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[Killing effect of Ad.TERT-TRAIL on tumor cell lines and its mechanism]
Yun-Xia Tang1, Yu Chen, Jin-Fa Gu
1Xin Yuan Institute of Medincine and Biotechnology, School of Life Science, Zhejiang Sci-Tech University, Hangzhou, Zhejiang, 310018, P.R.China. xyliu@sibs.ac.cn.
Background & Objective:
Oncolytic adenovirus Ad.TERT, a novel tumor-specific proliferating virus, has been constructed by replacing normal promoter of mild-type adenovirus E1A with promoter of human telomerase reverse transcriptase (hTERT). In vitro and in vivo experiments confirmed that Ad.TERT has antitumor effect. This study was to construct Ad.TERT-TRAIL through inserting apoptosis gene trail into Ad.TERT, and explore its antitumor effect and mechanism.
Methods:
Plasmid pZhTERT-trail and adenovirus packaging plasmid pBHGE3 were homologously recombined in HEK293 cells to construct Ad.TERT-TRAIL. Ad.TERT-TRAIL was identified by polymerase chain reaction (PCR), and confirmed by Western blot. Killing effects of Ad.TERT-TRAIL and Ad.TERT on 3 tumor cell lines, SW620, BEL-7404 and Bcap-37, and a normal cell line NHLF were detected by crystal violet dye method or MTT assay. Expression of Caspase-3 in Ad.TERT-TRAIL-, and Ad.TERT-transfected SW620 cells was detected by Western blotu cell apoptosis was detected by flow cytometry (FCM).
Results:
The 860 bp-length trail gene has been amplified by PCR. Western blot showed trail and E1A only expressed in tumor cells, which confirmed the successful construction of Ad.TERT-TRAIL. Killing effects of Ad.TERT-TRAIL on tumor cells were 10-100 times as strong as that of Ad.TERTu while both of them had little effects on normal cells. After 3 days infection (100 multiple of infection, MOI), survival rate of Ad.TERT-TRAIL-infected SW620 cells was 4%, but that of Ad.TERT-infected SW620 cells was 56%u both viruses had little effects on NHLF cells. Expression of Caspase-3 was higher in Ad.TERT-TRAIL-infected SW620 cells than in Ad.TERT-infected SW620 cells. Apoptosis rate of Ad.TERT-TRAIL-infected SW620 cells was 4 times as high as that of Ad.TERT-infected SW620 cells.
Conclusions:
Ad.TERT-TRAIL has much stronger antitumor effect than Ad.TERT. Its effect might relate with inducement effects of trail gene on expression of Caspase-3, and apoptosis of tumor cells.
