Diseases 54–61 Diseases of of the the Esophagus Esophagus (2016) (2014) 29, ••, ••–•• DOI: DOI: 10.1111/dote.12255 10.1111/dote.12255
Original article
Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro Z.-M. Wang,1,2* Y.-H. Kang,2* X. Yang,1* J.-F. Wang,2 Q. Zhang,2 B.-X. Yang,3 K.-L. Zhao,3 L.-P. Xu,1 L.-P. Yang,3 J.-X. Ma,2 G.-H. Huang,2 J. Cai,3 X.-C. Sun1 1
Department of Radiotherapy, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Department of Radiotherapy, The Second People’s Hospital of LianYungang, Lianyungang Hospital Affiliated to Bengbu Medical College, Lianyungang, and 3Department of Radiotherapy, Nantong Tumor Hospital Affiliated to Nantong University, Nantong, Jiangsu Province, China 2
SUMMARY. To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/ Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. KEY WORDS: andrographolide, esophageal cancer, radiosensitivity.
INTRODUCTION Esophageal cancer, one of the common malignant tumors in the world, is the sixth leading cause of malignant tumor mortality.1 Similar to radiotherapy, surgical care recorded only a 4% 5-year survival rate.2 The 5-year survival rate of postoperative esophageal Address correspondence to: Prof Xin-Chen Sun, PhD, MD Department of Radiotherapy, The First Affiliated Hospital of Nanjing Medical University, Guangzhou Road 300, Nanjing 210029, China. Email: [email protected] *These authors contributed equally to this work. Financial support: This work was supported by the Innovation Team (No. LJ201123 [EH11]), a project funded by the priority academic program development of Jiangsu Higher Education Institution (JX10231801), grants from Key Academic Discipline of Jiangsu Province ‘Medical Aspects of Specific Environments’, the Natural Science Foundation of China (No. 81272504), ‘333’ Project of Jiangsu Province (BRA2012210/RS12), and innovative research projects of Bengbu Medical College (Byycx1325). 54 © 2014 International Society for Diseases of the Esophagus
cancer was increased to 25% with the adoption of precise radiotherapy with chemotherapy.3 However, increasing the radiation dose does not yield better results.4 Enhancing the sensitivity of tumor cells to radiation is a major goal in radiotherapy, but the ideal radiation sensitizer should be determined. Therefore, the development of agents that can confer radiosensitivity on cancer cells with minimal radiation toxicity to normal cells is necessary. Andrographolide, a major ingredient of the medicinal herb Andrographis paniculata, exhibits a broad spectrum of pharmacological activities, including antioxidative,5 antiviral,6 anti-inflammatory,7 and anticancer effects.8–10 Zhou et al. showed that andrographolide can induce apoptosis in human cancer cells via the activation of caspase 8, Bid, and Bax.11 Although andrographolide can suppress the expression of multiple malignant cancers and some C 2014 International Society for Diseases of the Esophagus V 1
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tumor cells with radiotherapy sensitization, its function as a radiosensitizer in esophagus cancer cell line remains unknown. In the present study, we investigated andrographolide-mediated radiosensitization and its molecular mechanisms.
METHODS Reagents and cell line Andrographolide (98% purity), from Zelang Medical Food Technology Co., Ltd. (Nanjing, China), was dissolved in dimethyl sulfoxide (DMSO, Sigma, St Louis, MO, USA) as a concentrated stock solution. Neonatal calf serum (NCS), phosphate-buffered saline (PBS), and RPMI-1640 media were purchased from Gibco/Life Technologies (Carlsbad, CA, USA). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl2H-te trazolium (MTT), annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) reagent kit, nuclear and cytoplasmic protein extraction kit, β-actin, and Histon-3 were purchased from Beyotime Institute of Biotechnology (Shanghai, China). All Western blot analysis supplies were obtained from Bio-Rad (Hercules, CA, USA). Polyclonal antibodies against γ-H2AX, NF-κb, Cleaved-Caspase3, Bax, Bcl-2, and horseradish peroxidase-conjugated secondary antibody (goat-anti-rabbit or goat-antimouse) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The human esophageal squamous cell carcinoma cell line ECA109 was provided by Jiangsu Province Hospital. It was cultured in RPMI-1640 medium supplemented with 10% NCS, 100 U/mL penicillin, and 100 µg/mL streptomycin, and then maintained in a 5% CO2 humidified atmosphere at 37°C.
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control group/OD value of control group) × 100. Experiments were repeated three times. Clonogenic survival assays Clonogenic survival assays were performed following standard methods in triplicate wells using six-well plates. Growing cells were pretreated in six-well plates with andrographolide 120 µg/mL for 24 hours. The six-well plates were treated with a single dose of 0 (control), 2, 4, 6, or 8 Gy at approximately 2 Gy/ 40 s in a Precision X-ray 6Mv (Varian Clinac, 23EX, Milpitas, CA, USA), and then the medium was replaced to fresh medium. The cells were grown for 10 days to produce colonies of >50 cells/colony, washed once with 1 × PBS, and then stained with 0.25% crystal violet in 80% methanol. Finally, the plates were examined under the microscope, and the number of colonies with at least 50 cells was counted. Survival curves were fitted using a linear-quadratic model to estimate the sensitizer enhancement ratio (SER). Experiments were repeated three times. Cell cycle analysis ECA109 cells were incubated in a six-well cell culture cluster (1 × 106 per well) and then divided into the following groups: control (CG), simple drug (SG), radiotherapy (RG), and radiotherapy combined with drug (RCG). SG and RCG were pretreated. After culturing for 24 hours, all cells were collected, washed with PBS, and then suspended in 70% ethanol at 4°C overnight. The cells were incubated with 6 µL of 1 g/L RNase A, 1 mL of 1 mg/mL PI, and 400 µL of PBS at room temperature for 15 minutes. DNA content was analyzed using flow cytometry (FCM, BD, FACS Calibur, Franklin Lakes, NJ, USA). This procedure was repeated thrice.
