Pharmacodynamics of immediate and delayed effects of paclitaxel : Role of slow apoptosis and intracellular drug retention

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 = 12 h showed higher IC50s than longer treatments, whereas 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 pr