Frank Gaunitz

Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model

BackgroundIt was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy.ResultsA mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth factor receptor 2 (HER2/neu), were implanted into the dorsal skin of nude mice, and tumor growth in treated animals was compared to control mice. In two independent experiments nude mice that received tumor cells received a daily intra peritoneal injection of 500 μl of 1 M carnosine solution. Measurable tumors were detected 12 days after injection. Aggressive tumor growth in control animals, that received a daily intra peritoneal injection of NaCl solution started at day 16 whereas aggressive growth in mice treated with carnosine was delayed, starting around day 19. A significant effect of carnosine on tumor growth was observed up to day 24. Although carnosine was not able to completely prevent tumor growth, a microscopic examination of tumors revealed that those from carnosine treated animals had a significant lower number of mitosis (p < 0.0003) than untreated animals, confirming that carnosine affects proliferation in vivo.ConclusionAs a naturally occurring substance with a high potential to inhibit growth of malignant cells in vivo, carnosine should be considered as a potential anti-cancer drug. Further experiments should be performed in order to understand how carnosine acts at the molecular level.

Carnosine inhibits ATP production in cells from malignant glioma.

Abstract Objectives: Recently, it was revealed that carnosine inhibits growth of cells isolated from human malignant glioma. In order to understand how this effect is mediated, experiments were performed that addressed a possible influence of carnosine on energy metabolism. Methods: Cells from the glioma line T98G and primary cultured cells from human malignant glioma were cultivated in the presence of carnosine and inhibitors of cellular energy metabolism. As a specific inhibitor for anaerobic glycolysis, oxamate, and as an inhibitor for mitochondrial oxidative phosphorylation, potassium cyanide, were used, and the influence on ATP production was determined using cell-based assays. Results: The experiments identified glycolysis as crucial for ATP production in gliomas. In addition, ATP production by mitochondrial activity did not significantly contribute to ATP production and carnosine was identified to be an inhibitor of the vital anaerobic glycolysis. Discussion: Carnosine might be considered as a potential drug for the treatment of malignant glioma or other tumors since it inhibits the glycolytic energy metabolism that is crucial for cancer cells and malignant gliomas as shown in the current study. This is especially interesting since the dipeptide is a naturally occurring substance that should be well tolerated.

α2-Macroglobulin Inhibits the Malignant Properties of Astrocytoma Cells by Impeding β-Catenin Signaling

Targets that could improve the treatment of brain tumors remain important to define. This study of a transformation-associated isoform of alpha2-macroglobulin (A2M*) and its interaction with the low-density lipoprotein receptor-related protein-1 (LRP1) suggests a new mechanism for abrogating the malignant potential of astrocytoma cells. LRP1 bound A2M* found to be associated with an inhibition of tumor cell proliferation, migration, invasion, spheroid formation, and anchorage-independent growth. Transcriptional studies implicated effects on the Wnt/beta-catenin signaling pathway. Notably, LRP1 antibodies could phenocopy the effects of A2M*. Our findings suggest a pathway of tumor suppression in astrocytoma that might be tractable to therapeutic exploitation.

Cloning and expression of the rat BACE1 promoter.

The pathogenic processing of the amyloid precursor protein (APP) into β‐amyloid peptides, which give rise to β‐amyloid plaques in the brains of Alzheimers disease patients, requires the enzymatic activity of the beta‐site APP‐cleaving enzyme 1 (BACE1). We report the cloning and sequence of a 1.5‐kb DNA fragment upstream of the coding sequence of the rat BACE1 gene and the construction of a BACE1 promoter/luciferase reporter construct. The basal activity of this promoter construct was highest in neuronal cell lines such as BE(2)‐C and PC12 and in the pancreatic cell line AR42J, somewhat lower in rat primary neurons, and astrocytic and microglial cultures, very low in hepatocytes, and almost absent in fibroblasts and in the monocyte‐macrophage cell line RAW264.7. The first 600 bp of this promoter are highly conserved among rat, mouse, and human, suggesting that this region contains regulatory elements that modulate BACE1 transcription. Indeed, this fragment contains several putative transcription factor binding sites such as MZF1, Sp1, four GATA‐1 sites, and one YY1 site. Directed mutagenesis of GATA‐1 elements led to altered luciferase expression, indicating that these sites are involved in the regulation of BACE1 transcription. Additionally, the analysis of promoter activities of deletion mutants suggests the presence of activators of BACE1 transcription between bases −514 to −753 and of suppressor elements between bases −754 and −1541. The BACE1 promoter sequence data and the constructs described here will be useful to identify factors that influence the expression of BACE1 in experimental paradigms in vitro.