MTT assay MTT assays were performed to measure the cytotoxicity and radiosensitizing effects of andrographolide. ECA109 (5 × 103) were seeded into 96-well plates overnight. The cells were divided into eight groups, and each group comprised six wells. The cells were treated with various concentrations of andrographolide (0, 7.5, 15, 30, 60, 120, 240, 360, and 480 µg/mL) and cultured for 24, 48, and 72 hours. The medium was removed, and then the cells were incubated using MTT (5 mg/mL) for 4 hours under 5% CO2 at 37°C and lysed with solubilization solution (0.15 mL, DMSO). The optical density (OD) value at 490 nm was measured using an enzymelabeled meter (Bio Rad, 680, USA). The mean value of the OD values of the six wells was considered as the result. Cell viability percentage was calculated according to the following formula: cell viability percentage = (OD value of the drug groups except C 2014 International Society for Diseases of the Esophagus V
Annexin V-FITC/PI double-labeled FCM The pre-processing of the four groups was the same as the cell cycle analysis. After culturing for 24 hours, all cells were collected and washed with 1 × PBS twice. The cells were incubated with annexin-V-FITC in the dark at room temperature for 10 minutes. Then, PI was added, and the cells were incubated in the dark at room temperature for another 10 minutes. Cell apoptosis was measured using FCM. This procedure was repeated thrice. Western blot analysis The cells were washed with 1 × PBS and harvested on ice. Lysates were prepared in a Western lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, Ethylenediamine tetra-acetic acid, Na3VO4, and © 2014 International Society for Diseases of the Esophagus
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Fig. 1 (A) Effects of various concentrations of andrographolide on the proliferation of esophageal cell line ECA109. (B) Andrographolide sensitized ECA109 cells to radiation in vitro. Survival fractions were fitted with linear quadratic model to estimate sensitizer enhancement ratio. Andrographolide, 120 µg/mL; radiation 0, 2, 4, 6, 8 Gy. The bars presented means ± SD of three separate experiments. SER, sensitizer enhancement ratio.
leupeptin) by repeatedly passing cells through a pipette. Protein concentration was determined using the BCA protein assay kit (Beyotime). An equal amount of protein for each sample was resolved using 5% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred onto a Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane. The membrane was blocked with 5% skimmed milk and sequentially incubated with primary antibody and horseradish peroxidase-conjugated secondary antibody followed by ECL detection. The nucleoprotein was extracted using the nuclear and cytoplasmic protein extraction kit. Experiments were repeated three times.
γ-H2AX for testing number of highlights in the nuclei. Bax is detected in the cytoplasm. Experiments were repeated three times. Statistical analysis All data are expressed as means ± standard deviation. Data were analyzed using SPSS 17.0 software (Chicago, IL, USA). Survival curves were fitted using GraphPad Prism 5.0 (La Jolla, CA, USA). Student t-test was applied to compare the groups as necessary. Value of P < 0.05 indicated a significant difference.
RESULTS Total nuclear protein assay
Cytotoxicity of andrographolide on ECA109 cells
The cells were processed using the nuclear protein kits, and the nuclear protein was estimated using the BCA protein assay kit. Protein samples were analyzed using Western blot analysis. Histon-3 was used as internal control. Experiments were repeated three times.
The andrographolide induced ECA109 cell death in time- and dose-dependent manner. The effects of various concentrations of andrographolide on ECA109 after 24, 48, and 72 hours of incubation are shown in Figure 1A. Andrographolide from 30 to 480 µg/mL showed inhibitory effects on ECA109 cell growth. However, the 7.5 and 15 µg/mL concentrations exhibited hormesis. Then, at 24-hour posttreatment of 240 µg/mL andrographolide, the cell death rate reached almost 50% (Table 1). The ECA109 cells treated with andrographolide for 24 hours exhibited an IC20 value of 95.39 µg/mL. Therefore, the 120 µg/mL (about IC27%) concentration was used in the follow-up study.
Immunofluorescence staining of γ-H2AX and Bax The cells were treated as defined in the figure legends. Immunofluorescent staining was performed by fixing cells with pre-chilled (−20°C) acetone-methanol for 15 minutes. The primary antibodies, anti-γ-H2AX (Santa Cruz, 1 : 100) or anti-Bax (Santa Cruz, 1 : 100), were then added to the slides at 4°C overnight. After washing, the secondary antibodies, FITC-Goat antirabbit IgG (Santa Cruz, 1 : 200), were applied for 1 hour at room temperature. The slide was then covered with Beyotime antifade mounting medium with DAPI (Beyotime, 1 : 1000). Images were taken with a fluorescent microscope (Leica, TCS SP5). Images were captured by a charge coupled device camera. For each treatment condition, γ-H2AX foci were counted in at least 100 cells from randomly captured images. © 2014 International Society for Diseases of the Esophagus
Effects of andrographolide on clonogenic cell survival in ECA109 cells The radiosensitizing effects of andrographolide were initially measured using clonogenic assay. A 1.28 SER was calculated at a surviving fraction of 37%. The colony numbers clearly decreased after andrographolide with radiotherapy compared C 2014 International Society for Diseases of the Esophagus V
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Table 1 Effect of andrographolide on the viability rate of ECA109 cells Andrographolide (µg/mL)
n
24 hours
48 hours
72 hours
0 7.5 15 30 60 120 240 360 480
3 3 3 3 3 3 3 3 3
100.00 118.13 ± 8.61 106.43 ± 10.27 97.20 ± 10.60 78.22 ± 7.42 66.19 ± 7.63* 48.39 ± 14.20 34.52 ± 13.13 20.88 ± 12.73
100.00 97.22 ± 1.79 87.74 ± 2.81 54.82 ± 6.33 29.99 ± 5.91 13.87 ± 6.51* 8.73 ± 4.42 7.41 ± 2.33 3.69 ± 1.07
100.00 96.20 ± 3.06 85.43 ± 2.78 26.95 ± 4.04 13.38 ± 4.90 6.32 ± 1.36* 3.98 ± 1.01 2.33 ± 1.06 1.05 ± 0.55
*P < 0.01, compared with control, t = 7.68/22.92/119.30.
with those after independent radiotherapy or andrographolide (Fig. 1B). Effects of andrographolide on the formation of γ-H2AX nuclear foci in ECA109 cells Ionizing radiation kills cells by inflicting various types of damage to the genome. We speculate that the radiosensitizing effect of andrographolide on cancer cells may have been caused by the impairment in the repair of DNA double-strand breaks (DSBs). We, therefore, determined the levels of DSBs via immunofluorescence staining of γ-H2AX foci in ECA109 at different time points after exposure to X-rays. A higher number of ECA109 cells exhibited γ-H2AX nuclear foci at 0.5 hour, the majority of which was cleared at 2 hours after exposure to 6 Gy of X-ray (Fig. 2). Moreover, the average number of γ-H2AX foci subsided to near basal level at 24 hours. RCG always exhibited more γ-H2AX foci than RG.
