br Viral protein VP from CAV which induces apopto sis
Viral protein 3 (VP3) from CAV, which induces apopto-sis in chick thymocytes, was named as "Apoptin" because it can induce apoptosis of various HC 030031 [21,45,46]. Apoptin selectively induces apoptosis in a variety of cancer cells, but has no effect on normal cells [30,47−51]. Studies have shown that apoptin has different nuclear localizations in tumor cells and normal cells, explaining its ability to selec-tively kill tumor cells. Mutating the nuclear localization signal of apoptin suggested that its nuclear localization is closely related to the induction of tumor cell apoptosis . Studies have also shown that apoptin in cells has a tropism, transporting from high to low concentrations, and the abil-ity to induce apoptosis is dose-related . Other studies have shown that apoptin mainly accumulates in the nuclei of tumor cells and causes cell death, including in cells with a P53 mutation or Bcl-2 overexpression .
Telomerase is responsible for the extension of the ends of chromosomes in DNA synthesis, which occurs in germ cells as well as in most malignant cells. The constitutive expression of hTR and hTEP1 of the telomerase is mainly dependent on the transcription of hTERT. In the former, the expression is abnormal and the activity is high, while in the latter, it is almost not expressed and the activity is low. In most cancers, up-regulation of TERT mRNA expression and down-regulation of tumor suppressor genes, such as Rb and p16, may maintain the immortalization of cancer cells . Human hTERT act as tumor specific promoter for oncolytic adenoviruses. hTERT promoter fragment, which in the context of replication-conditional adenovirus Adeno-hTERT-E1A recapitulated high telomerase promoter-based E1A expression and viral activity in cancer cells .
In this study, the inhibitory effect of recombinant adeno-viruses on PC-3-luc cells was investigated by crystal violet staining and MTS assays. The results showed that Ad-VT, Ad-T, and Ad-vp3 all had different degrees of inhibitory effect on PC-3-luc cells, with the order of strength and weak-ness as Ad-VT > Ad-T > Ad-vp3, and had a certain time and dose effect; the inhibition induced by Ad-vp3 was the highest at 96 hours and reached 30%, and it is significantly higher than the Ad-mock. On the other hand, at 100 MOI, the inhi-bition rate of Ad-VT on PC-3-luc cells was the highest at 72 hours and reached 62.74%, while that of Ad-T was the high-est at 72 hours (47.77%). From the above results, we could see that the expression of apoptin protein played a very sig-nificant role in the inhibition effect of recombinant adenovi-ruses on PC-3-luc cells; although Ad-vp3 did not have the ability to replicate in tumor cells, but it expressed apoptin protein; the inhibition effect of Ad-vp3 was significantly higher than that of Ad-mock on PC-3-luc cells. While both Ad-VT and Ad-T contained the hTERT promoter, and there-fore both had the ability to replicate in tumor cells. It was found that the inhibitory effect of Ad-VT was significantly higher than that of Ad- T on PC-3-luc cells, possibly because Ad-VT has the ability to continuously express apoptin.
Subsequently, the inhibition pathway of recombinant adenovirus on PC-3-luc cells was analyzed using JC-1
staining, Hoechst staining, and Annexin V-FITC/PI flow assay. The results showed that Ad-VT, Ad-T, and Ad-vp3 could induce apoptosis to produce cytotoxicity. The Annexin V flow cytometry results showed that at 100 MOI and 72 hours, the apoptosis rate of Ad-VT group was 58.48%, and the apoptosis rate of Ad-T group was 44.13%, which was consistent with MTS results, further demonstrat-ing that Ad-VT, Ad-T, and Ad-vp3 exert their inhibitory effects mainly by inducing apoptosis. The effects of the recombinant adenoviruses on the migration and invasion of PC-3-luc cells were investigated using a scratch assay, Transwell migration, and invasion assay. The results showed that Ad-VT, Ad-T, and Ad-vp3 could decrease cell migration, invasion, and inhibition in the order Ad-VT > Ad-T > Ad-vp3, and the main reason was that Ad-VT, Ad-T, and Ad-vp3 could kill the cells or decreased the activity of some proteolytic enzymes, which could weaken cell migration and invasion. The specific mechanisms by which the adenoviruses affect cell migration and invasion require further research. In conclusion, Ad-VT, mainly by inducing apoptosis, inhibited the growth, migration, and invasion of PC-3-luc cells, to a greater extent than Ad-T and Ad-vp3, which is related to its specific replication and specific kill-ing ability. Ad-vp3 cannot specifically replicate in PC-3-luc cells, therefore, it can only produce a certain inhibitory effect over a short period.