Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Budapest, Hungary.
Bioconjug Chem. 2011 Oct 19;22(10):2154-65. doi: 10.1021/bc2004236. Epub 2011 Oct 5.
Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.
柔红霉素（Dau）是一种用于治疗各种类型癌症的 DNA 结合型抗肿瘤药物，如骨肉瘤和急性髓细胞性白血病。一种提高其选择性并降低副作用的方法是将 Dau 与寡肽载体偶联，这可能会改变药物摄取和细胞内命运。在这里，我们报告了一种新型偶联物的合成、表征和体外生物学特性，其中 Dau 通过肟键与从随机噬菌体肽文库中选择的一种癌症特异性小肽（LTVSPWY）连接。在表达不同水平 ErbB2 受体的各种人类癌细胞系上研究了 Dau═Aoa-LTVSPWY-NH（2）缀合物的体外细胞生长抑制作用和细胞摄取。我们发现，新的柔红霉素-肽缀合物具有高度的细胞生长抑制作用，并且可以有效地被研究的人类癌细胞摄取。然而，与游离药物本身相比，该缀合物的效果较差。RP-HPLC 数据表明，在 pH 2.5-7.0 范围的缓冲液以及细胞培养基中，该缀合物至少在 24 小时内是稳定的。如 LC-MS 分析所示，在存在大鼠肝溶酶体匀浆的情况下，缀合物可以被降解。鉴定并制备的最小的、含柔红霉素的代谢物（Dau═Aoa-Leu-OH）表达 DNA 结合能力。为了深入了解潜在的作用机制，我们比较了 HL-60 人白血病细胞在用游离和肽结合的柔红霉素处理后的蛋白质表达谱。蛋白质组学分析表明，几种蛋白质的表达发生了改变。这包括三种蛋白质，其表达水平较低（微管蛋白β链）或明显较高（增殖细胞核抗原和蛋白激酶 C 抑制剂蛋白 1）在用 Dau 缀合物处理的细胞中。
