Manipulation of tumor metabolism for therapeutic approaches: ovarian cancer-derived cell lines as a model system.

BACKGROUND: Malignant transformation of cells is often accompanied by up-regulation of glycolysis-related enzymes and transporters, as well as a distortion of mitochondrial respiration. As a consequence, most malignant tumors utilize high amounts of glucose and produce and accumulate high concentrations of lactate, even in the presence of oxygen. This phenomenon has been termed 'Warburg Effect'. Here, we aimed at resolving the interrelation between tumor metabolism, reactive oxygen species, double strand DNA breaks and radio-resistance in ovarian cancer-derived cells. METHODS: As a model system two ovarian cancer-derived cell lines, OC316 and IGROV-1, and its corresponding xenografts were used. First, the metabolic properties of the xenografts were tested to ensure that initial in vitro data might later be transferred to in vivo data. In parallel, three inhibitors of tumor cell metabolism, 2-deoxy-D-glucose, an inhibitor of glycolysis, oxamate, a pyruvate analogue and inhibitor of lactate dehydrogenase, and rotenone, a specific inhibitor of mitochondrial electron complex I, were tested for their effect on the metabolism and radio-sensitivity of the respective ovarian cancer-derived cell lines. RESULTS: We found that all three inhibitors tested led to significant changes in the tumor cell energy metabolism at non-cytotoxic concentrations. Furthermore, we found that inhibition of tumor glycolysis by 2-deoxy-D-glucose in combination with rotenone decreased the radio-resistance at a clinically relevant radiation dose. This apparent radio-sensitizing effect appears to be based on an increased level of double strand DNA breaks 1 h and 24 h after gamma irradiation. Both cancer-derived cell lines maintained their metabolic properties, as well as their protein expression profiles and levels of reactive oxygen species in xenografts, thus providing a suitable model system for further in vivo investigations. CONCLUSION: A combination of metabolic inhibitors and reactive oxygen species-generating therapies, such as irradiation, may effectively enhance the therapeutic response in particularly metabolically highly active (ovarian) tumors.

Protein profiles in human ovarian cancer cell lines correspond to their metabolic activity and to metabolic profiles of respective tumor xenografts.

Many solid tumors show a large variability in glycolytic activity and lactate accumulation, which has been correlated with different metastatic spread, radioresistance and patient survival. To investigate potential differences in protein profiles underlying these metabolic variances, the highly glycolytic human ovarian cancer cell line OC316 was investigated and compared with the less glycolytic line IGROV-1. Extracellular acidification and oxygen consumption were analyzed with an extracellular flux analyzer. Glycolysis-associated proteins, including specific membrane transporters, were quantified through in-cell western analyses. Metabolic properties of corresponding tumor xenografts were assessed via induced metabolic bioluminescence imaging. Extracellular flux analyses revealed elevated bioenergetics of OC316 cells. Hexokinase II, pyruvate kinase, pyruvate dehydrogenase E1 beta subunit and pyruvate dehydrogenase kinase 1, as well as the glucose transporter 1 and the monocarboxylate transporter 4, were overexpressed in these cells compared with IGROV-1. When generating tumor xenografts in SCID mice, cells maintained their glycolytic behavior, i.e. OC316 showed higher lactate concentrations than IGROV-1 tumors. In summary, a congruency between protein profiles and metabolic properties has been demonstrated in the human ovarian cancer lines investigated. Also, a perpetuation of glycolytic characteristics during the transition from in vitro to the in vivo situation has been documented. This model system could be useful for systematic studies on therapeutic intervention by manipulation of tumor glycolysis and associated pathways.

Lactate: a metabolic key player in cancer.

Increased glucose uptake and accumulation of lactate, even under normoxic conditions (i.e., aerobic glycolysis or the Warburg Effect), is a common feature of cancer cells. This phenomenon clearly indicates that lactate is not a surrogate of tumor hypoxia. Tumor lactate can predict for metastases and overall survival of patients, as shown by several studies of different entities. Metastasis of tumors is promoted by lactate-induced secretion of hyaluronan by tumor-associated fibroblasts that create a milieu favorable for migration. Lactate itself has been found to induce the migration of cells and cell clusters. Furthermore, radioresistance has been positively correlated with lactate concentrations, suggesting an antioxidative capacity of lactate. Findings on interactions of tumor metabolites with immune cells indicate a contribution of lactate to the immune escape. Furthermore, lactate bridges the gap between high lactate levels in wound healing, chronic inflammation, and cancer development. Tumor cells ensure sufficient oxygen and nutrient supply for proliferation through lactate-induced secretion of VEGF, resulting in the formation of new vessels. In summary, accumulation of lactate in solid tumors is a pivotal and early event in the development of malignancies. The determination of lactate should enter further clinical trials to confirm its relevance in cancer biology.

