Apoptosis- and necrosis-inducing potential of cladribine, cytarabine, cisplatin, and 5-fluorouracil in vitro: a quantitative pharmacodynamic model

Abstract

Purpose: The purpose of this study was to characterize the concentration-dependent induction of apoptosis by anticancer drugs in vitro. Methods: The apoptosis- and necrosis-inducing potential of the anticancer drugs cladribine (CDA), cytarabine (ARA-C), cisplatin (CDDP), and 5-fluorouracil (5FU) were studied in vitro in the human leukemia cell lines HSB2 and Jurkat using a flow-cytometry assay that permits the simultaneous quantification of vital, apoptotic, and necrotic cells by double-staining with fluorescein isothiocyanate (FITC)-labeled Annexin-V and propidium iodide. The results were fit to different multicompartmental models and the sensitivity of the cell lines to apoptosis and necrosis was estimated. Results: A time- and dose-dependent decrease in vital cells as well as an increase in apoptotic and necrotic cells was observed in HSB2 cells upon continuous incubation with 10⁻⁵–10⁻⁷ M CDA, 10⁻⁵–10⁻⁸ M ARA-C, 5 × 10⁻⁵–5 × 10⁻⁶ M CDDP, and 10⁻⁴–10⁻⁵ M 5FU, whereas no effect was observed relative to controls upon incubation with 10⁻⁸–10⁻⁹ M CDA, 10⁻⁹ M ARA-C, 10⁻⁷–10⁻⁸ M CDDP, or 10⁻⁶–10⁻⁹ M 5FU. In Jurkat cells, apoptosis- and necrosis-inducing effects were observed at 10⁻⁴–5 × 10⁻⁶ M CDA, 10⁻⁵–10⁻⁷ M ARA-C, 5 × 10⁻⁵–5 × 10⁻⁶ M CDDP, and 10⁻⁴–10⁻⁵ M 5FU. In all experiments, apoptotic cells reached a peak after 6–48 h of drug exposure. These data were best fit by a model in which vital cells became irreversibly apoptotic by a direct pathway and necrotic by an irreversible indirect pathway following the apoptotic state (mean R=0.9876; range 0.9510–0.9993; mean modified Akaike's information criterion 3.88; range 1.86–5.82) and the rate constants of either pathway (Kva and Kan, respectively) were assessed. The sensitivity of both cell lines to apoptosis and necrosis (expressed as EC₅₀ and E_max values) induced by the anticancer drugs could be calculated from the sigmoidal concentration-effect curves. Furthermore, it was shown that drug treatment (10⁻⁶ M CDA or 10⁻⁶ M ARA-C) potentiated the apoptosis-inducing effects of irradiation (6 Gy) but not its necrosis-inducing potential. Conclusion: This study demonstrates that CDA, ARA-C, CDDP, and 5FU possess concentration-dependent apoptosis-inducing potential in the cell lines studied. The cytotoxic mechanism and cell-killing potential of these drugs is different, which is reflected by different EC₅₀ and E_max values. Furthermore, a method for pharmacodynamic modeling is introduced that permits a quantitative approach for the assessment of the sensitivity of tumor cells to anticancer drugs and combined treatments.