gms | German Medical Science

Objective: Carnosine, a dipeptide composed of beta-alanine and L-histidine has anti-neoplastic effects as demonstrated in different tumor models including glioblastoma cell culture. Although its molecular targets are unknown, it has been demonstrated that carnosine induces mRNA expression of pyruvate dehydrogenase kinase 4 (PDK4) in glioblastoma cells. Here we examined whether carnosine induces PDK4 expression via transcription factors interacting within the promoter of the PDK4 gene or whether regulation is mediated by 3’-non-coding regions or by histone acetylation.

Methods: Glioblastoma cells from the line U87 were transfected with reporter genes carrying 5’- and 3’-regions from the PDK4 gene and gene expression was determined after addition of carnosine. In addition, viability of glioblastoma cells exposed to carnosine (50 mM) was compared to viability in the presence of substances known to inhibit histone acetylation (HDAC inhibitors), including valproic acid (1, 2 and 4 mM), belinostat and vorinostat (both employed at 1, 2 and 4 µM) and Trichostatin A (0.8 and 1.6 µM) using cell-based assays and CalceinAM/propidium iodide staining. PDK4-transcription was analyzed by qRT-PCR. Histone acetylation of the PDK4 promoter was analyzed by chromatin immunoprecipitation (ChIP).

Results: After incubation in the presence of HDAC inhibitors all substances employed reduced viability of U87 cells in a time and dose dependent manner. At the highest concentrations employed and an incubation time of 48 hours dead cells were detected in the presence of all HDAC inhibitors but not in the presence of carnosine. Reporter gene assays did not reveal a contribution of 5’ and 3’ sequences of the PDK4 gene to the effect of carnosine on PDK4 expression, whereas ChiP analysis demonstrated enhanced promoter acetylation of the endogenous PDK4 gene 6 hours after addition of the dipeptide, which coincided with the onset of PDK4 expression. 12, 18 and 24 hours after addition, the level of acetylation dropped again to values even lower than to the level observed before the addition of carnosine. Reduced acetylation was followed by a continuous decline of the amount of mRNA as detected by qRT-PCR though it remained significantly higher than before the addition of carnosine.

Conclusion: Carnosine’s anti-neoplastic effect on U87 cells is accompanied by enhanced acetylation of the PDK4 gene which indicates that it shares similarities with HDAC inhibitors.