Takahashi N, Asakura T, Ohkawa K
Department of Biochemistry (I), Jikei University School of Medicine, Tokyo, Japan.
Anticancer Drugs. 1996 Aug;7(6):687-96. doi: 10.1097/00001813-199608000-00010.
After treatment of AH66DR cells with the multidrug resistance (MDR) phenotype with bovine serum albumin (BSA)-conjugated [14C]doxorubicin (DXR), accumulation of the drug in the secondary lysosomal fraction increased as a function of time up to 24 h without any significant increase of the drug in other organellae. By contrast, AH66P cells showed a marked increase in accumulation of the drug in the mitochondrial fraction, and a moderate increase in the lysosomal and nuclear fractions. The intracellular degradation of the internalized conjugate was assessed by HPLC gel filtration as molecular change of the drug. The initial molecular mass (M(r)) of BSA-conjugated [14C]DXR was estimated to be 70 kDa; however, the secondary lysosomal fraction contained mainly three peaks of [14C]compounds ranging from 3 to 70 kDa. The [14C]compound extracted from the nuclear and mitochondrial fractions showed only one peak, which was estimated to be smaller than 2 kDa. By contrast, the cytosolic fraction contained mainly two peaks of [14C]compounds, which were smaller than 2 kDa and larger than 500 kDa. These results indicated that the intracellular distribution of the administered drug, based probably on the drug-traffic mechanism in the cells, was quite different between the two cell lines, but some of the biochemical characteristics of the degraded compounds from each subcellular fraction were similar because the degradation processes in each fraction might be almost identical. The possibility of lysosomal degradation of the protein-conjugated DXR leading to expression of cytotoxicity was also confirmed by the fact that only lysosomal digestable poly-L-lysine-conjugated DXR exhibited dose-dependent cytotoxicity against both cell lines in marked contrast to the cells treated with poly-D-lysine-conjugated DXR. It was concluded that lysosomal breakdown of protein-conjugated DXR, which had been taken up by endocytosis, and the liberation of the degraded active adducts of the conjugate without efflux by the MDR pump mechanism must be an essential stage in the development of the cytotoxicity against tumor cells with or without the MDR phenotype.
用牛血清白蛋白（BSA）偶联的[14C]阿霉素（DXR）处理具有多药耐药（MDR）表型的AH66DR细胞后，药物在次级溶酶体组分中的积累随时间增加，直至24小时，而在其他细胞器中药物没有显著增加。相比之下，AH66P细胞显示药物在线粒体组分中的积累显著增加，在溶酶体和核组分中适度增加。通过HPLC凝胶过滤评估内化偶联物的细胞内降解，作为药物的分子变化。BSA偶联的[14C]DXR的初始分子量（M(r)）估计为70 kDa；然而，次级溶酶体组分主要包含分子量范围为3至70 kDa的三个[14C]化合物峰。从核和线粒体组分中提取的[14C]化合物仅显示一个峰，估计小于2 kDa。相比之下，胞质组分主要包含两个[14C]化合物峰，小于2 kDa且大于500 kDa。这些结果表明，基于细胞内药物转运机制，给药药物在两种细胞系中的细胞内分布有很大差异，但来自每个亚细胞组分的降解化合物的一些生化特性相似，因为每个组分中的降解过程可能几乎相同。蛋白质偶联的DXR的溶酶体降解导致细胞毒性表达的可能性也通过以下事实得到证实：与用聚-D-赖氨酸偶联的DXR处理的细胞形成鲜明对比，只有溶酶体可消化的聚-L-赖氨酸偶联的DXR对两种细胞系均表现出剂量依赖性细胞毒性。得出的结论是，通过内吞作用摄取的蛋白质偶联的DXR的溶酶体分解，以及偶联物降解的活性加合物的释放而不通过MDR泵机制外排，必定是对具有或不具有MDR表型的肿瘤细胞产生细胞毒性的一个重要阶段。
