[CANCER RESEARCH 58. 2141-2148. May 15. 1998] Pharmacodynamics of Immediate and Delayed Effects of Pad itaxel: Role of Slow Apoptosis and Intrace!lular Drug Retention1 Jessie L-S. Au,2 Dong Li, Yuebo Gan, Xiang Gao, Andrew L. Johnson, Jeffrey Johnston, Nancy J. Millenbaugh, Seong H. Jang, Hyo-Jeong Kuh, Chiung-Tong Chen, and M. Guillaume Wientjes College of Pharmacy ¡J.L-S. A.. D. L. Y. C.. X. C., A. L. J.. J. J.. N. J. M.. S. H. J.. H-J. K.. C-T. C.. M. C. W.] and Comprehensive Cancer Center ¡J.L-S. A.. Y. G.. M. G. W.¡. The Ohio Slate Universin, Columbus. Ohio 43210 ABSTRACT The kinetics of the time-dependent ani ¡tumoreffects of paclitaxel are not fully understood; some literature reports indicate a higher activity by prolonging treatment durations, whereas other reports indicate no en hancement 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 epi thelial cancer cell lines (bladder K 14. breast Ml I 7, 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 cumula tive 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 i/o 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, apop- tosis 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 treat ment was terminated (delayed effect); (b) the immediate and delayed effects showed different pharmacodynamics. The immediate effect in creased with treatment duration and drug concentration. For the delayed effect, all treatments produced the same maximum effect at 96 h, although treatments for £12 h showed higher IC50s than longer treatments, whereas treatments for >24 h showed indistinguishable IC50s; (c) treat ment 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 con centrations reached an equilibrium at ~5 h, which rules out slow and/or insufficient uptake as the cause of the lower effects at shorter treatment times (i.e., <24 h); (c) upon removal of drug-containing medium, the amount of drug retained intracellularly was about 10% of the applied dose and was reduced to —¿ 0.5% after three successive washes, separated by 3-h equilibration periods; and (/) the delayed effect of the 3-h treatment was largely due to the drug retained intracellularly, whereas the delayed effect of the 24 h treatment was independent of the drug retained intra cellularly. In conclusion, in human epithelial cancer cells, paclitaxel- induced cytotoxicity occurred after termination of drug treatment, which Received 10/17/97; accepted 3/17/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by R37 CA49816 and ROI CA63363 from the National Cancer Institute. NIH. A. L. J. was supported in part by Grant T32CA09338 from the National Cancer Institute. D. L. was supported in part by the Pharmacia-Upjohn Fellowship. N. J. M. was supported in part by the American Foundation of Pharmaceutical Education Fellowship. 1 To whom requests for reprints should be addressed, at College of Pharmacy and Medicine. Ohio State University, 508 Vernal G. Riff Building. 500 West 12th Avenue. Columbus. OH 43210. Phone: (614)292-4244: Fax: (614)688-3223. was partly due to the slow manifestation of apoptosis and partly due to the significant amount of drug retained intracellularly. Based on these find ings and recognizing that some previous studies measured the immediate effect whereas the other studies measured the delayed effect, we propose that the conflicting data in the literature regarding the effect of treatment duration on paclitaxel activity under in vitro conditions are in part due to the different pharmacodynamics of the immediate and delayed drug effects. Furthermore, differences in the delayed effects for treatments of <24 h and the minimal differences for treatments of ?»24h indicate that the delayed effect is maximally elicited by 24-h drug exposure. INTRODUCTION Paclitaxel is one of the most important anticancer drugs developed in the past two decades. It has shown impressive activity against human solid tumors, i.e., ovarian, head and neck, bladder, breast, and lung cancers (1). Paclitaxel enhances tubulin polymerization, pro motes microtubule assembly, and stabilizes microtubule dynamics, resulting in inhibition of cell proliferation and apoptosis (2-4). One of the challenges regarding the clinical use of paclituxel is the identification of optimal treatment schedules. Multiple treatment schedules with different infusion durations (1,3, 24. and 96 h) and different treatment frequency (daily, weekly, and every 3 weeks) are under evaluation in patients. The one completed randomized study in patients to date compares two dose levels, 135 and 175 mg/m2, and two infusion durations (3 and 24 h). The results show no differences in the combined response rates at the two doses nor in the response rate for the two treatment durations at the 135 mg/m2 dose. However, at 175 mg/m2, the 24-h treatment arm shows a higher response rate of 24% compared with the 15% for the 3-h treatment arm, although the relatively small sample size precluded meaningful statistical analysis (5). The 3-h infusion schedule is approved by the Food and Drug Administration, and there is economic pressure to use this schedule because it can be given in an ambulatory setting. However, preclinical data indicate a greater efficacy for longer treatment durations. For example, increasing the exposure time from 2, 3, or 6 h to 24, 48, 72, or 96 h resulted in a reduced clonogenic survival, a greater G2-M block, and/or lower plating efficiency in human leukemia, ovarian, breast, lung, cervical, astrocytoma. colon, ovarian, and pancreatic tumor cells, and CHO1 cancer cells (6-12). On the other hand, the formation of polyploid cells or formation of microtubule bundles in human leukemic cells and the cytotoxicity in human ovarian cancer cells were not enhanced by prolonging the exposure time from 4 to 24 h (13, 14). These conflicting results may be caused by biological differences between different cell lines and/or different experimental conditions. Examples of biological differences among cell lines are: (a) lymphoid cells are known to undergo primed apoptosis, which occurs more rapidly than the unprimed apoptosis often found in epithelial cells (14, 15); (b) the paclitaxel-induced microtubule bun dling and abnormal aster formation in several leukemia cells show cell type specificity; some cells show an increased response to prolonged 1The abbreviations used are: CHO. Chinese hamster ovary: SRB. sulforhodamine B. 2141 on June 12, 2015. © 1998 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from