Figures & data
Figure 1 Synthesis scheme of conjugation strategy for mannan to adenovirus under the appropriate oxidizing conditions.
Figure 2 PAS staining to confirm the formation of mannan-modified adenovirus. The different groups subjected to PAS staining are medium-free mannan (A), PAS reagent (B), free Ad5 (C), low mannan-modified Ad5 (D), medium mannanmodified Ad5 (E), and high mannan-modified Ad5 (F), respectively. Mannan of different concentrations was conjugated to adenovirus as described in the Materials and methods section, and then the conjugates were subjected to PAS assay. At the same time, PAS reagent alone was used as the negative control. Both well (A) and well (F) were further observed under light microscopy (100×).
Abbreviations: PAS, periodic acid Schiff; Man, mannan; Ad5, recombinant adenovirus using the PTEN gene; PTEN, phosphatase and tensin homolog deleted on chromosome ten.
Figure 3 Antitumor effect of combined therapy on suppression of pre-existing subcutaneous mice tumors.
Notes: The tumor model were established by subcutaneous injection of murine H22 hepatoma cells (1.0 × 107) into the right mouse flank. When the mean tumor size reached 100–150 mm3, the mice were divided into five groups (at least n = 8) and injected intratumorally with phosphate-buffered solution, naked Ad5-PTEN, docetaxel alone, Man-Ad5-PTEN, or Man-Ad5-PTEN-docetaxel. Arrow indicates the time point of single injection. Tumor volumes were monitored every day and were estimated as: tumor volume V (mm3) = 0.52 × length (mm) × width (mm)2, which was compared in tumor-bearing mice after treatment with either phosphate-buffered solution, naked Ad5-PTEN, docetaxel alone, Man-Ad5-PTEN, or Man-Ad5-PTEN-docetaxel. The significance of the differences was evaluated using the Student’s t-test. Data are shown as the mean ± standard deviation for five mice in each group.
Abbreviations: Man, mannan; Ad5, recombinant adenovirus using the PTEN gene; PTEN, phosphatase and tensin homolog deleted on chromosome ten.
Figure 4 Effect of combination treatment on tumor weight. The H22-tumor bearing mice in each group were given the different formulations, respectively. Two weeks later, the mice were euthanized by nitrogen inhalation. Tumor images were taken by Nikon D70 digital camera (I) and the weight was also examined (II). The tumor weight was compared between phosphate-buffered solution (A) and docetaxel (B), Ad5-PTEN (C), Man-Ad5-PTEN (D), and Man-Ad5-PTEN-docetaxel (E).
Notes: The significance of the differences was evaluated using the Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001). The tumor inhibition rate was calculated and normalized to the phosphate-buffered solution group. Data are shown as the mean ± standard deviation in each group.
Abbreviations: Man, mannan; Ad5, recombinant adenovirus using the PTEN gene; PTEN, phosphatase and tensin homolog deleted on chromosome ten.
Figure 5 Flow cytometry analysis of cell apoptosis in murine hepatoma H22 cells obtained from tumor-bearing mice. The different groups of mice were treated with Ad5-PTEN, docetaxel, phosphate-buffered solution, Man-Ad5-PTEN, and Man-Ad5-PTEN-docetaxel, respectively. The single cell solution was prepared as described in the Materials and methods section and then stained with Annexin V and propidium iodide. The samples were analyzed by FACSCalibur within one hour. The percent of apoptotic cells was compared between the phosphate-buffered solution group (C) and Ad5-PTEN (A), docetaxel (B), Man-Ad5-PTEN (D), and Man-Ad5-PTEN-docetaxel (E).
Notes: The significance of the differences was evaluated using the Student’s t-test (*P < 0.05; ***P < 0.001). Data are expressed as the mean ± standard deviation (n = 3) in each group.
Abbreviations: FITC, fluorescein isothiocyanate; Man, mannan; Ad5, recombinant adenovirus using the PTEN gene; PTEN, phosphatase and tensin homolog deleted on chromosome ten.
Figure 6 In vivo tumor cell death study of Man-Ad5-PTEN in combination with docetaxel. The H22 tumor-bearing mice were treated as mentioned earlier with Ad5-PTEN, docetaxel, phosphate-buffered solution, Man-Ad5-PTEN, or Man-Ad5-PTEN-docetaxel. Tumor sections were excised, fixed, dewaxed, and followed by standard H&E staining for necrosis (A, 200×) and apoptosis (B, 400×). As the arrow indicates, condensation and fragmentation of nuclei were observed, which are the hallmarks of apoptosis. Detection of apoptotic cells were also carried out by TUNEL assay (C, 400×).
Abbreviations: Man, mannan; Ad5, recombinant adenovirus using the PTEN gene; PTEN, phosphatase and tensin homolog deleted on chromosome ten; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
