Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10327
RESEARCH ARTICLE
Effects of carbon-ion beam irradiation on the angiogenic response in lung adenocarcinoma A549 cells Yuanyuan Liu1,2,3, Yang Liu1,2,3, Hong Zhang1,2,3*, Chao Sun1,2,3, Qiuyue Zhao1,2,3, Cixia Di1,2,3, Hongyan Li1,2,3, Lu Gan1,2,3 and Yali Wang4 1 2 3 4
Department of Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, China Key Laboratory of Heavy Ion Radiation Medicine of Gansu Province, Lanzhou 730000, China School of Pharmacy, Lanzhou University, Lanzhou 730000, China
Abstract Radiotherapy has been focused mainly on killing cancer cells, and little attention has been paid to the process supporting tumor growth and metastasis, including the process of angiogenesis. To investigate the effects of carbon-ion irradiation on angiogenesis in lung cancer cells, we examined the expression of vascular endothelial growth factor and basic fibroblast growth factor in the tumor conditioned medium (TCM) of A549 cells exposed to carbon-ion or X-ray irradiation, as well as endothelial cell growth, invasion, and tube formation induced by TCM. No changes in vascular endothelial growth factor secretion were detected in the TCM of A549 cells exposed to carbon-ion irradiation at 2 or 4 Gy, whereas 1 Gy of irradiation significantly decreased vascular endothelial growth factor and basic fibroblast growth factor levels. Carbon-ion irradiation at 1 Gy inhibited endothelial cell invasion and tube formation. The TCM from A549 cells irradiated with X-ray promoted angiogenesis, whereas the TCM of A549 cells exposed to carbon-ion irradiation at 2 or 4 Gy had no effect. These findings suggest that carbon-ion irradiation at 1 Gy significantly suppressed the process of angiogenesis in vitro by inhibiting endothelial cell invasion and tube formation, which are related to vascular endothelial growth factor and basic fibroblast growth factor production. Keywords: angiogenesis; bFGF; carbon-ion irradiation; lung adenocarcinoma cells; VEGF
Introduction Ionizing radiation (IR) induces apoptosis of lung cancer cells (He et al., 2007; Han et al., 2012). However, the prognosis of lung cancer patients treated with radiotherapy is frequently poor because of distant metastasis. In addition, factors such as the type of radiation can have a detrimental effect on the prognosis of patients. X-ray irradiation increases A549 cell proliferation at 0.5 Gy, whereas carbon-ion irradiation at 0.25 Gy had no effect on cell proliferation and significantly reduced migration and invasion capabilities (Akino et al., 2009). Thus high linear energy transfer (LET) charged carbon-ion irradiation has a potential inhibitory effect on lung cancer metastases. Radiation therapy has been focused mainly on killing cancer cells, and little attention has been paid to the
process supporting tumor growth, including the process of angiogenesis. Angiogenesis, which is a crucial step for tumor growth, invasion, and metastasis because of its role in delivering oxygen and nutrients, includes endothelial cell migration, proliferation, and differentiation. Tumor cells promote angiogenesis by secreting growth factors and inflammatory cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietins (Angs), and interleukin-8 (Ferrara, 1996; Eckermann et al., 2011; Chang et al., 2013; Liu et al., 2013; Nieminen et al., 2014; Porta et al., 2013; Richey and Hutson, 2013). Overexpression of VEGF has been associated with poor prognosis in non-small cell lung cancer (NSCLC) (Imoto et al., 1998; Hu et al., 2013), suggesting that the downregulation of VEGF expression
Corresponding author: e-mail: [email protected] Abbreviations: VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; TCM, tumor conditioned medium; LET, linear energy transfer; Angs, angiopoietins; NSCLC, non-small cell lung cancer; HMEC-1, human microvascular endothelial cells
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Cell Biol Int 38 (2014) 1304–1310 © 2014 International Federation for Cell Biology
Y. Liu et al.
could be an effective approach to inhibit angiogenesis in lung cancer. The efficacy of radiation therapy is influenced by angiogenesis. Low LET (X-ray) irradiation increases the expression of survival factors such as VEGF (Gorski et al., 1999; Gupta et al., 2002), which exert proangiogenic effects and promote tumor regrowth (Sonveaux et al., 2003). To date, the effects of high LET charged carbon-ion irradiation on tumorigenesis remains largely unknown. Here, we used human microvascular endothelial cells (HMEC-1), which are a type of endothelial cells involved in clinically relevant angiogenesis, to investigate the effects of carbon-ion irradiation on angiogenesis in lung cancer cells. Materials and methods
Cell culture Human lung adenocarcinoma A549 cells were obtained from Lanzhou University and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS). HMEC-1 was obtained from ATCC and cultured in MCDB131 medium (Sigma, St. Louis, MO, USA) supplemented with 20% FBS, 1 mg/mL hydrocortisone (Sigma), and 10 ng/ml EGF (Sigma). Cells were maintained in an atmosphere of 5% CO2 at 37 C.
