Clinical and Translational Oncology

, Volume 18, Issue 2, pp 212–219 | Cite as

VEGFR2 inhibition by RNA interference affects cell proliferation, migration, invasion, and response to radiation in Calu-1 cells

  • Y. Liu
  • Y. Qiao
  • C. Hu
  • L. Liu
  • L. Zhou
  • B. Liu
  • H. Chen
  • X. JiangEmail author
Research Article



To investigate the role of the vascular endothelial growth factor receptor 2 (VEGFR2) in the proliferation, migration, invasion, and radiation-induced apoptosis of the non-small cell lung cancer (NSCLC) cell line Calu-1.


VEGFR2 gene was silenced by RNA interference in Calu-1 cells, and the expression of VEGFR2 was measured by qRT-PCR and Western blot analysis. The cells were divided into control, VEGF-treated, VEGFR2 knockdown, and VEGFR2 knockdown and VEGF-treated groups. A CCK8 assay and Transwell assay were performed to assess cell proliferation, migration, and invasion, respectively, after VEGFR2 knockdown. Western blot assays were used to detect signaling proteins downstream of VEGFR2. Cells in the groups listed above were also subjected to radiation treatment, followed by apoptosis analysis.


(1) RNA interference of VEGFR2 in Calu-1 cells reduced VEGFR2 mRNA (P < 0.01) and protein levels (P < 0.01). (2) VEGFR2 knockdown inhibited proliferation (P < 0.05), migration (P < 0.05), and invasion (P < 0.05) in Calu-1 cells. (3) VEGFR2 knockdown blocked the phosphorylation of protein kinase B (Akt, also known as PKB), extracellular regulated kinase (ERK) 1/2, and p38 mitogen-activated protein kinase (p38 MAPK) to various extent (P < 0.05), but did not change their total protein expression. (4) Knockdown of VEGFR2 suppressed HIF-1α protein synthesis (P < 0.05), and exacerbated apoptosis induced by radiation (P < 0.05).


VEGFR2 gene knockdown significantly suppressed a number of cellular activities in Calu-1 cells and increased radiation-induced cell death.


RNA interference VEGFR2 Proliferation Migration Radiation 



Vascular endothelial growth factor receptor 2


Hypoxia-inducible factor-1α


Vascular endothelial growth factor



This study was supported by the National Natural Science Foundation of China (NO. 81472792), Research Fund from Ministry of Health (W201210), and the National Natural Science Foundation of Jiangsu Province (BK2012661).

Compliance with the ethical standards

Our study did not refer to clinical trial and animal trial, so there was no ethics statement.

