Zhuo Zhang

Department of Radiotherapy, the Second Hospital of Dalian Medical University

Objective:To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular mechanism. Lung cancer is one of the most common malignant tumors. Radiotherapy is the basic method of choice for the treatment of lung cancer (1), especially for middle to late stage lung cancer. Radiation works by damaging the DNA of cancerous cell and altering apoptosis-related genes or proteins, leading to cellular death. Improving the radiosensitivity of tumor cells is a significant factor that would improve the efficacy of radiotherapy. DNA-dependent protein kinase (DNA-PK) is an important enzyme that participates in DNA damage repair and has become the main target of radiation sensitivity interventions (2-4). DNA-PK catalytic subunit (cs) is the catalytic subunit of DNA-PK, which affects cellular radiosensitivity by regulating the phosphorylation of DNA damage repair related proteins (5). Inhibition of DNA-PKcs gene expression can block DNA double-strand breaks (DSB) repair and improve the cellular radiosensitivity. Cell apoptosis is the core characteristic of radiotherapy, and its regulatory mechanism plays an important role in cellular radiosensitivity (6, 7). It has been proven that apoptosis related genes like phosphoprotein (p)53, p16, B-cell lymphoma-2 (Bcl-2), and erythroblastic leukemia viral oncogene homolog 2 (erbB-2) are associated with tumor radiosensitivity (8, 9), especially p53 and Bcl-2. It was previously reported that elemene interacts with the frontier orbital of DNA base to form complexes between DNA molecules. Jiang et al., (10) have shown that elemene increases the radiosensitivity of A549 cells, and its mechanism may be related to the upregulation of p53, downregulation of Bcl-2, and induction of cell apoptosis. Elemene, which is extracted from Zingiberaceae plants (Curcuma aromatica Salisb.), is a non-cytotoxic antitumor compound that can improve the radiosensitivity of tumor cells (11). Results of an in vitro study showed that elemene increases the radiosensitivity of renal carcinoma cells, tongue squamous cancer cells, and non-small cell lung cancer cells (10, 12, 13). Animal experiments show that elemene exhibits radiotherapy sensitization effects in many kinds of tumor cells such as mice transplanted tumor U14, kidney cancer GRC-1, and tongue squamous carcinoma Tca-8113 cells (13-15). Beta elemene enhances A549 cell radiosensitivity through the enhancement of DNA damage and suppression of DNA repair (16). In the present study, A549 cells were irradiated following elemene treatment, and the changes in the expression of the apoptosis-related genes bcl-2 and p53 as well as the double-stranded DNA damage repair-related gene DNA-PKcs, were observed. These experiments were conducted to further understand the molecular mechanisms of action of elemene, in enhancing radiation sensitivity of A549 cells. Method: Cell culture: The human lung adenocarcinoma A549 cell line was purchased from the Chinese Academy of Medical Sciences (CAMS) cell center and passaged by the Second Affiliated Hospital of Dalian Medical University Center Laboratory. The cells were cultured in RPMI 1640 medium containing 10% inactivated fetal bovine serum (FBS) at 37°C and under an atmosphere of 5% CO₂ and saturated humidity. The cells were subcultured when they reached the exponential phase. Reagents and Instruments: Elemene (0.1g/20ml), which was obtained from DaLian JinGang Pharmaceutical Co. Ltd. (China), was dissolved in RPMI 1640 medium to final working concentrations of 10 and 20µg/ml before use. RPMI 1640 medium was from Gibco (USA), FBS was from TianJin TBD Biotechnology Company (China), p53 and Bcl-2 were from Santa Cruz (USA, 1:1000), DNA-PKcs (1:2000), and the β-actin mouse monoclonal antibody against human and histone H1 internuclear internal reference antibodies (1:200) were from Neomarker (USA). The Jim-X half-dry transfer electrophoresis apparatus was from DaLian JingMai Biotechnology Co. Ltd. (China). The flow cytometer was purchased from the Gene Company (USA). The CK2 type inverted microscope was obtained from the Olympus Company (Japan). The BX51 type fluorescent microscope was obtained from the Olympus Company (Japan). Irradiation conditions: Cell irradiation was performed using the Varian 2300C/D medical linear accelerator (Varian Companies, USA) with a coverage field of 20 cm × 20 cm. The culture dish was placed in the radiation field above 1.5 cm of organic glass. The cell irradiation conditions were 6 MV X-ray irradiation; dosage rate, 300 cGy/min; rack angle, 180°; and source-to-surface distance (SSD), 100 cm. Clonogenic assay: Logarithmic-growth phase cells were inoculated in a 60-mm culture dish. After adherence, the cells in the drug and combined irradiation groups were cultured in the presence of 10 or 