Liang Xing-Jie, Shen Ding-Wu, Chen Kevin G, Wincovitch Stephen M, Garfield Susan H, Gottesman Michael M
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-4256, USA.
J Cell Physiol. 2005 Mar;202(3):635-41. doi: 10.1002/jcp.20253.
Cisplatin is a chemotherapeutic agent commonly used in the treatment of a wide variety of malignant tumors. Resistance to cisplatin represents a major obstacle to effective cancer therapy because clinically significant levels of resistance quickly emerge after treatment. Based on previous studies indicating abnormal plasma membrane protein trafficking in cisplatin-resistant (CP-r) cells, Fluorescence (Alexa Fluor)-labeled cisplatin was used to determine whether this defect altered the trafficking and localization of cisplatin by comparing drug sensitive KB-3-1 and KB-CP-r cells. Alexa Fluor-cisplatin was readily internalized and localized throughout the KB-3-1 cells, but overall fluorescence decreased in KB-CP-r cells, as detected by flow cytometry (FACS) and confocal microscopy. Only punctate cytoplasmic staining was observed in KB-CP-r cells with less fluorescence observed in the nucleus. Colocalization experiments with a Golgi-selective stain indicate the involvement of Golgi-like vesicles in initial intracellular processing of Alexa Fluor conjugated cisplatin complexes. As detected using an antibody to Alexa Fluor-cisplatin, cisplatin complex-binding proteins (CCBPs) were reduced in membrane fractions of single-step cisplatin-resistant KB-CP.5 cells, and increased in the cytoplasm of KB-CP.5 cells compared to KB-3-1 cells. CCBPs localized to lower density fractions in KB-CP.5 cells than in KB-3-1 cells as determined by iodixanol gradient centrifugation. In summary, inappropriate trafficking of CCBPs might explain resistance to cisplatin in cultured cancer cells, presumably because membrane binding proteins for cisplatin are not properly located on the cell surface in these cells, but are instead trapped in low density vesicles within the cytoplasm.
顺铂是一种常用于治疗多种恶性肿瘤的化疗药物。对顺铂产生耐药性是有效癌症治疗的主要障碍，因为治疗后临床上显著水平的耐药性会迅速出现。基于先前研究表明顺铂耐药（CP-r）细胞中存在异常的质膜蛋白转运，通过比较药物敏感的KB-3-1细胞和KB-CP-r细胞，使用荧光（Alexa Fluor）标记的顺铂来确定这种缺陷是否改变了顺铂的转运和定位。通过流式细胞术（FACS）和共聚焦显微镜检测发现，Alexa Fluor标记的顺铂很容易被KB-3-1细胞内化并分布于整个细胞，但在KB-CP-r细胞中整体荧光减少。在KB-CP-r细胞中仅观察到点状细胞质染色，细胞核中的荧光较少。与高尔基体选择性染料的共定位实验表明，类似高尔基体的囊泡参与了Alexa Fluor偶联顺铂复合物的初始细胞内加工过程。使用抗Alexa Fluor-顺铂抗体检测发现，与KB-3-1细胞相比，单步顺铂耐药的KB-CP.5细胞的膜组分中顺铂复合物结合蛋白（CCBP）减少，而在KB-CP.5细胞的细胞质中增加。通过碘克沙醇梯度离心法测定，KB-CP.5细胞中CCBP定位于比KB-3-1细胞更低密度的组分中。总之，CCBP的不适当转运可能解释了培养癌细胞对顺铂的耐药性，推测是因为这些细胞中顺铂的膜结合蛋白没有正确定位在细胞表面，而是被困在细胞质中的低密度囊泡中。
