Date of Award

2003

Degree Name

Biological Sciences

College

College of Science

Type of Degree

M.S.

Document Type

Thesis

First Advisor

Laura J. Jenski

Second Advisor

Frank Binder

Third Advisor

Nicola Locascio

Abstract

Traditional chemotherapeutic drugs are prone to toxicity and may result in secondary cancers. In recent years much attention has been garnered by alternate methods of cancer treatment with fewer side effects, including immunotherapy and administration of ω-3 fatty acids, both of which have been shown to cause apoptosis in cancer cells. Docosahexaenoic acid (DHA), a fatty acid, is a normal component of cell membranes and is safe for systemic administration. The cytokine TRAIL (tumor necrosis factorrelated apoptosis-inducing ligand) is able to induce apoptosis in cancer cells while sparing normal tissue. In this study, the alkylating agent chlorambucil (CLB) was combined with DHA and TRAIL in order to assess their ability to induce apoptosis synergistically in Jurkat human T-cell leukemia and NCI-H460 human non-small cell lung cancer cell lines. Dose response curves were created by treating each cell line with each agent individually and analyzing by a colorimetric cell proliferation assay. Sublethal concentrations of each agent were combined to determine the nature of their interaction, be it additive, sub-additive, or synergistic. Selected sublethal doses of each agent were combined and further analyzed by flow cytometry using Annexin V-FITC, a fluorescently-labeled molecule that binds to cells undergoing apoptosis. Jurkat was more sensitive than H460 to every combination tested, as well as to every individual agent except TRAIL. CLB combined with DHA proved synergistic in both cell lines by XTT. DHA is believed to increase fluidity of the membrane allowing CLB to diffuse more easily into the cell. CLB combined with TRAIL also was synergistic in both cell lines, but to a different degree perhaps due to TRAIL’s ability to use different pathways in each, either receptor-mediated in Jurkat or mitochondrial in H460. By XTT, TRAIL and DHA combined in an additive manner at best. Flow cytometry indicated that in both cell lines DHA and TRAIL inhibited each other, more strongly in H460 than in Jurkat. The combination of all three agents resulted in synergy in both cell lines by XTT, with only 20% of Jurkat cells surviving, but appeared sub-additive by flow cytometry by the Annexin V-FITC assay, which is specific for apoptotic cells and does not stain those that have died by necrosis. The actual percentage of cell death may be higher than indicated by this analysis.

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