Abstract
OBJECTIVE:
To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular mechanism.
METHODS:
Apoptosis of A549 cells was detected by flow cytometry and fluorescence microscopy. The effect of double-strand break (DSB) damage repair in A549 cells was evaluated using the neutral comet assay. Protein expression levels were detected using western blotting, and the correlation between protein levels was analyzed.
RESULTS:
Elemene exhibited a radiosensitizing effect on A549 cells. The level of apoptosis induced by elemene combined with radiation was significantly greater (p<0.01) than that elicited by either radiation or elemene alone. Following radiation and subsequent repair for 24 h, the tail intensity of A549 cells treated with a combination of elemene and radiation was greater than that of cells treated with either elemene or radiation alone (p<0.01). This result indicates that elemene inhibits cellular DSB repair. Both elemene combined with radiation and radiation alone decreased the protein expression of DNA-PKcs and Bcl-2 compared to elemene alone (p<0.01), while p53 protein expression was increased (p<0.01). A negative correlation was observed between DNA-PKcs and p53 expression (r=−0.569, p=0.040), while a positive correlation was found between DNA-PKcs and Bcl-2 expression (r=0.755, p=0.012).
CONCLUSIONS:
Elemene exhibits a radiosensitizing effect on A549 cells, and its underlying molecular mechanism of action may be related to the downregulation of DNA-PKcs gene expression.
Elemene; Radiosensitivity; A549 cells; DNA-PKcs; Bcl-2; p53
INTRODUCTION
Lung cancer is one of the most common malignant tumors, and radiotherapy is the
prevailing method of choice for treatment (11. Maciejczyk A, Skrzypczynska I, Janiszewska M. Lung cancer.
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), especially for middle- to late-stage lung cancer. Radiation works by
damaging the DNA of cancerous cells and altering apoptosis-related genes or
proteins, leading to cell 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. Collis SJ, Swartz MJ, Nelson WG, DeWeese TL. Enhanced radiation
and chemotherapy-mediated cell killing of human cancer cells by small inhibitory
RNA silencing of DNA repair factors. Cancer Res. 2003;
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hyper-radiosensitivity and increased radioresistance. Int J Radiat Biol.
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The catalytic subunit (cs) of DNA-PK affects cellular radiosensitivity by regulating
the phosphorylation of DNA damage repair-related proteins (55. Jackson SP. Sensing and repairing DNA double-strand breaks.
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). Thus, inhibition of DNA-PKcs gene expression can block DNA
double-strand break (DSB) repair and improve cellular radiosensitivity.
Cellular apoptosis is the core characteristic of radiotherapy, and regulation of this
process thus plays an important role in cellular radiosensitivity (66. Kurdoglu B, Cheong N, Guan J, Corn BW, Curran WJ, Jr, Iliakis G.
Apoptosis as a predictor of paclitaxel-induced radiosensitization in human tumor
cell lines. Clin Cancer Res. 1999;5(9):2580–7.,77. Wang LP, Liang K. Apotosis and Radiosensitivity of Cells. Foreign
Medical Science. 2000;24(2):75–8.).
Previous studies have shown that apoptosis-related genes, such as phosphoprotein
(p53), p16, B-cell lymphoma-2 (Bcl-2), and erythroblastic leukemia viral oncogene
homolog 2 (erbB-2), are associated with tumor radiosensitivity (88. Rosen EM, Fan S, Rockwell S, Goldberg ID. The molecular and
cellular basis of radiosensitivity: implications for understanding how normal
tissues and tumors respond to therapeutic radiation. Cancer Invest.
1999;17(1):56–72, 10.3109/07357909909011718.
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,99. Rosen EM, Fan S, Goldberg ID, Rockwell S. Biological basis of
radiation sensitivity. Part 2: Cellular and molecular determinants of
radiosensitivity. Oncology (Williston Park). 2000;14(5):741–57; discussion
757–8, 761–6.),
especially p53 and Bcl-2. It has also been reported that elemene interacts with the
frontier orbitals of DNA bases to form complexes between DNA molecules.