Effects of andrographolide on the protein expression of NF-κb, Cleaved-Caspase3, Bax, and Bcl-2 The expression of Cleaved-Caspase3, Bax, and Bcl-2 in the ECA109 cells was examined to further investigate the molecular mechanisms underlying the andrographolide inhibition of cell growth. Immunoblot analysis revealed that andrographolide combined with radiation could increased Bax and Cleaved-Caspase3 and decreased Bcl-2 expression compared with control or single treatment groups. We next analyzed the expression of NF-κb in nuclear and found that andrographolide could decreased NF-κb expression (Fig. 3A). Effects of andrographolide on the apoptosis protein of Bax in ECA109 cells Bax, a Bcl-2 family protein, induces cell death through the disruption of mitochondrial permeability
Fig. 2 Formation of γ-H2AX nuclear foci in andrographolide-treated ECA109 cells. ECA109 cells were pretreated with andrographolide for 24 hours, followed by double immunofluorescent staining for γ-H2AX (green). Nuclei were counter-stained with DAPI (blue). At least 200 cells in four randomly selected fields were counted to determine the percentage of cells positive for active γ-H2AX nuclear foci. The individual pictures for γ-H2AX were taken under a magnification of ×400. Experiments were repeated three times. C 2014 International Society for Diseases of the Esophagus V
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Fig. 3 (A) Western blotting analysis of expression of Cleaved-Caspase3, Bax, Bcl-2(i) and NF-κb nuclear(ii) protein. The ECA109 cells were treated with andrographolide at different time points after exposure to X-rays. The SG and RCG groups were pretreated with drug for 24 hours. Total cellular proteins were harvested and subjected to Western blotting analysis. β-actin (i) or Histon-3 (ii) was used as a loading control. Detection of β-actin in order to confirm the extraction of nucleoprotein high purity, very few cytoplasmic protein, eliminating influence. (B) Bax in ECA109 cells was immunostained using Bax as the primary antibody and phycoerythrin-conjugated IgG as the secondary antibody; more details were described under the Methods section. The individual pictures for Bax were taken under a magnification of ×400 using an immunofluorescence microscope. Representative data from three independent experiments are shown.
and subsequent release of cytochrome C. ECA109 cells were treated with 120 µg/mL andrographolide for 24 hours and the non-treated cells were used as control. The results were further confirmed by the immunofluorescence microscopic study, which showed that 120 µg/mL andrographolide increased Bax expression in ECA109 cells after 24 hours of treatment or with 6 Mv X-ray (Fig. 3B). Effects of andrographolide on cell cycle distribution The distribution of ECA109 cells in four groups of the cell cycle was analyzed using FCM after andrographolide treatment for 24 hours to determine whether andrographolide regulates the cell cycle. The percentage of cells in each phase of the cell cycle in the different groups is summarized in Figure 4A,B and Table 2. Compared with the control, an accumulation of ECA109 cells in the G0/G1, S, and G2/M phase were noted in the other groups. Effects of andrographolide on the apoptosis of ECA109 cells The rates of apoptotic cells and dead cells were analyzed with annexin V-FITC/PI double-labeled FCM 24-hour post-andrographolide treatment or radiation. RCG (32.5% ± 0.9%) showed a significantly higher apoptosis rate than RG (26.5% ± 2.2%, P < 0.05) and SG (20.8% ± 0.8%, P < 0.001). RCG (52.5% © 2014 International Society for Diseases of the Esophagus
± 4.9%) also showed significantly higher death rate than RG (30.8% ± 0.6%, P < 0.01) and SG (14.1% ± 0.5%, P < 0.001) (Fig. 4C,D and Table 3) .
DISCUSSION The acute toxicity of andrographolide in mice has demonstrated that the lethal dose of an intraperitoneal injection is 11.46 g/kg.8 In the present study, we found that low doses have proliferation effect determined by MTT experiment (Fig. 1A). Therefore, the dose of andrographolide we used for experiment in vitro (0–480 µg/mL) was within a safe range. Previous studies have shown that andrographolide inhibits malignant cancer phenotypes and induces apoptosis of cancer cells.9–11 Then, we chose the safe concentration of 120 µg/mL as the experimental dose. By clonogenic survival assays found that andrographolide can significantly sensitization esophageal cancer cells (SER = 1.28). We also found that simply using andrographolide death rate is not high; however, radiotherapy combined with drug group reflects significant death rate (Table 3). We further investigated the mechanism by which andrographolide inhibits cell growth and found that andrographolide treatment not only induces G0/G1 arrest in ECA109 cells but causes significant DNA damage, as shown in the immunofluorescence staining assay. Numerous studies have revealed that different molecular mechanisms correspond to DNA C 2014 International Society for Diseases of the Esophagus V
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Fig. 4 (A/B) Effects of andrographolide on cell cycle distribution in cultured ECA109 cells. Flow cytometric analysis of the DNA content in four groups cultured for 24 hours. (C/D) Effects of andrographolide on apoptosis rate in cultured ECA109 cells. Percentage of apoptotic cells was measured by flow cytometry after Annexin-V/propidium iodide (PI) staining. Q2 represented cell apoptosis rate; Q3 represented cell death rate.
damage checkpoints at different phases.12 The results of the current study demonstrate for the first time the radiosensitization activity of andrographolide, suggesting a potential new application of this compound in human esophageal cancer therapy. Further investigation is required to explore the clinical benefits and fully understand the mechanisms of andrographolide-mediated radiosensitization. The results of the current study show that andrographolide enhances radiation-induced apoptotic changes. Moreover, independent andrographolide
treatment was found to induce apoptosis. A similar study has shown that andrographolide induces apoptosis of human cancer cell lines (HepG2, HeLa, and MDA-MB-231).11 However, a previous study has shown that andrographolide did not induce apoptosis of H-ras-transformed rat kidney epithelial cell line.13 We speculate that such discrepancy is a result of differences in the drug concentration and incubation time (e.g. andrographolide 10 µM for 3 hours was used in a previous study, but 120 µg/mL for 24 hours was used in the current study).
