Effects of Cationic Microbubble Carrying CD/TK Double Suicide Gene and α(V)β(3) Integrin Antibody in Human Hepatocellular Carcinoma HepG2 Cells

OBJECTIVE: Hepatocellular carcinoma (HCC), mostly derived from hepatitis or cirrhosisis, is one of the most common types of liver cancer. T-cell mediated immune response elicited by CD/TK double suicide gene has shown a substantial antitumor effect in HCC. Integrin α(V)β(3) over expresssion has been suggested to regulate the biology behavior of HCC. In this study, we investigated the strategy of incorporating CD/TK double suicide gene and anti-α(V)β(3) integrin monoclonal antibodies into cationic microbubbles (CMBs(αvβ3)), and evaluated its killing effect in HCC cells. METHODS: To improve the transfection efficiency of targeted CD/TK double suicide gene, we adopted cationic microbubbles (CMBs), a cationic delivery agent with enhanced DNA-carrying capacity. The ultrasound and high speed shearing method was used to prepare the non-targeting cationic microbubbles (CMBs). Using the biotin-avidin bridge method, α(V)β(3) integrin antibody was conjugated to CMBs, and CMBs(αvβ3) was generated to specifically target to HepG2 cells. The morphology and physicochemical properties of the CMBs(αvβ3) was detected by optical microscope and zeta detector. The conjugation of plasmid and the antibody in CMBs(αvβ3) were examined by immunofluorescent microscopy and flow cytometry. The binding capacities of CMBs(αvβ3) and CMBs to HCC HepG2 and normal L-02 cells were compared using rosette formation assay. To detect EGFP fluorescence and examine the transfection efficiencies of CMBs(αvβ3) and CMBs in HCC cells, fluorescence microscope and contrast-enhanced sonography were adopted. mRNA and protein level of CD/TK gene were detected by RT-PCR and Western blot, respectively. To evaluate the anti-tumor effect of CMBs(αvβ3), HCC cells with CMBs(αvβ3) were exposed to 5-flurocytosine / ganciclovir (5-FC/GCV). Then, cell cycle distribution after treatment were detected by PI staining and flow cytometry. Apoptotic cells death were detected by optical microscope and assessed by MTT assay and TUNEL-staining assay. RESULTS: CMBs(αvβ3) had a regular shape and good dispersion. Compared to CMBs, CMBs(αvβ3) had more stable concentrations of α(V)β(3) ligand and pEGFP-KDRP-CD/TK, and CMBs(αvβ3) was much sticker to HepG2 HCC cells than normal liver L-02cells. Moreover, after exposed to anti-α(V)β(3) monoclonal antibody, the adhesion of CMBs(αvβ3) to HepG2 cells and L-02 cells were significantly reduced. Also, CMBs(αvβ3) demonstrated a substantially higher efficiency in pEGFP-KDRP-CD/TK plasmid transfection in HepG2 cells than CMBs. In addition, CMBs(αvβ3) could significantly facilitate 5-FC/GCV-induced cell cycle arrest in S phase. Moreover, treatment of 5-FC/GCV combined with CMBs(αvβ3) resulted in a marked apoptotic cell death in HepG2 and SK-Herp-1 HCC cells. In vitro, treatment of 5-FC/GCV combined with CMBs(αvβ3) suppresed cell proliferation. In nude mice model, 5-FU + GCV combined with plasmid + CMBs(αvβ3)were able to significantly suppress tumor volumes. CONCLUSION: Through biotin-avidin mediation system, CMBs(αvβ3) were successfully generated to specifically target HCC HepG2 cells. More importantly, CMBs(αvβ3) could significantly facilitate 5-FC/GCV-induced cell cycle arrest and apoptotic cell death in HepG2 cells. Our study demonstrated a potential strategy that could be translated clinically to improve liver tumor gene delivery.