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  • br Bioluminescent imaging BLI is a visualization tech nique

    2020-08-12


    Bioluminescent imaging (BLI) is a visualization tech-nique for tracing A-83-01 and tissue, and gene behavior in vivo [15]. BLI is characterized by light scattering and has unique imaging advantages. In addition, tissue and cells have almost no endogenous luminescence and low endogenous signal-to-noise ratio; therefore, background interference can be effectively eliminated, and the bioluminescence signal in complex organisms can be clearly observed [16]. With the rapid development of in vivo BLI technology, it has been widely used in research into various types of cancer, and has become a promising tool in the field of biomedicine. The luciferase gene is used as a reporter gene for in vivo biolu-minescence imaging. The firefly luciferase gene is the most widely used and has high sensitivity, low endogenous, and good stability. It is often used to label viruses, bacteria, and tumor cells [17]. The establishment of a luciferase-labeled animal tumor model enables the visual, real-time, and con-tinuous monitoring of the growth and metastasis of tumor cells in different tumor models, such as in situ tumors, meta-static tumors, and spontaneous tumors. Even subtle changes can be detected in a timely manner, thus BLI provides an ideal animal model for a more intuitive evaluation of the therapeutic effects of anticancer drugs [18].
    Apoptin was originally identified as an apoptosis-induc-ing protein derived from Chicken Anemia Virus (CAV), a single-stranded DNA virus of the Gyrovirus genus [19]. The CAV genome contains three partially overlapping open reading frames encoding viral proteins from a single poly-cistronic mRNA: VP1 (capsid protein), VP2 (protein phos-phatase, scaffold protein) and the death inducing protein VP3 [20]. Expression of VP3 alone was shown to be suffi-cient to trigger cell death in chicken lymphoblastoid T-cells and myeloid cells, but not in chicken fibroblasts, and it was therefore renamed apoptin [21]. The gene encoding apoptin was among the first tumor selective anticancer genes to be isolated, and has become a focus in cancer research because 
    of its ability to induce apoptosis in a variety of human tumor cells, including melanoma, lymphoma, colon carcinoma, and lung cancer, while leaving normal cells relatively unharmed [22−25]. Thus, apoptin seems to sense an early event of oncogenic transformation and induces cancer-spe-cific apoptosis, regardless of tumor type; thus, it represents a potential future anticancer therapeutic agent.
    Transcription of human telomerase reverse transcriptase (hTERT) is a major step in regulating telomerase activity [26]. Embryonic stem cells and induced pluripotent stem cells maintain their telomere length by expressing telome-rase. The expression of telomerase is also upregulated in 85% to 90% of malignant tumor cells, giving them unlimited proliferation ability. Thus, telomerase is essential for cancer cells to maintain their immortality. Therefore, by interfering with the telomerase enzyme activity, the growth of cancer cells can be inhibited [27−29]. The hTERT promoter is inac-tive in most normal cells, but exhibits high activity in many human cancers. In many studies, the high expression of a protein targeting tumor cells is also dependent on the high efficiency and specificity of the hTERT promoter, thus open-ing up new prospects for tumor therapy.
    In a previous study, we exploited the characteristics of apoptin to construct a dual cancer-specific oncolytic ade-novirus expressing apoptin (Ad-Apoptin-hTERTp-E1a, Ad-VT) [30], which allows adenovirus to specifically repli-cate in tumor cells, and enables the apoptin protein to be expressed in a large amounts in tumor cells, thereby effec-tively killing the tumor. We have demonstrated the remark-able tumor killing effect of the recombinant adenovirus in a variety of tumor cells [30−34].
    In the present study, luciferase-labeled human prostate cancer cell line PC-3-luc was constructed, and through the detection of growth characteristics and cell cycle of PC-3-luc cells and PC-3 cells, we verified that there were no significant differences in biological characteristics between the 2 types of cells. Subsequently, a series of different in vitro experi-ments and the establishment of a luciferase labeled BALB/c nude mouse subcutaneous tumor model were used to study the inhibitory effect of recombinant adenovirus Ad-VT on tumors, which provided a theoretical basis for the treatment of prostate cancer using the oncolytic adenovirus Ad-VT.
    2. Materials and methods
    2.1. Cells, viruses and animals
    PC-3 cells were cryopreserved cells purchased from the Shanghai Institute of Biology cell bank. PC-3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), with 10% fetal bovine serum, 1000 U/ml penicil-lin, and 100 U/ml streptomycin. All the reagents for cells culture were purchased from GE healthcare life sciences, Hyclone Laboratories, (Logan, UT).