Function And Mechanism Of E1A Gene On Radiosensitivity Of Nasopharyngeal Carcinoma

Objective:The purpose of this study determined the effect and mechanism of Ad-E1A gene therapy in vitro radiosensitivity to nasopharyngeal carcinoma cell.Methods:The human nasopharyngeal carcinoma CNE-2Z cell lines were investigated. The recombinant adenovirus vector containing E1A gene was used for this study. After CNE-2Z cells was treated with PBS, Ad-β-gal and Ad-E1A for 48h, the three groups were irradiated in different doses at 0,2,4,6 and 8Gy, The cytotoxicity was determined by MTT assay and cell cycle was analysis by flow cytometry. Clone forming assays were carried out, cell survival curves were drawn and the sensitivity enhancing ratio (SER) was calculated. The VEGF expression was evaluated by RT-PCR assay and immunocytochemical analysis. The wtp53 express was evaluated by RT-PCR.Results:RT-PCR confirmed that E1A gene had been integrated into positively transfected cells and stably expressed. Significant cell deaths by IR were observed in a dose dependent manner in the three group CNE-2Z cells. After transduction of the E1A gene into CNE-2Z cells, the sensitivity of these cells to radiation was enhanced than the PBS treated group and Ad-β-gal treated group. Cell growth inhibition in Ad-E1A treated group by IR was strongly enhanced than Ad-β-gal treated group and PBS treated group. Cell survival curves showed that the SER of Do, Dq and SF2 value was 1.37,1.95 and 1.46 with Ad-E1A treated group The Do, Dq and SF2 value was 1.57Gy,1.82Gy,0.89 in PBS treated group and 1.53Gy,1.78Gy,0.82 in Ad-β-gal treated group,respectively. RT-PCR assay and immunocytochemical analysis showed VEGF expression was downregulation in Ad-E1A treated group. Moreover, the expression of wtp53 gene was markedly enhanced in Ad-E1A treated group. The Flow cytometry showed more apotosis can be detected in the cells treated with Ad-E1A plus radiotherapy than those of the other treated groups.Conclusions:E1A gene therapy can effectively enhance the nasopharyngeal carcinoma cell sensitivity to the radiotherapy by down regulated VEGF expression and enhanced the expression of wtp53. Objective:To determine the effect of Ad-E1A gene therapy in vivo radiosensitivity to nasopharyngeal carcinoma.Methods:CNE-2Z cells (2 x 105) were injected subcutaneously into nude mice resulted in tumor development (1-3 mm) 6 days later. The tumor-bearing mice were then randomly divided into six groups (10 mice per group) for PBS treatment or treatment with radiotherapy, Ad-E1 A, or Ad-β-gal alone or radiotherapy in combination with Ad-E1A or Ad-β-gal. Animals were treated with Ad-E1A or Ad-β-gal (5 x 109 plaque forming units) by intratumoral injection twice weekly for 2 weeks at beginning of week 2. Animals treated with radiotherapy in combination with Ad-E1A or Ad-β-gal were received 2 Gy radiotherapy daily for 5 days following the first week of treatment with Ad-E1A or Ad-β-gal. Control animals received PBS therapy or radiotherapy only after tumor cells were injected. When the sizes of tumors exceeded 2 cm, the mice were killed and the tumors underwent immunohistochemical analysis for VEGF and CD34 expression and RT-PCR analysis for wtp53 expression and TUNEL assay for apoptosis and Western blot analysis for VEGF expressionResults:The growth delay time (TGD) was longest in the Ad-E1A plus radiotherapy group. Tumors treated with Ad-E1A plus radiotherapy were 4.7-fold smaller than those treated with radiotherapy alone and 5.3-fold smaller than those treated with Ad-E1A alone. The survival rate of tumor-bearing mice treated with Ad-E1A plus radiotherapy was significantly higher than that of other treated groups. The vessel density and the VEGF expression were significantly lower in the tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-E1A alone, Ad-β-gal alone or Ad-β-gal plus radiotherapy (P<0.01). wtp53 expression were significantly increased in the tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-β-gal alone or Ad-β-gal plus radiotherapy. TUNEL staining revealing apoptosis can be detected in Ad-E1A group, radiotherapy group, Ad-E1A plus radiotherapy group, and more apoptosis can be detected in the tumors treated with Ad-E1A plus radiotherapy than those of the other treated groups.Conclusion:E1A gene therapy can effectively enhance the nasopharyngeal carcinoma sensitivity to the radiotherapy by down-regulating VEGF expression increasing wtp53 expression and inducing apoptosis. These findings may pave the way for efficient radiation-gene therapy to NPC in future.