Coley H M, Amos W B, Twentyman P R, Workman P
MRC Clinical Oncology and Radiotherapeutics Unit, Cambridge, UK.
Br J Cancer. 1993 Jun;67(6):1316-23. doi: 10.1038/bjc.1993.244.
This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.
本研究强调了激光扫描共聚焦显微镜在检测肿瘤细胞系中蒽环类药物亚细胞分布方面的实用性。已在两对亲本细胞系和多药耐药（MDR）细胞系中研究了蒽环类化合物的分布。对于用阿霉素（DOX）处理的亲本EMT6小鼠乳腺肿瘤细胞系EMT6/P，蒽环类药物荧光主要显示在细胞核中，但有一些颗粒状细胞质荧光，且质膜染色水平极低。在相同实验中，过表达P-糖蛋白的EMT6/AR1.0 MDR亚系的荧光要弱得多。细胞核荧光的损失相对大于细胞质荧光的损失。对于人肺癌大细胞系COR-L23/P，细胞内DOX分布明显集中在细胞核，伴有核膜染色和弥漫性细胞质荧光。对于缺乏P-糖蛋白表达的MDR细胞系COR-L23/R，与亲本细胞系相比，细胞核中的DOX荧光减少，但可见核周强烈染色区域，这与定位于高尔基体一致。吗啉基取代类似物MR-DOX在亲本细胞系和MDR细胞系中均实现了非常相似的亚细胞分布，这与其在后者中保留活性一致。在蒽环类药物暴露期间加入维拉帕米可增加MDR细胞系中的荧光强度，尤其是在细胞核中。在亲本细胞系中观察到的影响相对较小。共聚焦显微镜提供了蒽环类药物在亲本细胞系和MDR细胞系中亚细胞分布的高分辨率图像。不同细胞系中药物分布的差异可能有助于深入了解多药耐药机制，并为其治疗规避提供策略。
