Burger A M, Jenkins T C, Double J A, Bibby M C
Tumor Biology Center at the University of Freiburg, Germany.
Br J Cancer. 1999 Sep;81(2):367-75. doi: 10.1038/sj.bjc.6690702.
C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy.
C1311是一种新型治疗药物，对实验性结直肠癌具有强大活性，已被选入临床试验。该化合物先前已被证明具有DNA结合特性，并能抑制拓扑异构酶II的催化活性。在本研究中，研究了C1311在完整结肠癌细胞中与DNA相互作用并发挥细胞毒性作用的细胞摄取及机制。选用HT29结肠癌细胞系，在药物浓度刚好抑制细胞增殖50%或100%的情况下，追踪C1311在24小时时间进程中的细胞分布。通过荧光显微镜检查C1311的核摄取以及与溶酶体或线粒体染料的共定位，并通过测量酸性磷酸酶水平、罗丹明123释放或DNA结合行为来确定对这些细胞区室的影响。通过热变性稳定性、直接滴定和宿主双链长度的粘度测定研究确定DNA结合的强度和模式。使用TUNEL检测和DNA片段化追踪凋亡的起始，以确定细胞死亡的因果关系。C1311对HT29细胞的生长抑制与药物在细胞核中的快速积累同时发生，在此背景下，我们表明该化合物通过嵌入与双链DNA结合，可能优先结合A/T序列。在溶酶体中也观察到药物摄取，导致溶酶体破裂，并且在暴露于导致完全生长抑制的C1311浓度8小时后酸性磷酸酶活性显著增加。此外，在这些浓度下，溶酶体肿胀和破裂先于凋亡，在暴露于药物后24小时内凋亡并不明显。因此，C1311的溶酶体趋向性作用似乎是这种抗癌药物的一个新特征。由于仅C1311诱导的DNA损伤不太可能足以产生细胞毒性活性，溶酶体破裂可能是治疗效果的关键组成部分。
