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Causes and consequences of tumour acidity and implications for treatment
MOLECULAR MEDICINE TODAY, JANUARY 2000 (VOL. 6)
Opinion
Causes and consequences of tumour acidity and implications for treatment Marion Stubbs, Paul M.J. McSheehy, John R. Griffiths and C. Lindsay Bashford
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. In the 1930s, Warburg noted ‘the remarkable extent to which living tumour cells are able to convert carbohydrate into lactic acid’1. Subsequently, for about 50 years, it was assumed that tumour cells would have an acidic intracellular pH (pHi). Microelectrode measurements of tumour pH appeared to confirm that the pH of tumour cells was low. However, when 31P magnetic resonance spectroscopy (MRS) was introduced for the study of tumours in situ2, it provided a simple, noninvasive means of estimating pHi, and tumour cells turned out to have neutral or slightly alkaline pHi values3. Like normal cells, tumour cells regulate their cytoplasmic pHi within a narrow range to provide a favourable environment for various intracellular activities. Indeed, many tumour cells have a high pHi, which is considered to be permissive for cell growth4. In tumours, it is the extracellular fluid that is relatively acid. It was mainly this compartment that the microelectrodes had been sampling, and such measurements are now acknowledged largely to reflect extracellular pH (pHe ). The result is a reverse or negative pH gradient (pHi . pHe) across the tumour-cell plasma membrane in situ compared with normal tissues where pHi (~7.2) is lower than pHe (~7.4) (Ref 5).
pH measurement by MRS pHi The MRS measurement of pHi is based on a pHdependent chemical shift difference between the 31P inorganic phosphate (Pi) signal and an
endogenous reference signal. At physiological pH, the position of the Pi signal reflects the relative concentrations of the two phosphate species (H2PO42 and HPO422) present. There is phosphate in both the intra- and extracellular compartments, so unless two Pi peaks can be resolved in the MRS spectrum, the MRS measurement of tissue pH is a weighted average of pHi and pHe. In normal tissue, the concentration of intra- and extracellular Pi is approximately the same. It is generally considered that the pH measured by MRS is intracellular because the extracellular volume, for example of liver, is less than 25% of the total cell volume and thus extracellular Pi is only a minor component. For tumours, however, this assumption might not apply because, in addition to having higher-than-normal Pi signals, their extracellular volume could also be high, owing to necrotic areas and cysts for example. Estimates of the proportion of Pi signal coming from the intracellular volume can be made if the total tumour volume and the fractional volume of extracellular water are known. Calculations show that if the extracellular volume does not exceed 55% then pH measured by MRS largely represents pHi (Ref. 5).
pHe Recently, several MR-specific extracellular markers for pHe have become available6,7 that allow the simultaneous measurement of pHi and pHe in tumours and normal tissue. Studies using these
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probes confirm that tumour pHe in many (but not all) animal models is lower than pHi. However, it should be noted that pHe comprises both the interstitial and vascular compartments, and the latter (pHb) usually has a pH of about 7.4.
Why do tumours have high rates of glycolysis? Many tumours have high rates of glycolysis, regardless of whether their supply of oxygen is good (Warburg’s original observation) or poor. Most tumours in vivo synthesize some ATP by oxidative metabolism, and some by glycolytic metabolism to lactate (aerobic glycolysis). Clearly, if the oxygen supply is removed (acute hypoxia), the tumour cells switch to anaerobic glycolysis, just as would normal tissue. Research on hepatomas has shown that the rate of tumour glycolysis appears to be associated with the differentiation status and growth rate of the tumour8. But the question as to why some of the energy demand is satisfied by aerobic glycolysis, rather than by mitochondrial oxidative phosphorylation, remains unanswered. Several normal cell-types that are not necessarily hypoxic (for example, leukocytes and enterocytes) also have high rates of glycolysis and produce lactic acid. Leukocytes, after many rounds of cell division, have fewer mitochondria than their stem-cell precursors, and some tumour cells contain about half the mitochondria of comparable, untransformed tissue. If there are not enough mitochondria to replace ATP 15
Opinion
MOLECULAR MEDICINE TODAY, JANUARY 2000 (VOL. 6)
pH Extracellular H+ store
H+ pump
6
pHe ~6.8 Intracellular H+ source 7
pHi ~7.2
Tumour Normal
f1
Blood H+ sink
(a) pHe ~7.3
f2
pHb ~7.4
(b) 8 Molecular Medicine Today
Figure 1. Factors affecting pHi and pHe in normal and tumour tissue. H1 produced by metabolism is pumped from the intracellular compartment into the interstitial compartment and subsequently flows into the blood. The concentration of H1 in the interstitial compartment is relatively high in tumours (low pHe ) compared with normal tissue. At steady state, the flow (f) of H1 between the compartments is equal (f1 5 f2). The increased interstitial acidity of tumour cells could be caused by (a) an increase in H1 pumping by, for example, expression in the plasma membrane of vacuolar-type H1 pumps or (b) an increase in resistance induced either by altered gene expression or by the action of cytokines on cells at the interstitial/vascular interface.
