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 SonoVuetrade mark was i.v. injected. Ultrasound (1 MHz, 2 W/cm(2), 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.
Department of Medical Ultrasonics, Institution of Diagnostic and Interventional Ultrasound, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China. niefang028@yahoo.com.cn
Fang
F
Nie
Hui-Xiong
HX
Xu
Ming-De
MD
Lu
Ying
Y
Wang
Qing
Q
Tang
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000098:blood supply / Q000628:therapy*; D045744:Cell Line, Tumor; D003287:Contrast Media; D005260:Female; D015316:Genetic Therapy; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000098:blood supply / Q000628:therapy*; D051379:Mice; D008810:Mice, Inbred C57BL; D045423:Microbubbles; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000628:therapy; D010743:Phospholipids; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018139:Simplexvirus / Q000201:enzymology; D013459:Sulfur Hexafluoride; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D014465:Ultrasonics
D003287:Contrast Media; D010743:Phospholipids; C420843:contrast agent BR1; D013937:Thymidine Kinase; D013459:Sulfur Hexafluoride