br First we used crystal violet
First, we used crystal violet staining to verify whether the recombinant adenoviruses could inhibit PC-3-luc cell
proliferation. Recombinant adenoviruses Ad-VT, Ad-T, Ad-vp3, and Ad-Mock were infected into PC-3-luc cells, separately, and stained with 0.4% crystal violet. As shown in Fig. 3A, recombinant adenoviruses Ad- VT, Ad-T, and Ad-vp3 significantly inhibited cell proliferation relative to Ad-mock, in a dose- and time-dependent manner. The inhibitory effect was strongest in the order Ad-VT >Ad-T
> Ad-vp3, indicating that Ad-VT, Ad-T, and Ad-vp3 have a certain killing effect on PC-3-luc cells.
Subsequently, we performed a quantitative experiment on the cell inhibition rate using the MTS assay. PC-3-luc cells were inoculated with recombinant adenoviruses Ad-VT, Ad-T, Ad-vp3, and Ad-Mock, separately, and added to MTS at 24, 48, 72, and 96 hours for detection. As shown in Fig. 3B, at 4 different time points (24, 48, 72, and 96 hours), Ad-VT, Ad-T, and Ad-vp3 inhibited PC-3-luc cell proliferation, while Ad-Mock had no inhibitory effect on the cells; at 1 MOI, the inhibitory effects of Ad-VT, Ad-T,
cells increased with increased infection time, showing a certain time effect (P < 0.05). From 48 to 72 hours, the inhibition of PC-3-luc proliferation by Ad-VT fluctuated between 25% and 38%, while the inhibition of Ad-T and Ad-vp3 on PC-3-luc cells was less than 25%. In general, the inhibition of cell proliferation by these 3 recombinant adenoviruses increased with increasing infection time; at 100 MOI, the inhibitory effect of Ad-VT, Ad-T, and Ad-
vp3 on cells increased significantly with time, showing an obvious time effect (P < 0.05). During infection, the per-
Ad-VT, Ad-T and Ad-vp3 increased with increasing infec-tion dose (100 MOI >10 MOI >1 MOI), showing a signifi-cant dose-effect relationship. The results showed that Ad-VT has a very significant tumor killing effect, and the killing effect has a time- and dose-effect relationship.
3.4. Recombinant adenoviruses induces selective apoptosis of prostate cancer cells
Hoechst is a dye that can enter the cell freely and combine with cell nucleic HC-030031 to display blue fluorescence, which effectively shows changes of the nucleus. The staining results at different time points are shown in Fig. 4A. PC-3-luc cells were infected with Ad-VT, Ad-T, Ad-vp3, and Ad-Mock, and the nuclei of PC-3-luc cells infected with Ad-VT, Ad-T, and Ad-vp3 presented different degrees of bright blue hyperchromatism or fragmentation, while the nuclei of Ad-mock group showed uniform blue fluorescence. This
Fig. 2. Screening and identification of PC-3-luc cells.
(A) After transfection of the pGL4.51 plasmid, the two cell clones with the highest luciferase activity were screened with 800 mg/ml of G418. (B) The
luciferase activity of each cell clone was detected using a ONE-GloTM Luciferase Assay System. Subsequently, the luciferase activity (RLU) of cell clones was detected every five generations using the luciferase assay kit to observe whether the luc gene was stably expressed. (C and D) The cell clone with the highest luciferase activity was inoculated in 1:2 ratio into a 96-well plate. After adding fluorescein, the relationship between bioluminescence intensity and cell number was observed. The cell bioluminescence intensity increased with the increase of cell number, and proved that Clone 17 could stably express luciferase. (E) The PC-3 and PC-3-luc cells were cultured in 96-well cell culture plates, and cell growth trends were detected at 1, 2, 3, 4, 5, 6, and 7 days.
(F) The PC-3 and PC-3-luc cells were cultured in 12-well cell culture plates, and cell cycles were detected using flow cytometry. (H) Western blotting analysis of PC-3 and PC-3-luc cells extracts for Cyclin D1 and CDK2.
Fig. 3. Effect of the four recombinant adenovirus strains on the cell proliferation in human prostate cells (PC-3-luc).
demonstrated that the cells showed certain apoptotic charac-teristics after infection with recombinant adenovirus. Other strong evidence for the induction of apoptosis by the recom-binant adenovirus in PC-3-luc cells was the Annexin V anal-ysis. As shown in Fig. 4B, the 3 recombinant adenoviruses Ad-VT, Ad-T, and Ad-vp3 could induce apoptosis of PC-3-luc at 3 different time points (24, 48, and 72 hours), but the
degree of apoptosis varied. Fig. 4B shows that the apoptosis rates of Ad-VT, Ad-T, and Ad-vp3 at different time points are: Ad-VT > Ad-T > Ad-vp3, in which Ad-VT induced the highest apoptosis rate at all 3 time points; Ad-VT- and Ad-T-induced apoptosis had a significant time effect (P < 0.05). The apoptosis rate of Ad-vp3 group was highest at 48 hours and decreased slightly at 72 hours. In summary, the