Ulrike Sattler

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.

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.

Hypoxia Inducible Factor-1α 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.

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 μmol/g tissue (dry weight). Based on the analysis of samples of given pyruvate concentrations, the assay exhibits a recovery with a deviation ≤15%. The minimal detectable amount was 0.02 pmol based on a 20 μm thick tissue section with an area of 1 cm2. 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.

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 and redox status in malignant tumors

ZusammenfassungDer vorliegende Beitrag fasst Ergebnisse der experimentellen und der klinischen Onkologie zusammen, die auf eine zentrale Bedeutung des Kohlenhydratstoffwechsels maligner Tumoren für Malignitätsgrad und Therapieansprechen hinweisen. In Primärtumoren, wie etwa in Zervixkarzinomen, Plattenephithelkarzinomen der Kopf-Hals-Region oder Adenokarzinomen des Rektums, ist bereits bei Erstdiagnose eine überdurchschnittliche Laktatakkumulation als Spiegel einer hohen Glykolyseaktivität mit erhöhter Metastasenbildung oder höherer Strahlenresistenz verknüpft. Der Zusammenhang zwischen therapeutischer Resistenz und Glykolyse beruht zumindest teilweise auf den Radikalfängereigenschaften glykolytischer Produkte, hauptsächlich Pyruvat und Laktat, sowie deren Verknüpfung mit dem Redoxstatus der Zelle. Vor diesem Hintergrund wurde das neue Verfahren der quantitativen Biolumineszenz zur bildgebenden Darstellung des Laktat/Pyruvat-Verhältnisses in Tumorbiopsien entwickelt. Künftige Forschungsinitiativen in der Onkologie sollten vermehrt auf den Redoxstatus solider Tumoren fokussieren.AbstractThis article summarizes data from experimental and clinical oncology which are indicative of a pivotal role of tumor carbohydrate metabolism in malignant behavior and outcome of treatment. In primary tumors, such as cervix carcinomas, head and neck squamous cell carcinomas or rectum adenocarcinomas, elevated lactate levels as a mirror of a high glycolytic activity, are correlated even at the initial diagnosis with a high level of malignancy as indicated by increased formation of metastases or an elevated radiotherapy resistance. The relationship between therapeutic resistance and glycolysis may at least partially be due to the radical scavenging potential of glycolytic intermediates, mainly pyruvate and lactate and to the link between these metabolites and the cellular redox status. On the basis of these data and other considerations, a novel technique has been developed for imaging the lactate/pyruvate ratio in tumor biopsies using quantitative bioluminescence. More research effort should, therefore, be focussed on the redox status of tumors in oncological studies in the future.

Laktat und Redoxstatus in malignen Tumoren

ZusammenfassungDer vorliegende Beitrag fasst Ergebnisse der experimentellen und der klinischen Onkologie zusammen, die auf eine zentrale Bedeutung des Kohlenhydratstoffwechsels maligner Tumoren für Malignitätsgrad und Therapieansprechen hinweisen. In Primärtumoren, wie etwa in Zervixkarzinomen, Plattenephithelkarzinomen der Kopf-Hals-Region oder Adenokarzinomen des Rektums, ist bereits bei Erstdiagnose eine überdurchschnittliche Laktatakkumulation als Spiegel einer hohen Glykolyseaktivität mit erhöhter Metastasenbildung oder höherer Strahlenresistenz verknüpft. Der Zusammenhang zwischen therapeutischer Resistenz und Glykolyse beruht zumindest teilweise auf den Radikalfängereigenschaften glykolytischer Produkte, hauptsächlich Pyruvat und Laktat, sowie deren Verknüpfung mit dem Redoxstatus der Zelle. Vor diesem Hintergrund wurde das neue Verfahren der quantitativen Biolumineszenz zur bildgebenden Darstellung des Laktat/Pyruvat-Verhältnisses in Tumorbiopsien entwickelt. Künftige Forschungsinitiativen in der Onkologie sollten vermehrt auf den Redoxstatus solider Tumoren fokussieren.AbstractThis article summarizes data from experimental and clinical oncology which are indicative of a pivotal role of tumor carbohydrate metabolism in malignant behavior and outcome of treatment. In primary tumors, such as cervix carcinomas, head and neck squamous cell carcinomas or rectum adenocarcinomas, elevated lactate levels as a mirror of a high glycolytic activity, are correlated even at the initial diagnosis with a high level of malignancy as indicated by increased formation of metastases or an elevated radiotherapy resistance. The relationship between therapeutic resistance and glycolysis may at least partially be due to the radical scavenging potential of glycolytic intermediates, mainly pyruvate and lactate and to the link between these metabolites and the cellular redox status. On the basis of these data and other considerations, a novel technique has been developed for imaging the lactate/pyruvate ratio in tumor biopsies using quantitative bioluminescence. More research effort should, therefore, be focussed on the redox status of tumors in oncological studies in the future.