Cis-Regulatory Sequences from the First Intron of the Rat Glutamine Synthetase Gene Are Involved in Hepatocyte Specific Expression of the Enzyme

In order to identify regulatory elements involved in the hepatocyte specific expression of the enzyme glutamine synthetase [GS (E.C. 6.3.1.2)] we analyzed the first intron of the rat GS gene. A sequence analysis detected clusters of potential transcription factor binding sites in regions that are hypersensitive for DNase I, including sites for Sp1, HNF3 and elements related to binding of members from the C/EBP family. By use of DNA fragments with putative regulatory elements, reporter genes have been constructed that were transfected into isolated hepatocytes in primary culture and into HepG2 hepatoblastoma cells. By these experiments we cold show that sequences from the first intron are able to enhance transcription specifically in hepatocytes but not in cells from the hepatoblastoma cell line. The existence of enhancer effects in the first intron of the GS gene and their restriction to hepatocytes demonstrates that aside from regulatory regions upstream of the transcription start point, there are also downstream regions involved in the specific expression of the gene. We conclude that intronic elements are involved in the pretranslational regulation of the expression of the GS as part of a complex interplay between different regions of the gene.

Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments

Background Glioblastoma multiforme is the most common lethal brain tumor in human adults, with no major therapeutic breakthroughs in recent decades. Research is based mostly on human tumor cell lines deprived of their organotypic environment or inserted into immune-deficient animals required for graft survival. Here, we describe how glioblastoma specimens obtained from surgical biopsy material can be sectioned and transferred into cultures within minutes. Methods Slices were kept in 6-well plates, allowing direct observation, application of temozolomide, and irradiation. At the end of experiments, slice cultures were processed for histological analysis including hematoxylin-eosin staining, detection of proliferation (Ki67), apoptosis/cell death (cleaved caspase 3, propidium iodide), DNA double-strand breaks (γH2AX), and neural subpopulations. First clinical trials employed irradiation with the heavy ion carbon for the treatment of glioblastoma patients, but the biological effects and most effective dose regimens remain to be established. Therefore, we developed an approach to expose glioblastoma slice cultures to 12C and X-rays. Results We found preservation of the individual histopathology over at least 16 days. Treatments resulted in activation of caspase 3, inhibition of proliferation, and cell loss. Irradiation induced γH2AX. In line with clinical observations, individual tumors differed significantly in their susceptibility to temozolomide (0.4%–2.5% apoptosis and 1%–15% cell loss). Conclusion Glioblastoma multiforme slice cultures provide a unique tool to explore susceptibility of individual tumors for specific therapies including heavy ions, thus potentially allowing more personalized treatments plus exploration of mechanisms of (and strategies to overcome) tumor resistance.

Comparative in vitro toxicity of seven zinc-salts towards neuronal PC12 cells

Currently much attention has been given to the neurotoxicity of zinc, yet little is known about the influence of the counterions present. Therefore, we investigated the influence of different Zn(2+)-salts (concentrations range 0.05-0.3 mM) on cell viability, ATP and glutathione concentration and caspase activation in differentiated PC12 cells as a model for neuronal cells. Generally, at concentrations of 0.05 mM most Zn(2+)-salts were not cytotoxic except for zinc-citrate. At concentrations between 0.1 and 0.3 mM Zn(2+) a significant decrease in GSH and ATP levels preceded cell death induced by all salts, except of zinc-histidinate. Zinc-citrate and zinc-sulphate turned out to be the most toxic salts particularly at low concentrations. Analyses of caspase 3/7 activity showed that dependent on the concentration and the type of the salt used cell death may show more or less signs of both, necrosis and apoptosis. Interestingly, the uptake of Zn(2+) from zinc-sulphate and zinc-citrate was significantly higher than that of other salts, implicating a correlation between uptake and toxicity. In conclusion, Zn(2+)-salts could be divided into three categories with high (zinc-citrate, zinc-sulphate), moderate (zinc-orotate, zinc-acetate, zinc-chloride(,) zinc-gluconate) and low cytotoxicity (zinc-histidinate).

Glucocorticoid induced expression of glutamine synthetase in hepatoma cells.

The enzyme glutamine synthetase (GS) ranks as one of the most remarkable glucocorticoid-inducible vertebrate genes. However, little is known about the responsible DNA elements and the mode of glucocorticoid action. This is especially the case for the induction of GS in hepatoma cells. In the work presented, the rat hepatoma cell line FAO was used as a model to study the induction of GS under the influence of glucocorticoids. FAO cells do not show GS activity in the absence of glucocorticoids and are strongly responding to their presence. Analyzing sequences of several thousand base pairs upstream and downstream from the transcriptional start point of the GS gene, a glucocorticoid responsible element was identified within the first intron of the gene. However, evidence is presented that aside from a primary effect on transcription glucocorticoids mediate their effect on the expression of GS also at the posttranscriptional level.

Identification of factors involved in the anti-tumor activity of carnosine on glioblastomas using a proteomics approach.

Human glioblastoma multiforme is the most malignant brain tumor in adults and is difficult to treat. Recently, it was demonstrated that the dipeptide carnosine inhibits tumor growth, but the main molecular targets are not known. Therefore, a proteomics study with glioblastoma cells treated with carnosine was performed. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight detected 31 proteins expressed differentially under the influence of carnosine. Finally, peptide mass fingerprinting identified the “BCL2-associated athanogene 2” and the “von Hippel-Lindau binding protein 1” among other proteins, linking the action of carnosine to protein folding and HIF-1α signalling.

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.