Experimental study of dual promoter baculovirus-mediated tumor-targeting radioiodine therapy

Abstract

Objective To construct a recombinant baculovirus dual expression vector containing NIS gene under the control of human telomerase reverse transcriptase (hTERT) promoter and plasminogen kringle 5 (K5) gene driven by early growth response 1 (Egr1) promoter, and to explore the feasibility of targeting both tumor and tumor vessel with combination of radioiodide and antiangiogenic therapy. Methods The hTERT-NIS gene and Egr1-K5 gene fragments were subcloned into baculovirus vector, then packaged and amplified in the sf9 cells to obtain recombinant baculovirus Bac-hTERT-NIS-Egr1-K5. Bac-CMV-NIS-Egr1-K5, Bac-hTERT-0-Egr1-K5 and Bac-hTERT-NIS-Egr1-0 were constructed as controls. The expression of NIS and K5 genes in human cervix cancers cells (HeLa) was examined by Western blot and quantitative real-time PCR. Functional NIS activity was confirmed by the uptake of 125I, the inhibition of NaClO4 and the cytotoxicity of 131I. The apoptotic effect of 131I-induced K5 on human umbilical veins endothelial cells (HUVEC) was analyzed by an apoptosis assay using flow cytometry. Statistical analysis was performed using the analysis of variance. Results The recombinant baculovirus Bac-hTERT-NIS-Egr1-K5 was successfully constructed. The NIS gene under the control of hTERT promoter was specifically expressed in HeLa cells. The baculovirus-infected HeLa cells showed a significant increase of 125I uptake, which was significantly inhibited by NaClO4(F=199.296, P<0.05). Furthermore, a notable decreased cell survival rate (38.3%) was found after 131I treatment. The expression of K5 gene induced by 131I was elevated in a dose or time dependent manner and resulted in obvious inhibition with cell survival rate of 30.8% in baculovirus-infected HUVEC cells, which was significantly higher than that in the control groups (11.2% and 10.9% respectively, F=19.926, 45.409; both P<0.05). Conclusions A recombinant baculovirus dual expression vector containing the NIS and K5 genes has been successfully constructed. This study suggests the feasibility of a synergistic strategy of NIS-based raidoiodide therapy and K5-based antiangiogenic therapy in vitro, and make it possible to perform in vivo study in the near future. Key words: Baculoviridae; Telomerase; Sodium/iodide symporter; Early growth response protein 1; Tumor cells, cultured; Endothelial cells, cultured; Iodine radioisotopes; Gene therapy