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PI: Kok, J.W.

University Medical Center Groningen

Research group JW Kok: Bioactivity of sphingolipids

 

This project is aimed at obtaining a better understanding of the functional (bioactive) role of sphingolipids in (patho-) physiological processes at the cellular level, in particular in relation to cancer and tumor cell biology. Sphingolipids are a functionally important class of membrane lipids, which are expressed in various cellular membranes and prominently in the plasma membrane of cells, where they usually face the cellular environment. In accordance with this localization, they play a role in intercellular communication processes, e.g. as receptor (modulator). In addition, they have in recent years become known for their involvement in intracellular communication, e.g. as second messengers in signal transduction pathways.

The expression of specific sphingolipids plays a prominent role in cancer cell physiology. Metabolism, intracellular transport and turnover of sphingolipids at specific subcellular sites together determine the expression of sphingolipids at particular membrane sites. Here, they function locally in creating a membrane environment (e.g. rafts, DIG’s, caveolae, etc.) well suited to either support or directly regulate important cellular processes involving relay of information. The sphingolipid composition changes dramatically during oncogenesis and novel sphingolipids, not detected in normal cells, have been isolated from tumors. This may be a result of “aberrant glycosylation”, i.e. the activation of genes coding for glycosyltransferases, or defective degradation (loss of function of hydrolases). Sphingolipids have thus been identified as tumor specific antigens. A crucial metabolic step is the turn over of ceramide. This sphingolipid can be generated from sphingomyelin through activation of a sphingomyelinase, or by de novo biosynthesis. Ceramide plays a pivotal role in signal transduction pathways leading to apoptosis triggered by anticancer agents or death receptors. Cells can metabolize this sphingolipid to sphingomyelin or glycolipids. A major problem in the treatment of cancer is the occurrence of resistance to the drug that initially stopped tumor growth. Often, this involves the synthesis of multidrug resistance proteins, such as P-glycoprotein (Pgp or ABC B1) and multidrug resistance protein 1 (MRP1 or ABC C1), both of which confer resistance to a large variety of chemically unrelated drugs. Recently, multidrug resistant cells were shown to contain abnormally high amounts of glucosylceramide and other simple glycosphingolipids. Glucosylceramide synthase, the enzyme that converts ceramide to glucosylceramide, received considerable attention as a potential anti-apoptotic key contributing to multidrug resistance. In addition, metastasis, which is a major problem in cancer treatment, appears to require glycosphingolipids. Thus, metastatic potential of tumor cells is enhanced by supplementation with glycosphingolipids. Finally, tumor cells can secrete (‘shed’) complex glycosphingolipids (gangliosides), which block the ability of the immune system to attack the tumor cell and the growth and development of normal cells in the tumor-surrounding tissue. In conclusion, one can envision many ‘strategies’ that tumor cells employ to increase their survival potential based on adaptation of their sphingolipid metabolism.

Our research attempts to integrate the bioactivity of sphingolipids as regulators of tumor cell physiology with their cellular metabolism and subcellular expression. Currently, activities focus on “The involvement of sphingolipids in multidrug resistance”. We have characterized the alterations in sphingolipid metabolism and ABC transporter status in a number of multidrug resistant human tumor cell lines.

Human colon HT29 cells were subjected to colchicine selection and developed high levels of MRP1 expression, in concert with and kinetically similar to up regulation of glucosylceramide. Both MRP1 and glucosylceramide were found enriched in Lubrol insoluble membrane domains (DIGs or rafts). MRP1 was up regulated at the transcriptional level, but glucosylceramide synthase expression and activity had not changed. Instead, overall up regulation of sphingolipid metabolism had occurred at the level of ceramide synthesis.

Also in human SK-N neuroblastoma cell lines, a correlation was found between the expression of functionally active ABC transporters and a high rate of sphingolipid metabolism, either in the pathway of sphingomyelin or glycolipid biosynthesis. With the glucosylceramide synthase inhibitor PDMP, we have established a striking synergistic inhibition of murine Neuro-2a neuroblastoma cell survival when used in combination with microtubule-affecting cytotoxic drugs, such as taxol. Cell cycle disregulation is involved in this effect and results in both apoptosis and multinucleation. We are currently establishing whether and to which extent ceramide plays a role. In a murine melanoma cell line lacking glucosylceramide synthase no increased sensitivity towards a large panel of cytostatics was observed, moderating the importance of glucosylceramide synthase in multidrug resistance.

In human ovarian 2780AD tumor cells, which over express P-glycoprotein, extensive remodelling of sphingolipid metabolism had occurred in comparison to the drug-sensitive parental cell line. The most striking difference was a decrease in the conversion of glucosylceramide to lactosylceramide, which resulted in increased levels of glucosylceramide and decreased levels of lactosylceramide as well as down stream gangliosides. The underlying mechanism was not a change in glucosylceramide or lactosylceramide synthase expression or activity, but rather a change in Golgi architecture, giving rise to metabolic uncoupling of glucosylceramide synthase and lactosylceramide synthase. These studies were reported in international, peer reviewed journals.


In addition, the project comprises the following themes:

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  • The involvement of glucosylceramide in cell differentiation

  • Intracellular transport of ceramide, in relation to its metabolism

  • · ER/Golgi membrane dynamics in relation to sphingolipid metabolism

  • · The role of tumor cell-derived gangliosides in suppression of the immune and hemopoietic system

  • The role of sphingolipids in metastasis of tumor cells

 

  A central working model in our research is depicted in this diagram:

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Key words: Sphingolipid metabolism, Ceramide, Gangliosides, Multidrug resistance, Apoptosis, ABC transporters, Rafts, Golgi dynamics, Tumor cell biology

Last modified:November 19, 2012 09:43
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