Cisplatin Uptake Mechanism In Drug-Sensitive Cancer Cell

药物敏感癌细胞中的顺铂摄取机制

• 批准号: 7146069
• 负责人: Richard D Leapman
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7146069
• 关键词: cisplatin; drug resistance; electron microscopy; enzyme activity; freeze substitution; liver neoplasms; neoplasm /cancer pharmacology; neoplastic cell; squamous cell carcinoma; transmission electron microscopy; trypsin

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.

该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。将药物敏感和耐药的肝癌细胞和表皮样癌细胞与浓度高达 400 微摩尔的顺铂一起孵育长达 4 小时。使用胰蛋白酶从培养皿中释放细胞并添加血清以终止胰蛋白酶活性后，使细胞恢复其钠/钾梯度。然后将细胞悬浮液转移至200微米直径的塑料毛细管中，在高压冷冻机中冷冻于2000巴，用含有戊二醛固定剂的丙酮冷冻替代，并在低温下用Lowicryl树脂包埋以最小化元素重新分布。将厚度为 200-300 纳米的塑料切片安装在金网上，并在透射电子显微镜中以 300 kV 束电压进行成像，然后发送到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 X 射线图与电子显微镜获得的超微结构以及我们之前的电子探针显微分析数据相关。在药物敏感细胞中，我们已经证明铂会积聚在细胞质内的囊泡结构中。正在分析数据以确定这种分布在耐药细胞中如何变化。