Conflict of interest



  1. 1.
    Ahmed K, Emran AA, Jesmin T, Mukti RF, Rahman MZ, Ahmed F. Early detection of lung cancer risk using data mining. Asian Pac J Cancer Prev. 2013;14(1):595–8.CrossRefPubMedGoogle Scholar
  2. 2.
    de Jong WK, Schaapveld M, Blaauwgeers JL, Groen HJ. Pulmonary tumours in the Netherlands: focus on temporal trends in histology and stage and on rare tumours. Thorax. 2008;63(12):1096–102.CrossRefPubMedGoogle Scholar
  3. 3.
    Gressen EL, Curran WJ. Hyperfractionated radiotherapy for lung cancer. Curr Oncol Rep. 2000;2(1):71–5.CrossRefPubMedGoogle Scholar
  4. 4.
    Chi A, Liao Z, Nguyen NP. Xu j, Stea B, Komaki R. Systemic review of the patterns of failure following stereotactic body radiation therapy in early-stage non-small-cell lung cancer: clinical implications. Radiother Oncol. 2010;94(1):1–11.CrossRefPubMedGoogle Scholar
  5. 5.
    Terman BI, Carrion ME, Kovacs E, Rasmussen BA, Eddy RL, Shows TB. Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene. 1991;6(9):1677–83.PubMedGoogle Scholar
  6. 6.
    Takahashi S. Vascular endothelial growth factor (VEGF), VEGF receptors and their inhibitors for antiangiogenic tumor therapy. Biol Pharm Bull. 2011;34(12):1785–8.CrossRefPubMedGoogle Scholar
  7. 7.
    Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science. 2005;307(5706):58–62.CrossRefPubMedGoogle Scholar
  8. 8.
    Winkler F, Kozin SV, Tong RT, Chae SS, Booth MF, Garkavtsev I, et al. Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, Angiopoietin-1, and matrix metalloproteinases. Cancer Cell. 2004;6:553–63.PubMedGoogle Scholar
  9. 9.
    Moeller BJ, Dewhirst MW. HIF-1 and tumour radiosensitivity. Br J Cancer. 2006;95(1):1–5.PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Jiang XD, Ding MH, Qiao Y, Liu Y, Liu L. Study on lung cancer cells expressing VEGFR2 and the impact on the effect of RHES combined with radiotherapy in the treatment of brain metastases. Clin Lung Cancer. 2014;15(2):e23–9.CrossRefPubMedGoogle Scholar
  11. 11.
    Xiang QF, Chen WQ, Ren M, Wang JN, Zhang HW, Deng David YB, et al. Cabozantinib suppresses tumor growth and metastasis in hepatocellular carcinoma by a dual blockade of VEGFR2 and MET. Clin Cancer Res. 2014;20(11):2959–70.CrossRefPubMedGoogle Scholar
  12. 12.
    Adham SA, Sher I, Coomber BL. Molecular blockade of VEGFR2 in human epithelial ovarian carcinoma cells. Lab Invest. 2010;90(5):709–23.PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Sun Chris K, Man K, Ng Kevin T, Ho Joanna W, Lim Zophia X, Cheng Q. Proline-rich tyrosine kinase 2 (Pyk2) promotes proliferation and invasiveness of hepatocellular carcinoma cells through c-Src/ERK activation. Carcinogenesis. 2008; 29(11): 2096–105.Google Scholar
  14. 14.
    Ghosh S, Kumar A, Tripathi RP, Chandna S. Connexin-43 regulates p38-mediated cell migration and invasion induced selectively in tumour cells by low doses of γ-radiation in an ERK-1/2-independent manner. Carcinogenesis. 2014;35(2):383–95.CrossRefPubMedGoogle Scholar
  15. 15.
    Gomes E, Rockwell P. p38 MAPK as a negative regulator of VEGF/VEGFR2 signaling pathway in serum deprived human SK-N-SH neuroblastoma cells. Neurosci Lett. 2008;431(2):95–100.PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Shi L, Zhang S, Wu H, Zhang L, Dai X, Hu J, Xue J, Liu T, Liang Y, Wu G. MiR-200c increases the radiosensitivity of non-small-cell lung cancer cell line A549 by targeting VEGF-VEGFR2 pathway. PLoS One. 2013;8(10):e78344.PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Brown JM, Giaccia AJ. The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res. 1998;58(7):1408–16.PubMedGoogle Scholar
  18. 18.
    Shachar I, Cohen S, Marom A, Becker HS. Regulation of CLL survival by hypoxia-inducible factor and its target genes. FEBS Lett. 2012;586(18):2906–10.CrossRefPubMedGoogle Scholar
  19. 19.
    Li J, Song X, YU J. Analysis of radiosensitivity of A549 lung cancer cells with HIF-1 silencing by RNAi. Chinese Journal of Radiological Medicine and Protection. 2008, 28(2):120-3.Google Scholar
  20. 20.
    Yang X, Yang B, Cai J, Zhang C, Zhang Q, Xu L, et al. Berberine enhances radiosensitivity of esophageal squamous cancer by targeting HIF-1α in vitro and in vivo. Cancer Biol Ther. 2013;14(11):1068–73.PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Nilsson MB, Zage PE, Zeng L, Xu L, Cascone T, Wu HK, et al. Multiple receptor tyrosine kinases regulate HIF-1alpha and HIF-2alpha in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors. Oncogene. 2010;29(20):2938–49.CrossRefPubMedGoogle Scholar
  22. 22.
    Alvarez TM, Alfranca A, Aragonés J, Vara A, Landázuri MO, del PL. Lack of evidence for the involvement of the phosphoinositide 3-kinase/Akt pathway in the activation of hypoxia-inducible factors by low oxygen tension. J Biol Chem. 2002;277(16):13508–17.CrossRefGoogle Scholar
  23. 23.
    Hofer T, Desbaillets I, Höpfl G, Gassmann M, Wenger RH. Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1alpha activation cascade: implications for HIF-1alpha gene therapy. FASEB J. 2001;15(14):2715–7.PubMedGoogle Scholar
  24. 24.
    Befani CD, Vlachostergios PJ, Hatzidaki E, Patrikidou A, Bonanou S, Somos G. Bortezomib represses HIF-1α protein expression and nuclear accumulation by inhibiting both PI3 K/AKT/TOR and MAPK pathways in prostate cancer cells. J Mol Med (Berl). 2012;90(1):45–54.CrossRefPubMedGoogle Scholar

Copyright information

© Federación de Sociedades Españolas de Oncología (FESEO) 2015

Authors and Affiliations

  • Y. Liu
    • 1
  • Y. Qiao
    • 2
  • C. Hu
    • 2
  • L. Liu
    • 1
  • L. Zhou
    • 2
  • B. Liu
    • 1
  • H. Chen
    • 1
  • X. Jiang
    • 2
    Email author
  1. 1.Xuzhou Medical College Graduate AcademyXuzhouChina
  2. 2.Department of Radiation OncologyLianyungang First People’s HospitalLianyungang CityChina

Personalised recommendations