Specifically, Jiang et al. (1010. Jiang H, Ma S, Feng J. In vitro study of radiosensitization of
β-elemene in A549 cell line from denocarcinoma of lung. Chinese-German J of Clin
Oncol. 2009;8(1):12–5, 10.1007/s10330-008-0139-3.
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) showed that
elemene increases the radiosensitivity of A549 cells, the mechanism for which may be
related to the upregulation of p53, downregulation of Bcl-2, and induction of
cellular 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 (1111. Zou K, Tong E, Xu Y, Deng X, Zou L. Down regulation of mammalian
target of rapamycin decreases HIF-1α and survivin expression in anoxic lung
adenocarcinoma A549 cell to elemene and/or irradiation. Tumor Biol.
2014;35(10):9735–41, 10.1007/s13277-014-2226-0.
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). Results of an in vitro study showed that elemene
increased the radiosensitivity of renal carcinoma cells, tongue squamous cancer
cells, and non-small cell lung cancer cells (1010. Jiang H, Ma S, Feng J. In vitro study of radiosensitization of
β-elemene in A549 cell line from denocarcinoma of lung. Chinese-German J of Clin
Oncol. 2009;8(1):12–5, 10.1007/s10330-008-0139-3.
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,1212. Cheng W, Li JP, Wang ZM, Song W, Huang C. The variation of gene
expression profile in radiosensitivity to kidney cancer cells induced by
elemenen. Chin J Urol. 2007;28(2):87–90.,1313. Wu DP, Li XM, Zhao JF, Wang HB, Zhao DQ. A Study of
Radiosensitivity of β-Elemene to Squamous Cell Carcinoma of Tongue Tca-8113 Cell
Line in Vitro. Journal of Basic and Clinical Oncology.
2006;19(2):116–7.). Animal experiments further showed that elemene exhibited
radiotherapy-sensitizing effects in many types of tumor cells, such as transplanted
murine U14 tumors, kidney cancer GRC-1 cells, and tongue squamous carcinoma Tca-8113
cells (13. Wu DP, Li XM, Zhao JF, Wang HB, Zhao DQ. A Study of
Radiosensitivity of β-Elemene to Squamous Cell Carcinoma of Tongue Tca-8113 Cell
Line in Vitro. Journal of Basic and Clinical Oncology.
2006;19(2):116–7.
14. Zou LJ, Sun XH, Xu XY. The study of the β-elemene effect on the
augment of radiation treatment in mouse inoculated U14 strain. Chin J
Radiological Medicine and Protection. 2004;24(3):254–5.-1515. She JJ, Wang ZM, Che XM, Pan CE. Radiosensitization of
beta-elemene on VX2 carcinoma transplanted on kidney in rabbits in vivo. Zhong
Xi Yi Jie He Xue Bao. 2006;4(4):392–6, 10.3736/jcim20060415.
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).
In addition, beta elemene enhances A549 cell radiosensitivity through enhancement of
DNA damage and suppression of DNA repair (1616. Li LJ, Zhong LF, Jiang LP, Geng CY, Zou LJ. β-Elemene
radiosensitizes lung cancer A549 cells by enhancing DNA damage and inhibiting
DNA repair. Phytother Res. 2011; 25(7):1095–7,
10.1002/ptr.v25.7.
http://dx.doi.org/10.1002/ptr.v25.7...
).
In the current study, A549 cells were irradiated following elemene treatment, and the changes in expression of the apoptosis-related genes Bcl-2 and p53 as well as the double-stranded DNA damage repair-related gene DNA-PKcs were evaluated. These experiments were conducted to further understand elemene’s molecular mechanism of action in enhancing radiation sensitivity of A549 cells.
MATERIALS AND METHODS
Cell culture
The human lung adenocarcinoma A549 cell line was purchased from the Chinese Academy of Medical Sciences (CAMS) Cell Center and passaged at 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 under an atmosphere of 5% CO2 and saturated humidity. The cells were subcultured when they reached the exponential phase.
