Enhanced Anti-tumor Activity By Adenovirus Mediated LIF And IL-24 Co-expression For Tumor Cells And Its Mechanism

Objective: To construct a recombinant adenoviral vector co-expressing leukaemia inhibitory factor (LIF) and interleukin-24 (IL-24) and study its enhanced anti-tumor effects on glioma cells and its mechanism.Methods: The polyA+promoter fusion fragments, LIF, and IL-24 fragments were amplified by PCR using pORF-mbcl-2a, pcDNA3.0-LIF, and pAdTrack-CMV-IL-24 plasmids as templates, respectively, and PacI enzyme site in polyA+promoter was mutated(polyA+promoter△). Then the polyA+promoter△(SalI,NotI), LIF (BglII,SalI), and IL-24 (XhoI,XbaI) fragments were subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-LIF-polyA+promoter△-IL-24 and identified by PCR, double endonuclease digestion, and DNA sequencing. The pAdTrack-CMV- LIF-polyA+promoter△-IL-24 transfer vector linearized with PmeI digestion and pAdEasy-1 backbone vector were further cotransfected into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy- 1-pAdTrack-CMV-LIF-polyA+promoter△-IL-24(pAd-LIF-polyA+promoter△- IL-24) homologous recombinant plasmids purified from the above BJ5183 cells were transfected into the bacterial DH5αcells competent cells to abundantly amplify pAd-LIF-polyA+promoter△-IL-24 plasmids. Then they were linearized with PacI digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by Lipofectaimne2000, leading to formation of the recombinant adenoviruses Ad-LIF-polyA+promoter△-IL-24(Ad-LIF-IL-24) co-expressing LIF and IL-24. The most sensitive cell and the best MOI of Ad-LIF-IL-24 was chosen by Ad-LIF-IL-24 infecting SMMC-7721, U251, HT29, A549, HL-60, K562 and WI-38 in vitro. The effect of enhanced growth-suppressing and apoptosis-inducing by Ad-LIF-IL-24 on the tumor cells in vitro were assessed by MTT assay and FCM, and the apoptosis of the cell nucleus were detected by Hochest staining. The expression of apoptosis- and angiogenesis-related genes (bcl-2,bax,ICE,p53 and HIF-1α) and the Cleaved Caspase3 in U251 glioma cells was determined by RT-PCR and Western blotting, respectively.Results: The adenoviral vector co-expressing LIF and IL-24, Ad-LIF- polyA+promoter△-IL-24, was successfully constructed. More than 95% of theses tumor cells (SMMC-7721, U251, HT29, and A549) and WI-38 cells were infected by Ad-LIF-IL-24 at MOI of 50, whereas HL-60 and K562 cells were hardly infected. The growth of U251 glioma cells was significantly inhibited by Ad-LIF-IL-24 at MOI of 100. Ad-LIF-IL-24- mediated LIF and IL-24 co-expression significantly inhibited U251 glioma cells growth, induced cells apoptosis, exhibiting additive effect. Ad-LIF-IL-24 significantly up-regulated the expression of ICE,bax,p53, and Cleaved Caspase-3 and down-regulated the expression of bcl-2 and HIF-1α.Conclusion: The adenoviral vector co-expressing LIF and IL-24 (Ad-LIF-IL-24) was successfully constructed. Ad-LIF-IL-24 had significant additive effect in suppressing U251 glioma cells growth and inducing cells apoptosis in vitro. Ad-LIF-IL -24 can up-regulate the expression of ICE,bax,p53 and down-regulate Bcl-2 resulting activation of Caspase-3 and down-regulate the expression of angiogenesis-related gene HIF-1αto exhibit significantly anti-tumor effect on U251 glioma cells in vitro .