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Type of Document Dissertation Author Ma, Rui Author's Email Address rma@nd.edu URN etd-12132007-164844 Title Apoptosis of Breast and Colon Carcinoma Cells by Inhibitors of Glycolipid and DNA Biosynthesis Degree Doctor of Philosophy Department Chemistry and Biochemistry Advisory Committee
Advisor Name Title Dr. Subhash Basu Committee Chair Dr. Crislyn DSouza-Schorey Committee Member Dr. Holly Goodson Committee Member Dr. Paul Huber Committee Member Keywords
- apoptosis
- anti-cancer
- signal transdcution
- fluorescent dye
- microarray
Date of Defense 2007-10-31 Availability restricted Abstract Apoptotic effects of potential anti-cancer agents (GSL-glycosphingolipid biosynthesis inhibitor L-PPMP, DNA biosynthsis inhibitor cis-Platin, disialosylgangliosides GD3 and GD1b, etc.) in human breast cancer cell lines were evaluated in this study. Different techniques including Trypan staining, phase contrast microscopy, DNA laddering, PSS-380/PI staining with phosphatidylserine, and AKS-0 staining with cell membranes system were used to characterize the apoptosis. The cell-killing action by these anticancer agents were detected to be dose-dependent with ED50 identification and time-dependent with monitoring PSS-380 between 6 to 24 hours of drug incubation. The PSS-380 and AKS-0 fluorescence dyes were found to be good molecular probes to characterize the changes during apoptosis.Activation of both extrinsic and intrinsic apoptotic pathways by cis-Platin was observed in human breast cancer cells (MCF-7, MDA-468, and SKBR-3) with western blot using caspase-3, caspase-8, and caspase-9 antibodies. However, only activation of intrinsic mitochondrial apoptotic pathway was found in L-PPMP-treated cells using caspase-3 and caspase-9 antibodies. All the tested anti-cancer agents and disialosylgangliosides were able to activate the effector caspase-3 to its processed forms, which suggests their apoptotic abilities. Changes in other signal transduction pathways of MAPKs and NF-κB cascades were also detected with L-PPMP and cis-Platin treatments. The activation profile was presented in a cell-type-dependent and drug-specific manner. Various regulations of signal transduction pathways may be caused by the presence of different cell surface receptors and proteins in these MCF-7, MDA-468, and SKBR-3 cells.
Using radioactive serine and sugar incorporation assay, the elevation of endogenous ceramide biosynthesis was detected in different cell lines in the presence of various apoptotic agents except for cis-Platin. This implies the mechanism of ceramide-involved mitochondrial outer membrane permeability changes during apoptosis. With radioactive sugar incorporation assay, the down-regulation of GSL glycosphingolipid biosynthesis in the human breast cancer cells by L-PPMP were also suggested.
Fresh pork liver was successfully used as a source of Fucokinase and Pyrophosphorylase to synthesize GDP-[3H]-L-Fucose which was used for complete biosynthesis study of regulation of fucosyltransfereases activities during apoptosis. An optimal condition and a simple procedure for in vitro biosynthesis (preparative) was worked out so that the radioactive GDP-[3H]-L-Fucose (commercially unavailable) could be prepared in large scale quantity for people who are working in the area of fucosyltransfereases. Our newly prepared radioactive GDP-[3H]-L-Fucose was used for present study.
Regulation of GSL glycosyltransferases (GalT-4, GalT-5, SAT-2, SAT-4, SAT-4’, and FucT-3) was studied with the human breast cancer cells treated with L-PPMP and cis-Platin. Under most conditions, the anti-cancer agents were found to be able to inhibit GSL glycosyltransferase activities with some exceptions of enhancement of enzyme activities. This reveals the drug-, cell type-, and time-dependent manner of regulation of GSL glycosyltransferases during apoptosis.
In addition, DNA-microarray techniques were used with human glyco-related gene chips to study the regulation of GSL glycosyltransferase gene expressions during the apoptosis induced by L-PPMP in both the early stage (2 hours of treatment) and the late stage (24 hours of treatment). Several genes encoding the enzymes involved in the sphingolipid glycosylation were detected significantly changed (up-regulated or down-regulated), which indicating the complicated response of all three breast cancer cell lines to the apoptosis signal induced by L-PPMP.
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