Table 2 Effects of andrographolide on cell cycle analysis Groups
G0/G1 (%)
S (%)
G2/M (%)
CG SG (andrographolide 120 µg/mL) RG (6Gy) RCG (andrographolide 120 µg/mL + 6Gy)
16.57 ± 0.8 18.24 ± 1.3* 39.72 ± 1.1** 54.72 ± 1.9**
53.51 ± 3.2 48.54 ± 0.8* 37.62 ± 5.2** 25.76 ± 0.9**
29.92 ± 4.1 33.24 ± 0.7* 22.65 ± 0.6** 19.52 ± 3.2**
*P > 0.05; **P < 0.05, compared with control. Analysis of DNA in ECA109 cells. The percentage of each cell cycle was determined by flow cytometry. Compared with the control, andrographolide could induce G2/M arrest in ECA109 cells. C 2014 International Society for Diseases of the Esophagus V
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Table 3 Effects of andrographolide on cell apoptosis rate analysis Groups
Q2 (%)
Q3 (%)
SG (andrographolide 120 µg/mL) RG (6Gy) RCG (andrographolide 120 µg/mL + 6Gy)
20.8 ± 0.8 26.5 ± 2.2 32.5 ± 0.9
14.1 ± 0.5 30.8 ± 0.6 52.5 ± 4.9
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cell line20 and increased radiosensitization in K-ras-transformed human prostate epithelial 267B1 cells.21 We speculate that andrographolide-mediated radiosensitization is a result of, at least in part, the attenuation of NF-κb activation.
CONCLUSION The regulation of cell cycle on the occurrence and development of tumors has important significance. Cellular radiosensitivity varies along with the cell cycle phase, G2/M phase sensitivity of the highest, followed by G0/G1 phase of the cell, the worst S phase.14 Inducing G0/G1 arrest can increase the radiosensitivity of cancer cells.15 In the current study, we found that andrographolide treatment induces G0/G1 arrest, decreases S proportion, and has no obvious impact to G2/M in ECA109 cells. This means that the radiotherapy sensitization is by inducing G0/G1 arrest. DNA damage checkpoints are biochemical pathways that delay or arrest cell cycle progression in response to genomic DNA damage. Andrographolide treatment cause significant DNA damage, as shown in the immunofluorescence staining assay. Markers of a constitutively active DNA damage response have been described in many types of malignant lesions in different tissues.16 Therefore, we analyzed the major kinases of the signaling pathways in andrographolide-treated ECA109 cells. Investigating the DNA damage checkpoints in tumor cells will not only help in understanding the regulatory mechanisms of a cell cycle but also provide a potential theory for the development of a new tumor therapy. The oncogene-derived protein Bcl-2 negative controls the cellular suicide machinery pathway. The Bcl-2 homologous protein Bax promotes cell death by competing with Bcl-2. Bcl-2, Bax regulate mitochondrial outer membrane permeability, causing the release of cytochrome C, activation CleavedCaspase3. Our study found that, the levels of Cleaved-Caspase3 and Bax were elevated after exposure to andrographolide alone, radiation alone, and their combination (2 hours and 24 hours), whereas the Bcl-2 levels were decreased. Thus, the cell apoptosis induced by andrographolide and radiation may involve activation of the Bax/Bcl-2 pathway. Multiple signaling pathways, including PI3K/Akt, Erk, and NF-κb, have been highly associated with the pharmacological activity of andrographolide.17 We observed that radiation induced NF-κb activity in ECA109 cells, and andrographolide co-treatment reduced radiation-induced NF-κb activation. Radiationinduced NF-κb activity has been shown to cause radioresistance of different cell types.18,19 Furthermore, inhibition of NF-κb activity led to significant radiosensitization in the HT1080 fibrosarcoma © 2014 International Society for Diseases of the Esophagus
The results of the current study demonstrated that andrographolide may be a potentially promising agent of natural resource to treat esophageal cancer and a novel radiosensitizer. Its potential application in radiotherapy merits further investigation.
References 1 Pisani P, Parkin D M, Bray F et al. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer 1999; 83: 18–29. 2 Earlam R, Cunha-Melo J R. Oesophageal squamous cell carcinoma: I. A critical review of surgery. Br J Surq 1980; 67: 381–90. 3 Chiu P W, Chan A C, Leung S F et al. Multicenter prospective randomized trial comparing standard esophagectomy with chemoradiotherapy for treatment of squamous esophageal cancer: early results from the Chinese University Research Group for Esophageal Cancer (CURE). J Gastrointest Surg 2005; 9: 794–802. 4 Minsky B D, Pajak T F, Ginsberg R J et al. INT 0123 (Radiation Therapy Oncology Group 94-05) phase III trial of combined-modality therapy for esophageal cancer: high-dose versus standard-dose radiation therapy. J Clin Oncol 2002; 20: 1167–74. 5 Batkhuu J, Hattori K, Takano F et al. Suppression of NO production in activated macrophages in vitro and ex vivo by neoandrographolide isolated from Andrographis paniculata. Biol Pharm Bull 2002; 25: 1169–74. 6 Wiart C, Kumar K, Yusof M Y et al. Antiviral properties of ent-labdene diterpenes of Andrographis paniculata nees, inhibitors of herpes simplex virus type 1. Phytother Res 2005; 19: 1069–70. 7 Xia Y F, Ye B Q, Li Y D et al. Andrographolide attenuates inflammation by inhibition of NF-kappa B activation through covalent modification of reduced cysteine 62 of p50. J Immunol 2004; 173: 4207–17. 8 Handa S S, Sharma A. Hepatoprotective activity of andrographolide from Andrographis paniculataagainst carbontetrachloride. Indian J Med Res 1990; 92: 276–83. 9 Liang F P, Lin C H, Kuo C D et al. Suppression of v-Src transformation by andrographolide via degradation of the v-Src protein and attenuation of the Erk signaling pathway. J Biol Chem 2008; 283: 5023–33. 10 Rajagopal S, Kumar R A, Deevi D S et al. Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata. J Exp Ther Oncol 2003; 3: 147–58. 11 Zhou J, Zhang S, Onq C N et al. Critical role of pro-apoptotic Bcl-2 family members in andrographolide-induced apoptosis in human cancer cells. Biochem Pharmacol 2006; 72: 132–44. 12 Sancar A, Lindsey-Boltz L A, Unsal-Kaçmaz K et al. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004; 73: 39–85. 13 Hung S K, Hung L C, Kuo C D et al. Angrogapholide Sensitizes Ras-transformed cells to radiation in vitro and in vivo. Int J Radiat Oncol Biol Phys 2010; 77: 1232–9. 14 Simoens C, Vermorken J B, Korst A E et al. Cell cycle effects of vinflunine, the most recent promising Vinca alkaloid, and its interaction with radiation, in vitro. Cancer Chemother Pharmacol 2006; 58: 210–8. 15 Wang B F, Dai Z J, Wang X J et al. Saikosaponin-d increases the radiosensitivity of smmc-7721 hepatocellular carcinoma C 2014 International Society for Diseases of the Esophagus V
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cells by adjusting the g0/g1 and g2/m checkpoints of the cell cycle. BMC Complement Altern Med 2013; 13: 263. 16 Bartkova J, Horejsi Z, Koed K et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005; 434: 864–70. 17 Tsai H R, Yang L M, Tsai W J et al. Andrographolide acts through inhibition of ERK1/2 and Akt phosphorylation to suppress chemotactic migration. Eur J Pharmacol 2004; 498: 45–52. 18 Criswell T, Leskov K, Miyamoto S et al. Transcription factors activated in mammalian cells after clinically relevant doses of ionizing radiation. Oncogene 2003; 22: 5813–27.
