Institut für Pharmazie, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany.
Bioorg Med Chem. 2013 Nov 15;21(22):7118-24. doi: 10.1016/j.bmc.2013.09.008. Epub 2013 Sep 14.
We studied the influence of structurally different saponins on the cholesterol content of cellular membranes. Therefore a cell culture model using ECV-304 urinary bladder carcinoma cells was developed. To measure the cholesterol content we used radiolabeled (3)H-cholesterol which is chemically and physiologically identical to natural cholesterol. The cells were pre-incubated with (3)H-cholesterol and after a medium change, they were treated with saponins to assess a saponin-induced cholesterol liberation from the cell membrane. In another experiment the cells were pre-incubated with saponins and after a medium change, they were treated with (3)H-cholesterol to assess a saponin-induced inhibition of cholesterol uptake into the cell membrane. Furthermore, the membrane toxicity of all applied saponins was analyzed using extracellular LDH quantification and the general cytotoxicity was analyzed using a colorimetric MTT-assay and DNA quantification. Our results revealed a correlation between membrane toxicity and general cytotoxicity. We also compared the results from the experiments on the saponin-induced cholesterol liberation as well as the saponin-induced inhibition of cholesterol uptake with the membrane toxicity. A significant reduction in the cell membrane cholesterol content was noted for those saponins who showed membrane toxicity (IC50 <60 μM). These potent membrane toxic saponins either liberated (3)H-cholesterol from intact cell membranes or blocked the integration of supplemented (3)H-cholesterol into the cell membrane. Saponins with little influence on the cell membrane (IC50 >100 μM) insignificantly altered the cell membrane cholesterol content. The results suggested that the general cytotoxicity of saponins is mainly dependent on their membrane toxicity and that the membrane toxicity might be caused by the loss of cholesterol from the cell membrane. We also analyzed the influence of a significantly membrane toxic saponin on the cholesterol content of intracellular membranes such as those of endosomes and lysosomes. In these experiments ECV-304 cells were either incubated with (3)H-cholesterol or with (3)H-cholesterol and 5 μM saponin. After isolation of the endosomes/lysosomes their (3)H-cholesterol content was measured. A significant influence of the saponins on the cholesterol content of endosomal/lysosomal membranes was not detected.
我们研究了结构不同的皂苷对细胞膜胆固醇含量的影响。因此，建立了使用 ECV-304 膀胱癌细胞的细胞培养模型。为了测量胆固醇含量，我们使用放射性标记的（3）H-胆固醇，其在化学和生理上与天然胆固醇相同。细胞用（3）H-胆固醇预孵育，换液后用皂苷处理，以评估皂苷诱导的胆固醇从细胞膜释放。在另一个实验中，细胞用皂苷预孵育，换液后用（3）H-胆固醇处理，以评估皂苷对胆固醇摄取到细胞膜的抑制作用。此外，通过测定细胞外 LDH 定量分析所有应用的皂苷的膜毒性，通过比色 MTT 测定法和 DNA 定量分析分析一般细胞毒性。我们的结果表明，膜毒性与一般细胞毒性之间存在相关性。我们还比较了皂苷诱导的胆固醇释放实验以及皂苷诱导的胆固醇摄取抑制实验的结果与膜毒性的关系。对于显示出膜毒性的皂苷（IC50<60μM），细胞质膜胆固醇含量显著降低。这些具有强大膜毒性的皂苷要么从完整的细胞膜中释放（3）H-胆固醇，要么阻止补充的（3）H-胆固醇整合到细胞膜中。对细胞膜影响较小的皂苷（IC50>100μM）对细胞膜胆固醇含量无显著影响。结果表明，皂苷的一般细胞毒性主要取决于其膜毒性，而膜毒性可能是由于胆固醇从细胞膜中丢失引起的。我们还分析了一种具有显著膜毒性的皂苷对细胞内膜（如内体和溶酶体）胆固醇含量的影响。在这些实验中，ECV-304 细胞用（3）H-胆固醇或（3）H-胆固醇和 5μM 皂苷孵育。分离出内体/溶酶体后，测量其（3）H-胆固醇含量。未检测到皂苷对内体/溶酶体膜胆固醇含量有显著影响。