Reagents and instruments
Elemene (0.1 g/20 mL), which was obtained from DaLian JinGang Pharmaceutical Co. Ltd. (China), was dissolved in RPMI 1640 medium to final working concentrations of 10 and 20 µg/mL before use. RPMI 1640 medium was obtained from Gibco (USA); FBS was obtained from TianJin TBD Biotechnology Company (China); p53 and Bcl-2 antibodies were obtained from Santa Cruz (USA, 1:1,000); and DNA-PKcs antibody (1:2,000), anti-human β-actin mouse monoclonal antibody and histone H1 internuclear internal reference antibodies (1:200) were obtained from Neomarker (USA). The Jim-X half-dry transfer electrophoresis apparatus was obtained 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 Olympus (Japan). The BX51 type fluorescent microscope was also obtained from Olympus (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. Cells with irradiated with 6 MV X-ray irradiation at a dosage rate of 300 cGy/min, a rack angle of 180°, and source-to-surface distance (SSD) of 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 20 μg/mL elemene and seeded in culture plates at 100 cells/well for 24 h. The cells were exposed to 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 calculated as follows: SER=control group (D0, Dq)/experimental group (D0, Dq).
Morphological assessment of apoptosis
The cells were treated as follows: the control group received RPMI 1640 medium; the radiation group received a radiation dose of 4 Gy; the drug group was treated with 10 or 20 µg/mL elemene; and the drug plus radiation group was treated with 10 or 20 µg/mL elemene followed by a radiation dose of 4 Gy. Following incubation with elemene, the exponentially growing cells were irradiated as described above. Samples of 3 × 105 cells were then collected from each group, treated with pancreatic enzyme digesting cells, rinsed twice with PBS, and centrifuged at 1,000 rpm for 5 min. The SF was determined using the following equation:
SF=colony number/(plating cell number × PE).
The dose survival curve was fitted using the linear-quadratic (LQ) function model
S=e-(αd+βd2) (1717. Lindblom E, Dasu A, Lax I, Toma-Dasu I. Survival and tumour
control probability in tumours withheterogeneous oxygenation: A comparison
between the linearquadratic and the universal survival curve models for high
doses. Acta Oncol. 2014;53(8):1035–40,
10.3109/0284186X.2014.925582.
http://dx.doi.org/10.3109/0284186X.2014....
)
for calculating radiobiological parameters, including the sensitivity
enhancement ratio (SER), SER of the mean lethal dose (D0) SERDq, and
SER of the quasi-threshold dose (Dq) SERD0. Nuclear morphology was
examined using fluorescence microscopy following Hoechst 33342 staining (final
concentration 8 mg/mL) for 15 min at 37°C. Imaging was performed 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 demonstrated 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 specified above. Then, samples of 3 × 105 cells were collected from each group, treated with pancreatic enzyme digesting cells, rinsed twice with PBS, and centrifuged at 1,000 rpm for 5 min. The cells were treated with 100 μL 2% Triton X-100 for 20 min, rinsed twice with PBS, and centrifuged at 1,000 rpm for 5 min. Next, 200 μL 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 1,000 rpm for 5 min. The cells were resuspended in PBS and 50 µg/mL propidium iodide (PI) reagent containing 480 μL of PBS, 5 μL of PI (5 mg /mL), and 5 μL of RNase (10 mg /mL), and 10 μL of Triton X-100 (10%) was added 30 s later. Single-cell suspensions were analyzed by flow cytometry to determine the cellular apoptosis rate.
Neutral comet assay
A549 cells were irradiated in the absence or presence of elemene (10 or 20 µg/mL)
and assayed immediately after radiation or returned to the incubator for 24 h to
permit repair. The comet assays were performed immediately after incubation with
elemene, irradiation or combination treatment. Tumor cells were grown on glass
microscope slides using a standard protocol (1818. Forchhammer L, Johansson C, Loft S, Moller L, Godschalk RW,
Langie SA, et al. Variation in the measurement of DNA damage by comet assay
measured by the ECVAG inter-laboratory validation trial. Mutagenesis.