20μg/ml elemene and seeded in culture plates at 100 cells/well for 24 h. They were then administered 0, 2, 4, 6, 8, and 10 Gy of irradiation and cultured for another 14 days. The number of cell clones viewed under a low magnification microscope was 50. The plating efficiency (PE) was calculated relative to the control group (0 Gy), and the survival fraction (SF) of each group was determined using the following equation: SF=colony number/(plating cell number × PE).The dose survival curve was fitted by the linear-quadratic (LQ) function model for calculating the radiobiological parameters, including sensitivity enhancement ratio (SER), SERDq, and SERD0. SER was calculated as follows: SER=control group (D0, Dq)/experimental group (D0, Dq).Morphological assessment of apoptosis: The cells were grouped as follows: control, received RPMI 1640 medium; radiation, received a radiation dose of 4 Gy; drug, treated with 10 or 20µg/ml elemene; and drug plus radiation, treated with 10 or 20µg/ml elemene, followed by a radiation dose of 4 Gy. Following incubation with elemene, the exponentially growing cells were irradiated as described in the above section. Samples of 3 × 105 cells were then collected from each group, treated with pancreatic enzyme digesting cells, rinsed twice with PBS, and centrifuged at 1000 rpm for 5 min. Nuclear morphology was examined using fluorescence microscopy following Hoechst 33342 staining (final concentration 8mg/ml) for 15 min at 37°C. Imaging was carried out using an Olympus BX-51 fluorescent microscope with appropriate filter cubes. The excitation and emission wavelengths were 350 nm 460 nm, respectively. Apoptosis standard: Normal cells showed uniform dispersion of low-density fluorescence, while apoptotic cells showed high-density fluorescence, characterized by a bright blue hue. Assessment of apoptosis: The cells used were grouped and treated as previously specified, in the above section. Then samples of 3 × 105 cells were collected from each group, treated with pancreatic enzyme digesting cells, rinsed twice with PBS, and centrifuged at 1000 rpm for 5 min. The cells were then treated with 100μL 2% Triton X-100 for 20 min, rinsed twice with PBS, and centrifuged at 1000 rpm for 5 min. Then, 200μL of DNA-Prep LPR reagent (Beckman-Coulter Ltd) was added for 20 min, and the cells were rinsed twice with PBS, followed by centrifugation at 1000 rpm for 5 min. The cells were then resuspended in PBS and 50µg/ml propidium iodide (PI) reagent containing 480μL of PBS, 5μL of PI (5mg/ml), 5μL of RNase (10mg/ml), and 10μL of Triton X-100 (10%) was added 30 s later. Single cell suspensions were analyzed by flow cytometry for the cellular apoptosis rate. Neutral comet assay: The slides were submerged in lysing solution containing 30 mM ethylenediaminetetraacetic acid (EDTA) and 0.5% sodium dodecyl sulfate (SDS, pH 8.3) for 1.5 h at 37°C. Following lysis, the slides were rinsed three times in Tris-borate-EDTA (TBE) buffer consisting of 90 mM Tris, 90 mM boric acid, 2 mM EDTA, pH 8.5, and stored overnight in TBE buffer at 4°C. Slides were then transferred to an electrophoresis unit with TBE buffer and electrophoresed at 1 V/cm for 20 min. Following electrophoresis, the slides were neutralized with 0.4 M Tris buffer (pH 7.5) and stained with ethidium bromide (20µg/ml). Finally, the slides were viewed using an Olympus BX‐51 fluorescent microscope (excitation filter 549 nm, barrier filter 590 nm). Images of 50 randomly selected cells from each slide were analyzed with Comet Assay Software Project casp‐1.2.2 (University of Wroclaw, Poland). The tail moment was used as a parameter to assess DNA damage. Assay was completed three separate times, and 50 cells were evaluated per experiment. Western blot assay: Western blotting was used to detect the expression levels of the DNA-PKcs, p53, and Bcl-2 genes. The cells were grouped and treated as previously specified above. After 24 h, western blot analysis was performed using cytosolic fractions as previously described (17). Equal amounts of cytosolic protein were separated on 8–12% SDS- polyacrylamide denaturing gels and then transferred to nitrocellulose membranes. The membranes were then blocked in TBS-Tween (TBS-T, 10 mM Tris-HCl, pH 7.4; 150 mM NaCl; and 0.1% Tween-20) with 5% non-fat milk for 2 h and incubated with specific primary antibodies overnight at 4°C. Finally, membranes were incubated with horseradish peroxidase (HRP)-linked secondary antibodies at 37°C for 2 h and assayed using an enhanced chemiluminescence plus detection system. Statistical analysis: Data were analyzed using the statistical package for the social sciences (SPSS) v13.0 software. Data are expressed as mean ± standard deviation (SD). The statistical significance of differences between groups was determined by a one-way analysis of variance (ANOVA), followed by post hoc