Irradiation procedure A549 cells were irradiated with carbon-ion beams at room temperature using the radiation equipment at the Heavy Ion Research Facility (Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China), with 300 MeV/mm carbon ion beams and a LET value of 49 keV/mm. The dose rate was 1 Gy/min. For X-ray irradiation, A549 cells were treated with an X-ray machine [Elekta BMEI (Beijing) Medical Equipment Co. Ltd, China] at 100 kpv at room temperature, with a dose rate of 1.2 Gy/min. The film-tosource distance was 40.6 cm. Cells were exposed to 0, 1, 2, or 4 Gy with carbon-ion or X-ray irradiation. Cells were irradiated in the exponential growth phase, and nonirradiated cultured cells (control) were handled in parallel with the irradiated samples.
Collection of the tumor conditioned medium (TCM) A549 cells exposed to carbon-ion or X-ray irradiation were cultured with 1.5 mL serum-free MCDB131 medium. After 24 h of incubation, the medium was spun down at 12,000g for 15 min to eliminate cell debris, and the supernatant was filtered using 0.22 mm filters and stored at 80 C until assayed.
Angiogenic response to carbon-ion beam irradiation
Measurement of angiogenic growth factors The amounts of VEGF or bFGF in the TCM of A549 cells irradiated with carbon-ion or X-ray irradiation were measured using an enzyme-linked immunosorbent assay (Excell Bio, China) according to the manufacturer’s protocol. Each sample was assessed in triplicate.
Cell proliferation assay The proliferation of HMEC-1 cells was examined with a CCK-8 kit (Beyotime, China) as described previously with slight modifications (Mao et al., 2013). Briefly, 1 104 HMEC-1 cells were seeded in 96-well plates. When adherent to the flasks, the cells were washed and cultured in 200 mL TCM for another 24 h. Then, CCK-8 solution (40 mL) was added to each well and the plate was incubated at 4 C for 1 h. The absorbance in each well was read with a microplate reader at 450 nm.
Cell invasion assay Endothelia cell invasion was measured as described previously with slight modifications (Shi et al., 2012). Briefly, 1 106 HMEC-1 cells were resuspended in 400 mL of serumand EGF-free culture medium and seeded in Matrigel (BD Biosciences, Bedford, MA, USA) coated Transwell upper chambers (Millipore, USA) with 8.0-mm polycarbonate filter inserts in 24-well plates, while the bottom chambers were filled with 600 mL TCM as chemoattractant. After incubation for 24 h, non-migrated cells and the Matrigel were scraped using a cotton swab. The bottom side of the membrane was fixed with ethanol and stained with Giemsa. The Transwell chambers were washed three times with PBS. Images of migrated cells were obtained using a microscope (Carl Zeiss, Germany) and at least five randomly selected microscopic fields were imaged at 100 magnification. The number of migrated cells was counted using Image-plus software. Data were expressed as the number of invasive cells relative to the control.
Tube formation assay Tube formation of endothelial cells was measured as described previously with slight modifications (Marchetti et al., 2008). HMEC-1 cells trypsinized in serum- and EGFfree culture medium were centrifuged, and 8 105 HMEC-1 cells were seeded in 100 mL of TCM on Matrigel coated 96well plates and incubated at 37 C and air plus 5% CO2. After 8 h, tube formation was examined under an inverted light microscope at 200 magnification (Carl Zeiss, Germany). Each treatment was performed in triplicate.
