Cisplatin Uptake Mechanism In Drug-Sensitive Cancer Cell

本项目的目的是阐明抗癌药物顺铂进入癌细胞的机制，并确定耐药性如何影响顺铂的亚细胞分布。将药物敏感性和耐药性肝癌和表皮样癌细胞与浓度高达400微摩尔的顺铂孵育长达4小时。在使用胰蛋白酶从培养皿中释放细胞并加入血清以终止胰蛋白酶活性后，使细胞恢复其钠/钾梯度。然后将细胞悬液转移到直径为200 μ m的塑料毛细管中，在高压冷冻机中于2000 bar下冷冻，在含戊二醛固定剂的丙酮中冷冻置换，并在低温下用Lowicryl树脂包埋以最大限度地减少元素再分布。将厚度为200-300纳米的塑料切片固定在金网格上，并在透射电子显微镜中以300 kV束电压成像，然后送至阿贡国家实验室的高级光子源进行X射线微探针分析。元素的X射线图与电子显微镜获得的超微结构，以及与我们以前的电子探针显微分析数据。在药物敏感的细胞中，我们已经表明，铂积累在细胞质内的囊泡结构。目前正在分析数据，以确定这种分布在耐药细胞中的变化。

The aim of this project is to elucidate the mechanism for entry of the anti-cancer drug cisplatin into cancer cells and to determine how drug resistance affects the subcellular distribution of cisplatin. Drug-sensitive and drug-resistant liver and epidermoid carcinoma cells were incubated with cisplatin at concentrations up to 400 micromolar for periods up to 4 hours. After using trypsin to release the cells from the culture dishes and adding serum to stop the trypsin activity, the cells were allowed to recover their sodium/potassium gradient. The cell suspension was then transferred into 200-micrometer diameter plastic capillary tubes, frozen at 2000 bar in a high-pressure freezing machine, freeze-substituted in acetone containing glutaraldehyde fixative, and embedded at low temperature with Lowicryl resin to minimize elemental re-distribution. Plastic sections of thickness 200-300 nanometers were mounted on gold grids and imaged at 300 kV beam voltage in a transmission electron microscope before being sent for x-ray microprobe analysis to the Advanced Photon Source at Argonne National Laboratory. Elemental x-ray maps were correlated with ultrastructure obtained by electron microscopy, as well as with our previous electron probe microanalytical data. In drug-sensitive cells, we have shown that platinum accumulates in vesicular structures within the cytoplasm. Data are being analyzed to determine how this distribution changes in drug-resistant cells.