gms | German Medical Science

Objective: Previous studies demonstrated an anti-neoplastic effect of carnosine (β-alanyl-L-histidine) on tumor cell growth in malignant gliomas. In different models influences on signal transduction were demonstrated but available data are fragmentary or even conflicting and not much is known about carnosine`s influence on signaling in glioblastoma cells. Therefore, we performed a phosphoproteomic and transcriptomic analysis with U87 glioblastoma cells in order to obtain a precise and coherent picture of carnosine's influence on signal transduction.

Methods: U87 cells were treated with 50 mM carnosine for 24 hours and protein and RNA were isolated and subjected to phosphoprotein specific antibody array analysis (with 1318 antibodies) and to whole transcriptome sequencing. Data was analyzed using Ingenuity Pathway Analysis. Finally, reporter gene assays were performed in order to validate the results obtained by data analysis.

Results: Expression of 728 genes was significantly up-regulated and that of 1270 genes significantly reduced under the influence of 50 mM carnosine. The array detected 25 sites with reduced phosphorylation and 17 sites with an increased one. Using the array data for pathway analysis potential effects of carnosine on PI3K/AKT, PTEN/growth hormone and MAPK signaling and on cell cycle control were detected. Combining data obtained by sequencing with array data revealed that carnosine should inhibit migration of tumor cells and should affect cell cycle progression and the synthesis of DNA. Further analysis identified the upstream regulators ERBB2, RABL6, MEK2, CDKN1B as potentially responsible for carnosine’s influence on PI3K/AKT and MAPK signaling and E2F6, FOXM1 and BRD4 as potentially responsible for cell cycle inhibition. Using reporter genes for transcription factors down-streaming of PI3K/AKT and MAPK signaling we observed a significant reduction of HIF, SRF and myc/max reporter gene activity in U87 cells in the presence of carnosine (66%±10.2, 30%±3.1 and 27%±13.9, respectively).

Conclusion: The experiments and the data analysis presented demonstrate that carnosine inhibits the transcriptional activity of SRF via PI3K/AKT and MAPK signaling in glioblastoma cells. This and additional insights, such as activation of E2F6 and CDKN1B, will promote the development of new therapeutic strategies for tumor growth inhibition. Furthermore, our approach can be used to identify the molecular mechanisms and pathways which are involved when new drugs are designed or have to be tested.