Targeting glucose transport and the NAD pathway in tumor cells with STF-31: a re-evaluation.

BACKGROUND: Targeting glucose metabolism is a promising way to interfere with tumor cell proliferation and survival. However, controversy exists about the specificity of some glucose metabolism targeting anticancer drugs. Especially the potency of STF-31 has been debated. Here, we aimed to assess the impact of the glucose transporter (GLUT) inhibitors fasentin and WZB117, and the nicotinamide phosphoribosyltransferase (NAMPT) inhibitors GMX1778 and STF-31 on tumor cell proliferation and survival, as well as on glucose uptake. METHODS: Tumor-derived A172 (glioblastoma), BHY (oral squamous cell carcinoma), HeLa (cervix adenocarcinoma), HN (head neck cancer), HT-29 (colon carcinoma) and MG-63 (osteosarcoma) cells were treated with fasentin, WZB117, GMX1778 and STF-31. Proliferation rates and cell viabilities were assessed using XTT, crystal violet and LDH assays. mRNA and protein expression of GLUT1 and NAPRT were assessed using qPCR and Western blotting, respectively. The effects of inhibiting compounds on glucose uptake were measured using [18F]-fluoro-deoxyglucose uptake experiments. RESULTS: Stimulation of tumor-derived cells with the different inhibitors tested revealed a complex pattern, whereby proliferation inhibiting and survival reducing concentrations varied in [18F]-fluoro-deoxyglucose uptake experiments more than one order of magnitude among the different cells tested. We found that the effects of GMX1778 and STF-31 could be partially abolished by (i) nicotinic acid (NA) only in nicotinic acid phosphoribosyltransferase (NAPRT) expressing cells and (ii) nicotinamide mononucleotide (NMN) in all cells tested, supporting the classification of these compounds as NAMPT inhibitors. In short-time [18F]-fluoro-deoxyglucose uptake experiments the application of WZB-117 was found to lead to an almost complete uptake inhibition in all cells tested, whereas the effect of fasentin was found to be cell type dependent with a maximum value of ~35% in A172, BHY, HeLa and HT-29 cells. We also found that STF-31 inhibited glucose uptake in all cells tested in a range of 25-50%. These data support the classification of STF-31 as a GLUT inhibitor. CONCLUSIONS: Our data reveal a dual mode of action of STF-31, serving either as a NAMPT or as a GLUT inhibitor, whereby the latter seems to be apparent only at higher STF-31 concentrations. The molecular basis of such a dual function and its appearance in compounds previously designated as NAMPT-specific inhibitors requires further investigation.

Insulin-like growth factor 1 (IGF1) affects proliferation and differentiation and wound healing processes in an inflammatory environment with p38 controlling early osteoblast differentiation in periodontal ligament cells.

OBJECTIVE: The objective of this study was to investigate effects of insulin-like growth factor 1 (IGF1) on proliferation, wound healing and differentiation processes of human periodontal ligament (PDL) cells under inflammatory conditions and whether the protective, anabolic effects of IGF1 can attenuate unfavorable effects of interleukin-1ß (IL-1ß). DESIGN: Inflammation was mimicked through cell stimulation with IL-1ß. PDL cells were characterized in respect to the presence of components of the IGF system and the responsive potential on IL-1ß incubation. Gene expression levels were analyzed by quantitative real-time PCR. Cellular localization of target proteins was visualized using fluorescent-based immunohistochemistry. Effects on cell division were investigated by proliferation assays. Wound healing was analyzed using light microscopic techniques. Differentiation was quantified by measuring biomineralization and osteoblast-specific alkaline phosphatase enzyme activity. RESULTS: PDL cell proliferation and wound healing were positively affected by IGF1 and the combination of IGF1 with IL-1ß, while only IL-1ß showed negative effects. Biomineralization was enhanced by IGF1, IL-1ß, and the combination of both stimulants. Osteoblast differentiation was increased by IL-1ß and the combination of IL-1ß with IGF1, whereas only IGF1 negatively affected ALP activity. Phosphorylation of p38 was regulated by IL-1ß and IGF1. CONCLUSIONS: The data presented in this work showed a potential of IGF1 to improve wound healing and proliferation processes and to sustain cell differentiation under inflammatory stimuli in PDL cells.

Cellular Distribution and Gene Expression Pattern of Metastasin (S100A4), Calgranulin A (S100A8), and Calgranulin B (S100A9) in Oral Lesions as Markers for Molecular Pathology.

The objective of this study was to analyze cellular localization and expression levels of oncologic relevant members of the S100 family in common oral lesions.Biopsies of various oral lesions were analyzed. S100A4 showed a higher expression rate in leukoplakias and oral squamous cell carcinomas. Transcript levels of S100A8 and S100A9 were significantly decreased in malignant OSCCs. A correlation could be drawn between the expression levels of these genes and the pathological characteristics of the investigated lesions. S100A4, A8, and A9 proteins represent promising marker genes to evaluate the risk potential of suspicious oral lesions in molecular pathology.

Expression Pattern of Matrix Metalloproteinase 20 (MMP20) in Human Tumors.

