As outlined in the introduction, we and others have shown that the naturally occurring dipeptide carnosine inhibits the growth of cancer cells in vitro and in vivo [
14], whereas beneficial effects have been observed in cultured human fibroblasts [
19]. Using a colorimetric assay (tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS]) and human dermal fibroblasts, Ansurudeen et al. also demonstrated a greater number of viable cells in the presence of 50 mM carnosine after 24 h incubation [
20]. Unfortunately, it is not traceable whether the fibroblasts used by Ansurudeen et al. were from juvenile foreskin or from adult skin. In addition, the experiments performed by Holliday and McFarland were done by using a fibroblast cell line established from human foreskin of a newborn male (HFF-1) and a second cell line established from lung tissue of a male at 14 weeks gestation (MRC-5). Considering using carnosine for the treatment of elderly patients the question had to be answered whether fibroblasts isolated from adults or even senescent patients may behave different to fibroblasts isolated from fetal or newborn human tissue. In our experiments presented in Fig.
1 we did not see a measurable beneficial effect of carnosine on fibroblast viability, although it is interesting to note, that fibroblasts cultivated in 50 mM carnosine appeared to be rejuvenated compared to fibroblasts cultivated in the absence of carnosine in accordance to the observations of McFarland and Holiday [
21]. We also did not see any correlation between age of the patient, the tissue of origin or gender, though it has to be realized that the number of samples may be too low for such an analysis and most of our patients have been of comparable age. More importantly, a prolonged cultivation of fibroblasts as in the co-culture experiments demonstrates that the fibroblasts are alive and able to occupy the space left by dying glioblastoma cells even under the highest concentration of carnosine employed (50 mM). It is also very interesting to note that we observed complete cell death of T98G tumor cells in long term culture incubating the cells at a concentration of 75 mM carnosine (Fig.
2). In previous experiments, cells were usually kept in the presence of carnosine for 24, 48 or 96 h [
13] but not for 2 weeks. This is interesting, as shorter exposure times in previous experiments resulted in reduced proliferation but not complete elimination of tumor cells. Which processes are responsible for reduced tumor cell proliferation are under the influence of carnosine are still unknown [
14]. However, it is an interesting question whether the processes which reduce proliferation after short term exposure may finally lead to cell death when the tumor cells are exposed to carnosine for a longer period of time.
In order to discriminate tumor cells from fibroblasts in ring co-culture experiments several markers were tested including glial fibrillary acidic protein (GFAP) which did also stained fibroblasts in accordance with observations made by others [
22]. We finally identified that nestin-staining was suitable to discriminate primary cultured tumor cells from patient-derived fibroblasts. Nestin is a class VI intermediate filament protein and a marker for neural stem cells. In addition, it has been reported as a cancer stem cell-specific marker [
23] and a recent meta-analysis performed by Lv et al. [
24] demonstrated that increased expression of nestin is positively associated with higher histological grade in glioma patients. This analysis also indicated that patients with higher nestin expression are prone to recurrence and glioma cell infiltration into intact brain tissue. Surprisingly, in our ring co-culture experiments T98G cells, which did not express nestin, as previously reported by other investigators [
25,
26], gave rise to many colonies in the surrounding fibroblast layer which was not the case when primary cultures with a high expression of nestin were cultivated with fibroblasts. Although speculative, one interpretation could be that the primary cultures we used were of very early passages (Passage 1 and 5) and not as rapidly growing as T98G cells which is also reflected by their higher resistance towards carnosine (Fig.
1) as carnosine exerts its action mainly on metabolic highly active tumor cells [
27]. Nonetheless, the results presented in Fig.
6 clearly demonstrate that colony formation is significantly inhibited when invasively grown glioblastoma cells are treated with carnosine. In the last years, a so-called “go or grow concept” has been discussed, assuming that proliferation and migration are mutually exclusive phenomena in cancer cells [
4]. Studies confirming this hypothesis are for example observations made in breast cancer cell lines in which overexpression of Homeobox Protein C9 (HOXC9) resulted in increased invasiveness but at the same time inhibited proliferation [
28]. Another example is the observation that enforced expression of Y-box binding protein-1 (YB-1) in non-invasive breast epithelial cells induces an epithelial–mesenchymal transition (EMT) resulting in an enhanced metastatic potential but at the same time reduces proliferation [
29]. Unfortunately, the exact mechanisms and down-stream targets responsible for either proliferation or invasion mediated by HOXC9 or YB-1 signaling are still unknown. More importantly, our results demonstrate that carnosine does not inversely influence proliferation and invasion. Up to now, the mechanisms responsible for the dipeptides anti-proliferative effect, which has been confirmed in several studies with different types of cancer cells [
11‐
13,
30] are still not understood. With regard to carnosine’s effect on migration and invasion it has been discussed that it involves regulation of matrix metalloproteinases (MMPs) [
15], but more experiments are certainly needed to properly address this question.