| Objective: To investigate the inhibitory effects and anti-cancer mechanisms of recombinant adenovirus-mediated hING4 gene and hIL-24 gene on the T24 bladder cancer cells transplanted tumor in athymic nude mice.Methods: (1) Transfected recombinant adenovirus-mediated hING4 (Ad-ING4), recombinant adenovirus-mediated hIL24 (Ad-IL24) and recombinant adenovirus-mediated hIL24 and hING4 (Ad-ING-4IL-24) into QBI-293 cells, after several times amplification and obtained high potency contain objective gene adenovirus. (2) Male athymic nude mice were subcutaneously inoculated on their armpits of the right anterior limbs with 4×107 T24 bladder cancer cells suspension to construct the transplanted tumor models. (3) 35 mice bearing T24 bladder cancer transplantation tumor were randomly divided into seven groups:phosphate buffered saline group( PBS), recombinant adenovirus-green fluorescent protein group(Ad-GFP), Ad-IL24 group,Ad-ING4 group,Ad-ING-4IL-24 group, mitomycin group(MMC) and Ad-ING-4IL-24+MMC group. The mice were intratumorally injected every other day, 6 times in total. Tumor progression and regression were monitored daily. Tumor volumes were measured with a caliper every week and calculated by the following formula: tumor size=ab2/2, where a is the larger and b is the smaller of the two dimensions. In addition, the tumor bearing mice were sacrificed 3 weeks at therapy, and the tumors were removed, weighted, fixed by 10% neutral formalin, and embedded in paraffin for hematoxylin and eosin staining and immunohistochemistry. Immunohistochemistry detection the expressions of survivin, Fas, Cox-2, Bcl-2, caspase-3, VEGF, Bax and CD34. Detected micro vessel density (MVD) as previously described by Weidner.Results: (1) High tite (109pfu/ml)Adenoviral vector of ING4 gene,IL-24gene﹑and ING4-IL-24 double gene were obtained. (2) Transplanted tumor model was successfully constructed; the Tumor formation rate was 87.5%. (3) After treated, the tumor weight of Ad-IL-24 group,Ad-ING4 group and Ad-ING-4-IL24 group were 0.96±0.05g, 0.93±0.06g, 0.62±0.04g,compared with PBS group(1.73±0.05g) and Ad-GFP (1.69±0.09g), respectively, the difference was statistically significant(P<0.05); and the tumor weight of Ad-ING-4-IL24 group(0.62±0.04g)compared with Ad-IL-24 group (0.96±0.05g) and Ad-ING4 group(0.93±0.06g) respectively, the difference was statistically significant (P<0.05); the tumor weight of Ad-ING-4-IL24+MMC group(0.32±0.04g)compared with Ad-ING-4-IL24 group(0.62±0.04g)and MMC group(0.56±0.04g) respectively, the difference was statistically significant(P<0.05). The ratios of tumor suppression of Ad-ING-4-IL24 group(0.63±0.05g) compared with Ad-IL-24 group (0.43±0.06g) and Ad-ING4 group(0.45±0.06g) respectively, the difference was statistically significant (P<0.05); The ratios of tumor weight suppression of Ad-ING-4-IL24+MMC group (0.82±0.07g) compared with Ad-ING-4-IL24 group (0.63±0.03g) and MMC group (0.67±0.05g) respectively, the difference was statistically significant(P<0.05). (4)The expressions of Fas, Bax and caspase-3 were up-regulated, meanwhile Bcl-2 and survivin were down-regulated, VEGF and CD34 which correlated with angiopoiesis were down-regulated and MVD was degraded in Ad-IL-24 group(P<0.05). In Ad-ING4 group, the expressions of Fas, Bax and caspase-3 were up-regulated, meanwhile Bcl-2 and survivin were down-regulated, Cox-2 and CD34 which correlated with angiopoiesis were down-regulated and MVD was degraded(P<0.05). In Ad-ING-4-IL24 group, the expressions of Fas﹑Bax and caspase-3 were up-regulated, meanwhile Bcl-2 and survivin were down-regulated, VEGF,Cox-2 and CD34 which correlated with angiopoiesis were down-regulated and MVD was degraded(P<0.05), and Ad-ING4 and Ad-IL-24 had distinguished synergistic effec(tP<0.05). In Ad-ING-4-IL24+MMC group, the expression of Fas﹑Bax and caspase-3 were up-regulated, meanwhile Bcl-2and survivin were down-regulated, furthermore, VEGF,Cox-2 and CD34 which correlated with angiopoiesis were down-regulated and MVD was degraded(P<0.05), Ad-ING-4-IL24 and MMC had distinguished synergistic effect(P<0.05).Conclusions: (1) Ad-IL-24, Ad-ING4, Ad-ING-4-IL24 and Ad-ING-4IL-24+MMC treatment can noticeable inhibit the growth of transplanted tumor. (2) The double genes therapeutic effect is batter then two single gene means synergistic effect of both single genes. (3) The group of double genes unite with MMC therapeutic effect is batter than the group of double genes and MMC means the double gene is one kind sensitizer of MMC。(4) The anti-cancer molecular mechanisms of Ad-ING-4-IL24 may be relative with apoptosis and angiopoiesis;the MMC chemotherapeutic sensitization molecular mechanisms of Ad-ING-4-IL24 may be relative with the expression up-regulations of Fas , caspase-3 and ratio of Bax/Bcl-2 and down-regulations of survivin and VEGF. |
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