Viability of Glioblastoma Cells and Fibroblasts in the Presence of Imidazole-Containing Compounds.

The naturally occurring dipeptide carnosine (ß-alanyl-L-histidine) specifically attenuates tumor growth. Here, we ask whether other small imidazole-containing compounds also affect the viability of tumor cells without affecting non-malignant cells and whether the formation of histamine is involved. Patient-derived fibroblasts and glioblastoma cells were treated with carnosine, L-alanyl-L-histidine (LA-LH), ß-alanyl-L-alanine, L-histidine, histamine, imidazole, ß-alanine, and L-alanine. Cell viability was assessed by cell-based assays and microscopy. The intracellular release of L-histidine and formation of histamine was investigated by high-performance liquid chromatography coupled to mass spectrometry. Carnosine and LA-LH inhibited tumor cell growth with minor effects on fibroblasts, and L-histidine, histamine, and imidazole affected viability in both cell types. Compounds without the imidazole moiety did not diminish viability. In the presence of LA-LH but not in the presence of carnosine, a significant rise in intracellular amounts of histidine was detected in all cells. The formation of histamine was not detectable in the presence of carnosine, LA-LH, or histidine. In conclusion, the imidazole moiety of carnosine contributes to its anti-neoplastic effect, which is also seen in the presence of histidine and LA-LH. Despite the fact that histamine has a strong effect on cell viability, the formation of histamine is not responsible for the effects on the cell viability of carnosine, LA-LH, and histidine.

D,L-Methadone does not improve radio- and chemotherapy in glioblastoma in vitro.

PURPOSE: Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. Median survival of glioblastoma patients under standard therapy including radiotherapy and chemotherapy using temozolomide (TMZ) is 14.6 months. As cell culture experiments combining D,L-methadone with doxorubicin demonstrated an increased reduction of cell viability of glioblastoma cells, the opioid has been discussed as a drug for the treatment of GBM. Despite lack of clinical and experimental evidence that D,L-methadone in combination with standard therapy will be beneficial, an increasing number of tumor patients medicating themselves with D,L-methadone present to the hospitals in Germany. METHODS: As a first step towards understanding whether D,L-methadone may increase the efficacy of standard therapy, we used a cell culture model of primary GBM and fibroblast cell cultures derived from GBM patients. The cultures were treated with different concentrations of D,L-methadone in combination with X-irradiation, TMZ or both. Cell viability was determined by measuring ATP in cell lysates and dehydrogenase activity in living cells. RESULTS: When only treated with D,L-methadone, 1 µM of the opioid was sufficient to reduce viability of fibroblasts, whereas 10 µM was needed to significantly reduce glioblastoma cell viability. In addition, D,L-methadone did not improve the anti-neoplastic effects of X-irradiation, temozolomide or both. CONCLUSIONS: As D,L-methadone reduces glioblastoma cell viability only when concentrations are used that had been reported to be toxic to patients and as there were no interactions observable combining it with standard therapy, a recommendation for the use of D,L-methadone in glioblastoma therapy cannot be given.

Carnosine's inhibitory effect on glioblastoma cell growth is independent of its cleavage.

The naturally occurring dipeptide carnosine (ß-alanyl-L-histidine) inhibits the growth of tumor cells. As its component L-histidine mimics the effect, we investigated whether cleavage of carnosine is required for its antineoplastic effect. Using ten glioblastoma cell lines and cell cultures derived from 21 patients suffering from this malignant brain tumor, we determined cell viability under the influence of carnosine and L-histidine. Moreover, we determined expression of carnosinases, the intracellular release of L-histidine from carnosine, and whether inhibition of carnosine cleavage attenuates carnosine's antineoplastic effect. We observed a significantly higher response of the cells to L-histidine than to carnosine with regard to cell viability in all cultures. In addition, we detected protein and mRNA expression of carnosinases and a low but significant release of L-histidine in cells incubated in the presence of 50 mM carnosine (p < 0.05), which did not correlate with carnosine's effect on viability. Furthermore, the carnosinase 2 inhibitor bestatin did not attenuate carnosine's effect on viability. Interestingly, we measured a ~ 40-fold higher intracellular abundance of L-histidine in the presence of 25 mM extracellular L-histidine compared to the amount of L-histidine in the presence of 50 mM carnosine, both resulting in a comparable decrease in viability. In addition, we also examined the expression of pyruvate dehydrogenase kinase 4 mRNA, which was comparably influenced by L-histidine and carnosine, but did not correlate with effects on viability. In conclusion, we demonstrate that the antineoplastic effect of carnosine is independent of its cleavage.

Hedgehog signaling in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant brain tumor in adults with a median survival of 14.6 mo under the best available treatment. New treatment strategies are therefore urgently required, for which a profound understanding of tumor biology is necessary. Much effort has been devoted to tumor-specific aberrant signaling processes. Recently it was discovered that the transcription factor Gli1, which is activated by hedgehog signaling, is a highly predictive marker in GBM, as determined by immunohistochemistry. To determine whether GBM cells have transcriptionally active Gli1, we performed experiments with reporter genes with cells isolated from surgically removed human tumors and cell lines. We also determined whether the hedgehog signaling inhibitor cyclopamine influences reporter gene expression and cell viability, and we determined the expression of Gli1, SHH and Patched1 by quantitative real-time RT-PCR. Reporter gene analysis of nine cultures and four cell lines demonstrated a significantly enhanced transcriptional activity in six tumor cell cultures and all cell lines. Analysis of cell viability in the presence of cyclopamine revealed a response of all cell cultures with the exception of one primary culture and one cell line, but only one cell line responded to cyclopamine with reduced hedgehog signaling activity. This indicates that the toxicity of cyclopamine toward GBM cells is independent from hedgehog signaling. Since no correlation between hedgehog activity and SHH, Gli1 and Patched1 mRNA levels was observed we conclude that other mechanisms aside from transcriptional regulation of these factors are responsible for hedgehog activity in tumor cells derived from GBM.