Role Of AlphaV Integrin In Multicellular Resistance To Radiotherapy Of Human NPC

BackgroundThe resistance to radiotherapy of solid tumor is a major reason for impacting on the effect of radiotherapy.The past researchs related to a single tumor cells for the study of basic units,overlooked the special micro-environment and the growth of tumor cells exchange of information and interaction,as the heterogeneous group of tumors.Recent studies have found that tumor cells in three-dimensional culture conditions exist multicellular resistance(MCR) phenomenon:the interaction between cells-cells and tumor cells-stromal cells lead to reduce of sensitivity to cytotoxic drugs and radiation and of such treatments.Integrin is a important cell adhesion molecules(CAM),mediated cells-cells and cells-ECM adhesion.Through participation in tumor invasion,metastasis,angiogenesis and cell survival process,integrin play its role in the process of tumor development.SAPK/JNK signal pathway is important for the signal transduction pathway in cells,extracellular stimulating factors which triggered apoptosis could active the signal pathway,which shows the signal pathway may play an important role in the process of the environment stress-induced apoptosis.Researchs related to MCR of tumor in domestic were rarely reported.This research chosen alphaV integrin as a target,and used MCSs,which intercellular adhesion is extensive and close,to simulate solid tumors,preliminary explored the role and mechanisms of integrin alphaV in multicellular resistance to radiotherapy of human nasopharyngeal carcinoma.To reverse MCR of tumor,enhance radiation sensitivity,the study may provide direction.Objectives1.To establish the model of MCSs of human nasopharyngeal carcinoma;2.To investigate the role and mechanisms of cell adhesion molecules alphaV integrin in multicellular resistance to radiotherapy of human nasopharyngeal carcinoma. Methods1.Human NPC cell line CNE-2Z were cultured as multicellular spheroids using liquid overlay technique or as monolayer cells using routine method.2.Flow cytometry was employed to detect the expression of alphaV integrin under different culture conditions.3.MTT assay and blood cell counter were employed to detect radiosensitivity of CNE-2Z cells before and after blocking alphaV integrin function under different culture conditions.4.X-ray induced cell apoptotisis in MCSs before and after blocking alphaV integrin function were detected by flow cytometry.5.Western blot was employed to analyzed X-ray induced activities of SAPK/JNK signaling pathsway in MCSs,and before and after blocking alphaV integrin function in MCSs,and Western blot was also employed to analyzed the expression of caspase-3 protein before and after specially blocking SAPK/JNK signaling pathsway.6.X-ray induced cell apoptotisis in MCSs before and after specially blocking SAPK/JNK signaling pathsway were detected by TNUEL.Results1.After 4-5 days of training can form MCSs in diameter of 100μm.2.Compared with MCs,the alphaV integrin expression of MCSs increased significantly (p＜0.05).3.Compared with MCs,MCSs radiosensitivity was significantly decreased(P＜0.05), after blocking alphaV integrin,MCSs radiosensitivity was increased(P＜0.05).4.MCSs have some spontaneous apoptosis,after 2.0Gy X-ray irradiation,MCSs apoptosis rate increase was not significant(P＞0.05),if prior blocking alphaV integrin,the apoptosis rate will be significantly higher[(16.17±1.04)%vs(35.26±3.02)%,P＜0.05].5.After 2.0Gy X-ray irradiation,the level of SAPK/JNK phosphorylation in MCSs was a dynamic course,and there was a phosphorylation peaks at 2h later(0.6906±0.0236), about 5h later restored the original level(0.1982±0.0175).6.Blocked alphaV integrin,2h later after 2.0Gy X-ray irradiation,the level of SAPK/JNK phosphorylation in MCSs was significantly lower than that of control [(0.2891±0.0206) vs(0.6631±0.0518)]. 7.Specific blocked SAPK/JNK signaling pathsway before 2.0Gy X-ray irradiation, apoptosis rate in MCSs will be significantly higher[(17.94±1.98)%vs(33.37±2.11)%, P＜0.05].8.Specific blocked SAPK/JNK signaling pathsway before 2.0Gy X-ray irradiation, the expression of caspase-3 protein were significantly increased(P＜0.05).Conclusion1.The model of MCSs of human nasopharyngeal carcinoma successfully established in vitro,and it had multicellular resistance as tumor in vivo.2.The cell adhesion molecules alphaV integrins plays a important role in multicellular resistance to radiotherapy of human nasopharyngeal carcinoma,that may depend upon alphaV integrins suppressing apoptosis induced by X-ray irradiation;the possible mechanisms may be associated with the transient activation of SAPK/JNK signaling pathsway,inhibiting the activation of the signaling pathway,we can suppress MCR and that may associate with the increased expression of caspase-3.