Enhanced Anti-Tumor Effect Of RGD-Modified Adenovirus Mediated LIF And IL-24 Co-Expression For Human Leukemia Cells In Vitro And Vivo.

Objective:Construct RGD-modified LIF and IL-24 gene co-expression adenovirus vector and detect its enhanced anti-tumor effect on human leukemia cells in vitro and vivo.Methods:Use the method of homologous recombination to construct the RGD-modified adenovirus, Ad.RGD-LIF, Ad.RGD-IL24 and Ad.RGD-LIF-IL24. Amplify the adenovirus in QBI-293 A cells, then detect the titer of adenovirus. Use RGD-modified adenovirus infect MEG01 and K562 leukemia cells and compare the efficiency with un-modified adenovirus by FCM. We choose MEG01 cells to do the next study. The experiment were divided into 5 groups, Ad.RGD, Ad.RGD-LIF, Ad.RGD-IL24, Ad.RGD-LIF-IL24 and PBS. Western blot method was used to detect the expression of LIF and IL-24 in MEG01 cells. The CCK-8 and PE-AnexinV/7-AAD methods were used to detect the growth inhibition and apoptosis of MEG01 cells. Then we detected p53, Bax, Bad and Bcl-2 gene expression after adenovirus infected. PI staining method was used to detect cell cycle of MEG01 cells. Real-time PCR method was used to detect P21 and E2F1 gene expression. In vivo experiment, MEG01 cells were inoculated subcutaneously into the flanks of 4-week-old female athymic nude mice to build the xenografted tumor. Once palpable, tumors were measured using vernier calipers at intervals, group of adenovirus Ad.RGD, Ad.RGD-LIF, Ad.RGD-IL24, Ad.RGD-LIF-IL24 was dissolved in sterile PBS at 1×109(pfu/mL) and administered every 2 days(total 5 times). After treatment, execute nude mice, remove the specimens of tumor, fix it at 4% paraformaldehyde. Then use the immunohistochemical method to detect P53, E2F1, Bax, Bcl-2 and Caspase-3 gene expression.Results:Compared with the unmodified adenovirus, RGD-modification significantly improved the adenovirus efficiency on MEG01 and K562 leukemia cells. Exogenous gene LIF and IL-24 can inhibit the growth of MEG01 cells, induce cell apoptosis and block the cell cycle through regulate Bax, Bcl-2, P53, P21 and E2F1 gene expression. Besides, the double gene co-expression has a synergistic effect. In vivo, construct the leukemia cell xenografted tumor model successfully. After Ad.RGD-LIF, Ad.RGD-IL24 and Ad.RGD-LIF-IL24 treatment, tumor growth were significantly suppressed, the tumor volume and weight decreased remarkably, two gene co-expression have enhanced anti-tumor activity. IHC result indicate that Ad.RGD-LIF-IL24 have enhanced effect in up-regulating the expression of Bax, Caspase-3, P53 and down-regulating the expression of Bcl-2, E2F1.Conclusion:1. Construct the RGD-modified adenovirus Ad.RGD-LIF、Ad.RGD-IL24 and Ad.RGD-LIF-IL24 successfully. After RGD modified, the efficiency of adenovirus vector on MEG01 and K562 leukemia cells improved significantly.2. Ad.RGD-LIF、Ad.RGD-IL24 and Ad.RGD-LIF-IL24 could significantly inhibit MEG01 cell growth in vitro and vivo.3. LIF and IL-24 co-expression could inhibit MEG01 cell invasion through regulate MMP-2 and MMP-9 gene expression.4. Ad.RGD-LIF-IL24 co-expression LIF and IL-24 showed enhanced effect in up-regulating the expression of Bax, Caspase-3, P53 and down-regulating the expression of Bcl-2, E2F1, which may be responsible for its synergetic anti-tumor effect on human MEG01 cells in vitro and in vivo.