Carnosine inhibits glioblastoma growth independent from PI3K/Akt/mTOR signaling.

Henry Oppermann,Jürgen Meixensberger,Ulrike Yamanishi,Helene Faust,Frank Gaunitz,Salvatore V Pizzo

doi:10.1371/journal.pone.0218972

Henry Oppermann, Jürgen Meixensberger + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0218972

Copy DOI

Abstract

Glioblastoma is a high-grade glioma with poor prognosis even after surgery and standard therapy. Here, we asked whether carnosine (β-alanyl-L-histidine), a naturally occurring dipeptide, exert its anti-neoplastic effect on glioblastoma cells via PI3K/Akt/mTOR signaling. Therefore, glioblastoma cells from the lines U87 and T98G were exposed to carnosine, to the mTOR inhibitor rapamycin and to the PI3K inhibitor Ly-294,002. Pyruvate dehydrogenase kinase (PDK4) expression, known to be a target of PI3K/Akt/mTOR, and which is also affected by carnosine, was analyzed by RT-qPCR, and reporter gene assays with the human PDK4 promoter were performed. Cell viability was assessed by cell-based assays and mTOR and Akt phosphorylation by Western blotting. Rapamycin and Ly-294,002 increased PDK4 mRNA expression in both cell lines but significance was only reached in U87. Carnosine significantly increased expression in both lines. A significant combinatorial effect of carnosine was only detected in U87 when the dipeptide was combined with Ly-294,002. Reporter gene assays revealed no specific effect of carnosine on the human PDK4 promoter, whereas both inhibitors increased reporter gene expression. Rapamycin reduced phosphorylation of mTOR, and Ly-294,002 that of Akt. A significant reduction of Akt phosphorylation was observed in the presence of carnosine in U87 but not in T98G, and carnosine had no effect on mTOR phosphorylation. Cell viability as determined by ATP in cell lysates was reduced only in the presence of carnosine. We conclude that carnosine’s anti-neoplastic effect is independent from PI3K/Akt/mTOR signaling. As the dipeptide reduced viability in tumor cells that do not respond to PI3K or mTOR inhibitors, it appears to be worth to further investigate the mechanisms by which carnosine exerts its anti-tumor effect and to consider it for therapy, especially as it is a naturally occurring compound that has already been used for the treatment of other diseases without indication of side-effects.

Highlights

The most common and aggressive primary tumor of the brain is designated Glioblastoma (GBM)
In a first series of experiments, we asked whether the effect of carnosine on the expression of pyruvate dehydrogenase kinase 4 (PDK4) in U87 and T98G glioblastoma cells, that was described by Letzien et al [24], can be detected using inhibitors of PI3K/Akt/mTOR signaling
Cells from the two lines were exposed for 24 hours to carnosine (50 mM), the PI3K inhibitor Ly-294,002 (5 μM), the mTORC1 inhibitor rapamycin (25 nM), and to combinations of the compounds

Summary

Introduction

The most common and aggressive primary tumor of the brain is designated Glioblastoma (GBM). There have been hints that the Akt/mTOR/ Ribosomal protein S6 kinase beta-1 pathway is involved in carnosine’s anti-neoplastic effect [22]. At this point, it is noteworthy, that other observations point towards the possibility that carnosine may be a mimic of rapamycin, which is an inhibitor of mTORC1 (mTOR complex 1) [23]. As PDK4 expression is regulated by transcription factors, such as FOXO1a and FOXO3a (Forkhead box protein O1a and O3a) [25], which are downstream effectors of PI3K/AKT/mTOR signaling, we hypothesized that carnosine’s anti-neoplastic effect may be mediated by an influence on this pathway. We compared the effect of carnosine on glioblastoma cell viability, PDK4 expression and signaling molecule phosphorylation with the PI3K inhibitor Ly294,002 and the mTOR inhibitor rapamycin in two cell lines derived from glioblastoma

Materials and methods

Results

Discussion