analysis using the least significant difference (LSD) for multiple comparisons. The Spearman test was used for the correlation analysis of the relationships between the expressions of genes. The level of significance was set at P<0.05 and P<0.01 for all statistical analysis. Result: Effects of elemene on cell radiosensitivity. The survival fraction of A549 cells decreased following treatment with different doses of radiation with the same concentration of elemene. Conversely, the A549 cell survival fraction decreased in the groups treated with the same dose of radiation in combination with increasing concentrations of elemene (Table 1). Following treatment with 10 or 20μg/ml elemene, the A549 cell survival curve shifted to the left, and the shoulder area diminished. The steepness of the curve increased (Figure 1). Based on the cell survival curve, the radiobiological parameters and radiosensitization ratio were obtained and are listed in Table 1. The data show that, compared with the control group, the SERD0 and SERDq values for the 10 and 20μg/ml elemene groups were greater than 1. In addition, the ratio gradually increased with increasing drug concentration (Table 2). Effect of elemene on A549 cell apoptosis Fluorescence microscopy showed that, compared with the control, the groups treated with radiation alone and elemene alone, had more apoptotic cells (Figure 2). Furthermore, significantly higher apoptotic levels were observed in the groups treated with radiation and elemene at 10 or 20μg/ml (Figure 2A). Flow cytometry revealed that compared with the control group, the apoptosis rate of the group treated with radiation alone appeared to increase, but this was not statistically significant (P>0.05). The apoptosis rate of the group treated with elemene alone, showed no change (P>0.05), while that for elemene plus radiation group increased significantly (P<0.01). The rate of apoptosis increased with increasing concentration of elemene (Figure 2B). Effect of elemene on DSB damage repair in A549 cells treated with 10 or 20µg/ml elemene for 24 h showed a statistically significant increase in the tail intensity compared with the control group (P<0.01). This result suggests that elemene induces the production of DSB in A549 cells. Immediately after the cells were exposed to irradiation, the tail intensity of the combined treatment groups, compared with the radiation only and drug only groups, were significantly increased (P<0.01). This result indicates that elemene combined with radiation increases the DSB damage in A549 cells. Following incubation for 24 h and irradiation, the tail intensity of the combination group was significantly reduced as compared with the radiation only and drug only groups. This result suggests that elemene combined with radiation effectively inhibits the DSB damage repair in A549 cells (P<0.01, Figure 3). Effects of elemene on DNA-PKcs, Bcl-2, and p53 protein expression in A549 cells Results are shown in Figure 4A. In the 10 and 20μg/ml combined treatment groups, significant decreases in the protein expression of DNA-PKcs (P<0.01, Figure 4B) and Bcl-2 (P<0.01, Figure 4C) were observed, while the p53 protein expression level was significantly increased (P<0.01, Figure 4C). Protein expression correlation analysis. The Spearman correlation analysis showed that DNA-PKcs and p53 protein expression had a negative correlation (r=-0.569, P<0.05), while DNA-PKcs was positively correlated with Bcl-2 protein expression (r=0.755, P<0.05). Conclusion: Basic research in radiation biology shows that radiation therapy works mainly by damaging tumor cell DNA and altering the expression of apoptosis-related genes and proteins. The radiosensitivity of tumor cells relates to their capability to repair double-stranded DNA breaks via the related genes DNA-PKcs, Ku70/80, and ataxia telangiectasia mutated (ATM). Other genes known to be involved in radiosensitivity and responsible for apoptosis regulation include p53, Bcl-2, c-mycproto-oncogene (c-myc), and survivin (9). Beta elemene, which is the active component of elemene, has recently been demonstrated to enhance the radiosensitivity of human cancer cell lines, in vitro and a specific animal tumor, in vivo (16, 18). Beta elemene enhances radiosensitivity by influencing the cell cycle distribution of the gastric cancer MKN28 cells. The mechanisms involved include induction of G2/M phase arrest, inhibition of the repair of sublethal damage, and induction of cell apoptosis, which enhances the killing effects of radioactive rays (19). The results of the present study showed that the SERD0 and SERDq values of A549 cells exposed to a low concentration of the cytotoxic elemene were greater than 1. In addition, elemene enhanced the sensitivity of A549 cells to radiotherapy. Cell apoptosis is fundamental to radiotherapy, and its regulatory