2010;25(2):113–23, 10.1093/mutage/gep048.
http://dx.doi.org/10.1093/mutage/gep048...
). 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, and 2
mM EDTA, pH 8.5, and stored overnight in TBE buffer at 4°C. Slides were
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 (20 µg/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. The assay was completed three separate times, and 50 cells
were evaluated per experiment.
Western blot assay
Western blotting was performed to detect the expression levels of DNA-PKcs, p53,
and Bcl-2. The cells were grouped and treated as specified above. After 24 h,
western blot analysis was performed using cytosolic fractions as previously
described (1919. Li Z, Berk M, McIntyre TM, Gores GJ, Feldstein AE. The
lysosomal-mitochondrial axis in free fatty acid-induced hepatic lipotoxicity.
Hepatology. 2008;47(5):1495–503, 10.1002/(ISSN)1527-3350.
http://dx.doi.org/10.1002/(ISSN)1527-335...
). Equal amounts of
cytosolic protein were separated on 8–12% SDS polyacrylamide denaturing gels and
transferred to nitrocellulose membranes. The membranes were 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, the membranes were incubated with horseradish peroxidase
(HRP)-conjugated 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 the mean ± standard deviation (SD). The statistical significance of differences between groups was determined by 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 expression levels of genes. The level of significance was set at p<0.05 and p<0.01 for all statistical analysis.
RESULTS
Effects of elemene on cell radiosensitivity
The survival fraction of A549 cells decreased following treatment with different doses of radiation and 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 20 μg/mL elemene, the A549 cell survival curve shifted to the left, the shoulder area was diminished, and 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. These data show that, compared with the control group, the SERD0 and SERDqvalues for the 10 and 20 μg/mL elemene treatment groups were greater than 1. Furthermore, the ratio gradually increased with an increasing drug concentration (Table 2).
Cell survival curves of A549 cells following treatment with 0, 10 or 20 µg/mL elemene and exposure to x-ray doses from 0 to 10 Gy.
Effect of elemene on A549 cell apoptosis
Fluorescence microscopy showed that compared with the control group, the groups treated with radiation alone and elemene alone contained more apoptotic cells (Figure 2). Furthermore, significantly higher apoptotic levels were observed in the groups treated with radiation and elemene at 10 or 20 μg/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 found to be statistically significant (p>0.05). The apoptosis rate of the group treated with elemene alone did not change (p>0.05), while the apoptosis rate of the elemene plus radiation group increased significantly (p<0.01). The rate of apoptosis increased with increasing concentrations of elemene (Figure 2B).
Elemene induces apoptosis in A549 cells. Cells were incubated with 10 or 20 μg/mL elemene for 24 h, followed by irradiation with 4 Gy X-rays and washing with PBS. (A) Cells were imaged with an Olympus BX-51 fluorescent microscope using appropriate filter cubes. (B) Chromatin condensation was analyzed by fluorescence microscopy after DNA staining with Hoechst 3334. The results are expressed as the mean ± SD of three independent experiments, n=3, **p<0.01 compared to control, ##p<0.01 compared to radiation alone.
Effect of elemene on DSB repair in A549 cells
Figure 3 (0 h) shows that both elemene and irradiation alone increased the tail intensity in A549 cells, and the degree of DSB was augmented as the elemene concentration increased. In particular, the results showed a higher number of DSB in the combination group than the irradiation alone and elemene alone groups (p<0.01). As shown in Figure 3 (24 h), following incubation for 24 h, the tail intensity in the irradiation alone group returned to background levels, while there was a significant increase in the amount of remaining tail intensity in the combination group compared with the irradiation alone and elemene alone groups (p<0.01).