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19 Yamagishi N, Miyakoshi J, Takebe H. Enhanced radiosensitivity by inhibition of nuclear factor kappa B activation in human malignant glioma cells. Int J Radiat Biol 1997; 72: 157–62. 20 Wang C Y, Mayo M W, Baldwin A S. TNF- and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-kappaB. Science 1996; 274: 784–7. 21 Kim B Y, Kim K A, Kwon O et al. NF-kappaB inhibition radiosensitizes Ki-Ras-transformed cells to ionizing radiation. Carcinogenesis 2005; 26: 1395–403.
© 2014 International Society for Diseases of the Esophagus
Original article
Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro Z.-M. Wang,1,2* Y.-H. Kang,2* X. Yang,1* J.-F. Wang,2 Q. Zhang,2 B.-X. Yang,3 K.-L. Zhao,3 L.-P. Xu,1 L.-P. Yang,3 J.-X. Ma,2 G.-H. Huang,2 J. Cai,3 X.-C. Sun1 1
Department of Radiotherapy, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Department of Radiotherapy, The Second People’s Hospital of LianYungang, Lianyungang Hospital Affiliated to Bengbu Medical College, Lianyungang, and 3Department of Radiotherapy, Nantong Tumor Hospital Affiliated to Nantong University, Nantong, Jiangsu Province, China 2
SUMMARY. To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/ Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. KEY WORDS: andrographolide, esophageal cancer, radiosensitivity.
INTRODUCTION Esophageal cancer, one of the common malignant tumors in the world, is the sixth leading cause of malignant tumor mortality.1 Similar to radiotherapy, surgical care recorded only a 4% 5-year survival rate.2 The 5-year survival rate of postoperative esophageal Address correspondence to: Prof Xin-Chen Sun, PhD, MD Department of Radiotherapy, The First Affiliated Hospital of Nanjing Medical University, Guangzhou Road 300, Nanjing 210029, China. Email: [email protected] *These authors contributed equally to this work. Financial support: This work was supported by the Innovation Team (No. LJ201123 [EH11]), a project funded by the priority academic program development of Jiangsu Higher Education Institution (JX10231801), grants from Key Academic Discipline of Jiangsu Province ‘Medical Aspects of Specific Environments’, the Natural Science Foundation of China (No. 81272504), ‘333’ Project of Jiangsu Province (BRA2012210/RS12), and innovative research projects of Bengbu Medical College (Byycx1325). 54 © 2014 International Society for Diseases of the Esophagus
cancer was increased to 25% with the adoption of precise radiotherapy with chemotherapy.3 However, increasing the radiation dose does not yield better results.4 Enhancing the sensitivity of tumor cells to radiation is a major goal in radiotherapy, but the ideal radiation sensitizer should be determined. Therefore, the development of agents that can confer radiosensitivity on cancer cells with minimal radiation toxicity to normal cells is necessary. Andrographolide, a major ingredient of the medicinal herb Andrographis paniculata, exhibits a broad spectrum of pharmacological activities, including antioxidative,5 antiviral,6 anti-inflammatory,7 and anticancer effects.8–10 Zhou et al. showed that andrographolide can induce apoptosis in human cancer cells via the activation of caspase 8, Bid, and Bax.11 Although andrographolide can suppress the expression of multiple malignant cancers and some C 2014 International Society for Diseases of the Esophagus V 1
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tumor cells with radiotherapy sensitization, its function as a radiosensitizer in esophagus cancer cell line remains unknown. In the present study, we investigated andrographolide-mediated radiosensitization and its molecular mechanisms.
METHODS Reagents and cell line Andrographolide (98% purity), from Zelang Medical Food Technology Co., Ltd. (Nanjing, China), was dissolved in dimethyl sulfoxide (DMSO, Sigma, St Louis, MO, USA) as a concentrated stock solution. Neonatal calf serum (NCS), phosphate-buffered saline (PBS), and RPMI-1640 media were purchased from Gibco/Life Technologies (Carlsbad, CA, USA). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl2H-te trazolium (MTT), annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) reagent kit, nuclear and cytoplasmic protein extraction kit, β-actin, and Histon-3 were purchased from Beyotime Institute of Biotechnology (Shanghai, China). All Western blot analysis supplies were obtained from Bio-Rad (Hercules, CA, USA). Polyclonal antibodies against γ-H2AX, NF-κb, Cleaved-Caspase3, Bax, Bcl-2, and horseradish peroxidase-conjugated secondary antibody (goat-anti-rabbit or goat-antimouse) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The human esophageal squamous cell carcinoma cell line ECA109 was provided by Jiangsu Province Hospital. It was cultured in RPMI-1640 medium supplemented with 10% NCS, 100 U/mL penicillin, and 100 µg/mL streptomycin, and then maintained in a 5% CO2 humidified atmosphere at 37°C.