BACKGROUND/AIM: Matrix metalloproteinase 20 (MMP20) is a member of the family of matrix metalloproteinases. Under normal conditions the expression of MMP20 is restricted to ameloblasts and odontoblasts. In order to identify a possible expression of MMP20 under pathological conditions, we investigated three major human tumor entities, i.e. colon, breast and lung tumors, on the mRNA and protein level. MATERIALS AND METHODS: Real-time RT-PCR and immunocytochemical analyses of established human tumor cell lines were employed for our study; immunohistochemical analysis was performed on both primary tumors and normal control tissues. RESULTS: MMP20 was identified on both the mRNA and the protein level in breast MCF-7, colon HT-29, and lung A549 cell lines. MMP20 was also detected in primary tumor tissue by immunohistochemistry. CONCLUSION: MMP20 is a new potential candidate for tumor diagnosis or therapy.

Ghrelin promotes oral tumor cell proliferation by modifying GLUT1 expression.

In our study, ghrelin was investigated with respect to its capacity on proliferative effects and molecular correlations on oral tumor cells. The presence of all molecular components of the ghrelin system, i.e., ghrelin and its receptors, was analyzed and could be detected using real-time PCR and immunohistochemistry. To examine cellular effects caused by ghrelin and to clarify downstream-regulatory mechanisms, two different oral tumor cell lines (BHY and HN) were used in cell culture experiments. Stimulation of either cell line with ghrelin led to a significantly increased proliferation. Signal transduction occurred through phosphorylation of GSK-3ß and nuclear translocation of ß-catenin. This effect could be inhibited by blocking protein kinase A. Glucose transporter1 (GLUT1), as an important factor for delivering sufficient amounts of glucose to tumor cells having high requirements for this carbohydrate (Warburg effect) was up-regulated by exogenous and endogenous ghrelin. Silencing intracellular ghrelin concentrations using siRNA led to a significant decreased expression of GLUT1 and proliferation. In conclusion, our study describes the role for the appetite-stimulating peptide hormone ghrelin in oral cancer proliferation under the particular aspect of glucose uptake: (1) tumor cells are a source of ghrelin. (2) Ghrelin affects tumor cell proliferation through autocrine and/or paracrine activity. (3) Ghrelin modulates GLUT1 expression and thus indirectly enhances tumor cell proliferation. These findings are of major relevance, because glucose uptake is assumed to be a promising target for cancer treatment.

Oncogenic relevant defensins: expression pattern and proliferation characteristics of human tumor cell lines.

The objective of this study was to investigate gene expression levels of oncogenic relevant human defensins and their impact on proliferation rates of 29 cell lines derived from main types of different tumor origins. Differential gene expression analysis of human defensins was performed by real-time PCR experiments. The proliferation rate of tumor cells that had been cultivated in the absence or presence of biologically active peptides was analyzed with a lactate dehydrogenase assay kit. At least one member of the defensin family was expressed in each tumor cell line, whereby α-defensin (DEFA1), DEFA2, or DEFA3 transcripts could be ubiquitously detected. Cell lines of neural origin (glioma, neuroblastoma, and small-cell lung carcinoma) expressed far less human ß-defensins (hBDs) in comparison to other tumor types. The expression level of a specific defensin in various cell lines could vary by more than five orders of magnitude. Compensatory mechanisms on the expression levels of the different defensins could not be strictly observed. Only in 3 out of 29 tumor cell lines the proliferation rate was affected after defensin stimulation. The variable appearance of defensins, as well as the cell line-restricted functional activity, argues for the integration of defensins in complex cellular and molecular networks that tolerate rather flexible expression patterns.

Human α-defensin (DEFA) gene expression helps to characterise benign and malignant salivary gland tumours.

BACKGROUND: Because of the infrequence of salivary gland tumours and their complex histopathological diagnosis it is still difficult to exactly predict their clinical course by means of recurrence, malignant progression and metastasis. In order to define new proliferation associated genes, purpose of this study was to investigate the expression of human α-defensins (DEFA) 1/3 and 4 in different tumour entities of the salivary glands with respect to malignancy. METHODS: Tissue of salivary glands (n=10), pleomorphic adenomas (n=10), cystadenolymphomas (n=10), adenocarcinomas (n=10), adenoidcystic carcinomas (n=10), and mucoepidermoid carcinomas (n=10) was obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of DEFA 1/3 and 4 were analyzed by quantitative realtime PCR and compared with healthy salivary gland tissue. Additionally, the proteins encoded by DEFA 1/3 and DEFA 4 were visualized in paraffin-embedded tissue sections by immunohistochemical staining. RESULTS: Human α-defensins are traceable in healthy as well as in pathological altered salivary gland tissue. In comparison with healthy tissue, the gene expression of DEFA 1/3 and 4 was significantly (p<0.05) increased in all tumours - except for a significant decrease of DEFA 4 gene expression in pleomorphic adenomas and a similar transcript level for DEFA 1/3 compared to healthy salivary glands. CONCLUSIONS: A decreased gene expression of DEFA 1/3 and 4 might protect pleomorphic adenomas from malignant transformation into adenocarcinomas. A similar expression pattern of DEFA-1/3 and -4 in cystadenolymphomas and inflamed salivary glands underlines a potential importance of immunological reactions during the formation of Warthin's tumour.