Marion Stubbs

Causes and consequences of tumour acidity and implications for treatment

Tumour cells have a lower extracellular pH (pHe) than normal cells; this is an intrinsic feature of the tumour phenotype, caused by alterations either in acid export from the tumour cells or in clearance of extracellular acid. Low pHe benefits tumour cells because it promotes invasiveness, whereas a high intracellular pH (pHi) gives them a competitive advantage over normal cells for growth. Molecular genetic approaches have revealed hypoxia-induced coordinated upregulation of glycolysis, a potentially important mechanism for establishing the metabolic phenotype of tumours. Understanding tumour acidity opens up new opportunities for therapy.

Measurement of the extracellular ph of solid tumours in mice by magnetic resonance spectroscopy : a comparison of exogenous 19F and 31P probes

Precise measurement of pHe in vivo may be of clinical value for both diagnosis and selection of therapy. pHe measurements made by the 31P probe 3‐aminopropylphosphonate (3‐APP) were compared with those made by the 19F probe, 3‐[N‐(4‐fluor‐2‐trifluoromethylphenyl)‐sulphamoyl]‐propionic acid (ZK‐150471) in three solid tumour types, human HT29 xenografts, murine RIF‐1 fibrosarcomas and Lettre tumours grown subcutaneously in mice. No significant differences were observed when probe measurements of pHe were compared at 20–60 min post‐administration, although very low pHe values (ca. 6.0) were recorded in two out of eight Lettre tumours by ZK‐150471. The more rapid pHe measurements possible using ZK‐150471 showed that during the first 20 min post‐administration significant increases occurred in pHe which were greatest in the more necrotic tumours. Since isolated cell experiments showed that ZK‐150471 was non‐toxic and did not enter the cells, this early increase in pHe may reflect gradual penetration by ZK‐150471 of the reportedly alkaline necrotic space in the tumours. The wide chemical shift range, improved signal‐to‐noise and absence of signal overlap allowed a more rapid and precise measurement of pHe by ZK‐150471 compared to 3‐APP. These characteristics suggest that ZK‐150471 is currently the preferred pHe probe for non‐invasive MRS. Copyright

Combined metabolomic and proteomic analysis of human atrial fibrillation.

OBJECTIVES We sought to decipher metabolic processes servicing the increased energy demand during persistent atrial fibrillation (AF) and to ascertain whether metabolic derangements might instigate this arrhythmia. BACKGROUND Whereas electrical, structural, and contractile remodeling processes are well-recognized contributors to the self-perpetuating nature of AF, the impact of cardiac metabolism upon the persistence/initiation of this resilient arrhythmia has not been explored in detail. METHODS Human atrial appendage tissues from matched cohorts in sinus rhythm (SR), from those who developed AF post-operatively, and from patients in persistent AF undergoing cardiac surgery were analyzed using a combined metabolomic and proteomic approach. RESULTS High-resolution proton nuclear magnetic resonance (NMR) spectroscopy of cardiac tissue from patients in persistent AF revealed a rise in beta-hydroxybutyrate, the major substrate in ketone body metabolism, along with an increase in ketogenic amino acids and glycine. These metabolomic findings were substantiated by proteomic experiments demonstrating differential expression of 3-oxoacid transferase, the key enzyme for ketolytic energy production. Notably, compared with the SR cohort, the group susceptible to post-operative AF showed a discordant regulation of energy metabolites. Combined principal component and linear discriminant analyses of metabolic profiles from proton NMR spectroscopy correctly classified more than 80% of patients at risk of AF at the time of coronary artery bypass grafting. CONCLUSIONS The present study characterized the metabolic adaptation to persistent AF, unraveling a potential role for ketone bodies, and demonstrated that discordant metabolic alterations are evident in individuals susceptible to post-operative AF.

Tumor vascular architecture and function evaluated by non-invasive susceptibility MRI methods and immunohistochemistry.

To investigate the physiological origins responsible for the varying blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) responses to carbogen (95% O2/5% CO2) breathing observed with different tumor types.