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control group/OD value of control group) × 100. Experiments were repeated three times. Clonogenic survival assays Clonogenic survival assays were performed following standard methods in triplicate wells using six-well plates. Growing cells were pretreated in six-well plates with andrographolide 120 µg/mL for 24 hours. The six-well plates were treated with a single dose of 0 (control), 2, 4, 6, or 8 Gy at approximately 2 Gy/ 40 s in a Precision X-ray 6Mv (Varian Clinac, 23EX, Milpitas, CA, USA), and then the medium was replaced to fresh medium. The cells were grown for 10 days to produce colonies of >50 cells/colony, washed once with 1 × PBS, and then stained with 0.25% crystal violet in 80% methanol. Finally, the plates were examined under the microscope, and the number of colonies with at least 50 cells was counted. Survival curves were fitted using a linear-quadratic model to estimate the sensitizer enhancement ratio (SER). Experiments were repeated three times. Cell cycle analysis ECA109 cells were incubated in a six-well cell culture cluster (1 × 106 per well) and then divided into the following groups: control (CG), simple drug (SG), radiotherapy (RG), and radiotherapy combined with drug (RCG). SG and RCG were pretreated. After culturing for 24 hours, all cells were collected, washed with PBS, and then suspended in 70% ethanol at 4°C overnight. The cells were incubated with 6 µL of 1 g/L RNase A, 1 mL of 1 mg/mL PI, and 400 µL of PBS at room temperature for 15 minutes. DNA content was analyzed using flow cytometry (FCM, BD, FACS Calibur, Franklin Lakes, NJ, USA). This procedure was repeated thrice.
MTT assay MTT assays were performed to measure the cytotoxicity and radiosensitizing effects of andrographolide. ECA109 (5 × 103) were seeded into 96-well plates overnight. The cells were divided into eight groups, and each group comprised six wells. The cells were treated with various concentrations of andrographolide (0, 7.5, 15, 30, 60, 120, 240, 360, and 480 µg/mL) and cultured for 24, 48, and 72 hours. The medium was removed, and then the cells were incubated using MTT (5 mg/mL) for 4 hours under 5% CO2 at 37°C and lysed with solubilization solution (0.15 mL, DMSO). The optical density (OD) value at 490 nm was measured using an enzymelabeled meter (Bio Rad, 680, USA). The mean value of the OD values of the six wells was considered as the result. Cell viability percentage was calculated according to the following formula: cell viability percentage = (OD value of the drug groups except C 2014 International Society for Diseases of the Esophagus V
Annexin V-FITC/PI double-labeled FCM The pre-processing of the four groups was the same as the cell cycle analysis. After culturing for 24 hours, all cells were collected and washed with 1 × PBS twice. The cells were incubated with annexin-V-FITC in the dark at room temperature for 10 minutes. Then, PI was added, and the cells were incubated in the dark at room temperature for another 10 minutes. Cell apoptosis was measured using FCM. This procedure was repeated thrice. Western blot analysis The cells were washed with 1 × PBS and harvested on ice. Lysates were prepared in a Western lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, Ethylenediamine tetra-acetic acid, Na3VO4, and © 2014 International Society for Diseases of the Esophagus
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Fig. 1 (A) Effects of various concentrations of andrographolide on the proliferation of esophageal cell line ECA109. (B) Andrographolide sensitized ECA109 cells to radiation in vitro. Survival fractions were fitted with linear quadratic model to estimate sensitizer enhancement ratio. Andrographolide, 120 µg/mL; radiation 0, 2, 4, 6, 8 Gy. The bars presented means ± SD of three separate experiments. SER, sensitizer enhancement ratio.
leupeptin) by repeatedly passing cells through a pipette. Protein concentration was determined using the BCA protein assay kit (Beyotime). An equal amount of protein for each sample was resolved using 5% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred onto a Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane. The membrane was blocked with 5% skimmed milk and sequentially incubated with primary antibody and horseradish peroxidase-conjugated secondary antibody followed by ECL detection. The nucleoprotein was extracted using the nuclear and cytoplasmic protein extraction kit. Experiments were repeated three times.
γ-H2AX for testing number of highlights in the nuclei. Bax is detected in the cytoplasm. Experiments were repeated three times. Statistical analysis All data are expressed as means ± standard deviation. Data were analyzed using SPSS 17.0 software (Chicago, IL, USA). Survival curves were fitted using GraphPad Prism 5.0 (La Jolla, CA, USA). Student t-test was applied to compare the groups as necessary. Value of P < 0.05 indicated a significant difference.
RESULTS Total nuclear protein assay
Cytotoxicity of andrographolide on ECA109 cells
The cells were processed using the nuclear protein kits, and the nuclear protein was estimated using the BCA protein assay kit. Protein samples were analyzed using Western blot analysis. Histon-3 was used as internal control. Experiments were repeated three times.
The andrographolide induced ECA109 cell death in time- and dose-dependent manner. The effects of various concentrations of andrographolide on ECA109 after 24, 48, and 72 hours of incubation are shown in Figure 1A. Andrographolide from 30 to 480 µg/mL showed inhibitory effects on ECA109 cell growth. However, the 7.5 and 15 µg/mL concentrations exhibited hormesis. Then, at 24-hour posttreatment of 240 µg/mL andrographolide, the cell death rate reached almost 50% (Table 1). The ECA109 cells treated with andrographolide for 24 hours exhibited an IC20 value of 95.39 µg/mL. Therefore, the 120 µg/mL (about IC27%) concentration was used in the follow-up study.
