTGTAACATCTCCCTT-3’ respectively, and the length of the amplified fragment was 226 bp. The conditions for PCR included predenaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, and elongation at 72 ℃ for 45 seconds for 35 cycles. PCR products were determined and semi-quantitative products were purified by 1.5% agarose gel electrophoresis. The amplicons were analyzed for optical density with gel imaging system and Multi Gauge V3.1.
Determination of TE-1 cell apoptosis with FCM Annexin V-PI double staining was used to detect the changes of TE-1 cell apoptosis 24 hours after treatment with BSO, radiation and BSO + radiation respectively. The cells were digested and collected, washed with a 4℃ pre-cooled PBS twice, and re-suspended with 1 mL buffer solution to achieve the concentration of 1 × 106 /mL. The 100 μL of the cell suspension was added into the 5 mL flow cytometry tube. The 5 μL Annexin V-FITC and 10 μL 20 μg/mL propidium iodide were added to each tube. The cells were incubated in the dark for 15 min, and determined with flow cytometry.
Determination of MnSOD protein with Western blot Western blot was used to determine the protein expression levels of MnSOD in TE-1 esophageal cancer cells treated with BSO, radiation and BSO + radiation for 24 h. RIPA protein lysate was used to extract 4 types of protein for cell collection. BCA protein levels were used to determine the total protein levels determined with the reagent kit. The 50 μg of protein was loaded onto each well. The proteins were purified by SDS-PAGE electrophoresis and placed in the stable ice bath. The proteins were then electrically transferred to the nitrocellulose membrane, enclosed with 5% skim milk for 2 hours, and incubated with the first antibody at 4 ℃ overnight (MnSOD 1:500, GAPDH 1:500). After 16 hours, goat anti-rabbit IgG and infrared fluorescent secondary antibodies (1:20 000) were added, and the proteins were incubated at room temperature for 1 hour. The Western blot bands were scanned and quantitatively analyzed with two-color infrared laser imaging system.
Determination of TE-1 cell cycle with FCM FCM was used to detect the changes of TE-1 cell apoptosis 24 h after treatment with BSO, radiation and BSO + radiation respectively. The cells were digested and collected and washed with a 4 ℃ pre-cooled PBS twice to adjust the concentration of 1 × 106 /mL. Propidium iodide (PI) dye containing RNase was added to the cells, and the cells stood in the dark at room temperature for 30 min. FCM was used to detect fluorescence intensity.
Statistical methods The experimental results were analyzed with SPSS 13.0 statistical software. The data were expressed with mean ± SD (χ ± s). Single-factor analysis of variance (F test) was used for cell growth inhibition rate. Chi-square test was used for cell apoptosis and cell cycle. Independent sample t-test was used for MnSOD mRNA and protein expression changes. P < 0.05 was determined to be statistically significant.
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Table 1 Effects of BSO on cell proliferation by MTT BSO concentration (mg/mL) 12 h 0.1 1.73 ± 0.23 0.25 2.72 ± 0.54 0.5 6.18 ± 0.70 0.75 10.14 ± 1.59 1.0 15.06 ± 1.08 1.5 21.89 ± 1.81 2.0 28.52 ± 2.15 2.5 32.67 ± 2.76 ▲
Inhibition fraction (%) 24 h 48 h 4.53 ± 0.53 14.71 ± 0.63▼ 9.26 ± 0.82▲ 21.63 ± 0.74▼ 13.83 ± 1.03▲ 26.18 ± 1.30▼ 18.09 ± 1.39▲ 30.14 ± 1.59▼ ▲ 23.73 ± 2.06 32.06 ± 1.08▼ ▲ 29.53 ± 2.57 35.38 ± 1.69▼ ▲ 32.21 ± 2.07 38.52 ± 1.15▼ 36.42 ± 2.23▲ 40.53 ± 1.76▼
72 h 17.39 ± 1.21● 25.52 ± 2.00● 33.37 ± 2.52● 39.94 ± 2.86● 44.47 ± 3.75● 48.37 ± 3.06● 56.43 ± 3.87● 63.76 ± 4.07●
, P < 0.05 vs 12 h; ▼, P < 0.05 vs 12 h and 24 h; ●, P < 0.05 vs 12 h, 24 h and 48 h
Table 2
Effects of BSO on cell proliferation by MTT
Radiation dose (Gy) 1 2 4 6 8
12 h 1.53 ± 0.21 2.75 ± 0.38 7.67 ± 1.06 9.49 ± 1.36 13.26 ± 1.70
Inhibition fraction (%) 24 h 48 h 12.53 ± 0.61▼ 4.82 ± 0.38▲ 11.48 ± 0.69▲ 16.75 ± 0.78▼ ▲ 15.52 ± 1.15 25.67 ± 1.06▼ ▲ 21.31 ± 1.83 29.49 ± 1.36▼ 29.28 ± 1.54▲ 32.35 ± 1.03▼
72 h 13.98 ± 0.53 19.36 ± 0.69● 29.39 ± 1.08● 34.47 ± 2.03● 37.82 ± 2.49●
, P < 0.05 compared to 12 h; ▼, P < 0.05 compared to 12 h and 24 h; ●, P < 0.05 compared to 12 h, 24 h and 48 h
▲
Results The inhibitory effect of BSO on the proliferation of TE-1 esophageal cells MTT assay results showed that with increasing concentration (0.10–2.50 mg/mL) and time the inhibitory effect of BSO was increased. This indicated that BSO inhibited the growth of TE-1 cells in a time- and concentrationdependent manner, and the relationship was dose- and time-dependent. There were statistically significant differences in the inhibition rates among 12 h, 24 h, 48 h and 72 h (P < 0.05; Fig. 1 and Table 1).
