Chemotherapeutic drug cytotoxicity enhancement in human cells in culture

This thesis examined the effect of combinations of different drugs on toxicity to cancer cells in vitro. Combinations of a known anticancer agent with one of a series of coded non-toxic compounds known to be safe for human use were assayed. Significant enhancement of toxicity of adriamycin and vincristine but not 5- fluorouracil, was observed with three compounds from the series. Studies in a wide range of cellular models showed that this combination effect was not observed in cells overexpressing P-glycoprotein and that the mechanism involved was not inhibition of P-glycoprotein. An assay was developed to analyse how synergy was affected when cells were exposed to the compounds at different times relative to chemotherapeutic drug pulse exposure. Activity of the active compounds was observed only during 24 hours subsequent to 2 hours drug exposure. Pre-treatment or treatment at later times was ineffective. Flow cytometric analysis was used to determine the cell cycle distribution of these test compounds in DLKP-SQ, a human lung carcinoma clonal cell line. U-l (active in the combination assay) on its own was shown to induce a transient G/S arrest. There was no apparent effect on the cell cycle distribution using A-l (inactive) or N-l (active). A combination of adriamycin and U-l caused an increased delay in the G, phase and the S phase compared to adriamycin alone. Vincristine in combination with U-l displayed an increased G2/M arrest compared to vincristine treated cells alone. DLKP-SQ treated with 5-fluorouracil alone and in combination showed no effect on cell cycle distribution. Time lapse videomicroscopic studies demonstrated that vincristine induced apoptosis within 24 hours and the rate of apoptosis increased in the combination. Cells underwent apoptosis 24 hours after treatment with adriamycin but surprisingly there was no significant difference in the rate of apoptosis between adriamycin alone and in combination. To investigate if the combination effect was due to interference with proteins controlling cell cycle progress, the levels of cyclin E and CDK2 were determined using western blotting; Cyclin E/CDK2 kinase activity was also measured.