Immunofluorescence staining of γ-H2AX and Bax The cells were treated as defined in the figure legends. Immunofluorescent staining was performed by fixing cells with pre-chilled (−20°C) acetone-methanol for 15 minutes. The primary antibodies, anti-γ-H2AX (Santa Cruz, 1 : 100) or anti-Bax (Santa Cruz, 1 : 100), were then added to the slides at 4°C overnight. After washing, the secondary antibodies, FITC-Goat antirabbit IgG (Santa Cruz, 1 : 200), were applied for 1 hour at room temperature. The slide was then covered with Beyotime antifade mounting medium with DAPI (Beyotime, 1 : 1000). Images were taken with a fluorescent microscope (Leica, TCS SP5). Images were captured by a charge coupled device camera. For each treatment condition, γ-H2AX foci were counted in at least 100 cells from randomly captured images. © 2014 International Society for Diseases of the Esophagus
Effects of andrographolide on clonogenic cell survival in ECA109 cells The radiosensitizing effects of andrographolide were initially measured using clonogenic assay. A 1.28 SER was calculated at a surviving fraction of 37%. The colony numbers clearly decreased after andrographolide with radiotherapy compared C 2014 International Society for Diseases of the Esophagus V
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Table 1 Effect of andrographolide on the viability rate of ECA109 cells Andrographolide (µg/mL)
n
24 hours
48 hours
72 hours
0 7.5 15 30 60 120 240 360 480
3 3 3 3 3 3 3 3 3
100.00 118.13 ± 8.61 106.43 ± 10.27 97.20 ± 10.60 78.22 ± 7.42 66.19 ± 7.63* 48.39 ± 14.20 34.52 ± 13.13 20.88 ± 12.73
100.00 97.22 ± 1.79 87.74 ± 2.81 54.82 ± 6.33 29.99 ± 5.91 13.87 ± 6.51* 8.73 ± 4.42 7.41 ± 2.33 3.69 ± 1.07
100.00 96.20 ± 3.06 85.43 ± 2.78 26.95 ± 4.04 13.38 ± 4.90 6.32 ± 1.36* 3.98 ± 1.01 2.33 ± 1.06 1.05 ± 0.55
*P < 0.01, compared with control, t = 7.68/22.92/119.30.
with those after independent radiotherapy or andrographolide (Fig. 1B). Effects of andrographolide on the formation of γ-H2AX nuclear foci in ECA109 cells Ionizing radiation kills cells by inflicting various types of damage to the genome. We speculate that the radiosensitizing effect of andrographolide on cancer cells may have been caused by the impairment in the repair of DNA double-strand breaks (DSBs). We, therefore, determined the levels of DSBs via immunofluorescence staining of γ-H2AX foci in ECA109 at different time points after exposure to X-rays. A higher number of ECA109 cells exhibited γ-H2AX nuclear foci at 0.5 hour, the majority of which was cleared at 2 hours after exposure to 6 Gy of X-ray (Fig. 2). Moreover, the average number of γ-H2AX foci subsided to near basal level at 24 hours. RCG always exhibited more γ-H2AX foci than RG.
Effects of andrographolide on the protein expression of NF-κb, Cleaved-Caspase3, Bax, and Bcl-2 The expression of Cleaved-Caspase3, Bax, and Bcl-2 in the ECA109 cells was examined to further investigate the molecular mechanisms underlying the andrographolide inhibition of cell growth. Immunoblot analysis revealed that andrographolide combined with radiation could increased Bax and Cleaved-Caspase3 and decreased Bcl-2 expression compared with control or single treatment groups. We next analyzed the expression of NF-κb in nuclear and found that andrographolide could decreased NF-κb expression (Fig. 3A). Effects of andrographolide on the apoptosis protein of Bax in ECA109 cells Bax, a Bcl-2 family protein, induces cell death through the disruption of mitochondrial permeability
Fig. 2 Formation of γ-H2AX nuclear foci in andrographolide-treated ECA109 cells. ECA109 cells were pretreated with andrographolide for 24 hours, followed by double immunofluorescent staining for γ-H2AX (green). Nuclei were counter-stained with DAPI (blue). At least 200 cells in four randomly selected fields were counted to determine the percentage of cells positive for active γ-H2AX nuclear foci. The individual pictures for γ-H2AX were taken under a magnification of ×400. Experiments were repeated three times. C 2014 International Society for Diseases of the Esophagus V
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Fig. 3 (A) Western blotting analysis of expression of Cleaved-Caspase3, Bax, Bcl-2(i) and NF-κb nuclear(ii) protein. The ECA109 cells were treated with andrographolide at different time points after exposure to X-rays. The SG and RCG groups were pretreated with drug for 24 hours. Total cellular proteins were harvested and subjected to Western blotting analysis. β-actin (i) or Histon-3 (ii) was used as a loading control. Detection of β-actin in order to confirm the extraction of nucleoprotein high purity, very few cytoplasmic protein, eliminating influence. (B) Bax in ECA109 cells was immunostained using Bax as the primary antibody and phycoerythrin-conjugated IgG as the secondary antibody; more details were described under the Methods section. The individual pictures for Bax were taken under a magnification of ×400 using an immunofluorescence microscope. Representative data from three independent experiments are shown.
and subsequent release of cytochrome C. ECA109 cells were treated with 120 µg/mL andrographolide for 24 hours and the non-treated cells were used as control. The results were further confirmed by the immunofluorescence microscopic study, which showed that 120 µg/mL andrographolide increased Bax expression in ECA109 cells after 24 hours of treatment or with 6 Mv X-ray (Fig. 3B). Effects of andrographolide on cell cycle distribution The distribution of ECA109 cells in four groups of the cell cycle was analyzed using FCM after andrographolide treatment for 24 hours to determine whether andrographolide regulates the cell cycle. The percentage of cells in each phase of the cell cycle in the different groups is summarized in Figure 4A,B and Table 2. Compared with the control, an accumulation of ECA109 cells in the G0/G1, S, and G2/M phase were noted in the other groups. Effects of andrographolide on the apoptosis of ECA109 cells The rates of apoptotic cells and dead cells were analyzed with annexin V-FITC/PI double-labeled FCM 24-hour post-andrographolide treatment or radiation. RCG (32.5% ± 0.9%) showed a significantly higher apoptosis rate than RG (26.5% ± 2.2%, P < 0.05) and SG (20.8% ± 0.8%, P < 0.001). RCG (52.5% © 2014 International Society for Diseases of the Esophagus
± 4.9%) also showed significantly higher death rate than RG (30.8% ± 0.6%, P < 0.01) and SG (14.1% ± 0.5%, P < 0.001) (Fig. 4C,D and Table 3) .