Glycolytic phenotype and AMP kinase modify the pathologic response of tumor xenografts to VEGF neutralization.

VEGF antagonists are now widely used cancer therapeutics, but predictive biomarkers of response or toxicity remain unavailable. In this study, we analyzed the effects of anti-VEGF therapy on tumor metabolism and therapeutic response by using an integrated set of imaging techniques, including bioluminescence metabolic imaging, 18-fluorodeoxyglucose positron emission tomography, and MRI imaging and spectroscopy. Our results revealed that anti-VEGF therapy caused a dramatic depletion of glucose and an exhaustion of ATP levels in tumors, although glucose uptake was maintained. These metabolic changes selectively accompanied the presence of large necrotic areas and partial tumor regression in highly glycolytic tumors. In addition, we found that the central metabolic protein kinase AMP-activated protein kinase (AMPK)-a cellular sensor of ATP levels that supports cell viability in response to energy stress-was activated by anti-VEGF therapy in experimental tumors. AMPK-α2 attenuation increased glucose consumption, tumor cell sensitivity to glucose starvation, and tumor necrosis following anti-VEGF therapy. Taken together, our findings reveal functional links between the Warburg effect and the AMPK pathway with therapeutic responses to VEGF neutralization in tumor xenograft models.

Association between the herpes simplex virus-1 DNA polymerase and uracil DNA glycosylase.

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIalpha-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions -1 and -2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.

Glycolytic metabolism and tumour response to fractionated irradiation.

BACKGROUND AND PURPOSE: To study whether pre-therapeutic lactate or pyruvate predict for tumour response to fractionated irradiation and to identify possible coherencies between intermediates of glycolysis and expression levels of selected proteins. MATERIALS AND METHODS: Concentrations of lactate, pyruvate, glucose and ATP were quantified via bioluminescence imaging in tumour xenografts derived from 10 human head and neck squamous cell carcinoma (HNSCC) lines. Tumours were irradiated with 30 fractions within 6 weeks. Expression levels of the selected proteins in tumours were measured at the mRNA and protein level. Tumour-infiltrating leucocytes were quantified after staining for CD45. RESULTS: Lactate but not pyruvate concentrations were significantly correlated with tumour response to fractionated irradiation. Lactate concentrations in vivo did not reflect lactate production rates in vitro. Metabolite concentrations did not correlate with GLUT1, PFK-L or LDH-A at the transcriptional or protein level. CD45-positive cell infiltration was low in the majority of tumours and did not correlate with lactate concentration. CONCLUSIONS: Our data support the hypothesis that the antioxidative capacity of lactate may contribute to radioresistance in malignant tumours. Non-invasive imaging of lactate to monitor radiation response and testing inhibitors of glycolysis to improve outcome after fractionated radiotherapy warrant further investigations.

The anti-oxidant capacity of tumour glycolysis.

PURPOSE: In this mini-review data are summarised which provide evidence for the biological and clinical significance of tumour glycolysis and of its relationship to the redox state of cancer cells. RESULTS: Malignant transformation is associated with an overexpression of numerous glycolysis-related genes in the vast majority of human cancers. At the same time, glycolytic activity and glycolysis-linked metabolic milieu are often variable between individual tumours which induces large variations in treatment response and aggressiveness. Currently, there is no genetic or proteomic marker for the prediction of the therapeutic response for individual tumours, but the prognostic value of tumour lactate accumulation for the emergence of metastasis, for patient survival and for radioresistance has been documented in a number of studies. CONCLUSIONS: Transactivation of tumour glycolyis appears to generate a chemically reduced milieu associated with an inhibition of ROS (reactive oxygen species) -mediated fixation of DNA damage and induction of radioresistance. Furthermore, highly glycolytic cells enhance the antioxidant defense via glutathione, and pyruvate can be decarboxylated non-enzymatically upon reducing hydrogen peroxide. The summary of data given here emphasises the importance of further research efforts on the link between carbohydrate metabolism and redox state of cancer cells.

Hypoxia inducible factor-1alpha inactivation unveils a link between tumor cell metabolism and hypoxia-induced cell death.

Hypoxia and the acquisition of a glycolytic phenotype are intrinsic features of the tumor microenvironment. The hypoxia inducible factor-1alpha (HIF-1alpha) pathway is activated under hypoxic conditions and orchestrates a complex transcriptional program that enhances cell survival. Although the consequences of HIF-1alpha inactivation in cancer cells have been widely investigated, only a few studies have addressed the role of HIF-1alpha in the survival of cancer cells endowed with different glycolytic capacities. In this study, we investigated this aspect in ovarian cancer cells. Hypoxia-induced toxicity was increased in highly glycolytic cells compared with poorly glycolytic cells; it was also associated with a sharp decrease in intracellular ATP levels and was prevented by glucose supplementation. Stable HIF-1alpha silencing enhanced hypoxia-induced cell death in vitro due to a lack of cell cycle arrest. Tumors bearing attenuated HIF-1alpha levels had similar growth rates and vascularization as did controls, but tumors showed higher proliferation levels and increased necrosis. Moreover, tumors formed by HIF-1alpha deficient cells had higher levels of lactate and lower ATP concentrations than controls as shown by metabolic imaging. The findings that such metabolic properties can affect the survival of cancer cells under hypoxic conditions and that these properties contribute to the determination of the consequences of HIF-1alpha inactivation could have important implications on the understanding of the effects of anti-angiogenic and HIF-1alpha-targeting drugs in cancer.