at the required rate, enhanced glycolysis would be an obvious metabolic solution, and many tumour cells appear to adopt a mixed metabolic economy of simultaneous oxidative phosphorylation and glycolysis. In most normal cells, the levels of ADP and Pi needed to sustain a high glycolytic rate would be sufficient to maximally activate oxidative phosphorylation. The changes in metabolic control that allow both pathways to be active, but not saturated, in tumours (and mitochondrially deficient normal cells) remain uncertain. The observation that overexpression of key glycolytic enzymes in yeast abolishes the Pasteur effect9 might suggest that the oncogenic events that switch on uncontrolled DNA replication in the nucleus fail to cause an equivalent increase in replication of mitochondrial DNA, and permit overexpression of glycolytic enzymes.
How might the activities of the enzymes participating in glycolysis be raised? Modern metabolic control analysis has shown that the control of metabolic pathways is distributed over many enzymes; if glycolytic flux is increased, many of the glycolytic enzymes are likely to have raised activities10. Recently, a widespread system of oxygen-related gene expression, based on the activation of the transcription factor hypoxia inducible factor-1 (HIF-1), has been defined11. Intriguingly, these molecular genetic approaches 16
have revealed that sequences that are 59 to the coding region of genes for glycolytic enzymes contain a common motif12, and this could provide the basis for coordinated upregulation of the pathway. Chronic hypoxia also upregulates the expression of a number of ‘stress’ proteins, several of which are glycolytic enzymes. For example, the major hypoxic stress protein p34 is an isozyme of lactate dehydrogenase. These findings suggest that hypoxia is a trigger that coordinates induction of gene expression and may well be one of the factors that determines the metabolic phenotype of tumours. Thus, it is possible to imagine that the genetic disruptions that produce tumour cells might also stimulate the HIF-1 system without the need for the hypoxic stimulus. Indeed, HIF-1 is activated constitutively in cells that are defective in an important tumour suppressor, the von Hippel–Lindau gene product13. The modified cells would have the metabolic phenotype associated with tumours and could induce the chaotic vasculature typically found in tumours. Subsequent hypoxia would reinforce this pattern by super-induction of the HIF-1 system12.
Why is pHe of tumours acidic? Does excess lactate production cause extracellular acidity? Following glycolysis, the major pathway of lactate export from cells is the H1-monocarboxylate cotransporter. The steady-state intracellular lactate
concentration of tumour cells tends to be at least twofold higher than the extracellular concentration. This follows from the intimate association between lactate and H1 gradients and the direction of the H1 gradient across the tumour-cell plasma membrane14. One obvious hypothesis to explain the low pHe in solid tumours is that metabolic acids (lactate and/or CO2) exported from the cancer cells into the interstitial fluid cannot be exported to the blood rapidly enough. This poor H1 clearance could be due to the disorganized vasculature of tumours, poor lymphatic drainage and elevated interstitial pressure. However, when a cancer cell line that produced large amounts of lactic acid was compared with a mutant line that had a defective glycolytic pathway and produced very small amounts of lactic acid, solid tumours grown up from both the wild-type and mutant lines still had an acidic interstitial pH (Ref. 15). One possible explanation for this is that the mutant cells had produced large amounts of CO2 by oxidative metabolism, which would also acidify the extracellular compartment. However, other mutants defective in the glycolytic pathway produced similar amounts of CO2 to the normal cells. This implies that the acidity of tumours is not caused simply by excessive production of lactate and CO2 (Ref. 16).