Influence of elemene on radiation-induced DSB. A549 cells were irradiated with 4 Gy X-rays in the absence or presence of elemene (10 or 20 μg/mL) and assayed immediately after radiation or returned to the incubator for 24 h to permit repair. Comet assays were performed immediately after incubation with elemene treatment, irradiation, or combination treatment. Columns represent the means of the tail moments from three independent experiments, and the bars represent the SD, **p<0.01 vs. control, ##p<0.01 compared to radiation alone.
Effects of elemene on DNA-PKcs, Bcl-2, and p53 protein expression in A549 cells
The results shown in Figure 4A revealed that in the 10 and 20 μg/mL combined treatment groups, a significant decrease in the protein expression of DNA-PKcs (p<0.01, Figure 4B) and Bcl-2 (p<0.01, Figure 4C) was observed, while the p53 protein expression level was significantly increased (p<0.01, Figure 4C).
Western blot analysis of the protein levels of Bcl-2, p53 and DNA-PKcs. A549 cells were irradiated with 4 Gy X-rays following treatment with 10 or 20 μg/mL elemene for 24 h. Proteins were extracted and separated by SDS-PAGE. (A) Levels of Bcl-2, p53 and DNA-PKcs were quantitated by densitometry, and the ratios of the three proteins are displayed. Values represent the mean ± SD, n=3, **p<0.01 compared to the control, ##p<0.01 compared to radiation alone. Elemene inhibited the protein expression of (B)DNA-PKcs and (C) Bcl-2 and promoted the protein expression of p53. Key: 1, Control; 2, irradiation; 3, 10 μg/mL; 4, 20 μg/mL; 5, 10 μg/mL + irradiation; 6, 20 μg/mL + irradiation.
Protein expression correlation analysis
Spearman correlation analysis showed that DNA-PKcs and p53 protein expression was negatively correlated (r=−0.569, p<0.05), while DNA-PKcs expression was positively correlated with Bcl-2 protein expression (r=0.755, p<0.05).
DISCUSSION
Basic research in radiation biology has shown 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 capacity to repair
DSB 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-myc proto-oncogene (c-myc), and survivin
(99. Rosen EM, Fan S, Goldberg ID, Rockwell S. Biological basis of
radiation sensitivity. Part 2: Cellular and molecular determinants of
radiosensitivity. Oncology (Williston Park). 2000;14(5):741–57; discussion
757–8, 761–6.). 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 in an animal tumor model
in vivo (1616. Li LJ, Zhong LF, Jiang LP, Geng CY, Zou LJ. β-Elemene
radiosensitizes lung cancer A549 cells by enhancing DNA damage and inhibiting
DNA repair. Phytother Res. 2011; 25(7):1095–7,
10.1002/ptr.v25.7.
http://dx.doi.org/10.1002/ptr.v25.7...
,2020. Li G, Xie B, Li X, Chen Y, Wang Q, Xu Y, et al. Down-regulation
of survivin and hypoxia-inducible factor-1 alpha by beta-elemene enhances the
radiosensitivity of lung adenocarcinoma xenograft. Cancer Biother Radiopharm.
2012;27(1):56–64, 10.1089/cbr.2011.1003.
http://dx.doi.org/10.1089/cbr.2011.1003...
). In particular, beta elemene was found to
enhance radiosensitivity by influencing the cell cycle distribution of gastric
cancer MKN28 cells, and the mechanisms responsible for this effect include the
induction of G2/M phase arrest, inhibition of sublethal damage repair, and induction
of cell apoptosis, which enhances the killing effects of radioactive rays (2121. He S, Liu J, Zhang Z, Che X, Fan L, Chang S, et al. Enhancement
of gastric cancer MKN28 cell line radiosensitivity induced by beta-elemene.
