Studies on the Protection Mechanisms Against Doxorubicin Toxicity in Resistant and Sensitive Human Tumour Cells

Al-Kabban, Muzahim M (1989) Studies on the Protection Mechanisms Against Doxorubicin Toxicity in Resistant and Sensitive Human Tumour Cells. PhD thesis, University of Glasgow.

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A rapid, sensitive and selective HPLC method for the quantitation of glutathione (GSH) at the cellular level was developed by the author. Glutathione is resolved isocratically by ion-pair high-performance liquid chromatography and detected by UV at 200 - 210 nm. The mobile phase consisted of an aqueous buffer of methanol containing 0.1% tetrabutyl ammonium hydroxide adjusted to pH 3.5 by 10% v/v orthophosphoric acid. This method is able to detect GSH in a small amount of cells and can be adapted for quantitative determination of biological thiols and some other cellular compounds of special interest such as amino acids at small tissue volumes. The non invasive nuclear magnetic resonance (NMR) technique which was developed for this study (1H spin echo NMR) is capable of detecting certain small molecules and structural entities in intact tumour cells. This method is specific and selective, providing information on the concentration and conformation of such molecules as glutathione, phosphorylcholine, lactate, mobile triglyceride, acetyl choline etc in the living cells. The technique has the advantage that it is non-invasive, providing detailed structural information on individual species present in the cell matrix. It has been used in this case to study the rate of energy consumption following the activation of the glycolytic pathway with glucose. The signals and patterns observed have been used in a preliminary way to study changes in glutathione metabolism and in lactate production when challenged by therapeutic agents. The effect of doxorubicin on the cellular biochemistry of Hela tumour cells using 1H spin echo NMR of the intact and viable cell in conjunction with the dual wave length HPLC of cell lysate is reported here. Dose-related changes were observed in lactate and reduced glutathione concentration. Doxorubicin induces a time-dependent depletion of the cytosolic pool of glutathione and a change in the glycolytic pattern of the cells. The glutathione depletion could be partially reversed by controlled pretreatment of the cells with N-acetylcysteine and cysteine, the protection being linked to the intracellular concentration of the thiol. Glutathione was also measured in other doxorubicin-sensitive cells from small cell carcinoma of lung (GLC4 210), and the levels compared with those in cells with acquired resistance and a line of resistant non-small-cell adenocarcinoma of lung A549 (alveolar type 2). The effect of different doxorubicin concentrations on GSH was measured using the HPLC method which has been shown to correlate with the NMR studies in live cells. 1H spin echo NMR of the leukaemia cell line (J111) is also reported here for studying cellular glycolysis. The concentration of cells in the NMR tube is high (approx. 10e9 cells in 0.4 ml) and as a result the available oxygen is restricted, making the NMR experiment a plausible in vitro tumour model in which kinetics in the living cell can be probed in a non-invasive manner. Treatment with pharmacological concentrations of doxorubicin produced immediate enhanced anaerobic glycolysis and eventual cell death. Glutathione-S-transferase (GST) activity in the different lung tumour cells was linked with their content of glutathione and the effect of doxorubicin on such activity was studied as well. High levels of lipid peroxidation were noticed in the two sensitive cell lines (Hela and GLC4 210 [S]) as doxorubicin readily passes into these cells and interacts with glutathione. N-acetylcysteine pre-treatment of Hela cells showed little protection from the effect of doxorubicin. The effect of doxorubicin on the viability of the different tumour cells was studied using MTT dye reduction by living but not dead cells. An increase in A549 sensitivity to doxorubicin was produced using Buthionine-S,R-Sulfuximine at a non-toxic concentration. The inherent resistant of A549 tumour cells toward doxorubicin was circumvented by using one of the antiarrythmic drugs (amiodarone) which trap the drug in the cells by decreasing its efflux. This was indicated by measuring intracellular doxorubicin and amiodarone after harvesting of the cells in two different ways (scraping and trypsinization). These results were confirmed by the HPLC measurement of GSH in these cells after amiodarone and doxorubicin treatment and by using a chemosensitivity assay.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Medicine, Analytical chemistry
Date of Award: 1989
Depositing User: Enlighten Team
Unique ID: glathesis:1989-77845
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53

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