Altered intracellular distribution of weak base anticancer drugs owing to lysosomal sequestration is one purported mechanism contributing to chemotherapy resistance. This has often been demonstrated with the example of daunorubicin (DNR), chemotherapy with its characteristic red fluorescence used to trace it in cellular compartments.

Here we addressed the question whether image analysis of DNR fluorescence can reflect its real intracellular distribution. We observed that the relationship between the intensity of the DNR fluorescence and its concentration in water solutions with or without proteins is far from linear. In contrast, nucleic acids, RNA and DNA in particular, dramatically diminish the DNR fluorescence, however, the intensity was proportional to the amount. Therefore, image analysis reflects the composition of different cell compartments (i.e., the presence of proteins and nucleic acids) rather than the actual concentration of DNR in these compartments. In line with these results, we observed highly fluorescent lysosomes and low fluorescent nucleus in sensitive cancer cells treated with low DNR concentrations, a fluorescence pattern thought to be found only in resistant cancer cells. Importantly, LC/MS/MS analysis of extracts from sensitive cells treated with DNR or DNR in combination with an inhibitor of vacuolar ATPase, concanamycin A, indicated that lysosomal accumulation of DNR increased with increasing extracellular concentration. However, even the highest lysosomal accumulation of DNR failed to reduce its extralysosomal concentration and thus change the cell sensitivity to the drug.

In conclusion, our results strongly suggest that DNR fluorescence within cells does not indicate the real drug distribution. Further they suggested that lysosomal sequestration of DNR can hardly contribute to its resistance in cancer cells in vitro.

由于溶酶体隔离，弱碱性抗癌药物在细胞内的分布发生改变，这是导致化疗耐药的一种据称机制。这通常以柔红霉素（DNR）的化学疗法为例，柔红霉素具有特征性的红色荧光，可在细胞隔室中对其进行追踪。

在这里，我们解决了DNR荧光图像分析是否可以反映其实际细胞内分布的问题。我们观察到DNR荧光强度与其在含或不含蛋白质的水溶液中的浓度之间的关系远非线性。相反，核酸，尤其是RNA和DNA，会大大降低DNR荧光，但是强度与荧光强度成正比。因此，图像分析反映的是不同细胞区室的组成（即蛋白质和核酸的存在），而不是这些区室中DNR的实际浓度。与这些结果一致，我们在用低DNR浓度处理的敏感癌细胞中观察到了高荧光溶酶体和低荧光核，仅在抗性癌细胞中发现的荧光模式。重要的是，对用DNR或DNR与液泡ATPase抑制剂伴刀豆球蛋白A结合处理的敏感细胞提取物的LC / MS / MS分析表明，DNR的溶酶体积累随细胞外浓度的增加而增加。但是，即使DNR的最高溶酶体积累也未能降低其溶酶体浓度，从而改变了细胞对药物的敏感性。

总之，我们的结果强烈表明细胞内的DNR荧光不能指示药物的真实分布。进一步他们认为DNR的溶酶体隔离几乎不能促进其在体外对癌细胞的抗性。