title: Suppression of hepatocellular carcinoma cell proliferation by short hairpin RNA of frizzled 2 with Sonazoid-enhanced irradiation.
abstract: Short-hairpin RNA of frizzled-2 (shRNA-Fz2) is known to suppress the proliferation of hepatocellular carcinoma (HCC) cells; however, its effect on HCC cell motility is unknown. In this study, suppression of HCC cell motility by shRNA-Fz2 was analyzed, and introduction of shRNA-Fz2 into HCC cells was facilitated with ultrasound (US) irradiation generated from a diagnostic US device, which was enhanced by the contrast-enhanced US reagent Sonazoid. The HCC cell lines HLF and PLC/PRF/5 that were transfected with shRNA-Fz2 were plated to form monolayers, following which the cell monolayers were scratched with a sterile razor. After 48 h, the cells were stained with hematoxylin and eosin, and the distance between the growing edge of the cell layer and the scratch lines was measured. Total RNA from the cells was isolated and subjected to real-time quantitative PCR to quantify matrix metalloproteinase 9 expression at 48 h after transfection of shRNA-Fz2. Starch-iodide method was applied to analyze the generation of H2O2 following US irradiation with the addition of Sonazoid in the liquid, and cell proliferation was analyzed 72 h later. The distances between the growing edge of the cell layer and the scratch lines and MMP9 expression levels were significantly decreased with transfection of shRNA-Fz2 (P<0.05). In the starch-iodide method, absorbance significantly decreased with the addition of Sonazoid (P<0.05), which suggested that US irradiation with Sonazoid generated H2O2 and enhanced sonoporation. ShRNA-Fz2 suppressed cell proliferation of both cell lines at a mechanical index of 0.4. Motility of HLF cells and PLC/PRF/5 cells was suppressed by shRNA-FZ2. Sonazoid enhanced sonoporation of the cells with the diagnostic US device and the suppression of proliferation of both HCC cell lines by shRNA-Fz2.
authors.affiliation: Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba 284-0003, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Tomizawa
authors.affiliation: Department of Radiology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba 284-0003, Japan.
authors.forename: Fuminobu
authors.initials: F
authors.lastname: Shinozaki
authors.affiliation: Department of Neurology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba 284-0003, Japan.
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Motoyoshi
authors.affiliation: Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba 284-0003, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Sugiyama
authors.affiliation: Department of Pediatrics, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba 284-0003, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Yamamoto
authors.affiliation: Department of Neurosurgery, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba 284-0003, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Ishige
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000528:radiation effects; D049109:Cell Proliferation / Q000235:genetics* / Q000528:radiation effects; D005290:Ferric Compounds / Q000008:administration & dosage; D051157:Frizzled Receptors / Q000037:antagonists & inhibitors / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007501:Iron / Q000008:administration & dosage; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000235:genetics; D010087:Oxides / Q000008:administration & dosage; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance / Q000187:drug effects; D000069453:Ultrasonic Waves
chemical_list: C489742:FZD2 protein, human; D005290:Ferric Compounds; D051157:Frizzled Receptors; D010087:Oxides; D034741:RNA, Small Interfering; C069727:Sonazoid; D007501:Iron; D020780:Matrix Metalloproteinase 9