Pharmacodynamics of immediate and delayed effects of paclitaxel: role of slow apoptosis and intracellular drug retention.
Pharmacodynamics of immediate and delayed effects of paclitaxel: role of slow apoptosis and intracellular drug retention.
复制标题
DOI：
--
复制
发表时间：
1998-05
期刊：
影响因子：
11.2
通讯作者：
J. L. Au;D. Li;Y. Gan;X. Gao;A. L. Johnson;J. Johnston;N. Millenbaugh;S. Jang;H. Kuh;C. T. Chen;M. Wientjes
中科院分区：
文献类型：
--
作者：
J. L. Au;D. Li;Y. Gan;X. Gao;A. L. Johnson;J. Johnston;N. Millenbaugh;S. Jang;H. Kuh;C. T. Chen;M. Wientjes
紫杉醇的时间依赖性抗肿瘤作用的动力学机制还不完全清楚；一些文献报道表明通过延长治疗时间具有更高的活性，而另一些报告则表明在体外条件下没有增强作用。本研究旨在解决这一争议，并确定与较长治疗时间相关的较高细胞毒性的机制。6株人上皮癌细胞系(膀胱RT4、乳腺MCF7、咽部FaDu、卵巢SKOV3、前列腺癌PC3和DU145)。为了确定在较长时间内观察到的较高活性是否是由于延迟了药物效应的显现和/或反映了需要持续药物暴露的累积效应，用紫杉醇处理细胞3-96h，然后：(A)立即进行药物效应测量；或(B)洗涤，在无药物介质中孵育，并在96h进行药物效应测量。此外，为了确定细胞凋亡是否存在时间延迟，在药物处理不同时间后立即收集的细胞和药物处理3h但在随后的时间点收集的细胞进行了凋亡率测定。琼脂糖凝胶电泳法检测细胞凋亡率，酶联免疫吸附试验检测胞浆DNA-组蛋白复合体。通过对细胞内药物摄取和外排动力学的表征以及对细胞内药物清除效果的考察，研究了细胞内药物滞留对药物延迟效应的贡献。所有6个细胞株都显示出相似的结果，如下：(A)紫杉醇产生细胞毒性，在治疗后立即(即时效应)和治疗终止后(延迟效应)；(B)即时和延迟效应表现出不同的药效学。即刻效应随治疗时间和药物浓度的增加而增加。对于延迟效应，所有处理在96小时产生相同的最大效果，尽管处理或=24小时显示出没有区别的IC50；(C)短至3小时的处理足以诱导细胞凋亡，其滞后时间约为24小时，尽管较长的处理产生更大程度的凋亡；(D)细胞内和细胞外的浓度在大约5小时达到平衡，这排除了较短的处理时间(即，或=24小时表明)较低效果的原因是缓慢和/或吸收不足
The kinetics of the time-dependent antitumor effects of paclitaxel are not fully understood; some literature reports indicate a higher activity by prolonging treatment durations, whereas other reports indicate no enhancement under in vitro conditions. The present study was designed to address this controversy and to determine the mechanism of the higher cytotoxicity associated with longer treatment durations. Six human epithelial cancer cell lines (bladder RT4, breast MCF7, pharynx FaDu, ovarian SKOV3, and prostate PC3 and DU145) were used. To determine whether the higher activity observed for the longer treatment durations is due to a delayed exhibition of drug effects and/or a reflection of cumulative effects that required a continuous drug exposure, cells were treated with paclitaxel for 3-96 h and then either: (a) immediately processed for drug effect measurement; or (b) washed, incubated in drug-free medium, and processed for drug effect measurement at 96 h. The overall drug effect (i.e., combination of cytostatic and apoptotic effects) was determined by the sulforhodamine B assay, which measures the cellular protein. In addition, to determine whether apoptosis occurs with a time delay, apoptosis was measured in cells that were collected immediately after drug treatment for various durations or in cells that were treated with drugs for 3 h but collected at later time points. Apoptosis was determined using agarose gel electrophoresis and by measuring the cytoplasmic DNA-histone complex using ELISA. The contribution of the intracellularly retained drug to the delayed drug effect was studied by characterizing the kinetics of cellular drug uptake and efflux and by examining the effect of removal of the intracellularly retained drug. All six cell lines showed similar results, as follows: (a) paclitaxel produced cytotoxicity that was exhibited immediately after treatment (immediate effect) and after treatment was terminated (delayed effect); (b) the immediate and delayed effects showed different pharmacodynamics. The immediate effect increased with treatment duration and drug concentration. For the delayed effect, all treatments produced the same maximum effect at 96 h, although treatments for or = 24 h showed indistinguishable IC50s; (c) treatment for as brief as 3 h was sufficient to induce apoptosis, which occurred with a lag time of about 24 h, although longer treatments produced a greater extent of apoptosis; (d) The intracellular and extracellular concentrations reached an equilibrium at approximately 5 h, which rules out slow and/or insufficient uptake as the cause of the lower effects at shorter treatment times (i.e., or = 24 h indicate that th