Department of Nuclear Medicine, Kyungpook National University School of Medicine, 50 Samduk-dong 2-ga, Jung Gu, Daegu, 700-721, Korea.
Nucl Med Biol. 2013 Nov;40(8):987-93. doi: 10.1016/j.nucmedbio.2013.08.004. Epub 2013 Sep 17.
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.
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.
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.
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.
本研究旨在探讨联合腺嘌呤核苷酸转位酶-2（ANT2）基因 RNA 干扰（RNAi）和更昔洛韦（GCV）治疗对动物模型中肝癌细胞（Huh7）的抗癌作用。
通过将人甲状腺钠碘转运体（hNIS）、单纯疱疹病毒 1 胸苷激酶（HSV1-sr39tk）和增强型绿色荧光蛋白（EGFP）融合基因的载体转染到 Huh7 细胞中，建立了 Huh7/NTG 稳定细胞系。通过 RT-PCR 分析评估这些基因的 mRNA 表达。通过（125）I 摄取和（3）H-喷昔洛韦（PCV）摄取试验验证 hNIS 和 HSV1-sr39tk 的功能。通过免疫细胞化学染色后共聚焦显微镜观察，证实 EGFP 和 hNIS 的表达。我们用 ANT2 shRNA 或 GCV 或 ANT2 shRNA 和 GCV 联合处理肿瘤细胞，并将 Huh7/NTG 肿瘤异种移植物用于体内小鼠模型。通过卡尺测量和使用（99m）Tc-过锝酸盐进行伽马相机成像评估体内研究的治疗效果。
与亲本 Huh7 细胞相比，Huh7/NTG 细胞的（125）I 摄取呈细胞数量依赖性增加，（3）H-PCV 摄取增加了 24 倍。与转染 scramble shRNA 的细胞相比，转染 ANT2 shRNA 的 Huh7/NTG 细胞的 ANT2 mRNA 表达降低，增殖活性受损更大。GCV 处理也抑制了 Huh7/NTG 细胞的增殖。在体外研究中，联合 GCV 和 ANT2 shRNA 治疗进一步抑制了细胞增殖。在小鼠模型中，联合 GCV 和 ANT2 shRNA 治疗进一步降低了肿瘤生长。
我们的结果表明，与单独的 GCV 治疗或 ANT2 shRNA 治疗相比，联合 RNA 干扰抑制腺嘌呤核苷酸转位酶-2 和 GCV 治疗在体外和体内更能抑制肝癌细胞增殖。因此，它可用于治疗无法治愈的肝癌。
