Cisplatin Uptake Mechanism in Drug Sensitive and Drug Resistant Cancer Cells

该项目的目的是确定 抗癌药物的入境机制 顺二氨二氯铂II（顺铂）进入癌细胞，和 这种进入是如何影响耐药细胞的。药敏和 将耐药肝癌细胞孵育不同时间， 5小时、1小时、2小时、4小时）， 100微克/毫升氯化铁。将细胞沉淀，并迅速 在液态乙烷中冷冻，但很难获得高质量的冷冻切片 从而导致可用冷冻切片的产量非常低。它 因此，有必要通过以下方式改进冷冻切片技术： 在使用低温电子的制备的每个步骤之后， 显微镜将合适的样品转移到分析仪中。 扫描透射电子显微镜（STEM），冷冻干燥， 在低剂量下成像，然后通过能量色散X射线分析 谱从不同的细胞器记录光谱 包括线粒体、细胞核和内体。元素 使用参比光谱和多重线性最小二乘法定量 拟合用400微摩尔顺铂处理0.5小时， 在敏感细胞中发现破坏Na +/K+梯度，但在 抗性细胞测量表明，敏感细胞含有 铁含量升高的细胞器（约20 mmol/kg干重） 并且较低量的铂（大约，1 mmol/kg干重） 在这些隔间中有共同定位。结果表明 含铁细胞器可能是内体， 服用顺铂。耐药细胞似乎缺乏 含有铁或铂的细胞器。这是一个延续 校内研究项目Z01-RR-10491 - 01 BEI。

The aim of this project is to determine the mechanism for entry of the anti-cancer drug cis-diamminedichloroplatinum II (cisplatin) into cancer cells, and how this entry is affected in drug-resistant cells. Drug-sensitive and drug-resistant liver carcinoma cells were incubated for different times (0.5 hr, 1 hr, 2 hr, 4 hr) with 400 micromolar cisplatin and 100 micrograms/ml ferric chloride. Cells were pelleted and rapidly frozen in liquid ethane but high quality cryosectioning was difficult to achieve resulting in a very low yield of usable cryosections. It was therefore necessary to improve the cryosectioning technique by following each step of the preparation using a cryo-electron microscope. Suitable specimens were transferred into an analytical scanning transmission electron microscope (STEM), freeze-dried, imaged at low dose and then analyzed by energy- dispersive x-ray spectroscopy. Spectra were recorded from different organelles including mitochondria, nuclei and endosomes. Elements were quantified using reference spectra and multiple linear least squares fitting. Treatment with 400 micromolar cisplatin for 0.5 hr was found to destroy the Na+/K+ gradient in sensitive cells but not in resistant cells. Measurements indicated that sensitive cells contained organelles with elevated iron levels (approx. 20 mmol/kg dry wt.) and that lower amounts of platinum (approx. 1 mmol/kg dry wt.) were co- localized in some of these compartments. Results suggest that the iron-containing organelles may be endosomes which also take up cisplatin. Resistant cells appeared to be deficient in organelles containing iron or platinum. This is a continuation of Intramural Research Project Z01-RR-10491-01 BEI.