Cisplatin Uptake Mechanism in Drug Sensitive and Drug Resistant Cancer Cells

该项目的目的是确定 抗癌药物进入的机制 顺二胺氯铂II(顺铂)进入癌细胞 这种进入在耐药细胞中是如何影响的。对药物敏感， 耐药肝癌细胞经不同浓度不同时间孵育 时间(0.5小时，1小时，2小时，4小时)400微摩尔顺铂和 100微克/毫升三氯化铁。细胞被制粒，并迅速 冰冻在液态乙烷中，但高质量的冷冻切片是困难的 要达到这一目的，可使用的冷冻切片产量非常低。它 因此，有必要通过以下方式改进冷冻切割技术 在使用冷冻电子进行制备的每一步之后 显微镜。合适的样本被转移到一个分析 冷冻干燥扫描电子显微镜(STEM)， 低剂量成像，然后用能量色散x射线分析 光谱学。从不同的细胞器记录了光谱 包括线粒体、细胞核和内小体。元素是 使用参考光谱和多线性最小二乘进行量化 很合身。用400微摩尔顺铂治疗0.5小时 在敏感细胞中发现破坏Na+/K+梯度，但在 耐药细胞。测量表明，敏感细胞含有 含铁水平升高的细胞器(约20 mmol/kg干重。) 以及较低数量的铂金(约1 mmol/kg干重。) 在其中一些隔间里是共同定位的。结果表明 含铁细胞器可能是内小体，它也 服用顺铂。抗性细胞似乎缺乏 含有铁或铂的细胞器。这是一种延续 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.