Date of Award

12-1-2009

Embargo Period

1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Pharmaceutical Sciences

College

College of Graduate Studies

First Advisor

Craig E. Crosson

Abstract

Sphingosine kinases (SK) are promising therapeutic targets for cancer because they regulate the balance between pro-apoptotic ceramides and mitogenic sphingosine-1 phosphate (S1P); however, the functions of the two isoenzymes (SK1 and Sk2) in tumor cells are not well defined, and the systemically comparison of the anti-cancer effects from SK isoform selective inhibitors is elusive. Therefore, firstly, RNA interference was used to assess the individual roles of SK1 and SK2 in tumor cell sphingolipid metabolism, proliferation and migration/invasion. Treatment of A498, Caki-1 or MDA-MB-231 cells with siRNA specific for SK1 and SK2 effectively suppressed the expression of the target mRNA and protein. Ablation of SK1 did not affect mRNA or protein levels of SK2, and reduced intracellular levels of S1P while elevating ceramide levels. In contrast, ablation of SK2 elevated mRNA, protein and activity levels of SK1, and increased cellular S1P levels. Interestingly, cell proliferation and migration/invasion were suppressed more by SK2-selective ablation than by SK1-selective ablation, demonstrating that the increased S1P does not rescue these phenotypes. Similarly, exogenous S1P did not rescue the cells from the anti-proliferative or anti-migratory effects of the siRNAs. Consistent with these results, differential affects of SK1- and SK2-selective siRNAs on signaling proteins including p53, p21, ERK1, ERK2, FAK and VCAM1 indicate the SK1 and SK2 have only partially overlapping functions in tumor cells. Secondly, several SK inhibitors are identified and classified according to their inhibiting target as following: SKI-II (Ki SK1 16 μM, Ki SK2 7.9 μM) and ABC2947353 (sphingosine competitive, Ki SK1 2.5 μM, Ki SK2 3.5 μM) are SK1/2-dual inhibitors; CB5468139 (Ki SK1 0.25 μM) is a SK1-selective inhibitor; ABC294640 (sphingosine competitive, Ki SK2 10 μM) is a SK2-selective inhibitor. Together with DMS as the positive control, we compared the effects of the SK inhibitors on A498 kidney cancer cells. In terms of SK1 and SK2 mRNA transcription, cell cycle arrest, amount of ceramide species and FBS-induced mobility, the SK2-selective inhibitor, ABC294640, demonstrated the highest inhibition against proliferation and migration/invasion. The SK2-selective inhibitor also down-regulated the expressions and/or activation of signal proteins such as STAT3, AKT, ERK, p21, p53 and FAK. These effects were equivalent or superior to those of SK1/2-dual inhibitors. IT also induces autophagy marked by increased expression of Beclin1 and LC3. Overall, these results suggests that loss of SK2 has stronger anticancer effects than does suppression of SK1, and the SK2-selective inhibitor, ABC294640, has stronger anticancer properties with less toxicity than SK1/2-dual or SK1-selective inhibitors.

Rights

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