The role of the VPS4a-exosome pathway in the intrinsic egress route of a DNA-binding anticancer drug
The role of the VPS4a-exosome pathway in the intrinsic egress route of a DNA-binding anticancer drug
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DOI：
10.1007/s11095-006-9043-0
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发表时间：
2006-08-01
影响因子：
3.7
通讯作者：
Rosania, Gus R.
中科院分区：
文献类型：
--
作者：
Chen, Vivien Y.;Posada, Maria M.;Rosania, Gus R.
目的。本文研究了肿瘤细胞内药物外排的亚细胞药代动力学，探讨了多泡体（multivesicular body， MVB）在促进阿霉素（一种广泛应用的dna靶向抗癌药物）从细胞核外排中的作用。人红细胞K562细胞用阿霉素脉冲，然后在无药培养基中追逐，以允许外排。利用显微镜和生化技术可视化药物的亚细胞定位，并测量药物在外排期间的含量和分布。为了探讨MVB在阿霉素外排中的作用，我们用VPS4a-GFP嵌合的显性阴性突变体转染K562细胞。虽然细胞内药物浓度超过细胞外浓度，但阿霉素在活细胞内的核外排比阿霉素从离体细胞核脱离进入无药缓冲液的速度要快。在显性表达VPS4a阴性的细胞中，多柔比星在VPS4a阳性的囊泡中积累，药物的隔离被抑制，直接暗示了多柔比星在该细胞中输出途径中的MVB通路。细胞膜是K562细胞中阿霉素外排机制的一个组成部分。显性阴性GFP嵌合突变体可用于阐明特定膜运输途径在亚细胞药物运输途径中的作用。
Purpose. This study investigates the subcellular pharmacokinetics of drug efflux in cancer cells and explores the role of the multivesicular body (MVB) in facilitating efflux of doxorubicin, a widely used DNA-targeting anticancer agent, from the nucleus.Methods. Human erythroleukemic K562 cells were pulsed with doxorubicin and then chased in drug-free media to allow for efflux. Microscopy and biochemical techniques were used to visualize the subcellular localization of the drug and measure drug content and distribution during the efflux period. To explore the role of the MVB in doxorubicin efflux, K562 cells were transfected with dominant negative mutant forms of VPS4a-GFP chimeras.Results. Although the intracellular concentration of drug exceeds the extracellular concentration, nuclear efflux of doxorubicin occurs in living cells at a faster rate than doxorubicin unbinding from isolated nuclei into drug-free buffer. In cells expressing dominant negative VPS4a, doxorubicin accumulates in VPS4a-positive vesicles and drug sequestration is inhibited, directly implicating the MVB pathway in the egress route of doxorubicin in this cell type.Conclusions. Cellular membranes are a component of the doxorubicin efflux mechanism in K562 cells. Dominant-negative GFP chimeric mutants can be used to elucidate the role of specific membrane trafficking pathways in subcellular drug transport routes.