Comparison of cytotoxic effect and cellular uptake of 1-beta-D-arabinofuranosylcytosine and its N4-acyl derivatives, using cultured KB cells.
Comparison of cytotoxic effect and cellular uptake of 1-beta-D-arabinofuranosylcytosine and its N4-acyl derivatives, using cultured KB cells.
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使用培养的 KB 细胞比较 1-β-D-阿拉伯呋喃糖基胞嘧啶及其 N4-酰基衍生物的细胞毒性作用和细胞摄取。
作者：
T. Tsuruo;H. Iida;S. Tsukagoshi;Y. Sakurai
摘要在N4位引入C4~C22碳原子的1-棕榈酰基-d-阿拉伯呋喃胞嘧啶(Ara-C)的N-4-酰基衍生物中，N4--pentadecanoyl-1-β-d-arabinofuranosylcytosine，和N4-β-1-β-d-Ara-C对培养的KB细胞具有最强的细胞毒作用。N4-丁酰基-1-β-d-阿拉伯糖胞嘧啶、N4-苯乙酰基-1-β-d-阿拉伯糖胞嘧啶、N4-月桂酰基-1-β-d-阿拉伯糖胞嘧啶、N4-硬脂酰基-1-β-d-阿拉伯糖胞嘧啶、N4-硬脂酰基-1-β-d-阿拉伯糖胞嘧啶、N 4-苯乙酰基-1-FU-d-阿拉伯糖胞嘧啶、N4-硬脂酰基-1-β-d-阿拉伯糖胞嘧啶、N 4-苯乙酰基-1-FU-d-阿拉伯糖胞嘧啶、N-4-月桂酰基-1-FU-d-阿拉伯糖胞嘧啶、N-4-硬脂酰基-1-FU-d-阿拉伯糖胞嘧啶、N-4-苯乙酰基-1-FU-d-阿糖胞嘧啶和阿糖胞苷对药物的摄取速率分别为0.067、1.47、1.87、3.89、4.89和1.85pmoL/106个细胞/分钟。N4-苯乙酰-1-β-d-阿拉伯氟尿嘧啶、N4-硬脂酰基-1-β-d-阿拉伯氟尿嘧啶和N4-棕榈酰-阿糖胞苷的细胞内药物浓度随着孵育时间的延长而增加，32h时分别达到最大值192、214和450pmoL/106个细胞。N4-月桂酰基-1-β-d-阿拉伯氟尿嘧啶和N4-丁酰基-1-β-d-阿拉伯氟尿嘧啶的摄取分别在23小时和5小时达到最高水平，分别为110和9.2pmol/106细胞，并在治疗后32h保持不变。Ara-C被迅速转运到细胞内。细胞内Ara-C浓度在处理后5h达最大值(93pmol/106个细胞)。然而，此后随着KB细胞对Ara-C的脱氨基作用，它逐渐下降，在治疗后23小时达到基础水平。N4-Palmitoyl-Ara-C在治疗后32小时的药物摄取率最高(450pmol/106个细胞)，表明大约25%的添加药物被转运到细胞内。此外，还观察到N4-棕榈酰基-Ara-C具有最高的药物掺入细胞DNA。N4-丁酰基-1-β-d-阿拉伯呋喃胞苷、N4-苯乙酰-1-β-d-阿拉伯呋喃胞嘧啶、N4-硬脂酰基-1-β-d-阿拉伯呋喃胞嘧啶、N4-月桂酰基-1-β-d-阿拉伯糖胞嘧啶和N-棕榈酰基-阿糖胞嘧啶的DNA掺入量分别为1.73、1.90、3.20、4.43和7.79pmoL/106个细胞，而23小时和47小时则分别为3.89和2.23pmo1/106细胞分别进行了分析。Ara-C的细胞毒作用主要依赖于细胞密度，在2100个/平方厘米生长面接种的细胞与21,000个/平方厘米生长面接种的细胞相比，50%抑制所需的药物浓度相差17,000倍。KB细胞能将阿糖胞苷迅速降解为1-β-d-阿拉伯呋喃尿苷，该反应在细胞内被催化。1微米Ara-C处理24小时后，在21,000个/平方厘米生长面接种的培养液中未检测到Ara-C，而在2,100个/平方厘米生长面接种的培养液中仍有30%的Ara-C存在。N4-Palmitoyl-Ara-C具有较强的细胞毒作用，其原因可能是药物摄取增强以及被掺入药物对脱氨酶的抗性。
Abstract Among the N 4 -acyl derivatives of 1-β-d-arabinofuranosylcytosine (ara-C), in which linear saturated fatty acids with C 4 to C 22 carbon atoms were introduced into the N 4 -position, N 4 -myristoyl-1-β-d-arabinofuranosylcytosine, N 4 -pentadecanoyl-1-β-d-arabinofuranosylcytosine, and N 4 -palmitoyl-1-β-d-arabinofuranosylcytosine (N 4 -palmitoyl-ara-C) showed the highest cytotoxic effect against cultured KB cells. The uptake rates of the drugs during the initial 15 min of incubation were 0.067, 1.47, 1.87, 3.89, 4.89, and 1.85 pmol/10 6 cells/min for N 4 -butyryl-1-β-d-arabinofuranosylcytosine, N 4 -behenoyl-1-β-d-arabinofuranosylcytosine, N 4 -lauroyl-1-β-d-arabinofuranosylcytosine, N 4 -stearoyl-1-β-d-arabinofuranosylcytosine, N 4 -palmitoyl-ara-C, and ara-C, respectively. The intracellular drug concentrations of N 4 -behenoyl-1-β-d-arabinofuranosylcytosine, N 4 -stearoyl-1-β-d-arabinofuranosylcytosine, and N 4 -palmitoyl-ara-C increased with a prolonged period of incubation, reaching maximum levels of 192, 214, and 450 pmol/10 6 cells, respectively, at 32 hr after treatment. The uptake of N 4 -lauroyl-1-β-d-arabinofuranosylcytosine, and N 4 -butyryl-1-β-d-arabinofuranosylcytosine reached maximum levels of 110 and 9.2 pmol/10 6 cells at about 23 and 5 hr, respectively, and the levels remained constant up to 32 hr after treatment. ara-C was rapidly transported into the cells. The intracellular concentration of ara-C reached maximum (93 pmol/10 6 cells) at 5 hr after treatment. However, it decreased gradually thereafter, reaching the basal level at 23 hr after treatment as the deamination of ara-C by KB cells occurred. The highest uptake of the drug (450 pmol/10 6 cells) observed for N 4 -palmitoyl-ara-C at 32 hr after treatment indicated that approximately 25% of the added drug was transported into the cells. Also, N 4 -palmitoyl-ara-C was observed to have the highest incorporation of the drug into cellular DNA. The amounts incorporated into cellular DNA at 47 hr after treatment were 1.73, 1.90, 3.20, 4.43, and 7.79 pmol/10 6 cells for N 4 -butyryl-1-β-d-arabinofuranosylcytosine, N 4 -behenoyl-1-β-d-arabinofuranosylcytosine, N 4 -stearoyl-1-β-d-arabinofuranosylcytosine, N 4 -lauroyl-1-β-d-arabinofuranosylcytosine, and N 4 -palmitoyl-ara-C, respectively, whereas the amounts of ara-C incorporated into DNA were 3.89 and 2.23 pmol/10 6 cells at 23 and 47 hr, respectively. The cytotoxic effect of ara-C was greatly dependent on the cell density, and a 17,000-fold difference in the drug concentration required for 50% inhibition was observed between the cultures seeded at 2,100 cells/sq cm growth surface and 21,000 cells/sq cm growth surface. KB cells rapidly degraded ara-C to 1-β-d-arabinofuranosyluracil, and the reaction was catalyzed intracellularly. At 24 hr after treatment with ara-C at 1 µm, no ara-C was detected in the culture medium seeded at 21,000 cells/sq cm growth surface, whereas 30% of ara-C remained in the culture seeded at 2,100 cells/sq cm growth surface. The strong cytotoxic effect of N 4 -palmitoyl-ara-C could be explained by the enhanced uptake of the drug and by the resistance of the incorporated drug to deaminase.