Zhonghua Wai Ke Za Zhi. 2014;52(6):442–5.). The results of the current study show that
the SERD0 and SERDq values of A549 cells exposed to a low
concentration of cytotoxic elemene were greater than 1. In addition, elemene
enhanced the sensitivity of A549 cells to radiotherapy. Cellular 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 progression of rapid apoptosis induced by
radiation therapy. For instance, a previous study showed that the levels of the
antiapoptotic genes Bcl-2 and Bcl-xl in A549 cells decreased, while p53 expression
and the production of exosomes increased, following elemene treatment (2222. Li J, JunYu, Liu A, Wang Y. β-Elemene against human lung cancer
via up-regulation of P53 protein expression to promote the release of exosome.
Lung Cancer. 2014;86(2):144–50, 10.1016/j.lungcan.2014.08.015.
http://dx.doi.org/10.1016/j.lungcan.2014...
). This result demonstrates that both p53 and
Bcl-2 have important regulatory actions in cervical cancer cell apoptosis induced by
radiation. A number of experimental studies have further shown that elemene is
involved in regulating the expression of Bax, c-myc, p53, poly (ADP-ribose)
polymerase (PARP), survivin, and livin as well as inducing tumor cell apoptosis
(23. Zhang Y, Zhao MF, Liu YP. The effects of β-elemene on the
activation of Akt and the expressions of apoptosis-related proteins in gastric
cancer. Shanxi Oncology Medicine. 2012;3:451–4.
24. Zhou XQ, Qiu XJ, Zhao HY. Influence on apoptosis relevant
protein expression of human lung adenocarcinoma A549 cell line managed by
Elemene. Journal of Modern Oncology. 2012; 20(10):2027–30.
25. Yang WZ, Chen CM, Shi SS. The Study on inhibition of the
expression of c-myc gene of human glioma U251cell line and the effect mechanism
of effect on cellular apoptosis by elemene. Chin J Clin Oncol.
2005;32(13):763–6.-2626. Zhao QT, Yang Y, Sun CB. Effect of β-elemene combined with
radiotherapy on the expression of Livin mRNA and apoptosis of lung
adenocarcinoma cell line A549. Journal of Modern Oncology. 2013;
21(2):257–60.).
Our results showed that, compared with the exposure alone group, the group that
received elemene combined with irradiation exhibited increased p53 gene expression
and significantly decreased Bcl-2 gene expression, and the expression of both genes
was significantly correlated. Furthermore, elemene was shown to regulate expression
of the apoptosis-related genes Bcl-2 and p53 and induce A549 cell apoptosis, thereby
increasing cell radiosensitivity.
Interestingly, when Bcl-2 and p53 gene expression was significantly altered, DNA-PKcs
protein expression was significantly decreased in the combined treatment group. This
result indicates that elemene is also involved in regulating DNA damage repair
pathways. Protein kinase activation leads to the phosphorylation of downstream DNA
repair proteins, which initiate DNA chain fracture repair (2727. Calsou P, Delteil C, Frit P, Drouet J, Salles B. Coordinated
assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends
is necessary for XRCC4-ligase IV recruitment. J Mol Biol. 2003;326(1):93–103,
10.1016/S0022-2836(02)01328-1.
http://dx.doi.org/10.1016/S0022-2836(02)...
), and the relationship between DNA-PKcs and radiotherapy
sensitivity has been a topic of significant research in recent years. It is well
established that inhibiting tumor cell expression of DNA-PKcs increases radiation
sensitivity. Panet al. (2828. Pan Y, Li WX, Li JM, Zhu JQ, Liang YQ, Guo AL. Correlation of
DNA-dependent protein kinase catalytic subunit expression to radiosensitivity of
non-small cell lung cancer cell lines. Ai Zheng.
2009;28(7):714–7.) studied the
relationship between DNA-PKcs expression and radiation sensitivity in non-small cell
lung cancer cell lines, and in adenocarcinomas and large cell carcinomas, DNA-PKcs
was shown to be an important component regulating cellular radiosensitivity. This
result indicates that DNA-PKcs may be predictive of non-small cell lung cancer cell
radiosensitivity. Zou et al. (2929. Zou W, Che J, Wang CJ. DNA-PKcs silencing inhibit the DNA repair
induced by low dose radiation on human breast epithelial cells. Chin J Biotech.
