Evaluation der Schlüsselkomponenten des Glycin-Serin- und Sphingolipid-Stoffwechsels in Hinblick auf die Identifizierung neuer metabolischer Tumormarker für Nephroblastome der Hochrisikogruppe
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Despite the generally good prognosis achieved through standardized multimodal treatment strategies, children with high-risk nephroblastoma still have a comparatively poor prognosis. One possible reason for this could be the lack of valid prognostic and metabolic tumor markers. Consequently, this dissertation has investigated the significance of selected key components of glycine-serine and sphingolipid metabolism, such as glycine cleavage system protein H (GCSH) and sphingosine-1-phosphate lyase 1 (SGPL1), in high-risk nephroblastoma cells and examined whether the metabolites of these metabolic pathways could be suitable as prognostic and/or metabolic tumor markers. In addition, a brief digression tested possible NCAM expression in high-risk nephroblastoma cells.
SGPL1 and GCSH expression was analyzed using immunofluorescence, RT-PCR, and Western blot to determine whether there was a difference between the highly malignant nephroblastoma cells and healthy kidney cells. No relevant difference in GCSH expression was found in vitro, either in immunofluorescence microscopy or at the transcript or protein level. In contrast, the SGPL1 expression status differed in immunofluorescence microscopy in both 2D and 3D cell culture systems based on its localization. While SGPL1 was located more in the cell periphery and less near the nucleus in healthy kidney cells, SGPL1 was found exclusively in the immediate vicinity of the nucleus in nephroblastoma cells of the high-risk group. In addition, at the protein level, immuno-blotting revealed significantly lower SGPL1 protein expression in the two tumor cell lines compared to the control cell line.
Since glycine-serine metabolism does not appear to play a primary role in the energy production of nephroblastoma, based on the lack of difference in GCSH expression status in high risk nephroblastoma cells compared to healthy kidney cells, only metabolites of sphingolipid metabolism were considered in the investigations of metastasis and proliferation behavior. A scratch assay was performed to evaluate proliferation behavior with the addition of sphingosine-1-phosphate (S1P) and dihydrosphingosine (DHS). Wound healing occurred significantly faster in the nephroblastoma cells of the high-risk group with the addition of both S1P and DHS. Extracellular enrichment with S1P and DHS caused an increase in proliferation and in cell mortality. Thus, these two metabolites of sphingolipid metabolism appear to be suitable prognostic markers for proliferation in vitro.
In addition, the influence of sphingosine-1-phosphate on the effectiveness of chemotherapy was investigated. S1P led to a lower response of high-risk nephroblastoma cells to chemotherapy treatment with actinomycin D and vincristine, thereby causing a type of drug resistance and promoting the proliferation of tumor cells. Due to the drug resistance triggered by S1P, this metabolite of sphingolipid metabolism could be suitable as a marker for the metastasis of high-risk nephroblastoma cells.
Furthermore, NCAM was detected in the nephroblastoma cells of the high-risk group in vitro using immunofluorescence microscopy and immuno-blotting.
While the key components of the glycine-serine metabolic pathway did not differ in their expression status compared to healthy kidney cells and therefore do not appear to be suitable as prognostic and/or metabolic tumor markers, the sphingolipid metabolism showed significantly reduced expression of the key component SGPL1 and, due to the metabolite S1P, increased proliferation behavior and drug resistance. It therefore seems worthwhile to investigate other enzymes and metabolites of sphingolipid metabolism. Sphingolipid metabolism should be given greater consideration in relation to nephroblastomas in the future.
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