The In Vitro And In Vivo Radiosensitization For Human Nasopharyngeal Carcinoma Cell By Ad.RGD-ING4Gene Therapy

Objective:1. The in vitro and in vivo radiosensitization for human nasopharyngealcarcinoma cell by adenovirus-mediated ING4with RGD.2. To investigate the possiblemechanism of radiosensitization for human nasopharyngeal carcinoma cell byadenovirus-mediated ING4with RGD.Methods: In vitro: Ad.RGD－ING4was completed the amplification and the titrationwas detected in QBI-293A cells. Detected infection efficiency of Ad.RGD－ING4in thehuman nasopharyngeal carcinoma cell to determine the Optimal multiplicity of infection.The human nasopharyngeal carcinoma cell was infected by Ad.RGD－ING4according tothe Optimal multiplicity of infection. Using RT-PCR and Western blot method to detectING4gene transcription and expression in the CNE. CNE were irradiated with differentdoses of radiation, then using Annexin-V-PE/7-AAD double staining to detect apoptoticrate to find suitable experimental radiation dose. MTT assay was used to measured thecell growth inhibition by Ad.RGD-ING4combined with radiotherapy. The affect of thecell cycle and apoptosis were detected by flow cytometry.RT-PCR was used to detect thetranscription of Bax,Bcl-2and P21. The expression of Survivin and Caspase-3wasdetected by Western blot. In vivo: We established CNE human nasopharyngeal carcinomatransplanted tumor in athymic nude mouse model, then used Ad.RGD-ING4combinedwith radiotherapy to detect the tumor volume and weight,to detect the expression of P21、Bax、Bcl-2、VEGF、CD34、Cox2and Survivin in human nasopharyngeal carcinoma celltransplanted tumor by immunohistochemistry.Results:1.RT-PCR and Western blot showed that adenovirus-mediated ING4withRGD can transcript and translate in CNE human nasopharyngeal carcinoma cells stably.2.Ad.RGD-ING4combined with radiotherapy can significantly inhibit the growth of CNE cells, resulting in cell G2/M phase arrest and induction of apoptosis in vitro, and theresults were significantly better than Ad.RGD-ING4group and radiotherapy group (P<0.05). Experiments In vivo showed that Ad.RGD-ING4combined with radiotherapy cansignificantly inhibit the growth of human nasopharyngeal carcinoma tumor growth, theresults were significantly better than Ad.RGD-ING4group and radiotherapy group (P<0.05), and had anti-tumor growth additive effect efficiency (Q=1.04). The detection ofcellular and molecular mechanisms found that Ad.RGD-ING4combined with radiotherapycan significantly up-regulate the expression of P21, Bax, cleaved Caspase-3and down-regulate the expression of Bcl-2, Survivin, CD34, VEGF in CNE human nasopharyngealcarcinoma cell, and its effect is more significant than Ad.RGD-ING4group andradiotherapy group.Conclusion: Ad.RGD-ING4combined with radiotherapy can significantly inhibithuman nasopharyngeal carcinoma cells and induce apoptosis in vitro and in vivo(P <0.05),which may be related with up-regulating the expression of P21, Bax, cleaved Caspase-3and down-regulating the expression of Bcl-2, CD34, Survivin, VEGF, then further inducedapoptosis.