Cell Biol Int 38 (2014) 1304–1310 © 2014 International Federation for Cell Biology
1305
Y. Liu et al.
Angiogenic response to carbon-ion beam irradiation
Statistical analysis Data are expressed as means standard error (SE). Statistical significance was evaluated by ANOVA. P values
RESEARCH ARTICLE
Effects of carbon-ion beam irradiation on the angiogenic response in lung adenocarcinoma A549 cells Yuanyuan Liu1,2,3, Yang Liu1,2,3, Hong Zhang1,2,3*, Chao Sun1,2,3, Qiuyue Zhao1,2,3, Cixia Di1,2,3, Hongyan Li1,2,3, Lu Gan1,2,3 and Yali Wang4 1 2 3 4
Department of Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000, China Key Laboratory of Heavy Ion Radiation Medicine of Gansu Province, Lanzhou 730000, China School of Pharmacy, Lanzhou University, Lanzhou 730000, China
Abstract Radiotherapy has been focused mainly on killing cancer cells, and little attention has been paid to the process supporting tumor growth and metastasis, including the process of angiogenesis. To investigate the effects of carbon-ion irradiation on angiogenesis in lung cancer cells, we examined the expression of vascular endothelial growth factor and basic fibroblast growth factor in the tumor conditioned medium (TCM) of A549 cells exposed to carbon-ion or X-ray irradiation, as well as endothelial cell growth, invasion, and tube formation induced by TCM. No changes in vascular endothelial growth factor secretion were detected in the TCM of A549 cells exposed to carbon-ion irradiation at 2 or 4 Gy, whereas 1 Gy of irradiation significantly decreased vascular endothelial growth factor and basic fibroblast growth factor levels. Carbon-ion irradiation at 1 Gy inhibited endothelial cell invasion and tube formation. The TCM from A549 cells irradiated with X-ray promoted angiogenesis, whereas the TCM of A549 cells exposed to carbon-ion irradiation at 2 or 4 Gy had no effect. These findings suggest that carbon-ion irradiation at 1 Gy significantly suppressed the process of angiogenesis in vitro by inhibiting endothelial cell invasion and tube formation, which are related to vascular endothelial growth factor and basic fibroblast growth factor production. Keywords: angiogenesis; bFGF; carbon-ion irradiation; lung adenocarcinoma cells; VEGF
Introduction Ionizing radiation (IR) induces apoptosis of lung cancer cells (He et al., 2007; Han et al., 2012). However, the prognosis of lung cancer patients treated with radiotherapy is frequently poor because of distant metastasis. In addition, factors such as the type of radiation can have a detrimental effect on the prognosis of patients. X-ray irradiation increases A549 cell proliferation at 0.5 Gy, whereas carbon-ion irradiation at 0.25 Gy had no effect on cell proliferation and significantly reduced migration and invasion capabilities (Akino et al., 2009). Thus high linear energy transfer (LET) charged carbon-ion irradiation has a potential inhibitory effect on lung cancer metastases. Radiation therapy has been focused mainly on killing cancer cells, and little attention has been paid to the
process supporting tumor growth, including the process of angiogenesis. Angiogenesis, which is a crucial step for tumor growth, invasion, and metastasis because of its role in delivering oxygen and nutrients, includes endothelial cell migration, proliferation, and differentiation. Tumor cells promote angiogenesis by secreting growth factors and inflammatory cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietins (Angs), and interleukin-8 (Ferrara, 1996; Eckermann et al., 2011; Chang et al., 2013; Liu et al., 2013; Nieminen et al., 2014; Porta et al., 2013; Richey and Hutson, 2013). Overexpression of VEGF has been associated with poor prognosis in non-small cell lung cancer (NSCLC) (Imoto et al., 1998; Hu et al., 2013), suggesting that the downregulation of VEGF expression
Corresponding author: e-mail: [email protected] Abbreviations: VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; TCM, tumor conditioned medium; LET, linear energy transfer; Angs, angiopoietins; NSCLC, non-small cell lung cancer; HMEC-1, human microvascular endothelial cells
1304
Cell Biol Int 38 (2014) 1304–1310 © 2014 International Federation for Cell Biology
Y. Liu et al.
could be an effective approach to inhibit angiogenesis in lung cancer. The efficacy of radiation therapy is influenced by angiogenesis. Low LET (X-ray) irradiation increases the expression of survival factors such as VEGF (Gorski et al., 1999; Gupta et al., 2002), which exert proangiogenic effects and promote tumor regrowth (Sonveaux et al., 2003). To date, the effects of high LET charged carbon-ion irradiation on tumorigenesis remains largely unknown. Here, we used human microvascular endothelial cells (HMEC-1), which are a type of endothelial cells involved in clinically relevant angiogenesis, to investigate the effects of carbon-ion irradiation on angiogenesis in lung cancer cells. Materials and methods
Cell culture Human lung adenocarcinoma A549 cells were obtained from Lanzhou University and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS). HMEC-1 was obtained from ATCC and cultured in MCDB131 medium (Sigma, St. Louis, MO, USA) supplemented with 20% FBS, 1 mg/mL hydrocortisone (Sigma), and 10 ng/ml EGF (Sigma). Cells were maintained in an atmosphere of 5% CO2 at 37 C.
