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  • br A Changes in red and

    2020-08-12


    (A) Changes in red and green HC030031 under fluorescence microscopy after JC-1 staining. Increased apoptosis results in a decreased ratio of red fluorescence/ green fluorescence. (B) We analyzed the change in the ratio of red to green after JC-1 staining quantitatively. Ad-VT, Ad-T, and Ad-vp3 can cause changes in the mitochondrial membrane potential, and Ad-VT had the strongest ability to induce apoptosis by affecting the mitochondrial membrane potential. (C)
    Cells were treated with the four kinds of recombinant adenoviruses for 48 hours at an MOI of 100. The vertical coordinate for the content of caspases was in units of mg/ml. Data are the mean § SD. * P < 0.05, ** P < 0.01, compared with the control.
    The invasion result demonstrated that Ad-VT had strongest ability to inhibit PC-3-luc cells invasion. These results indi-cated that Ad-VT could significantly inhibit the migration and invasion of PC-3-luc cells in a short period of time.
    3.7. Antitumor effect of recombinant adenovirus in vivo
    We examined the antitumor potential of recombinant adenovirus in a PC-3-luc tumor model.
    From the week 0 after the injection of the viruses, the change in the bioluminescence intensity of the tumor was observed using a living body imaging system, and was con-tinuously observed for 5 weeks. The results are shown in Fig. 9A and B. At 0 to 2 weeks, there was no significant dif-ference in the average bioluminescence intensity of the 
    tumors in each treatment group (P > 0.05); at 2 to 3 weeks, the average bioluminescence intensity of the tumors in the Ad-VT treatment group, the Ad-T treatment group, and the Ad-vp3 treatment group increased slightly, but with no sig-nificant difference (P > 0.05), while the average biolumi-nescence intensity of the Ad-Mock treatment group and the control group increased rapidly. At 3 to 5 weeks, the aver-age bioluminescence intensity of the Ad-VT treatment group was always lower than that of the other treatment groups; at 4 to 5 weeks, the average bioluminescence inten-sity of the Ad-VT and Ad-T treatment groups was signifi-cantly lower than that of Ad-Mock treatment group and the control group (P < 0.05).
    After subcutaneous tumor formation, the tumor size was measured continuously for 5 weeks. The results are shown
    Fig. 6. Identification of PC-3-luc cell mobility induced by recombinant adenovirus with cell scratch assay.
    (A and B) The PC-3-luc cells were infected with recombinant adenovirus at 1 and 10 MOI. The width of the scratches in the cell layer at each time point was measured. All measurements were performed in triplicate. (C and D) The cell migration rate was calculated according to the following formula: Cell mobility = (0 hour scratch width ¡ 24/48 hours scratch width)/0 hour scratch width. Data are presented as the means § standard deviation (SD). * P < 0.05, ** P < 0.01, compared with the control.
    Fig. 7. Migration suppression effects on PC-3-luc cells assessed using the Transwell assay.
    (A) The migration suppression of PC-3-luc cells infected with 10 and 100 MOI recombinant adenovirus at 24 and 48 hours. (B and C) Cells that had
    migrated through the membrane were counted under a microscope after they were fixated with carbinol and stained with crystal violet. Cells infected with Ad-VT showed the lowest migration at 24 and 48 hours. Data are presented as the means § standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the control.
    in Fig. 9C and D. The average tumor growth rate between the control group and the Ad-Mock treatment group was not significantly different (P > 0.05); the tumor growth rate of the Ad-T treatment group and Ad-vp3 treatment group became slower at 3 to 4 weeks, and had no significant difference (P > 0.05). After 4 weeks, the average growth rate of the tumor in the Ad-T treatment group was slower; however, the average growth rate of tumors in the Ad-vp3 treatment group did not slow down, and was always larger than that in the Ad-T treatment group. Compared with the 
    Ad-Mock treatment group and the control group, the aver-age growth rate of the Ad-VT treatment group was signifi-cantly lower at 4 to 5 weeks (P < 0.05). The average growth rate of the Ad-VT treatment group was always lower than that of the other groups at 3 to 5 weeks, indicat-ing that Ad-VT could effectively inhibit the growth of PC-3-luc tumors.
    From the tumor growth inhibition curve, it can be seen that the average tumor inhibition rate of the control group, Ad-Mock treatment group, and Ad-vp3 treatment group
    Fig. 8. Invasion suppression effects on PC-3-luc cells assessed using the Transwell assay.
    (A) The invasion suppression of PC-3-luc cells infected with 10 and 100 MOI recombinant adenovirus at 24 and 48 hours. (B and C) Cells that had passed
    through the membrane were counted under a microscope after they were fixated with carbinol and stained with crystal violet. Cells infected with Ad-VT showed the lowest invasion at 24 and 48 hours. Data are presented as the means § standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the control.