mechanism plays an important role in cellular radiosensitivity. Apoptosis-related genes such as p53 and Bcl-2 have important regulatory functions in the process of rapid apoptosis induced by radiation therapy. A previous study has shown that the levels of the antiapoptotic genes Bcl-2 and Bcl-xl in A549 cells decreased while the expression of p53 and the production of exosomes increased following elemene treatment (20). This shows that both p53 and Bcl-2 have important regulatory actions in cervical cancer cell apoptosis induced by radiation. A number of experimental studies show that elemene is involved in regulating the expression of Bax, C-myc, p53, poly (ADP-ribose) polymerase (PARP), survivin, and livin, and induction of tumor cells apoptosis (21-24). Our results showed that, compared with the pure exposure group, the group that received elemene combined with irradiation, exhibited increased p53 gene expression and significantly decreased Bcl-2 gene expression. The expressions of both genes were significantly correlated. Furthermore, elemene was shown to regulate the expression of the apoptosis-related genes Bcl-2 and p53 and induce A549 cell apoptosis, thereby increasing the radiosensitivity of cells. It is noteworthy that when Bcl-2 and p53 gene expression was significantly altered, DNA-PKcs protein expression decreased significantly in the A549 cells in the combined treatment group. This indicates that elemene is also involved in the regulation of DNA damage repair pathways. The activation of protein kinase subsequently increases its levels and leads to the phosphorylation of the downstream DNA repair proteins, which initiate the DNA chain fracture repair (25). The relationship between DNA-PKcs and radiotherapy sensitivity has been under scrutiny in recent years. It has been established that inhibiting tumor cell expression of DNA-PKcs increases their radiation sensitivity. Pan (26) studied the relationship between the expression of DNA-PKcs and radiation sensitivity in non-small cell lung cancer cell lines. In adenocarcinomas and large cell carcinomas, DNA-PKcs is an important component in the cellular radiosensitivity. This indicates that DNA-PKcs might be a predictive index of non-small cell lung cancer cell radiosensitivity. Zou (27) silenced the DNA-PKcs gene of human mammary epithelial cells (MCF10F) by using small interfering RNA (siRNA) technology. Simultaneously, the expression of DNA repair-related proteins, such as DNA-PKcs, Ku80, ATM, and p53 decreased in the cells, and the sensitivity of the cells increased with low doses of radiation. Small molecule inhibitors of DNA-PKcs enhance the radiation sensitivity of cervical cancer cells (28). Our experimental results showed that elemene inhibits DNA-PKcs expression in A549 cells, reduces DNA damage repair, and increases cellular radiosensitivity. DNA-PKcs is a protein with a wide range of functions and is involved in DNA damage repair, apoptosis, and V(D)J recombination (29). Yu (30) found that in non-small cell lung cancer, high expression of DNA-PKcs increased the activity of DNA damage repair system. In addition, the apoptosis inhibition caused by mutant p53 and Bcl-2 expression exhibited a combined effect and influenced each other. This may be the major cause of development of resistance to radiotherapy in small-cell lung cancer. Daido (31) indicated that following exposure to low doses of radiation, human malignant glioma M059J cells that lack DNA-PKcs underwent massive autophagic cell death that significantly increased after exposure to DNA-PKcs inhibitors. Furthermore, DNA-PKcs inhibitors exert radiotherapy sensitization effects on glioma cells by enhancing typeⅡprogrammed cell death. Li (32) found that the p53-inducible gene 3 (PIG3) molecule is involved in apoptosis caused by p53 activation and can regulate the expression of DNA-PKcs. The knock down of PIG3 decreases the level of DNA-PKcs in cells. This experiment further studied the correlation between DNA-PKcs, Bcl-2, and p53 expressions. The results showed that DNA-PKcs expression was significantly positively correlated with that of Bcl-2 (r=0.755, P<0.05) and was significantly negatively correlated with p53 expression (r=0.569, P<0.05). It was further shown that DNA-PKcs is closely related to apoptosis and that elemene increases the apoptosis of A549 cells and strengthens the cellular radiosensitivity by inhibiting the expression of DNA-PKcs. In summary, elemene has radiotherapy sensitization effects on lung adenocarcinoma A549 cells, and its mechanism of action involves upregulation of p53 and downregulation of Bcl-2 gene expressions to promote cell apoptosis, as well as downregulation of the expression of DNA-PKcs to inhibit the repair of double-strand DNA breaks. The specific mechanism of action requires further elucidation.

Key Words: elemene  Radiosensitivity