Background Alterations in cellular metabolism are considered as hallmarks of cancers, however, to recognize these alterations and understand their mechanisms appropriate techniques are required. Our hypothesis was to determine whether dominant bioenergetic mechanism may be estimated by comparing the substrate utilisation with different methods to detect the labelled carbon incorporation and their application in tumour cells. Methods To define the bioenergetic pathways different metabolic tests were applied: (a) measuring CO 2 production from [1- 14 C]-glucose and [1- 14 C]-acetate; (b) studying the effect of glucose and acetate on adenylate energy charge; (c) analysing glycolytic and TCA cycle metabolites and the number of incorporated 13 C atoms after [U- 13 C]-glucose/[2- 13 C]-acetate labelling. Based on [1- 14 C]-substrate oxidation two selected cell lines out of seven were analysed in details, in which the highest difference was detected at their substrate utilization. To elucidate the relevance of metabolic characterisation the expression of certain regulatory factors, bioenergetic enzymes, mammalian target of rapamycin (mTOR) complexes (C1/C2) and related targets as important elements at the crossroad of cellular signalling network were also investigated. Results Both [U- 13 C]-glucose and [1- 14 C]-substrate labelling indicated high glycolytic capacity of tumour cells. However, the ratio of certain 13 C-labelled metabolites showed detailed metabolic differences in the two selected cell lines in further characterisation. The detected differences of GAPDH, β-F1-ATP-ase expression and adenylate energy charge in HT-1080 and ZR-75.1 tumour cells also confirmed the altered metabolism. Moreover, the highly limited labelling of citrate by [2- 13 C]-acetate—representing a novel functional test in malignant cells—confirmed the defect of TCA cycle of HT-1080 in contrast to ZR-75.1 cells. Noteworthy, the impaired TCA cycle in HT-1080 cells were associated with high mTORC1 activity, negligible protein level and activity of mTORC2, high expression of interleukin-1β, interleukin-6 and heme oxygenase-1 which may contribute to the compensatory mechanism of TCA deficiency. Conclusions The applied methods of energy substrate utilisation and other measurements represent simple assay system using 13 C-acetate and glucose to recognize dominant bioenergetic pathways in tumour cells. These may offer a possibility to characterise metabolic subtypes of human tumours and provide guidelines to find biomarkers for prediction and development of new metabolism related targets in personalized therapy.

19h and 28h.All cell cultures were negative for CD90 and a-SMA, positive for cytokeratin, whereas vimentin expression varied among cultures.In vivo tumorigenicity was evaluated for 5 primary cell cultures so far using the s.c.xenograft model.Tumor taking rates were 100% for 3 cell cultures, and 80% and 60% for 2 cell cultures.Tumor volumes reached ~1000 mm 3 after 40 days (4 cell cultures) and 90 days (1 cell culture).Mutational profiling identified PTEN, PIK3CA, KRAS, NRAS, BRAF and p53 mutations in the cell cultures.Interestingly, mutation profiles remained identical in the primary tumor, the established cell culture, and xenograft tumor. Conclusion:We here present the first evidence for the establishment of primary EC cell cultures.Moreover, mutation profiles remained identical in established cell cultures, and xenograft tumors, as compared to the primary patient tumor.Hence, we provide an alternative, primary tumor-based model for in vitro and in vivo (targeted) therapeutic drug screening in EC.

Cancer progression is mediated by overexpression of oncogenes and downregulation or loss of tumor suppressors. Proteins, which were traditionally categorized into these groups, have been recently joined by a species of RNA molecules known as microRNAs (miRNAs). miRNAs belong to a class of approximately 22-nt-long non-coding RNAs found in eukaryotes that hinder gene expression by inducing degradation or inhibiting translation of select mRNAs. A growing number of miRNAs have been implicated in promoting or suppressing tumorigenesis in a variety of tissues. The supporting evidence ranges from suggestive expression profiling data to direct functional validation using methods of forward and reverse genetics. We discuss the nature of published results, as well as the merits and pitfalls of various approaches aimed at identification of cancer-related miRNAs and their mRNA targets.

The metabolism of CCl4 initiates the peroxidation of polyunsaturated fatty acids producing α,β-unsaturated aldehydes, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The facile reactivity of these electrophilic aldehydic products suggests they play a role in the toxicity of compounds like CCl4. To determine the rate at which CCl4-initiated lipid peroxidation results in the formation of 4-HNE and/or MDA hepatic protein adducts, rats were given an intragastric dose of CCl4 (1.0 ml/kg) and euthanized 0–72 h after administration. Rabbit polyclonal antisera directed toward 4-HNE- or MDA-protein epitopes were employed in immuno-histochemical and immuno-precipitation/Western analyses to detect 4-HNE and MDA-protein adducts in paraffin-embedded liver sections and liver homogenates. As early as 6 h post CCl4 exposure, 4-HNE and MDA adducts were detected immuno-histochemically in hepatocytes localized to zone 2 of the hepatic acinus. Liver injury was progressive to 24 h as lipid peroxidation and hepatocellular necrosis increased. The hallmark of CCl4 hepatotoxicity, zone 3 necrosis, was observed 24 h after CCl4 administration and immuno-positive hepatocytes were observed in zone 2 as well as zone 3. Immuno-positive cells were no longer visible by 36 to 72 h post CCl4 administration. From 6 to 48 h after CCl4 administration, at least four adducted proteins were immuno-precipitated from liver homogenates with the anti-MDA or anti-4HNE serum, which corresponded to molecular weights of 80, 150, 205, and greater than 205 kDa. These results demonstrate that 4-HNE and MDA alkylate specific hepatic proteins in a time-dependent manner, which appears to be associated with hepatocellular injury following CCl4 exposure.