Irradiation procedure A549 cells were irradiated with carbon-ion beams at room temperature using the radiation equipment at the Heavy Ion Research Facility (Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China), with 300 MeV/mm carbon ion beams and a LET value of 49 keV/mm. The dose rate was 1 Gy/min. For X-ray irradiation, A549 cells were treated with an X-ray machine [Elekta BMEI (Beijing) Medical Equipment Co. Ltd, China] at 100 kpv at room temperature, with a dose rate of 1.2 Gy/min. The film-tosource distance was 40.6 cm. Cells were exposed to 0, 1, 2, or 4 Gy with carbon-ion or X-ray irradiation. Cells were irradiated in the exponential growth phase, and nonirradiated cultured cells (control) were handled in parallel with the irradiated samples.
Collection of the tumor conditioned medium (TCM) A549 cells exposed to carbon-ion or X-ray irradiation were cultured with 1.5 mL serum-free MCDB131 medium. After 24 h of incubation, the medium was spun down at 12,000g for 15 min to eliminate cell debris, and the supernatant was filtered using 0.22 mm filters and stored at 80 C until assayed.
Angiogenic response to carbon-ion beam irradiation
Measurement of angiogenic growth factors The amounts of VEGF or bFGF in the TCM of A549 cells irradiated with carbon-ion or X-ray irradiation were measured using an enzyme-linked immunosorbent assay (Excell Bio, China) according to the manufacturer’s protocol. Each sample was assessed in triplicate.
Cell proliferation assay The proliferation of HMEC-1 cells was examined with a CCK-8 kit (Beyotime, China) as described previously with slight modifications (Mao et al., 2013). Briefly, 1 104 HMEC-1 cells were seeded in 96-well plates. When adherent to the flasks, the cells were washed and cultured in 200 mL TCM for another 24 h. Then, CCK-8 solution (40 mL) was added to each well and the plate was incubated at 4 C for 1 h. The absorbance in each well was read with a microplate reader at 450 nm.
Cell invasion assay Endothelia cell invasion was measured as described previously with slight modifications (Shi et al., 2012). Briefly, 1 106 HMEC-1 cells were resuspended in 400 mL of serumand EGF-free culture medium and seeded in Matrigel (BD Biosciences, Bedford, MA, USA) coated Transwell upper chambers (Millipore, USA) with 8.0-mm polycarbonate filter inserts in 24-well plates, while the bottom chambers were filled with 600 mL TCM as chemoattractant. After incubation for 24 h, non-migrated cells and the Matrigel were scraped using a cotton swab. The bottom side of the membrane was fixed with ethanol and stained with Giemsa. The Transwell chambers were washed three times with PBS. Images of migrated cells were obtained using a microscope (Carl Zeiss, Germany) and at least five randomly selected microscopic fields were imaged at 100 magnification. The number of migrated cells was counted using Image-plus software. Data were expressed as the number of invasive cells relative to the control.
Tube formation assay Tube formation of endothelial cells was measured as described previously with slight modifications (Marchetti et al., 2008). HMEC-1 cells trypsinized in serum- and EGFfree culture medium were centrifuged, and 8 105 HMEC-1 cells were seeded in 100 mL of TCM on Matrigel coated 96well plates and incubated at 37 C and air plus 5% CO2. After 8 h, tube formation was examined under an inverted light microscope at 200 magnification (Carl Zeiss, Germany). Each treatment was performed in triplicate.
Cell Biol Int 38 (2014) 1304–1310 © 2014 International Federation for Cell Biology
1305
Y. Liu et al.
Angiogenic response to carbon-ion beam irradiation
Statistical analysis Data are expressed as means standard error (SE). Statistical significance was evaluated by ANOVA. P values
