Sano Y, Deen D F, Oredsson S M, Marton L J
Cancer Res. 1984 Feb;44(2):577-81.
9L rat brain tumor cell spheroids were treated with alpha-difluoromethylornithine (DFMO) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone and in combination. In contrast to results obtained with 9L monolayer culture cells, very low concentrations of DFMO killed spheroid cell within 0.5 day after the start of treatment; cell kill was maximum within 2 to 3 days. DFMO cytotoxicity could be prevented by adding putrescine (to a final concentration of 1 mM) to the culture medium. DFMO also significantly slowed and eventually stopped the growth of spheroids in a dose-dependent manner. Cells in all regions of DFMO-pretreated spheroids were sensitized to BCNU as measured by colony-forming efficiency; this sensitization was prevented when putrescine was added to culture medium before BCNU treatment. When used as single agents, either a 3-day treatment with 10 mM DFMO or a 1-hr treatment with BCNU (1.5 micrograms/ml) produced similar growth delay, but used in combination, the two agents produced a much longer growth delay than produced by either agent alone. Growth of spheroids treated continuously for up to 28 days with 10 mM DFMO ceased at approximately 7 times the volume at the time of treatment. When spheroid cells were treated for 1 hr with BCNU (1.5 micrograms/ml) and then were treated continuously with DFMO, growth plateaued at approximately 3.5 times the volume at the time of treatment; when spheroids were treated first with DFMO for 3 days, then with BCNU (1.5 micrograms/ml) for 1 hr, and then treated continuously with DFMO, growth plateaued at approximately 1.5 times the volume at the time of treatment. The number of clonogenic cells per spheroid that survived combination treatment also reflected the cytotoxic effects of the two drugs. Thus, the combined drug treatment was very effective in inhibiting the growth of spheroids and in preventing an increase in the number of clonogenic cells per spheroid.
用α-二氟甲基鸟氨酸（DFMO）和1,3-双（2-氯乙基）-1-亚硝基脲（BCNU）单独及联合处理9L大鼠脑肿瘤细胞球体。与9L单层培养细胞的结果相反，极低浓度的DFMO在处理开始后0.5天内就能杀死球体细胞；细胞杀伤在2至3天内达到最大值。向培养基中添加腐胺（终浓度为1 mM）可防止DFMO的细胞毒性。DFMO还以剂量依赖的方式显著减缓并最终停止球体的生长。通过集落形成效率测量，DFMO预处理球体所有区域的细胞对BCNU均敏感；在BCNU处理前向培养基中添加腐胺可防止这种敏感性。当单独使用时，10 mM DFMO的3天处理或BCNU（1.5微克/毫升）的1小时处理产生相似的生长延迟，但联合使用时，这两种药物产生的生长延迟比单独使用任何一种药物都长得多。用10 mM DFMO连续处理长达28天的球体生长在处理时体积的约7倍时停止。当球体细胞用BCNU（1.5微克/毫升）处理1小时，然后连续用DFMO处理时，生长在处理时体积的约3.5倍时达到平台期；当球体先经DFMO处理3天，然后用BCNU（1.5微克/毫升）处理1小时，再连续用DFMO处理时，生长在处理时体积的约1.5倍时达到平台期。联合处理后每个球体存活的克隆形成细胞数量也反映了两种药物的细胞毒性作用。因此，联合药物处理在抑制球体生长和防止每个球体克隆形成细胞数量增加方面非常有效。