DISCUSSION The acute toxicity of andrographolide in mice has demonstrated that the lethal dose of an intraperitoneal injection is 11.46 g/kg.8 In the present study, we found that low doses have proliferation effect determined by MTT experiment (Fig. 1A). Therefore, the dose of andrographolide we used for experiment in vitro (0–480 µg/mL) was within a safe range. Previous studies have shown that andrographolide inhibits malignant cancer phenotypes and induces apoptosis of cancer cells.9–11 Then, we chose the safe concentration of 120 µg/mL as the experimental dose. By clonogenic survival assays found that andrographolide can significantly sensitization esophageal cancer cells (SER = 1.28). We also found that simply using andrographolide death rate is not high; however, radiotherapy combined with drug group reflects significant death rate (Table 3). We further investigated the mechanism by which andrographolide inhibits cell growth and found that andrographolide treatment not only induces G0/G1 arrest in ECA109 cells but causes significant DNA damage, as shown in the immunofluorescence staining assay. Numerous studies have revealed that different molecular mechanisms correspond to DNA C 2014 International Society for Diseases of the Esophagus V
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Fig. 4 (A/B) Effects of andrographolide on cell cycle distribution in cultured ECA109 cells. Flow cytometric analysis of the DNA content in four groups cultured for 24 hours. (C/D) Effects of andrographolide on apoptosis rate in cultured ECA109 cells. Percentage of apoptotic cells was measured by flow cytometry after Annexin-V/propidium iodide (PI) staining. Q2 represented cell apoptosis rate; Q3 represented cell death rate.
damage checkpoints at different phases.12 The results of the current study demonstrate for the first time the radiosensitization activity of andrographolide, suggesting a potential new application of this compound in human esophageal cancer therapy. Further investigation is required to explore the clinical benefits and fully understand the mechanisms of andrographolide-mediated radiosensitization. The results of the current study show that andrographolide enhances radiation-induced apoptotic changes. Moreover, independent andrographolide
treatment was found to induce apoptosis. A similar study has shown that andrographolide induces apoptosis of human cancer cell lines (HepG2, HeLa, and MDA-MB-231).11 However, a previous study has shown that andrographolide did not induce apoptosis of H-ras-transformed rat kidney epithelial cell line.13 We speculate that such discrepancy is a result of differences in the drug concentration and incubation time (e.g. andrographolide 10 µM for 3 hours was used in a previous study, but 120 µg/mL for 24 hours was used in the current study).
Table 2 Effects of andrographolide on cell cycle analysis Groups
G0/G1 (%)
S (%)
G2/M (%)
CG SG (andrographolide 120 µg/mL) RG (6Gy) RCG (andrographolide 120 µg/mL + 6Gy)
16.57 ± 0.8 18.24 ± 1.3* 39.72 ± 1.1** 54.72 ± 1.9**
53.51 ± 3.2 48.54 ± 0.8* 37.62 ± 5.2** 25.76 ± 0.9**
29.92 ± 4.1 33.24 ± 0.7* 22.65 ± 0.6** 19.52 ± 3.2**
*P > 0.05; **P < 0.05, compared with control. Analysis of DNA in ECA109 cells. The percentage of each cell cycle was determined by flow cytometry. Compared with the control, andrographolide could induce G2/M arrest in ECA109 cells. C 2014 International Society for Diseases of the Esophagus V
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Table 3 Effects of andrographolide on cell apoptosis rate analysis Groups
Q2 (%)
Q3 (%)
SG (andrographolide 120 µg/mL) RG (6Gy) RCG (andrographolide 120 µg/mL + 6Gy)
20.8 ± 0.8 26.5 ± 2.2 32.5 ± 0.9
14.1 ± 0.5 30.8 ± 0.6 52.5 ± 4.9
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cell line20 and increased radiosensitization in K-ras-transformed human prostate epithelial 267B1 cells.21 We speculate that andrographolide-mediated radiosensitization is a result of, at least in part, the attenuation of NF-κb activation.
CONCLUSION The regulation of cell cycle on the occurrence and development of tumors has important significance. Cellular radiosensitivity varies along with the cell cycle phase, G2/M phase sensitivity of the highest, followed by G0/G1 phase of the cell, the worst S phase.14 Inducing G0/G1 arrest can increase the radiosensitivity of cancer cells.15 In the current study, we found that andrographolide treatment induces G0/G1 arrest, decreases S proportion, and has no obvious impact to G2/M in ECA109 cells. This means that the radiotherapy sensitization is by inducing G0/G1 arrest. DNA damage checkpoints are biochemical pathways that delay or arrest cell cycle progression in response to genomic DNA damage. Andrographolide treatment cause significant DNA damage, as shown in the immunofluorescence staining assay. Markers of a constitutively active DNA damage response have been described in many types of malignant lesions in different tissues.16 Therefore, we analyzed the major kinases of the signaling pathways in andrographolide-treated ECA109 cells. Investigating the DNA damage checkpoints in tumor cells will not only help in understanding the regulatory mechanisms of a cell cycle but also provide a potential theory for the development of a new tumor therapy. The oncogene-derived protein Bcl-2 negative controls the cellular suicide machinery pathway. The Bcl-2 homologous protein Bax promotes cell death by competing with Bcl-2. Bcl-2, Bax regulate mitochondrial outer membrane permeability, causing the release of cytochrome C, activation CleavedCaspase3. Our study found that, the levels of Cleaved-Caspase3 and Bax were elevated after exposure to andrographolide alone, radiation alone, and their combination (2 hours and 24 hours), whereas the Bcl-2 levels were decreased. Thus, the cell apoptosis induced by andrographolide and radiation may involve activation of the Bax/Bcl-2 pathway. Multiple signaling pathways, including PI3K/Akt, Erk, and NF-κb, have been highly associated with the pharmacological activity of andrographolide.17 We observed that radiation induced NF-κb activity in ECA109 cells, and andrographolide co-treatment reduced radiation-induced NF-κb activation. Radiationinduced NF-κb activity has been shown to cause radioresistance of different cell types.18,19 Furthermore, inhibition of NF-κb activity led to significant radiosensitization in the HT1080 fibrosarcoma © 2014 International Society for Diseases of the Esophagus
The results of the current study demonstrated that andrographolide may be a potentially promising agent of natural resource to treat esophageal cancer and a novel radiosensitizer. Its potential application in radiotherapy merits further investigation.
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19 Yamagishi N, Miyakoshi J, Takebe H. Enhanced radiosensitivity by inhibition of nuclear factor kappa B activation in human malignant glioma cells. Int J Radiat Biol 1997; 72: 157–62. 20 Wang C Y, Mayo M W, Baldwin A S. TNF- and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-kappaB. Science 1996; 274: 784–7. 21 Kim B Y, Kim K A, Kwon O et al. NF-kappaB inhibition radiosensitizes Ki-Ras-transformed cells to ionizing radiation. Carcinogenesis 2005; 26: 1395–403.
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