Regulation of cellular metabolism by intracellular phosphate

Abstract 1. Incubation of liver cell suspensions with 10 mM glycerol or 10 mM fructose caused a decrease in intracellular [Pi] and a fall in the [ ATP ] [ ADP ] while the respiratory rate, the redox state of the intramitochondrial NAD couple, and the [ ATP ] [ ADP ] · [ P i ] remained essentially unaltered. 2. The concentration of intracellular phosphate in yeast Candida utilis was found to be dependent on phosphate concentration in the growth medium: it was 25–30 μmol/g wet weight in cells grown in the presence of 50 mM Pi, 10–12 μmol/g wet weight in cells grown in 5 mM Pi, and 3–4 μmol/g wet weight in cells grown with 1 mM Pi. 3. The [ ATP ] [ ADP ] ratios were found to be highest in yeast cells grown in the presence of 50 mM Pi (⩾ 30) and lowest in cells grown with 1 mM Pi (8–9). The increase in [ ATP ] [ ADP ] was due to an increase in [ATP]. 4. The endogenous levels of glucose 6-phosphate in washed and aerated cells were lowest in cells grown in the presence of 50 mM Pi and highest in those grown in the presence of 1 mM Pi. A suggestion is made that the high intracellular [Pi] stimulates phosphorolytic breakdown of glycogen and depletes the cellular stores of this substrate. 5. It is postulated that the cellular respiratory rate and the NADH generation rate are regulated by [ ATP ] [ ADP ] · [ P i ] and not by the “energy charge” as defined by Atkinson (Biochemistry 7, 4030–4034, 1968). 6. The concentration of intracellular [Pi] is implicated as an important contributor to the regulatory mechanisms of cellular metabolism.

Magnetic resonance imaging techniques for monitoring changes in tumor oxygenation and blood flow

The application of functional magnetic resonance (MR) imaging techniques to the measurement of oxygenation and blood flow in tumors is described. Gradient recalled echo MR imaging (GRE-MRI) offers a real-time noninvasive method for monitoring tumor response to vasomodulators such as carbogen (95% O2/5% CO2) breathing in attempts to overcome tumor hypoxia and improve treatment efficacy. Although the response is tumor-type dependent, increases in signal intensity of up to 100% have been observed in several animal tumor types. Responses are also seen in human tumors. The observed increases in GRE-MRI signal intensity are due to a combination of a reduction of deoxyhemoglobin in the blood causing changes in the MR imaging relaxation time T2* and changes in blood flow and may also reflect the capillary density. Thus, the magnitude of the GRE image intensity change gives an indication of the potential response of an individual tumor to treatments that aim to improve tissue oxygenation and therefore how the tumor may respond to therapy. In addition, carbogen breathing by the host has been shown to increase the uptake and efficacy of chemotherapeutic agents in animal tumors.

Causes and consequences of acidic pH in tumors: a magnetic resonance study

A consequence of metabolism in any tissue is the formation of hydrogen ions, which are actively transported out of the cell. However, although most solid tumors maintain their intracellular pH (pHi) within a narrow range to provide a favorable environment for various intracellular activities, their extracellular pH (pHe) is on average about 0.2 pH units more acid. It is important to understand the relationship between tumor metabolism and pH, and how it differs from that of normal tissue and to ask the question: How does an understanding of pH and tumor metabolism affect strategies for therapeutic approaches? Although, in vitro, isolated cell experiments have shown positive correlations between pHi and pHe the relationship is complex and somewhat dependent on experimental conditions. Measurement of pHi in solid tumors by non-invasive Magnetic Resonance Spectroscopy (MRS) has been possible for some time now and recently several specific markers for measuring pHe have become available. As a result we have been able to study the relationship between pHi and pHe in vivo in several different solid tumor types. In only one tumor type (HT29 xenografts) was there a significant correlation between pHi and pHe; in 3 other tumor types (RIF-1 in mice, GH3 prolactinomas and H9618a in rats) there was no correlation. A significant correlation between pHi and NTP/Pi ratios was seen across all tumor types. Theoretical considerations of causes of tumor acidity, hypotheses to explain extracellular acidity and the possibility that low pHe might be an intrinsic feature of the tumor phenotype and not merely the consequence of metabolic activity have been discussed. In addition the consquences for concepts of treatment based on pH are considered.

Glut-1 as a therapeutic target: increased chemoresistance and HIF-1-independent link with cell turnover is revealed through COMPARE analysis and metabolomic studies