2009;25(5):727–32.) silenced
the DNA-PKcs gene of human mammary epithelial cells (MCF10F) using small interfering
RNA (siRNA) technology. Simultaneously, the expression of DNA repair-related
proteins, such as DNA-PKcs, Ku80, ATM, and p53, was decreased in these cells, while
their sensitivity increased with low doses of radiation. Small molecule inhibitors
of DNA-PKcs were also shown to enhance radiation sensitivity of cervical cancer
cells (3030. An J, Sui JL, Xu QZ. Inhibition of DNA-PKcs by siRNA and its
effect on the growth of HeLa cells. Carcinogenesis, Teratogenesis &
Mutagenesis. 2005;17(6):327–31.). Our experimental results showed
that elemene inhibited DNA-PKcs expression in A549 cells, reduced DNA damage repair,
and increased 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 (3131. Yan YQ, Zhou PK. Mechanism of non-homologous end joing and its
bilolgical implications. Journal of Medical Molecular Biology. 2006;
3(1):69–72.). Yu et al. (3232. Yu S, Xiong Y, Tian S. The expression of DNA-PKcs in non-small
cell lung cancer and its relationship with apoptosis associated proteins.
Zhongguo Fei Ai Za Zhi. 2003;6(5):356–9.) found that in
non-small cell lung cancer, high expression of DNA-PKcs increased the activity of
the DNA damage repair system. In addition, apoptosis inhibition caused by mutant p53
and Bcl-2 expression exhibited a combined effect, which may explain the development
of resistance to radiotherapy in small-cell lung cancer. Daido et al. (3333. Daido S, Yamamoto A, Fujiwara K, Sawaya R, Kondo S, Kondo Y.
Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes
malignant glioma cells by inducing autophagy. Cancer Res. 2005;65(10):4368–75,
10.1158/0008-5472.CAN-04-4202.
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) indicated that following exposure to low
doses of radiation, human malignant glioma M059J cells that lack DNA-PKcs underwent
massive autophagic cell death that was significantly increased after exposure to
DNA-PKcs inhibitors. Furthermore, DNA-PKcs inhibitors exert radiotherapy-sensitizing
effects on glioma cells by enhancing type II programmed cell death. Lee et al.
(3434. Lee JH, Kang Y, Khare V, Jin ZY, Kang MY, Yoon Y, et al. The
p53-inducible gene 3 (PIG3) contributes to early cellular response to DNA
damage. Oncogene. 2010;29(10):1431–50, 10.1038/onc.2009.438.
http://dx.doi.org/10.1038/onc.2009.438...
) found that p53-inducible gene 3
(PIG3) is involved in apoptosis caused by p53 activation and that this molecule can
regulate DNA-PKcs expression. Moreover, knockdown of PIG3 was shown to decrease the
level of DNA-PKcs in cells. Our study further addressed the correlation between
DNA-PKcs, Bcl-2, and p53 expression, and the results showed that DNA-PKcs expression
was significantly positively correlated with that of Bcl-2 (r=0.755,
p<0.05) and significantly negatively correlated with p53
(r=0.569, p<0.05). We further showed that DNA-PKcs was closely
related to apoptosis and that elemene increased apoptosis of A549 cells and
strengthened cellular radiosensitivity by inhibiting DNA-PKcs expression.
In summary, elemene exhibits radiotherapy-sensitizing effects on lung adenocarcinoma A549 cells, and its mechanism of action involves the upregulation of p53 and downregulation of Bcl-2 to promote cell apoptosis, as well as the downregulation of DNA-PKcs to inhibit DSB repair. However, the specific mechanism of action of elemene requires further elucidation.
ACKNOWLEDGEMENT
This study was supported by grants from the National Natural Science Foundation of China (No. 81473452).
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Publication Dates
-
Publication in this collection
Aug 2015
History
-
Received
6 Jan 2015 -
Reviewed
27 Feb 2015 -
Accepted
1 June 2015