creased, and the relationship was dose- and time-dependent. This indicated that radiation inhibited the growth of TE-1 cells in a time- and dose-dependent manner. There were statistically significant differences in the inhibition rates among 12 h, 24 h, 48 h and 72 h (P < 0.05; Fig. 2 and Table 2).
The inhibitory effect of radiation on the proliferation of TE-1 esophageal cells MTT assay results showed that with the increase of dose (1.0–8 Gy), the inhibitory effect of radiation in-
Radiosensitivity experiments The cell survival curves of both groups fitted to a multi-target single-hit model showed that after treatment of TE-1 cells with radiation for 24 h, the values of D0, Dq and N were 20.91, 1.09 and 22.774 respectively. After treatment with 1.0 mmol/mL NO with 1 Gy, 2 Gy, 4 Gy and 6 Gy radiation for 24 h, the values of D0, Dq and N were decreased relative to original values to 9.11, 0.52 and 4.71. The sensitization enhancement ratio (SER) was 2.29, indicating that NO had the effect of radiosensitiza-
Fig. 1
Fig. 2
Effects of BSO on cell proliferation
Effects of radiation on cell proliferation
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Cell cycles distribution detected by flow cytometry Cell phase Groups G0 + G1 G2 + M S BSO 28.90 ± 1.70 17.70 ± 1.30 53.40 ± 2.50 Radiation 29.00 ± 1.60 16.30 ± 0.80 54.70 ± 2.90 BSO + radiation 25.80 ± 1.30▲ 30.70 ± 1.50▲ 42.50 ± 2.10▲ ▲ , P < 0.05, vs BSO or radiation
Fig. 3
Multi-target single-hit model-fitted dose-survival curve of cells
tion enhancement and there was statistically significant difference between both groups (P < 0.05; Fig. 3). Determination of the effect of BSO and radiation on cell apoptosis with FCM After treatment of TE-1 cells with BSO, radiation and BSO + radiation for 24 hours, the scatterplots of bivariate flow cytometry showed the lower right quadrant was early apoptotic cells. The apoptotic cell ratio under the action of BSO, radiation, and BSO + radiation was (3.3 ± 0.4)%, (3.0 ± 0.2)% and (9.6 ± 0.7)% respectively. There
was a statistically significant difference between the apoptotic cell ratio under the action of BSO/radiation and the apoptotic cell ratio under the action of BSO + radiation (P < 0.05; Fig. 4). Determination of the effect of BSO and radiation on cell cycle with FCM Table 3 and Fig. 5 showed after treatment of TE-1 cells with BSO, radiation and BSO + radiation for 24 h, the ratios of TE-1 cells in the G0 + G1 and S phases under the action of BSO/radiation were higher than that under the action of BSO + radiation, while the ratio of TE-1 cells in the G2 + M phase was relatively low. There was a statistically significant difference between the ratios of the cell cycle under the action of BSO + radiation and under the
Fig. 4 The apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. (a): BSO; (b): Radiation; (c): BSO + radiation
Fig. 5
Cell cycles distribution detected by flow cytometry. (a): BSO; (b): Radiation; (c): BSO + radiation
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action of BSO/radiation (P < 0.05; Fig. 5 and Table 3). Determination of MnSOD mRNA expression changes with RT-PCR RT-PCR results showed (Fig. 6) that after treatment of TE-1 cells with BSO, radiation and BSO + radiation for 24 h, the relative expression levels of MnSOD mRNA of TE-1 cells were 0.14 ± 0.01, 0.25 ± 0.03 and 0.23 ± 0.01 respectively. There was a statistically significant difference between the expression levels of MnSOD mRNA under the action of BSO + radiation and under the action of BSO/radiation (P < 0.05). Determination of MnSOD protein expression changes with RT-PCR RT-PCR results showed (Fig. 7) that after treatment of TE-1 cells with BSO, radiation and BSO + radiation for 24 h, the relative expression levels of MnSOD mRNA of TE-1 cells were 0.21 ± 0.01, 0.28 ± 0.03 and 0.26 ± 0.02 respectively. There was a statistically significant difference between the expression levels of MnSOD mRNA under the action of BSO + radiation and under the action of BSO/radiation (P < 0.05).
