Effects of Cationic Microbubble Carrying CD/TK Double Suicide Gene and αVβ3 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_Vβ₃ 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_Vβ₃ 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_Vβ₃ 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_Vβ₃ 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_Vβ₃ 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_αvβ3were 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. [ABSTRACT FROM AUTHOR]