Gene Therapy Selectively Enhanced Radiosensitization Effect In NPC

Objective: Construct radiosensitive novel fusion promoter E6hTERTp. Observe the effect of radiosensitive enhancer to hTERT promoter's specifity and radiosensitivuty after different dose of radiation; construct vector carrying E6hTERTp and fusion suicide gene CD/UPRT.UL49, induce the expression of the suicide gene in CNE-2 cells with different dose of radiation to find the radiosensitization effect of the radio -suicide gene /prodrug system in vitro and vivo to lay a solid experimental foundation for a new stratety for radio-gene therapy.Method and resultsThe first part: Research on hTERTp's tumor-specific promotive effect enhanced by radiosensitive enhancer E6Method: The fusion gene E6.hTERTp and CD/UPRT.UL49 were constructed by overlap PCR. The combined promoter was inserted into PGL3 basic plasmid. PGL3-hTERTp and pGL3-EGR-lp were also constructed for comparison. All the plasmids together with pRL-SV40 plasmids were transfected into CNE-2 cells and HDF cells with liposome .The promotive effect of recombinant promoter E6.hTERTp in CNE-2 cells and HDF cells were measured by Dual Luciferase Reporter Gene Assay system and was compared with EGR-1p and hTERTp for difference between every two groups under different dose of radiation to find the enhancing effect of radiosensitive enhancer to hTERT promoter and its influence to specifity after different dose of radiation. Construct suicide gene vector pcDNA3.1(-)E6.hTERTp. CD/UPRT and pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49; transfect the plasmids into CNE-2 cells with liposome . after different dose (0Gy, 2Gy,6Gy,10Gy ) of radiation, the expression of suicide gene CD/UPRT.UL49 and CD/UPRT were analyzed by half -quantitive RT-PCR and WEST BLOTTING.Results:Dual Luciferase Reporter Gene Assay system revealed that the promotive effects of hTERT promoter and E6.hTERTp remains low in HDF cells with any dose of radiation ;the value between all radiation groups are not significantly different (p>0.05) ; While in CNE-2 cells ,increase of promotive effects in accordance with the radiation dose can be observed in all groups . The promotive effect of E6.hTERTp group were 18.8184±4.1969 , 102.6512±20.5879 , 291.7274±35.4752 , 407.5505±27.1526, increased by5.4, 15.2 and 21.7 times compared with baseline(OGy), the promotive effect of different radiation groups are statisticaly significant (p<0.05) . When the dose of radiation were 2Gy, 6Gy and 10Gy , the promotive effect of E6.hTERTp increased to2.4, 3.5 and 2.8 times the value of hTERTp group ,which proved that radiosensitive enhancer E6 increase the radiosensitive promotive effect of hTERTp in CNE-2 cells and do not affect its NPC specific character. When Suicide gene pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 and pcDNA3.1 (-) E6.hTERTp.CD/UPRT were transfected into CNE-2 cells, expression of the novel fusion suicide gene can be detected by western blotting test .RT-PCR shows that after radiation ,the amount of mRNA were significantly higher than that of 0Gy group (p<0.05). the amount of mRNA of pcDNA3.1 (-) E6.hTERTp.CD/UPRT reach the peak in 6Gy group; The amount is not signifieantly different for pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 in 6Gy and 10Gy group (p>0.05) but is significantly higher than 2Gy group (p<0.05) . The result revealed that radiation can evoke the activity of E6hTERTP and enhance the expression of downstream suicide gene.The second part: radiosensitization effect by enhanced expression of novel fusion gene CD/UPRT.UL49 and CD/UPRTgene mediated by E6hTERTp in NPCMethod: Suicide gene pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 and pcDNA3.1 (-) E6.hTERTp.CD/UPRT were transfected into CNE-2 cells , and the transform rate of prodrug 5-Fc to 5-FU for the two suicide gene groups under different dose of radiation (0Gy, 2Gy,6Gy, 10Gy )were measured by HPLC.The killing effect of both suicide gene/prodrug system were tested by MTT after different dose of radiation with different 5-Fc concentration and were analyzed for difference between the two treatment groups and simple radiation -prodrug groups with Spss software.Results: After treated with prodrug 5-Fc and radiation, 5-FU can be detected from the supernatant of the culture media of CNE-2 transfected with both suicide gene ,which shows that after incorporation of VP22 segment ,the CD/UPRT protein maintained its bioactivity of transforming 5-Fc.The MTT analysis showed that when the prodrug concentration was 200 (ug/ml) , the survival rate of wild CNE-2 group was 93.04,;while after radiation the survival rate of gene therapy group decrease significantly, the tendency of decrease is more obvious in CNE-2/pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 group (p<0.05) in 2Gy and 10Gy group . The third part: Research on the radiosensitization effect of prodrug suicide gene therapy in situMethod : CNE-2 cells in log phase were inoculated subcutaneously in nude mice to construct a nude mouse model of NPC ,and an in situ gene therapy was performed with plasmid packed by plasmid. When the size of tumors reaching 1.0-0.7cm, the nude mice were randomized to different grouped, and was given radiation , system administration of produg and in situ gene injection, the suppression of tumor growth was observed. Biopsy of the tumor were performed to find the difference between treatment groups .Then targeted RNA was isolated from tumor tissues and RT-PCR was performed to define the expression of suicide gene.Results 9 days after inoculation, all the nude mouse developed transplanted tumor. In situ gene therapy experiment showed no suppressing effect of gene /prodrug system without radiation; compared with other radiated groups , growth of transplant tumor of radio-pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 /prodrug group was distinctly suppressed(P<0.05) ,the growth suppressing rate is 93.7579 While the radio-pcDNA3.1 (-) E6.hTERTp.CD/UPRT /prodrug group showed no sign of more suppressing effect compared with other radiation groups (p>0.05) ,the growth suppressing rate is88.86427.biopsy shows severe necrosis of all radiated group .Conclusions:The suicide gene E6.hTERTp.CD/UPRT.UL49/prodrug system, is both radiosensitive and tumor specifically effective in anticancer therapy, and exert powerful killing effect to CNE-2 cells than E6.hTERTp.CD/UPRT / prodrug system under radiation. From this study, we established a new strategy for radio-gene therapy of NPC.