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  • br In this study the inhibitory

    2022-05-22


    In this study, the inhibitory effect of recombinant adeno-viruses on PC-3-luc Melatonin was investigated by crystal violet staining and MTS assays. The results showed that Ad-VT, Ad-T, and Ad-vp3 all Melatonin 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.
    In this study, a subcutaneous tumor-bearing model of nude mice labeled with luciferase was successfully estab-lished. The in vivo inhibitory effect of recombinant adeno-virus on PC-3-luc-induced tumors was investigated using a living imaging system. The results of in vivo imaging in nude mice showed that the average bioluminescence inten-sity of the tumors in the Ad-VT treatment group was always lower than that in the other groups at 2 to 5 weeks. The average tumor growth curve and the average tumor inhibi-tion curve showed that Ad-VT had a significant antitumor effect, with the inhibition rate reaching 66% at 5 weeks. The survival curve of nude mice showed that the Ad-VT treatment group could significantly prolong the survival of mice: The survival rate was 66.67% at 6 weeks. These results showed that Ad-VT has antitumor effect and signifi-cantly prolongs mouse survival.
    In short, through a series of in vitro and in vivo experi-ments demonstrated that Ad-VT has a significant inhibitory effect on the prostate cancer cell line PC-3.