Park Seung-Yoon, Kwak Wonjung, Thapa Narendra, Jung Mi-Yeon, Nam Ju-Ock, So In-Seop, Kim So-Youn, Yoo Jeongsoo, Lee Jaetae, Kim In-San
Department of Biochemistry, School of Medicine, Dongguk University, Kyungju, Republic of Korea.
J Nucl Med. 2008 Sep;49(9):1480-8. doi: 10.2967/jnumed.108.050963. Epub 2008 Aug 14.
We investigated the feasibility of using combination gene therapy and noninvasive nuclear imaging after expression of the human sodium iodide symporter (hNIS) and inhibition of the multidrug resistance (MDR1) gene in colon cancer cells.
HCT-15 cells were stably transfected with a dual expression vector, in which the hNIS gene, driven by a constitutive cytomegalovirus promoter, has been coupled to an MDR1 short hairpin RNA (shRNA) cassette. Cell lines stably expressing the hNIS gene and MDR1 shRNA (designated MN-61 and MN-62) were produced, and the expression of the NIS gene and MDR1 shRNA was examined by Western blotting, reverse transcription-polymerase chain reaction, and immunostaining. The functional activities of MDR1 shRNA were determined by paclitaxel uptake and sensitivity to doxorubicin. Functional NIS expression was confirmed by the uptake and efflux of (125)I and the cytotoxicity of (131)I. The effect of the combination of (131)I and doxorubicin was determined by an in vitro clonogenic assay. In vivo NIS expression was examined by small-animal PET with (124)I.
The shMDR-NIS-expressing cells showed a significant decrease in the expression of MDR1 messenger RNA and its translated product, P-glycoprotein. The inhibition of P-glycoprotein expression by shRNA enhanced the intracellular accumulation of paclitaxel, the cellular retention of which is mediated by P-glycoprotein, thereby increasing sensitivity to the anticancer drug. The shMDR-NIS-expressing cells showed a significant increase of (125)I uptake, which was completely inhibited by KClO(4). Although the iodide efflux rate was rapid, the cell survival rate was markedly reduced by (131)I treatment. Interestingly, the combination of doxorubicin and a radioiodide ((131)I) displayed synergistic cytotoxicity that correlated with MDR1 inhibition and NIS expression in shMDR-NIS-expressing cells. Furthermore, in mice with shMDR-NIS-expressing tumor xenografts, small-animal PET with (124)I clearly visualized shMDR1-NIS-expressing tumors.
We developed a dual expression vector with the NIS gene and MDR1 shRNA. This study represents a promising first step in investigations of the potential use of a combination of the NIS gene and MDR1 shRNA as a new therapeutic strategy allowing RNA interference-based gene therapy, NIS-based radioiodine therapy, and in vivo monitoring based on NIS imaging.
我们研究了在人钠碘同向转运体（hNIS）表达及多药耐药（MDR1）基因抑制后，联合基因治疗和非侵入性核成像用于结肠癌细胞的可行性。
用双表达载体稳定转染HCT - 15细胞，其中由组成型巨细胞病毒启动子驱动的hNIS基因与MDR1短发夹RNA（shRNA）盒相连。构建稳定表达hNIS基因和MDR1 shRNA的细胞系（命名为MN - 61和MN - 62），通过蛋白质免疫印迹法、逆转录 - 聚合酶链反应和免疫染色检测NIS基因和MDR1 shRNA的表达。通过紫杉醇摄取和对阿霉素的敏感性测定MDR1 shRNA的功能活性。通过（125）I的摄取和流出以及（131）I的细胞毒性确认功能性NIS表达。通过体外克隆形成试验测定（131）I和阿霉素联合的效果。用（124）I通过小动物PET检测体内NIS表达。
表达shMDR - NIS的细胞显示MDR1信使核糖核酸及其翻译产物P - 糖蛋白的表达显著降低。shRNA对P - 糖蛋白表达的抑制增强了紫杉醇的细胞内积累，其细胞内潴留由P - 糖蛋白介导，从而增加了对抗癌药物的敏感性。表达shMDR - NIS的细胞显示（125）I摄取显著增加，这被高氯酸钾完全抑制。虽然碘流出率很快，但（131）I处理使细胞存活率明显降低。有趣的是，阿霉素和放射性碘（（131）I）联合显示出协同细胞毒性，这与表达shMDR - NIS的细胞中MDR1抑制和NIS表达相关。此外，在表达shMDR - NIS的肿瘤异种移植小鼠中，用（124）I的小动物PET清晰地显示了表达shMDR1 - NIS的肿瘤。
我们构建了一种带有NIS基因和MDR1 shRNA的双表达载体。本研究代表了将NIS基因和MDR1 shRNA联合作为一种新的治疗策略进行研究的有前景的第一步，该策略允许基于RNA干扰的基因治疗、基于NIS的放射性碘治疗以及基于NIS成像的体内监测。