Discussion Esophageal cancer is a common human gastrointestinal malignancy with a 5-year survival rate of only about 20%. The main reason for the failure of radiotherapy is uncontrolled or recurrent tumors in the local radiation field, suggesting the presence of radiation-resistant tumor cells. Therefore, there has been significant interest among researchers to improve the therapeutic gain ratio and to find an effective radiosensitizer to increase the radiosensitivity of esophageal cancer cells. BSO is a potent inhibitor of glutathione synthesis ratelimiting enzyme (γ-glutamyl-cysteine synthase). BSO reduces glutathione biosynthesis and intracellular glutathione levels, thereby increasing the killing effect of radiation on the tumor cells [1]. Studies have shown that BSO has different degrees of radiosensitizing effect on malignant melanoma [2], rat C6 glioma cells [3] and ovarian cancer cells [4]. We have shown that different levels of BSO and different doses of radiation can significantly inhibit the proliferation of esophageal cancer, promote apoptosis, and increase its SER, which were consistent with the above results [2–4]. We used FCM to determine that the cycle of TE-1 esophageal cancer cells treated with BSO and radiation changed, and the ratio of cells in the G2 + M phase under the action of BSO + radiation was higher than that under the action of BSO/radiation, while the ratios of TE-1 cells in the G0 + G1 phase and S phase were decreased. We further confirmed by Western blot that after the esophageal cancer cells were exposed to BSO, the
Fig. 6 BSO or/and radiation-induced MnSOD expression. A: 1.0 mg/ mL BSO; B: 2 Gy radiation; C: 1.0 mmol/L BSO + 2 Gy radiation
Fig. 7 BSO or/and radiation-induced MnSOD expression. A: 1.0 mg/ mL BSO; B: 2 Gy radiation; C: 1.0 mmol/L BSO + 2 Gy radiation
expression level of manganese superoxide dismutase (MnSOD) was reduced, and more obvious under the action of BSO + radiation. We also found that the radiosensitization effect could only be maximized under the action of BSO + radiation of a certain concentration. MnSOD present in mitochondria is an important antioxidant enzyme in human cells, which can convert superoxide radical O2– into H2O2, which is decomposed into water and oxygen under the action of peroxidase (GPx) and catalase (CAT) as part of cellular detoxification activity. It was found in the preliminary study conducted with immunohistochemistry and RT-PCR that the expression level of MnSOD was significantly lower in esophageal cancer tissues than in normal esophageal tissues [5], and that moderate expression can inhibit the proliferation of esophageal cancer cells and promote their apoptosis, while high expression had the opposite effect [6, 7]. Komatsu [8] and Wang [9] et al studied osteosarcoma cell lines (SaOS2), and found that the sensitivity of SaOS2 to doxorubicine at different levels of MnSOD showed bidirectional regulation mechanism, that is, high overexpression decreases sensitivity by decreasing apoptosis while low overexpression increases sensitivity by increasing apoptosis. This was assumed to be because the different proportions of reactive oxygen species produced induce their reaction products. These may help illustrate that different biological effects are produced from different expression levels of MnSOD under the action of BSO and radiation and that the appropriate levels of BSO combined with radiation are more conducive to killing tumor cells. In summary, we found in this study that BSO can inhibit esophageal cancer cell proliferation, promote apoptosis and enhance radiosensitivity. This effect may be related to the ability of BSO to arrest of cell cycle, inhibit MnSOD mRNA or protein expression, and influence reactive oxygen species levels in the cells.
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