Role of the NRP-1-mediated VEGFR2-independent pathway on radiation sensitivity of non-small cell lung cancer cells
To determine if inhibiting neuropilin-1 (NRP-1) affects the radiosensitivity of NSCLC cells through a vascular endothelial growth factor receptor 2 (VEGFR2)-independent pathway, and to assess the underlying mechanisms.
The expression of VEGFR2, NRP-1, related signaling molecules, abelson murine leukemia viral oncogene homolog 1 (ABL-1), and RAD51 were determined by RT-PCR and Western blotting, respectively. Radiosensitivity was assessed using the colony-forming assay, and the cell apoptosis were analyzed by flow cytometry.
We selected two cell lines with high expression levels of VEGFR2, including Calu-1 cells that have high NRP-1 expression, and H358 cells that have low NRP-1 expression. Upon inhibition of p-VEGFR2 by apatinib in Calu-1 cells, the expression of NRP-1 protein and other related proteins in the pathway was still high. Upon NRP-1 siRNA treatment, the expression of both NRP-1 and RAD51 decreased (p < 0.01; p < 0.05). Upon ABL-1 siRNA treatment, the expression of NRP-1 was increased and the expression of RAD51 was unchanged. Calu-1 cells treated with NRP-1 siRNA exhibited significantly higher apoptosis and radiation sensitivity in radiation therapy compared to Calu-1 cells treated with apatinib alone (p < 0.01; p < 0.01). The apoptosis and radiation sensitivity in H358 cells with NRP-1 overexpression was similar to the control group regardless of VEGFR2 inhibition.
We demonstrated that when VEGFR2 was inhibited, NRP-1 appeared to regulate RAD51 expression through the VEGFR2-independent ABL-1 pathway, consequently regulating radiation sensitivity. In addition, the combined inhibition of VEGFR2 and NRP-1 appears to sensitize cancer cells to radiation.
KeywordsNRP-1 VEGFR2 Non-small cell lung cancer Radiosensitivity
This study was supported by the National Natural Science Foundation of China (no. 81472792), the Natural Science Foundation of Jiangsu Province, China (no. BK20151279), the Youth Medical Talent Project of Lianyungang (QNRC2016499), the Health Family Planning Technology Project of Lianyungang (QN1602), and the Youth Talents Found of Lianyungang First People’s Hospital (QN150101, QN140202).
Compliance with ethical standards
Conflict of interest
We have read and understood the Journal of Cancer Research and Clinical Oncology’s policy on disclosing conflicts of interest and declare that we have none.
The use of the tissue microarray was approved by the Medical ethics committee.
- Cao S, Yaqoob U, Das A, Shergill U, Jagavelu K, Huebert RC, Routray C, Abdelmoneim S, Vasdev M, Leof E, Charlton M, Watts RJ, Mukhopadhyay D, Shah VH (2010) Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-beta signaling in hepatic stellate cells. J Clin Invest 120(7):2379–2394CrossRefPubMedPubMedCentralGoogle Scholar
- Chatterjee S, Heukamp LC, Siobal M, Schöttle J, Wieczorek C, Peifer M, Frasca D, Koker M, König K, Meder L, Rauh D, Buettner R, Wolf J, Brekken RA, Neumaier B, Christofori G, Thomas RK, Ullrich RT (2013) Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer. J Clin Invest 123(4):1732–1740CrossRefPubMedPubMedCentralGoogle Scholar
- Evans IM, Yamaji M, Britton G, Pellet-Many C, Lockie C, Zachary IC, Frankel P (2011) Neuropilin-1 signaling through p130Cas tyrosine phosphorylation is essential for growth factor-dependent migration of glioma and endothelial cells. Mol Cell Biol 31(6):1174–1185CrossRefPubMedPubMedCentralGoogle Scholar
- Ko JC, Ciou SC, Cheng CM, Wang LH, Hong JH, Jheng MY, Ling ST, Lin YW (2008) Involvement of Rad51 in cytotoxicity induced by epidermal growth factor receptor inhibitor (gefitinib, IressaR) and chemotherapeutic agents in human lung cancer cells. Carcinogenesis 29(7):1448–1458CrossRefPubMedGoogle Scholar