br Fig Identification of PC luc
Fig. 4. Identification of PC-3-luc cell apoptosis induced by recombinant adenovirus using Hoechst and Annexin V staining.
(A) Morphological changes under fluorescence microscopy after Hoechst staining. PC-3-luc HC 030031 were treated with Ad-VT, Ad-T, Ad-vp3, and Ad-mock for 24, 48, and 72 hours and compared with untreated cells. After the cells were infected with the recombinant adenovirus, in addition to the mock group, the phenomenon of nuclear thickening and nuclear fragmentation increased significantly with the prolongation of time. (B) The PC-3-luc cells were analyzed by
flow cytometry after Annexin-V and PI staining. We quantified the percentage of apoptotic cells: The PC-3-luc cells infected with Ad-VT, Ad-T, and Ad-vp3 all showed overt apoptosis. Data are shown as the mean § SD. * P < 0.05, ** P < 0.01, compared with the control.
recombinant adenovirus Ad-VT induced apoptosis in PC-3-luc cells specifically.
3.5. Recombinant adenovirus induces apoptosis through the mitochondrial pathway
The activation of caspases was then determined (Fig. 5C). Infection of PC-3-luc cells with Ad-VT, Ad-T, and Ad-vp3 caused a marked increase in the levels of cas-pases−3, 6, and 7. We also evaluated the effects of recom-binant adenoviruses on the MMP. The 4 recombinant adenoviruses, Ad-VT, Ad-T, Ad-vp3, and Ad-Mock, were inoculated into PC-3-luc cells, respectively, and stained with JC-1 at 24, 48, and 72 hours, respectively.
As shown in Fig. 5A and B, compared with the ad-mock, the inhibitory effect of Ad-VT, Ad-T, and Ad-vp3 on PC-3-luc cells was demonstrated by changes in the MMP at dif-ferent time points. The different recombinant adenoviruses have varying abilities to induce apoptosis, as reflected in the different degrees of MMP depolarization. The ability of Ad-VT and Ad-T to induce apoptosis increased with time. The apoptotic cells gradually increased, and the JC-1 grad-ually changed from the initial red aggregate to the green monomer. By 72 hours, the number of apoptotic cells was the largest, and the ratio of red fluorescence to green fluo-rescence was the most significant (P < 0.05). However, the ability of Ad-vp3 to induce apoptosis did not change signif-icantly with increased time. At 48 hours, the number of apoptotic cells was the largest, that is, the ratio of red fluo-rescence to green fluorescence decreased, but at 72 hours, the ratio of red fluorescence to green fluorescence increased slightly. At all 3 different time points, the ratio of red fluo-rescence to green fluorescence is: Ad-VT < Ad-T < Ad-vp3 < Ad-Mock. Ad-VT had the strongest ability to induce apoptosis through MMP changes, with the highest number of apoptotic cells and the most significant decrease in the red/green fluorescence ratio.
3.6. Recombinant adenovirus inhibits migration and invasion of prostate cancer cells
After the scratches were formed, photographs were taken at different time points (0, 24, and 48 hours) (at the same position), and the width of the scratch was calculated. The cell mobility was reflected by the change in the scratch width. The changes were studied to investigate the effect of the 4 recombinant adenoviruses on the migration of PC-3-luc cells. As shown in Fig. 6A-C, the migration of cells infected with Ad-VT and Ad-T was significantly lower than that of cells infected with Ad-vp3 and Ad-Mock; Ad-VT induced the strongest inhibition of cell migration, and at 48 hours, the migration of PC-3-luc cells was 12.11%, which was significantly lower (P < 0.05) than that of Ad-Mock group and the control group. Inhibition of migration was strongest in the order: Ad-VT > Ad-T > Ad-vp3 > Ad-Mock. The cell scratch assay demonstrated that the migration ability of PC-3-luc cells was inhibited after infection with recombinant adenovirus. In the transwell migration assay, PC-3-luc cells were treated with 4 recom-binant adenoviruses for 24 hours and 48 hours, respectively, and then transferred into the chamber. At each time point, we stained the chamber with crystal violet, observed the cell adherence on the bottom wall of the chamber, and cal-culated the number of cells. As shown in Fig. 7A-C, Ad-VT, Ad-T, and Ad-vp3, but not Ad-Mock, could inhibit the migration ability of PC-3-luc cells, and the extent of inhibi-tion was different. At 24 hours or 48 hours after infection, the inhibitory effect of Ad-VT and Ad-T on cell migration ability showed a significant dose effect (P < 0.05), and the inhibition of migration was strongest in the order: Ad-VT > Ad-T > Ad-vp3 > Ad-Mock. At 100 MOI and 10 MOI, cells infected with Ad-VT, Ad-T, and Ad-vp3 showed a lower cell migration rate at 48 hours than at 24 hours, indi-cating a time effect relationship. Similar results were also observed in the Transwell invasion assay (Fig. 8A-C).
Fig. 5. Analysis of mitochondrial membrane potential of recombinant adenovirus-treated PC-3-luc cells by JC-1 staining and analysis of caspase activation in PC-3-luc cells treated with the recombinant adenoviruses.