Multon E, Riou J F, LeFevre D, Ahomadegbe J C, Riou G
Laboratoire de Pharmacologie Clinique, Moléculaire Institut Gustave Roussy, Villejuif, France.
Biochem Pharmacol. 1989 Jul 1;38(13):2077-86. doi: 10.1016/0006-2952(89)90060-9.
Ellipticine derivatives have been shown to induce DNA strand breaks by trapping DNA-topoisomerase II (Topo II) in an intermediary covalent complex between Topo II and DNA which could be related to their cytotoxic effects. We report here that Celiptium and Detalliptinium, two ellipticine derivatives clinically used for their antitumoral properties against breast cancer, exhibit the highest in vitro activity on Topo II DNA cleavage reaction and decatenation among a series of 14 ellipticine derivatives. The in vitro cleavage site specificity in pBR 322 plasmid DNA and in a human c-myc gene inserted in a lambda phage DNA is identical for both ellipticines, but different from m-AMSA, another Topo II related antitumoral agent. Recently, it has been shown that the ellipticine derivative Celiptium presents a strong cytotoxic activity in vitro on different human tumors including small cell lung carcinoma (SCLC). However, the studies that involved Topo II as a target for ellipticine derivatives have been performed only by using animal tumor cell lines. Therefore we have studied the in vivo DNA cleavage activity of Celiptium and Detalliptinium on a human SCLC cell line, NCI N417, comparatively to that obtained with m-AMSA. The respective IC50 on cell growth are 9, 8 and 1 microM for Celiptium, Detalliptinium and m-AMSA, respectively. Using the alkaline elution technique, we have observed that Celiptium and Detalliptinium exhibit a weak cleavage activity on genomic DNA from whole cells. The ellipticines are about 50 times less potent than m-AMSA in inducing DNA strand breaks. Analysis of in vivo c-myc gene cleavage by Southern blot hybridization also demonstrates a lack of activity of the ellipticine derivatives as no gene cleavage could be detected up to 50 microM of the drug. With m-AMSA, c-myc gene cleavage is detected at a concentration of 0.2 microM, which indicates that this methodology is less sensitive in detecting DNA strand breaks than is the alkaline elution. Further studies of the drug effect on isolated nuclei by alkaline elution also show that the DNA cleavage activity of Celiptium and Detalliptinium is increased when compared to whole cells. Our data indicate that these two drugs have a weaker cytotoxic effect than m-AMSA on NCI N417 cell line, due to a limited access to the cell nucleus rather than to a lack of activity on Topo II as assessed by in vitro and isolated nuclei experiments.
椭圆玫瑰树碱衍生物已被证明可通过将DNA拓扑异构酶II（Topo II）捕获在Topo II与DNA之间的中间共价复合物中来诱导DNA链断裂，这可能与其细胞毒性作用有关。我们在此报告，西利替姆和地他替姆这两种临床上用于抗乳腺癌的椭圆玫瑰树碱衍生物，在14种椭圆玫瑰树碱衍生物系列中，对Topo II DNA切割反应和解连环作用表现出最高的体外活性。两种椭圆玫瑰树碱在pBR 322质粒DNA和插入λ噬菌体DNA中的人类c-myc基因中的体外切割位点特异性相同，但与另一种与Topo II相关的抗肿瘤药物m-AMSA不同。最近，已表明椭圆玫瑰树碱衍生物西利替姆在体外对包括小细胞肺癌（SCLC）在内的不同人类肿瘤具有很强的细胞毒性活性。然而，涉及将Topo II作为椭圆玫瑰树碱衍生物靶点的研究仅使用动物肿瘤细胞系进行。因此，我们研究了西利替姆和地他替姆对人SCLC细胞系NCI N417的体内DNA切割活性，并与用m-AMSA获得的结果进行了比较。西利替姆、地他替姆和m-AMSA对细胞生长的各自IC50分别为9、8和1 microM。使用碱性洗脱技术，我们观察到西利替姆和地他替姆对全细胞基因组DNA表现出较弱的切割活性。椭圆玫瑰树碱在诱导DNA链断裂方面的效力比m-AMSA低约50倍。通过Southern印迹杂交分析体内c-myc基因切割也表明椭圆玫瑰树碱衍生物缺乏活性，因为在高达50 microM的药物浓度下未检测到基因切割。对于m-AMSA，在0.2 microM的浓度下检测到c-myc基因切割，这表明该方法在检测DNA链断裂方面不如碱性洗脱敏感。通过碱性洗脱对分离细胞核上药物作用的进一步研究还表明，与全细胞相比，西利替姆和地他替姆的DNA切割活性有所增加。我们的数据表明，这两种药物对NCI N417细胞系的细胞毒性作用比m-AMSA弱，这是由于进入细胞核的机会有限，而不是如体外和分离细胞核实验所评估的那样对Topo II缺乏活性。
