Ganglioside overexpression and shedding is a characteristic of tumors believed to contribute to tumor survival and progression. This study was directed to determine the effect of ganglioside expression changes on cell function. Initially, we knocked down GM3 synthase, the initial enzyme in the ganglioside synthesis pathway, by 70% using siRNA in Daoy human medulloblastoma cells. After several generations, however, the level of cellular gangliosides recovered, despite the fact that the cells continued to exhibit a 70-80% decrease in GM3 synthase at the mRNA level, as well as a decreased level of surface GM3 ganglioside. This suggests that the cells have an as yet unknown compensatory mechanism, and that restoring a normal level of gangliosides is an adaptive mechanism for the cells.
In subsequent studies, we used pharmacological inhibitors of glucosylceramide synthase, an enzyme two steps upstream of GM3 synthase, to inhibit ganglioside synthesis and to study the effects of ganglioside depletion in human breast (MCF-7) and colon cancer (HCT-116) cells. Cell proliferation was significantly inhibited by ganglioside depletion in MCF-7 and HCT-116 cells under a variety of growth conditions, once again supporting an important role for glycosphingolipid synthesis in enhancing cell survival and proliferation. The converse, addition of exogenous gangliosides GM3 and GD1a, did not show a statistically significant effect on cell proliferation, possibly because the cells were already proliferating at a near-maximal rate. This was the case even in cells that were ganglioside-depleted using a chemical inhibitor, which suggests that exogenous gangliosides may not be able to fully replicate the effects of those produced endogenously in tumor cells.
In an initial attempt to pinpoint the mechanism for this differential effect on cell growth, phosphorylation of insulin receptor, EGFR and HER2, as well as their downstream effectors Akt and MAPK, was measured in ganglioside-replete and ganglioside-depleted tumor cells (MCF-7, HCT-116, and Daoy), both while starving and after stimulation with varying levels of insulin or EGF. In contrast to the findings in studies of signaling in ganglioside-depleted normal human dermal fibroblasts, no change in signaling was observed in the tumor cells in the presence of a glucosylceramide synthase inhibitor, nor did exogenous gangliosides affect signaling. Overall, it can be concluded that the deleterious effect of ganglioside-depleting drugs on cancer cell proliferation is independent of the insulin and EGFR/HER-2 pathway, as well as Akt and MAPK pathways in general.
These findings suggest that tumor cells respond differently to ganglioside-depleting treatment as compared to normal cells, and that glycosphingolipid depletion affects tumor cell proliferation, but not by insulin receptor, EGFR, or other signaling pathways dependent on MAPK and Akt activation. Further studies would need to be undertaken in order to determine how glycosphingolipid depletion affects tumor cell proliferation, since this could be a potential therapeutic target.
|Commitee:||Ceryak, Susan, Kumar, Ajit, Leitenberg, David, MacDonald, Tobey, Noonan, Frances|
|School:||The George Washington University|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 68/11, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Oncology|
|Keywords:||Breast cancer, Cell proliferation, Colon cancer, Ganglioside depletion, Gangliosides, Glucosylceramide synthase, Medulloblastoma|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be