The Construction Of Adenovirus-Mediated IL-24/OSM Co-expression And The In Vitro And In Vivo Antitumor Activity For The Human Melanoma Cells

Objective:To construct a recombinant adenviral vector co-expressing interleukin24(IL-24)and oncostainM (OSM) and study its antitumor activity for the human melanoma cellsin vitro and in vivo.Methods:The recombinant Ad-IL-24-OSM was obtained by gene recombination and in vitropackaging technique.Firstly,between the restriction enzyme cutting sites Bgl II and Sal Ias well as Not I and XhoI,the IL-24and OSM fragments were inserted respectively,atthe foundation of pAdTrack-CMV-polyA-promoter,then we got the pAdTrack-CMV-IL-24-polyA-promoter-OSM. Secondly, the recombinant adenovirus Ad-IL-24-OSM,which co-expressing IL-24and OSM can be obtained from homologous recombination,package and amplification.A375cells were infected with Ad-IL-24-OSM and RT-PCRwas used to detect the transcription of IL-24and OSM in the cells. Western blot wasused to detect the expression of IL-24and OSM in the cells. Morphologic changes wereobserved by light microscope and fluorescence microscope. Cell growth inhibition wasdetected by MTT assay. The cell cycle and apoptosis were detected by flow cytometryand RT-PCR was used to detect the transcription of P21, P53,Bax and Bcl-2. Theexpression of Caspase-3of related genes of apoptosis was detected by Western blot. Invivo enhanced anti-tumor effect on malignant melanoma cells was further observedusing A375human malignant melanoma transplanted tumor in athymic nude mousemodel. The expression of cell cycle and apoptosis-and angiogenesis-related proteins(P21, P53, Cox-2,Bcl-2, Bax, Caspase-3and CD34) in A375human malignantmelanoma cells and A375transplanted tumor was determined by immunohistochemistryanalysis. Results:1. Ad-IL-24-OSM was proved in correct construction by double digests andRT-PCR results.2. Expression of IL-24and OSM in A375cells were identified by western blotand RT-PCR.3. Adenovirus-mediated IL-24and OSM co-expression can significantly inhibitA375human malignant melanoma cell growth, induce cell apoptosis and excelobviously Ad-IL-24, Ad-OSM single work(P<0.05)4. A375human malignant melanoma transplanted tumor growth in athymic nudemouse, exhibited enhanced anti-tumor effect(Q=1.02)。5. By testing the molecular mechanism of cellular apoptosis, we foundAd-IL-24-OSM had more marked effect in up-regulating the expression of P21,P53,Bax,Caspase-3and down-regulating the expression of Cox-2, Bcl-2, CD34compared with Ad-IL-24or Ad-OSM (p<0.05).Conclution:Ad-IL-24-OSM was proved in correct construction. Adenovirus-mediated IL-24and OSM co-expression significantly inhibited A375human malignant melanoma cellgrowth, induced cell apoptosis in vitro and in vivo,which may result in up-regulating theexpression of P21, P53, Bax and down-regulating the expression of Cox-2,CD34andBcl-2and induced cell apoptosis by activating Caspase-3pathway and inhibitingneovascularization.