Anti-angiogenic gene therapy for hepatocellular carcinoma mediated by microbubble-enhanced ultrasound exposure: An in vivo experimental study

Objective: The aim of this study was to assess the effect of anti-angiogenic gene therapy for hepatocelluar carcinoma (HCC) treated by microbubble-enhanced ultrasound exposure.

Methods: Forty C57BL/6J female mice were inoculated s.c. with Hepa1-6 tumor cell line. Herpes simplex virus thymidine kinase under the control of kinase domain-containing receptor (KDR, angiogenic growth factor's corresponding receptor) promoter was used. Plasmid DNA with or without microbubble contrast agent of SonoVue? was i.v. injected. Ultrasound (1 MHz, 2 W/cm², 5 min) was delivered to hepatic carcinomas in mice. The KDR-tk gene transfer was followed by ganciclovir (GCV) injection for 10 days and then the diameters of tumors were measured every 4 days till 28 days. The survivals of tumor-bearing mice were observed. PCR analysis and immunohistochemistry measurements revealed expression of the transfected gene. Transferase-mediated dUTP nick end labeling staining was used to detect apoptotic cells.

Results: Compared with the group treated by ultrasound alone, KDR-tk gene treatment treated by ultrasound combined with SonoVue restrained tumor growth and increased survival time of tumor-bearing mice; microvessel density in group mediated by ultrasound and SonoVue was significantly lower than that in group ultrasound alone (12.3 ± 1.4 vs. 27.4 ± 3.2, P < 0.05). An apoptosis index increased in the group treated by ultrasound and SonoVue compared with the group treated by ultrasound alone (25 ± 3.6 vs. 36 ± 3.8, P < 0.05), whereas there was no significant difference between group mediated by SonoVue alone and group phosphate-buffered saline alone (17 ± 1.8 vs. 14 ± 1.2, P>0.05).

Conclusions: Gene therapy mediated by ultrasound exposure enhanced by a microbubble contrast agent may become a new treatment option for persistent HCC.