The facilitative glucose transporter Glut-1 is overexpressed and confers poor prognosis in a wide range of solid tumours. The peri-necrotic pattern of expression often seen in human tumour samples is linked with its transcriptional control in hypoxic conditions by hypoxia-inducible factor HIF-1 or through a reduced rate of oxidative phosphorylation. Hypoxia-regulated genes offer promise as novel therapeutic targets as a means of preventing the proliferation and eventual metastatic spread of tissue originating from residual chemically and radio resistant hypoxic cells that have survived treatment. Inhibiting the expression or functionality of Glut-1 may be a way of specifically targeting hypoxic cells within the tumour that depend upon a high rate of glucose uptake for anaerobic glycolysis. We used an array of formalin-fixed, paraffin-embedded samples of the NCI-60 panel of cell lines to carry out immunohistochemical detection of Glut-1 and to select possible candidate lead compounds by COMPARE analysis with agents from the NCI diversity screen, which may work via inhibition of Glut-1 or Glut-1-dependent processes. “Positive” COMPARE hits were mostly conjugated Pseudomonas toxins binding the epidermal growth factor receptor (EGFR). However, correlations with standard anticancer agents were virtually all negative, indicating a link between Glut-1 and chemoresistance. MTT proliferation assays carried out using stable, Glut-1 overexpressing cell lines generated from the bladder EJ138, human fibrosarcoma HT 1080 and the hepatoma wild type Hepa and HIF-1B-deficient c4 tumour cell lines revealed a cell line-dependent increase in chemoresistance to dacarbazine, vincristine and the bioreductive agent EO9 in Glut-1 overexpressing EJ138 relative to WT and empty vector controls. Metabolomic analysis (31P-MRS and 1H MRS) carried out using cell lysates and xenografts generated from Glut-1 overexpressing Hepa and c4 cell lines showed higher glucose levels in Glut-1 overxpressing c4 relative to parental tumour extracts occurred in the absence of an increase in lactate levels, which were in turn significantly higher in the Glut-1 overexpressing Hepa xenografts. This implies that Glut-1 over-expression without a co-ordinate increase in HIF-1-regulated glycolytic enzymes increases glucose uptake but not the rate of glycolysis. Glut-1 overexpressing xenografts also showed higher levels of phosphodiester (PDE), which relates to the metabolite turnover of phospholipids and is involved in membrane lipid degradation, indicating a mechanism by which Glut-1 may increase cell turnover.

Effects of HIF-1alpha and HIF2alpha on Growth and Metabolism of Clear-Cell Renal Cell Carcinoma 786-0 Xenografts.

In cultured clear-cell renal carcinoma (CCRCC) 786-0 cells transfected with HIF1α (HIF-1+), HIF-2α (HIF-2+), or empty vector (EV), no significant differences were observed in the growth rates in vitro, but when grown in vivo as xenografts HIF-2α significantly increased, and HIF-1α significantly decreased growth rates, compared to EV tumors. Factors associated with proliferation were increased and factors associated with cell death were decreased in HIF-2+ tumors. Metabolite profiles showed higher glucose and lower lactate and alanine levels in the HIF-2+ tumors whilst immunostaining demonstrated higher pyruvate dehydrogenase and lower pyruvate dehydrogenase kinase 1, compared to control tumors. Taken together, these results suggest that overexpression of HIF-2α in CCRCC 786-0 tumors regulated growth both by maintaining a low level of glycolysis and by allowing more mitochondrial metabolism and tolerance to ROS induced DNA damage. The growth profiles observed may be mediated by adaptive changes to a more oxidative phenotype.

The altered metabolism of tumors: HIF-1 and its role in the Warburg effect.

Almost 90 years ago, Warburg (1930) first reported the propensity for cancer cells to convert glucose to lactate even in the presence of oxygen, a phenomenon he had discovered in his work on tissue slices. Since that time the cause of the ‘‘Warburg Effect’’ has been hotly debated, and it is still not well understood. Interest in this phenomenon is at the present time enjoying high profile attention, as evidenced by an article in The Economist in 2007 as well as comprehensive reviews in Science (Vander Heiden et al., 2009), Nature Cancer Reviews (Denko, 2008) and a complete volume of Seminars in Cancer Biology (The Warburg Effect, 2009). This is not only because the Warburg effect underlies the clinical success of FDG-PET as a diagnostic tool in oncology, but also because one of the transcription factors that promotes tumor metabolism, growth, and angiogenesis seems to have a significant role in the Warburg effect. HIF-1 is an oxygen-sensing transcription factor that regulates several hundred genes concerned with many aspects of tumor metabolism and progression. Under hypoxic conditions, a common feature of the tumor environment, the HIF-1dimer is formed and (amongst other things) it enhances aerobic glycolysis through co-ordinated up-regulation of glycolytic enzymes and downregulation of mitochondrial oxidative metabolism, suggesting the possibility of a role in the Warburg effect (Semenza, 2007). Whatever cause underlies the cancer (e.g. virus, mutation, DNA damage etc), tumor metabolism appears to be similar across a broad range of cancer types (Griffiths and Stubbs, 2005). In this review we discuss HIF-1 targets that relate specifically to tumor metabolism and cellular energy production, factors that must ultimately determine tumor progression. We also consider the conventional assumption that ‘‘to generate the energy needed for cellular processes, most cancer cells rely on aerobic glycolysis’’ (Vander Heiden et al., 2009) and what proportion of total tumor ATP might be made by this pathway.