Departments of Medicine and Biochemistry and Molecular Biology, James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202, USA.
Cancer Res. 2010 Nov 1;70(21):8617-29. doi: 10.1158/0008-5472.CAN-10-0920. Epub 2010 Sep 21.
AS1411 is a first-in-class anticancer agent, currently in phase II clinical trials. It is a quadruplex-forming oligodeoxynucleotide that binds to nucleolin as an aptamer, but its mechanism of action is not completely understood. Mechanistic insights could lead to clinically useful markers for AS1411 response and to novel targeted therapies. Previously, we proposed a model where cell surface nucleolin serves as the receptor for AS1411, leading to selective uptake in cancer cells. Here, we compare uptake of fluorophore-labeled AS1411 (FL-AS1411) in DU145 prostate cancer cells (sensitive to AS1411) and Hs27 nonmalignant skin fibroblasts (resistant to AS1411). Uptake of FL-AS1411 occurred by endocytosis in both cell types and was much more efficient than an inactive, nonquadruplex oligonucleotide. Unexpectedly, uptake of FL-AS1411 was lower in cancer cells compared with Hs27 cells. However, the mechanism of uptake was different, occurring by macropinocytosis in cancer cells, but by a nonmacropinocytic pathway in Hs27 cells. Additionally, treatment of various cancer cells with AS1411 caused hyperstimulation of macropinocytosis, provoking an increase in its own uptake, whereas no stimulation was observed for nonmalignant cells. Nucleolin was not required for initial FL-AS1411 uptake in DU145 cells but was necessary for induced macropinocytosis and FL-AS1411 uptake at later times. Our results are inconsistent with the previous mechanistic model but confirm that nucleolin plays a role in mediating AS1411 effects. The data suggest a new model for AS1411 action as well as a new role for nucleolin in stimulating macropinocytosis, a process with potential applications in drug delivery.
AS1411 是一种首创的抗癌药物，目前正在进行 II 期临床试验。它是一种形成四链体的寡脱氧核苷酸，作为适体与核仁素结合，但作用机制尚不完全清楚。对其作用机制的深入了解可能会为 AS1411 反应提供有临床价值的标志物，并为新的靶向治疗提供依据。此前，我们提出了一个模型，即细胞表面核仁素作为 AS1411 的受体，导致其在癌细胞中选择性摄取。在这里，我们比较了荧光标记的 AS1411（FL-AS1411）在前列腺癌细胞（对 AS1411 敏感）和 Hs27 非恶性皮肤成纤维细胞（对 AS1411 耐药）中的摄取。两种细胞类型均通过内吞作用摄取 FL-AS1411，其摄取效率明显高于非四链体寡核苷酸。出乎意料的是，与 Hs27 细胞相比，FL-AS1411 在癌细胞中的摄取量较低。然而，摄取机制不同，在癌细胞中通过巨胞饮作用摄取，而在 Hs27 细胞中则通过非巨胞饮途径摄取。此外，用 AS1411 处理各种癌细胞会引起巨胞饮的过度刺激，从而增加其自身的摄取量，而对非恶性细胞则没有观察到刺激作用。核仁素不是 DU145 细胞摄取初始 FL-AS1411 的必需条件，但在诱导巨胞饮和随后摄取 FL-AS1411 时是必需的。我们的结果与之前的机制模型不一致，但证实核仁素在介导 AS1411 作用中发挥作用。这些数据为 AS1411 的作用提出了一个新模型，也为核仁素在刺激巨胞饮中的新作用提供了依据，巨胞饮在药物输送中有潜在的应用。
