Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada.
PLoS One. 2013;8(2):e56423. doi: 10.1371/journal.pone.0056423. Epub 2013 Feb 18.
Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system--capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors--increased hCNT3 mRNA and (3)H-gemcitabine uptake by >2,000- and 3,400-fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative nucleoside transporters and hCNT3 displayed 5% of (3)H-gemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative nucleoside transporters caused significant efflux of (3)H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of (3)H-gemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little nucleoside transport activity which are resistant to almost all current anticancer therapies.
健择是一种亲水性临床抗癌药物，需要核苷转运体穿过质膜进入细胞。核苷转运体水平低的胰腺腺癌通常对健择有抗药性，目前是临床问题。我们测试了用超声和脂质稳定的微泡转染人高亲和性核苷转运体 3(hCNT3)是否可以增加用二氮嗪进行药物处理使核苷转运缺陷的 HEK293 细胞摄取和对健择的敏感性。据我们所知，目前尚无关于使用 hCNT3 作为治疗基因逆转健择耐药性的文献报道。我们的超声转染系统能够转染细胞培养物、小鼠肌肉和异种移植的 CEM/araC 肿瘤，使二氮嗪处理的 HEK293 细胞中 hCNT3mRNA 和(3)H-健择的摄取分别增加了 2000 倍和 3400 倍。有趣的是，具有功能性人平衡核苷转运体和 hCNT3 的 HEK293 细胞显示出仅具有功能性 hCNT3 的细胞中观察到的(3)H-健择摄取的 5%，这表明平衡核苷转运体导致(3)H-健择的大量外排。外排测定证实，二氮嗪可以抑制 HEK293 细胞中(3)H-健择的大部分外排，这表明 hENT 负责测试细胞的大部分外排。卵母细胞摄取转运测定也进行了，并为我们的假说提供了支持。在胰腺癌细胞 AsPC-1 和 MIA PaCa-2 中也进行了健择摄取和外排测定，结果与 HEK293 细胞相似。使用 MTS 增殖测定，与未用二氮嗪处理的 HEK293 细胞相比，用二氮嗪处理的 HEK293 细胞对健择的耐药性增加了 13 倍，而这种耐药性可以通过转染 hCNT3 cDNA 逆转。我们提出，用超声和微泡转染 hCNT3 cDNA 可能是逆转具有低核苷转运活性的胰腺肿瘤对健择耐药的一种方法，这些肿瘤对几乎所有现有的抗癌疗法都有耐药性。
