Enhanced Anti-tumor Activity By Adenovirus-mediated ING4 And IL-24 Co-expression Combined With Anticancer Drug Cisplatin For Hepatocellular Carcinoma And Its Mechanism

Objective:To construct a recombinant adenoviral vector co-expressing inhibitor of growth 4 (ING4) and interleukin-24 (IL-24) and explore its enhanced anti-tumor and chemosensitivity to anticancer drug cisplatin (CDDP) effects on hepatocellular carcinoma and its mechanism.Methods:The internal ribosome entry site (IRES), IL-24, and ING4 fragments were amplified by PCR using pGEZ-Term, pAdTrack-CMV-IL-24, and pAdTrack-CMV-ING4 plasmids as templates, respectively. The IRES (SalI,NotI), IL-24 (XhoI,XbaI), and ING4 (BglII,SalI) fragments were subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-IRES-IL-24 and identified by PCR, double endonuclease digestion, and DNA sequencing. The pAdTrack-CMV-ING4-IRES-IL-24 transfer vector linearized with PmeI digestion and pAdEasy-1 backbone vector were further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy-1-pAdTrack-CMV-ING4-IRES-IL-24 (pAd-ING4-IRES-IL-24) homologous recombinant plasmids purified from the above BJ5183 cells were transformed into the bacteria DH5αcompetent cells to abundantly amplify pAd-ING4-IRES-IL-24 plasmids. Then they were linearized with PacI digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by Lipofectamine2000, leading to formation of the recombinant adenoviruses Ad-ING4-IRES-IL-24 co-expressing ING4 and IL-24. The other kind of recombinant adenoviruses Ad-ING4-polyA+promoter-IL-24 was similarly prepared as described above, except that the polyA+promoter fusion fragments were obtained by SOE-PCR using pORF-mbcl-2a plasmids as templates. The in vitro enhanced growth-suppressing, apoptosis-inducing, and chemosensitivity to anticancer drug cisplatin (CDDP) effect of Ad-ING4-IL-24 co-expressing ING4 and IL-24 on SMMC-7721 and HepG2 human hepatocellular carcinoma cells were assessed by MTT assay and FCM, and its in vivo enhanced anti-tumor effect on hepatocellular carcinoma was further observed using SMMC-7721 human hepatocellular carcinoma transplanted tumor in athymic nude mouse model. The in vitro effect of Ad-ING4-IL-24 on SMMC-7721 cells'invasive ability was addressed using a Transwell Chamber system. The expression of cell cycle and apoptosis- and angiogenesis-related proteins (P53, P21, P27, Cox-2, Fas, Bcl-2, Bax, Caspase-3, VEGF, IL-8, and CD34) in SMMC-7721 hepatocellular carcinoma cells and SMMC-7721 transplanted tumor was determined by Western blot, ELISA, semi-quantitative RT-PCR, and immunohistochemistry analysis, respectively. The effect of Ad-ING4-IL-24 on the expression of metastasis-related factors (MMP-2 and MMP-9) in SMMC-7721 hepatocellular carcinoma cells was also examined by semi-quantitative RT-PCR.Results:Two kinds of adenoviral vector co-expressing ING4 and IL-24, Ad-ING4-IRES-IL-24 and Ad-ING4-polyA+promoter-IL-24, were successfully constructed. Adenovirus-mediated ING4 and IL-24 co-expression significantly inhibited SMMC-7721 and HepG2 hepatocellular carcinoma cell growth, induced cell apoptosis, suppressed SMMC-7721 cell invasiveness, and retarded SMMC-7721 human hepatocellular carcinoma transplanted tumor growth in athymic nude mouse, exhibiting synergetic anti-tumor effect. Furthermore, Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug CDDP in SMMC-7721 and HepG2 cells in vitro. Ad-ING4-IL-24 had more marked effect in up-regulating the expression of P53, P21, P27, Fas, Bax, and Caspase-3 and down-regulating the expression of Cox-2, Bcl-2, VEGF, IL-8, CD34, MMP-2, and MMP-9 compared with Ad-ING4 or Ad-IL-24.Conclusions:1. Two kinds of adenoviral vector co-expressing two suppressor gene of ING4 and IL-24 (Ad-ING4-IRES-IL-24 and Ad-ING4-polyA+promoter-IL-24), for the first time, were successfully constructed at home and abroad.2. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had significant synergetic and enhanced chemosensitivity to CDDP effect in suppressing SMMC-7721 and HepG2 human hepatocellular carcinoma cell growth and inducing cell apoptosis in vitro.3. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had significant synergetic effect in retarding SMMC-7721 human hepatocellular carcinoma transplanted tumor growth in athymic nude mouse.4. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had more potent effect in up-regulating the expression of P53, P21, P27, Fas, Bax, and Caspase-3 and down-regulating the expression of Cox-2 and Bcl-2, which may be responsible for its synergetic anti-tumor effect on SMMC-7721 human hepatocellular carcinoma cells in vitro and in vivo in athymic nude mouse model.5. Ad-ING4-IL-24 co-expressing ING4 and IL-24 had more potent effect in down-regulating the expression of angiogenesis-related factors VEGF, IL-8, and CD34, which may be another important mechanism involved in its synergetic effect in suppressing SMMC-7721 human hepatocellular carcinoma transplanted tumor growth in vivo in athymic nude mouse model.6. Ad-ING4-IL-24 had more potent effect in suppressing in vitro SMMC-7721 human hepatocellular carcinoma cell's invasiveness, which may be associated with down-regulating the expression of metastasis-related factors MMP-2 and MMP-9.