Understanding and using the meaning of statements in a bio-ontology: recasting the Gene Ontology in OWL.

The bio-ontology community falls into two camps: first we have biology domain experts, who actually hold the knowledge we wish to capture in ontologies; second, we have ontology specialists, who hold knowledge about techniques and best practice on ontology development. In the bio-ontology domain, these two camps have often come into conflict, especially where pragmatism comes into conflict with perceived best practice. One of these areas is the insistence of computer scientists on a well-defined semantic basis for the Knowledge Representation language being used. In this article, we will first describe why this community is so insistent. Second, we will illustrate this by examining the semantics of the Web Ontology Language and the semantics placed on the Directed Acyclic Graph as used by the Gene Ontology. Finally we will reconcile the two representations, including the broader Open Biomedical Ontologies format. The ability to exchange between the two representations means that we can capitalise on the features of both languages. Such utility can only arise by the understanding of the semantics of the languages being used. By this illustration of the usefulness of a clear, well-defined language semantics, we wish to promote a wider understanding of the computer science perspective amongst potential users within the biological community.

A bioluminescence technique for quantitative and structure-associated imaging of pyruvate.

A novel bioluminescence assay has been developed for measuring pyruvate within sections of snap-frozen tissue in a quantitative manner as well as with a spatial resolution on a microscopical level. The assay was verified via HPLC and two independent photometric tests. The novel assay makes it possible to determine pyruvate concentrations in cryosections in the range of 0-5.0 micromol/g tissue (dry weight). Based on the analysis of samples of given pyruvate concentrations, the assay exhibits a recovery with a deviation < or =15%. The minimal detectable amount was 0.02 pmol based on a 20 microm thick tissue section with an area of 1 cm(2). Combination of the already established imaging bioluminescence techniques for ATP, glucose, and lactate with the novel pyruvate assay allows for a comprehensive characterization of the metabolic profile of individual tumors. As the redox state of cancer cells can be critical for the efficiency of irradiation and a number of chemotherapeutics, and as pyruvate and lactate are known to have radical scavenger functions, we hypothesize that the novel bioluminescence assay may be used for measuring the pretherapeutic lactate-to-pyruvate ratio which may predict the radiosensitivity of individual malignancies.

DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain.

The postreplicative repair of double-strand breaks (DSBs) is thought to require sister chromatid cohesion, provided by the cohesin complex along the chromosome arms. A further specialized role for cohesin in DSB repair is suggested by its de novo recruitment to regions of DNA damage in mammals. Here, we show in budding yeast that a single DSB induces the formation of a approximately 100 kb cohesin domain around the lesion. Our analyses suggest that the primary DNA damage checkpoint kinases Mec1p and Tel1p phosphorylate histone H2AX to generate a large domain, which is permissive for cohesin binding. Cohesin binding to the phospho-H2AX domain is enabled by Mre11p, a component of a critical repair complex, and Scc2p, a component of the cohesin loading machinery that is necessary for sister chromatid cohesion. We also provide evidence that the DSB-induced cohesin domain functions in postreplicative repair.

The expression level of the orphan nuclear receptor GCNF (germ cell nuclear factor) is critical for neuronal differentiation.

The germ cell nuclear factor (GCNF) is essential for normal embryonic development and gametogenesis. To test the prediction that GCNF is additionally required for neuronal differentiation, we used the mouse embryonal carcinoma cell line PCC7-Mz1, which represents an advantageous model to study neuronal cells from the stage of fate choice until the acquirement of functional competence. We generated stable transfectants that express gcnf sense or antisense RNA under the control of a tetracycline-regulated promoter. After retinoic acid-induced withdrawal from the cell cycle, sense clones developed a neuron network with changed properties, and the time course of neuron maturation was shortened. Consistent with these data, differentiation of neuronal precursor cells was impaired in antisense cultures. This involved a delay in 1) the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells and stem cells of the central nervous system, and 2) up-regulation of the somatodendritic protein microtubule-associated protein 2 and the synaptic vesicle protein synaptophysin. Neuronal cells in the antisense cultures acquired functional competence, although with a significant delay. Our data propose that the level of GCNF is critical for differentiation and maturation of neuronal precursor cells.

Long-patch DNA repair synthesis during base excision repair in mammalian cells.

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway.