Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Pharmacology and Toxicology

Supervisor

Dr. John Di Guglielmo

Abstract

Although cancer survival rates have significantly improved over the past few decades, the improvements are primarily due to early diagnosis and inhibiting cancer growth. Limited progress has been made in the treatment of cancer metastasis, which contributes to 90% of cancer related deaths, and therapeutic agents targeting the various aspects of metastasis are lacking. One potential approach is to utilize small pharmacological compounds to inhibit tumour cell motility, as a strategy against tumour cell migration, invasion, and metastasis. The acetylenic tricyclic bis-(cyano enone), TBE-31, has been shown to be a promising chemopreventative compound. However, its effects on cell migration are unknown. This thesis focuses on deciphering the molecular mechanisms TBE-31 utilizes to inhibit cell migration. I demonstrated that TBE-31 binds with cysteine 374 of actin, inhibits actin polymerization and stress fiber formation. These findings were applied to a model of epithelial-to-mesenchymal transition, a precursor event to metastasis, where I determined TBE-31 was able to inhibit the crucial rearrangement of cortical actin to form actin stress fibers, which prime tumour cells for migration. In addition to the actin cytoskeleton, I demonstrated that TBE-31 alters microtubule dynamics and organization. Microtubule-dependent trafficking was also shown to be disrupted by TBE-31, and the localization of the polarity proteins Rac1, IQGAP and Tiam1 were altered from the leading edge of migrating cells. Lastly, TBE-31 was shown to inhibit Rat2 and NIH3T3 fibroblast as well as H1299 non-small cell lung cancer tumour cell migration. Taken together, my work provides novel insights on the underlying mechanisms by which TBE-31 utilizes to inhibit cell migration and provide important knowledge for developing therapeutic compounds that target tumour cell motility in metastasis.

Video 2-1 TBE-31 inhibits NSCLC tumor cell migration.avi (10062 kB)
Confluent monolayers of H1299 lung tumor cells were scratched and incubated at 37°C in media containing either DMSO (top), 1 μM TBE-31 (middle) or 1 μM CDDO-Im (bottom) for 18 h. Representative bright field microscopy images were taken at 10x magnification every 15 min using an Olympus IX81 microscope to create a movie.

Video 2-2 TBE-31 inhibits NIH 3T3 cell migration.avi (8699 kB)
Single cell tracking of NIH 3T3 cells was carried out using time-lapse microscopy with an Olympus IX81 inverted microscope equipped with a custom chamber which allowed us to maintain a temperature of 37°C and an atmosphere containing 5% CO2. Bright field images were collected at 10 min intervals over 18 h. Distance of migration was determined by tracking the positions of cell nuceli over 18 h using the MtrackJ plugin (32) for imageJ software (33).

Video 3-1 EB1 Movies - DMSO.avi (2555 kB)
Subconfluent Mv1Lu cells transiently expressing GFP-EB1 were incubated in DMSO and imaged using an Olympus IX81 fluorescent microscope equipped with a cell chamber. GFP-EB1 mobility was visualized over 2.5 min.

Video 3-2 EB1 Movies -TBE-31.avi (4071 kB)
Subconfluent Mv1Lu cells transiently expressing GFP-EB1 were incubated in 2 µM TBE-31 and imaged using an Olympus IX81 fluorescent microscope equipped with a cell chamber. GFP-EB1 mobility was visualized over 2.5 min.

Available for download on Tuesday, January 01, 2019


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