Date of Award

Summer 2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Christopher J. Osgood

Committee Member

Keith A. Carson

Committee Member

R. James Swanson

Committee Member

Lloyd Wolfinbarger, Jr.

Abstract

Cancer is the second leading killer in the United States. Anticancer drug development is always based on the understanding of molecular and cellular mechanisms of carcinogenesis, as well as comprehensive knowledge of potential anticancer drugs. Arglabin-dimethylaminohydrochloride (arglabin-DMA) represents one of the new classes of anti-cancer agents that have shown promise in suppressing the growth of various tumor cells. However, the cellular mechanism of arglabin-DMA cytotoxic effects on tumor cells is still unclear. The current study was to determine the farnesyltransferase (FTase) inhibitory activity of arglabin-DMA and to investigate the effects of arglabin-DMA on three proteins: Ras, Rho and cyclin kinase inhibitor p21/WAF1/CIP1.

In vitro FTase assays were used to study the effect of arglabin-DMA on FTase activity. The FTase assay using expressed FTase showed that the 50% inhibition concentration of arglabin-DMA was 2.9 mM. The FTase assay using lysates of Ras/3T3 cells that had been incubated with arglabin-DMA showed that the highest FTase inhibition was 59% as compared to the no treatment control, and this inhibition plateaued when the arglabin-DMA concentration was 100 nM or higher. These results suggested that arglabin-DMA was transformed in cells and that the transformed arglabin-DMA inhibited FTase activity in Ras/3T3 cells. However, this inhibition was limited by the substrate availability in cells that may transform arglabin-DMA to an FTase inhibitor.

The study of the effects of arglabin-DMA on Ras protein, Rho protein, and cyclin kinase inhibitor p21/WAF1/CIP1 was performed using Ras/3T3 cells incubated with arglabin-DMA or FTI-277 (positive control) for 24 or 72 hours. Western blots and densitometry showed a decreased ratio of processed to unprocessed H-Ras protein in cell lysates incubated with 100 μM of arglabin-DMA. Western blotting of active H-Ras protein from GTPase pull-down assays showed significant reduction of active H-Ras after incubation with 50 μM of arglabin-DMA for 24 hours, or 1 μM of arglabin-DMA for 72 hours. Only unprocessed RhoA was detected by Western blot after incubation with 10 μM (or higher) arglabin-DMA or FTI-277. Western blots showed a trend of increased p21/WAF1/CIP1 production after 72 hours of incubation with arglabin-DMA and this increase was not found in the FTI-277 control.

Comments

A Dissertation Submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/b9ha-qz06

ISBN

9780542855498

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