Combined RNA interference of adenine nucleotide translocase-2 and ganciclovir therapy in hepatocellular carcinoma.
OBJECTIVE
The purpose of this study was to investigate the anticancer effects of combined RNA interference (RNAi) of the adenine nucleotide translocase-2 (ANT2) gene and ganciclovir (GCV) therapy for treatment of hepatocellular carcinoma cells (Huh 7) in an animal model.


METHODS
The Huh 7/NTG stable cell line was established by transfection of a vector with the human sodium iodide symporter (hNIS), HSV1-sr39 thymidine kinase (tk), and enhanced green florescent protein (EGFP) fusion gene into Huh 7 cells. mRNA expressions of these genes were evaluated by RT-PCR analysis. The functions of hNIS and HSV1-sr39tk were verified with (125)I uptake and (3)H-penciclovir (PCV) uptake tests. EGFP and hNIS expression was confirmed with confocal microscopy after immunocytochemical staining. We treated the tumor cells with ANT2 shRNA or GCV or both ANT2 shRNA and GCV and treated the in vivo mouse model with a Huh 7/NTG tumor xenograft. The therapeutic effects of the in vivo study were assessed with caliper measurements and gamma camera imaging using (99m)Tc-pertechnetate.


RESULTS
Huh 7/NTG cells showed a cell number-dependent increase in (125)I uptake and a 24-fold higher (3)H-PCV uptake compared to parent Huh 7 cells. Huh 7/NTG cells transfected with ANT2 shRNA had lower ANT2 mRNA expression and more impaired proliferation activity than cells transfected with scramble shRNA. Proliferation of Huh 7/NTG cells was also inhibited by GCV treatment. Combined GCV and ANT2 shRNA therapy further inhibited cell proliferation in the in vitro study. The combined therapy with GCV and ANT2 shRNA showed a further decrease in tumor growth in the mouse model.


CONCLUSIONS
Our results suggest that the combined RNA interference with ANT2 and GCV therapy inhibited hepatocellular carcinoma cell proliferation more than single GCV therapy or ANT2 shRNA therapy in vitro and in vivo. Therefore it could be applied treating incurable hepatocellular carcinoma.
Department of Nuclear Medicine, Kyungpook National University School of Medicine, 50 Samduk-dong 2-ga, Jung Gu, Daegu, 700-721, Korea. Electronic address: gene7302@hotmail.com.
Jung Eun
JE
Kim
Mi-Hye
MH
Hwang
Ho Won
HW
Lee
Sang-Woo
SW
Lee
Jaetae
J
Lee
Byeong-Cheol
BC
Ahn
D033742:Adenine Nucleotide Translocator 2 / Q000172:deficiency* / Q000235:genetics*; D000818:Animals; D001692:Biological Transport; D006528:Carcinoma, Hepatocellular / Q000000981:diagnostic imaging / Q000188:drug therapy / Q000235:genetics / Q000628:therapy*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002471:Cell Transformation, Neoplastic; D015774:Ganciclovir / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000000981:diagnostic imaging / Q000188:drug therapy / Q000235:genetics / Q000628:therapy*; D051379:Mice; D011355:Prodrugs / Q000494:pharmacology; D034622:RNA Interference; D011877:Radionuclide Imaging; D013670:Sodium Pertechnetate Tc 99m / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics
D033742:Adenine Nucleotide Translocator 2; D011355:Prodrugs; D013670:Sodium Pertechnetate Tc 99m; D013937:Thymidine Kinase; D015774:Ganciclovir
Adenine nucleotide translocase-2; Combination therapy; Ganciclovir therapy; HSV1-sr39 thymidine kinase; Hepatocellular carcinoma; Huh 7/NTG cells; Huh 7/hNIS-HSV1-sr39tk-EGFP; Huh 7/human sodium iodide symporter gene; Huh 7/human sodium iodide symporter gene-Herpes simplex virus-sr39 thymidine kinase-green fluorescence protein; RNA interference; enhanced green fluorescence protein