APOPTOSIS-INDUCING AND NECROSIS-INDUCING POTENTIAL OF CLADRIBINE, CYTARABINE, CISPLATIN, AND 5-FLUOROURACIL IN-VITRO - A QUANTITATIVE PHARMACODYNAMIC MODEL

Purpose. The purpose of this study was to characterize the concentrati on-dependent induction of apoptosis by anticancer drugs in vitro. Meth ods: The apoptosis-and necrosis-inducing potential of the anticancer d rugs cladribine (CDA), cytarabine (ARA-C), cisplatin (CDDP), and 5-flu orouracil (5FU) were studied in vitro in the human leukemia cell lines HSB2 and Jurkat using a flow-cytometry assay that permits the simulta neous 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 multicompartmenta l models and the sensitivity of the cell lines to apoptosis and necros is was estimated. Results: A time- and dose-dependent decrease in vita l cells as cell as an increase in apoptotic and necrotic cells was obs erved in HSB2 cells upon continuous incubation with 10(-5)-10(-7) M CD A, 10(-5)-10(-8) MARA-C, 5 x 10(-5) x 10(-6) M CDDP, and 10(-4)-10(-5) M 5FU, whereas no effect was observed relative to controls upon incub ation 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-induc ing effects were observed at 10(-4) 5 x 10(-6) M CDA, 10(-5)-10(-7) M ARA-C, 5 x 10(-5)-5 x 10(-6) M CDDP, and 10(-4)-10(-5) M 5FU. In all e xperiments, apoptotic cells reached a peak after 6-48 h of drug exposu re. These data were best fit by a model in which vital cells became ir reversibly apoptotic by a direct pathway and necrotic by an irreversib le indirect pathway following the apoptotic state (mean R = 0.9876; ra nge 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 a poptosis and necrosis (expressed as EC50 and E-max values) induced by the anticancer drugs could be calculated from the sigmoidal concentrat ion-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. Conclu sion: This study demonstrates that CDA, ARA-C,CDDP, and 5FU possess co ncentration-dependent apoptosis-inducing potential in the cell lines s tudied. The cytotoxic mechanism and cell-killing potential of these dr ugs is different, which is reflected by different EC50 and E-max value s. Furthermore, a method for pharmacodynamic modeling is introduced th at permits a quantitative approach for the assessment of the sensitivi ty of tumor cells to anticancer drugs and combined treatments.