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 activ ity by prolonging treatment durations, whereas other reports indicate no enhancement under in vitro conditions. The present study was design ed to address this controversy and to determine the mechanism of the h igher cytotoxicity associated with longer treatment durations, Six hum an epithelial cancer cell lines (bladder RT4, breast MCF7, pharynx FaD u, ovarian SKOV3, and prostate PC3 and DU145) were used, To determine whether the higher activity observed for the longer treatment duration s is due to a delayed exhibition of drug effects and/or a reflection o f cumulative effects that required a continuous drug exposure, cells w ere treated with paclitaxel for 3-96 h and then either: (a) immediatel y processed for drug effect measurement; or (b) washed, incubated in d rug-free medium, and processed for drug effect measurement at 96 h, Th e overall drug effect (i.e., combination of cytostatic and apoptotic e ffects) was determined by the sulforhodamine B assay, which measures t he cellular protein, In addition, to determine whether apoptosis occur s with a time delay, apoptosis was measured in cells that were collect ed immediately after drug treatment for various durations or in cells that were treated with drugs for 3 h but collected at later time point s. Apoptosis was determined using agarose gel electrophoresis and by m easuring the cytoplasmic DNA-histone complex using ELISA. The contribu tion of the intracellularly retained drug to the delayed drug effect w as studied by characterizing the kinetics of cellular drug uptake and efflux and by examining the effect of removal of the intracellularly r etained drug. All six cell lines showed similar results, as follows: ( a) paclitaxel produced cytotoxicity that was exhibited immediately aft er treatment (immediate effect) and after treatment was terminated (de layed effect); (b) the immediate and delayed effects showed different pharmacodynamics, The immediate effect increased with treatment durati on and drug concentration. For the delayed effect, all treatments prod uced the same maximum effect at 96 h, although treatments for less tha n or equal to 12 h showed higher IC(50)s than longer treatments, where as treatments for greater than or equal to 24 h showed indistinguishab le IC(50)s; (c) treatment for as brief as 3 h was sufficient to induce apoptosis, which occurred with a lag time of about 24 h, although lon ger treatments produced a greater extent of apoptosis; (d) The intrace llular and extracellular concentrations reached an equilibrium at simi lar to 5 h, which rules out stow and/or insufficient uptake as the cau se of the lower effects at shorter treatment times (i.e., <24 h); (e) upon removal of drug-containing medium, the amount of drug retained in tracellularly was about 10% of the applied dose and was reduced to sim ilar to 0.5% after three successive washes, separated by 3-h equilibra tion periods; and (f) the delayed effect of the 3-h treatment was larg ely due to the drug retained intracellularly, whereas the delayed effe ct of the 24 h treatment was independent of the drug retained intracel lularly. In conclusion, in human epithelial cancer cells, paclitaxel-i nduced cytotoxicity occurred after termination of drug treatment, whic h partly due to the slow manifestation of apoptosis and partly due to the significant amount of drug retained intracellularly. Based on thes e findings and recognizing that some previous studies measured the imm ediate effect whereas the other studies measured the delayed effect, w e propose that the conflicting data in the literature regarding the ef fect of treatment duration on paclitaxel activity under in vitro condi tions are in part due to the different pharmacodynamics of the immedia te and delayed drug effects, Furthermore, differences in the delayed e ffects for treatments of <24 h and the minimal differences for treatme nts of greater than or equal to 24 h indicate that the delayed effect is maximally elicited by 24-h drug exposure.