Cisplatin Uptake Mechanism in Drug Sensitive and Drug Resistant Cancer Cells

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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.