Takemura Y, Kobayashi H, Miyachi H, Hayashi K, Sekiguchi S, Ohnuma T
Department of Laboratory Medicine, National Defense Medical College, Saitama, Japan.
Cancer Chemother Pharmacol. 1991;27(6):417-22. doi: 10.1007/BF00685154.
The effect of tumor cell density on the cellular pharmacokinetics of doxorubicin (DXR) and cisplatin (CDDP) was studied using MOLT-3 human acute lymphoblastic leukemia cells. As determined by the MTT assay, the growth-inhibitory effect of DXR was approx. 40 times lower when cell density was increased from 10(6) to 10(8) cells/ml (positive inoculum effect), whereas little or no influence of cell density was observed in CDDP-induced cell-growth inhibition. As measured by high-performance liquid chromatography using a fluorescence detector, the cellular accumulation of DXR showed 6- and 18-fold decreases after 1 h incubation when the cells were concentrated from 10(6) to 10(7) and 10(8) cells/ml, respectively. Only at low cell density (10(6) cells/ml) did the amount of DXR in the cells increase with increasing exposure times of up to 6 h. The DXR concentration in the supernatant that was separated from a cell suspension showing a density of 10(8) cells/ml fell to 20% of that obtained at 10(6) cells/ml. The metabolites of DXR, including Adriamycinol and Adriamycinone, were not detectable in the cell extracts or supernatants at any cell density examined. In contrast, the cellular accumulation of CDDP calculated from the platinum concentration, which was measured with a flameless atomic absorption spectrophotometer, was essentially identical at all cell densities examined; moreover, extension of the exposure period resulted in a linear increase in the amount of CDDP in the cells. CDDP concentrations in the supernatants were equally retained, irrespective of cell densities. These observations indicate that the positive inoculum effect shown in DXR-induced cell-growth inhibition results from the decreased cellular accumulation of the drug at high cell densities. We found no influence for cell density on the cellular accumulation of CDDP that might be relevant to the therapeutic potentiation of this drug at high tumor-cell density.
使用MOLT-3人急性淋巴细胞白血病细胞研究了肿瘤细胞密度对阿霉素（DXR）和顺铂（CDDP）细胞药代动力学的影响。通过MTT法测定，当细胞密度从10⁶增加到10⁸个细胞/毫升时（阳性接种效应），DXR的生长抑制作用降低了约40倍，而在CDDP诱导的细胞生长抑制中未观察到细胞密度的影响。使用荧光检测器通过高效液相色谱法测量，当细胞分别从10⁶浓缩到10⁷和10⁸个细胞/毫升时，孵育1小时后DXR的细胞积累分别下降了6倍和18倍。仅在低细胞密度（10⁶个细胞/毫升）下，细胞内DXR的量才会随着长达6小时的暴露时间增加而增加。从密度为10⁸个细胞/毫升的细胞悬液中分离出的上清液中DXR浓度降至10⁶个细胞/毫升时的20%。在任何检测的细胞密度下，细胞提取物或上清液中均未检测到DXR的代谢产物，包括阿霉素醇和阿霉素酮。相反，通过无火焰原子吸收分光光度计测量的铂浓度计算得出的CDDP细胞积累在所有检测的细胞密度下基本相同；此外，延长暴露时间导致细胞内CDDP量呈线性增加。上清液中的CDDP浓度无论细胞密度如何均能同等保留。这些观察结果表明，DXR诱导的细胞生长抑制中显示的阳性接种效应是由于高细胞密度下药物的细胞积累减少所致。我们发现细胞密度对CDDP的细胞积累没有影响可能与该药物在高肿瘤细胞密度下的治疗增强作用有关。
