Defense Date

2008

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmacology & Toxicology

First Advisor

David Gewirtz

Abstract

Microtubule poisons have proven to be effective in the treatment of a variety of malignancies. Although taxol-based derivatives promote microtubule stabilization, there is continuing interest in compounds that, like colchicines, act as microtubule destabilizing agents. Previous work from this laboratory showed that the novel microtubule poison, JG- 03-14, was active against breast tumor cells, promoting autophagic cell death. In the current work, we studied the influence of JG-03-14 on p53 wild type HCT116 colon carcinoma cells. A crystal violet sensitivity assay indicated that JG-03-14 induced growth inhibition, with 75% suppression of growth evident at a concentration of 500 nM. Time course studies of drug effects on cell viability indicated that JG-03-14 also produced cell killing. FACS analysis demonstrated that the HCT-116 cells arrested in the G2/M stage; furthermore, there was evidence of a hyperdiploid population that would be consistent with failure of the cells to divide despite completion of DNA synthesis. Finally, there was evidence of a small sub G0/G1cell population, indicating that the cells were not dying primarily by apoptosis, and suggesting that JG-03-14 induces an alternative mode of cell death. In contrast to cell shrinkage and nuclear fragmentation that was evident after treatment with taxol ( a positive control for apoptosis), DAPI staining of HCT-116 cells treated with JG-03-14 showed intact and enlarged nuclei, again consistent with the absence of apoptosis. Furthermore, there was no evidence of mitotic catastrophe (micronuclei in binucleated cells). Based on previous studies in MCF-7 and MDA-MB231 cell lines that demonstrated a substantial population of autophagic cells, HCT-116 cells were subjected to staining with acridine orange and monodansylcadaverine after treatment with JG-03-14. While control cells tended to show a single large autophagic vesicle closely associated with the cell nucleus, treatment with JG-03-14 resulted in extensive distribution of small acidic vesicles within the cytoplasm, indicative of autophagy. GFP-LC3 transfected cells incubated with JG-03-14 showed punctuated patterns that were also consistent with the promotion of autophagy. Finally, activation of the DNA damage response pathway was ruled out by the lack of induction of p53 and p21 in cells treated with JG-03-14. In summary, our studies indicate that the JG-03-14 induces both growth arrest and autophagic cell death in HCT116 colon carcinoma cells. The possibility of an alternative mode of cell death induced by JG-03-14 makes it a potentially usefull candidate as a chemotherapeutic drug that could be used to treat cancers resistant to apoptosis. Our result also suggested that JG-03-14 failed to induce bone marrow toxicity adding to its potential for clinical use.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2008

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