Guchelaar H J, Vermes I, Koopmans R P, Reutelingsperger C P, Haanen C
Department of Pharmacy, Academic Medical Center, University of Amsterdam, The Netherlands.
Cancer Chemother Pharmacol. 1998;42(1):77-83. doi: 10.1007/s002800050788.
The purpose of this study was to characterize the concentration-dependent induction of apoptosis by anticancer drugs in vitro.
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.
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) MCDA, 10(-5)-10(-8) MARA-C, 5 x10(-5)-5 x 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 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 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 Emax 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.
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 Emax 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.
本研究旨在表征抗癌药物在体外诱导细胞凋亡的浓度依赖性。
使用流式细胞术检测法，通过异硫氰酸荧光素（FITC）标记的膜联蛋白V和碘化丙啶双重染色，同时对存活、凋亡和坏死细胞进行定量分析，研究了抗癌药物克拉屈滨（CDA）、阿糖胞苷（ARA - C）、顺铂（CDDP）和5 - 氟尿嘧啶（5FU）在人白血病细胞系HSB2和Jurkat中的体外凋亡和坏死诱导潜力。将结果拟合到不同的多室模型中，并估计细胞系对凋亡和坏死的敏感性。
在HSB2细胞中，用10⁻⁵ - 10⁻⁷M CDA、10⁻⁵ - 10⁻⁸M ARA - C、5×10⁻⁵ - 5×10⁻⁶M CDDP和10⁻⁴ - 10⁻⁵M 5FU持续孵育后，观察到存活细胞呈时间和剂量依赖性减少，凋亡和坏死细胞增加；而用10⁻⁸ - 10⁻⁹M CDA、10⁻⁹M ARA - C、10⁻⁷ - 10⁻⁸M CDDP或10⁻⁶ - 10⁻⁹M 5FU孵育时，相对于对照组未观察到影响。在Jurkat细胞中，在10⁻⁴ - 5×10⁻⁶M CDA、10⁻⁵ - 10⁻⁷M ARA - C、5×10⁻⁵ - 5×10⁻⁶M CDDP和10⁻⁴ - 10⁻⁵M 5FU时观察到凋亡和坏死诱导作用。在所有实验中，凋亡细胞在药物暴露6 - 48小时后达到峰值。这些数据最适合一个模型，即存活细胞通过直接途径不可逆地转变为凋亡细胞，并在凋亡状态后通过不可逆的间接途径转变为坏死细胞（平均R = 0.9876；范围0.9510 - 0.9993；平均修正赤池信息准则3.88；范围1.86 - 5.82），并评估了任一途径的速率常数（分别为Kva和Kan）。可以从S形浓度 - 效应曲线计算出两种细胞系对抗癌药物诱导的凋亡和坏死的敏感性（以EC50和Emax值表示）。此外，研究表明药物处理（10⁻⁶M CDA或10⁻⁶M ARA - C）增强了辐射（6 Gy）的凋亡诱导作用，但未增强其坏死诱导潜力。
本研究表明，CDA、ARA - C、CDDP和5FU在所研究的细胞系中具有浓度依赖性凋亡诱导潜力。这些药物的细胞毒性机制和细胞杀伤潜力不同，这通过不同的EC50和Emax值反映出来。此外，引入了一种药效学建模方法，该方法允许采用定量方法评估肿瘤细胞对抗癌药物和联合治疗的敏感性。