Does changing the set point of pHi regulation cause extracellular acidity? Tumour cells might raise their set point for pHi by increasing the export of H1 from the intracellular compartment4. Increased H1 export could permit faster overall production of acid by tumour metabolism (‘H1 source’ in Fig.1) and lead indirectly to increased extracellular acidity. The increased H1 export could be achieved via activation of the mitogen-sensitive Na1/H1exchanger, or via increased functional expression in the plasma membrane of H1-pumping ATPases. Functional expression of H1-pumping ATPases has been measured on the cell surface of some tumour cells, particularly those with a higher pHi, and it is associated with greater ATP turnover, increased glycolysis and decreased protein degradation4, all features of the tumour metabolic phenotype.
Is pHe regulated and does changing the set point of pHe cause extracellular acidity? The pH of any compartment at steady-state is determined by the balance between H1 entering and leaving that compartment (Fig.1) and the nature of the internal buffers. pHi and pHe correlate strongly in cell culture experiments in which the pH of the medium has been manipulated. However in four rodent models of solid tumours (including one human xenograft) in which pHi
Opinion
MOLECULAR MEDICINE TODAY, JANUARY 2000 (VOL. 6)
Glossary
Tumour cell plasma membrane
(a)
Calcification – The deposition of calcium salts in tissue.
pHe ~6.8 H+
Chemical shift – A shift in resonating frequency between molecules that depends on the electron shielding of their chemical bonds. For example, HPO422 resonates at a slightly different frequency to that of HPO42.
(f) Ca2+
pHi ~7.2
Na+
Na+ ADP+Pi ATP
H+
ATP ADP+Pi K+
(e)
(b)
Lactate−
HCO3− Glycolysis Cl−
H+
(c)
Na+ ?
(d)
Endothelial cells Glucose
? (g)
pHb ~7.4 Molecular Medicine Today
Figure 2. Mechanisms involved in maintenance of intracellular neutrality in tumours. When lactic acid is produced from glucose (a non-electrolyte), the lactate ion and H1 can pass into the extracellular fluid via the monocarboxylate carrier (a). Some lactate ions accumulate in the cell and contribute to the high lactate levels commonly observed in tumours. The Na1/H1 antiport (b), which is activated in transformed cells, exports H1 and imports Na1 contributing to the high intracellular Na1, another common tumour-cell feature. There is also some buffering by HCO32, which enters the cell by the Na1 dependent HCO32 /Cl2exchanger (c). There are many additional mechanisms which may also play a role in the regulation of maintenance of neutral pHi including the ATP-dependent Na1/K1 antiport (d) and vacuolar H1-pump (e). The low energy of the Na1 gradient causes Ca21 to accumulate (f) and together with high Pi may initiate tumour calcification. Also shown is a hypothetical exchanger at the tumour–endothelial cell interface (g) which might regulate pHe.
and pHe were measured simultaneously in vivo (using 3-aminopropylphosphonate6 or ZK150471 (Ref. 7) as extracellular pH markers), only one model (a xenograft of human HT29 cells) showed a correlation between pHi and pHe, whereas the other three models (transplanted rodent tumours) showed no correlation between these two parameters14. Does this simply reflect a higher output of hydrogen ions by the HT29 cells or is there usually regulation of pH in the extracellular space that is somehow subverted by the tumour cell? If, like pHi, tumour pHe is regulated, any correlation between these two parameters would depend on factors other than pHi, as was observed in the three rodent tumour models. Under these circumstances, the acidic pHe of tumours could be a consequence of some interaction between the tumour cell and its host-cell matrix. For example, the set point for pHe might be altered at the intersti-
tial–vascular interface (Figs 1 and 2); tumour endothelial cells are known to release growth factors and cytokines that regulate tumour cell function and vice versa17. Human cytokines released by the HT29 xenograft might have been unable to modulate the host (rodent) stromal cells.
Is low pH an intrinsic feature of the cancer phenotype? e
These considerations imply that acidic pHe, a feature of tumours that has been proposed to facilitate tumour progression18, might not be just a consequence of tumour metabolism, but an intrinsic tumour property. For example, tumour cells might change their pHe set point either as a result of tumour gene-expression, for example by overexpression of carbonic anhydrase isozymes caused by inactivation of the VHL tumour suppressor gene19, or as a result of a tumour cell–host-matrix interaction. Low pHe has been
Cytokines – Locally produced proteins that regulate the differentiation, proliferation and activities of cells. Glycolysis – The sequence of metabolic reactions that transforms glucose into pyruvic acid with the concomitant formation of two molecules of ATP from ADP and Pi. Pyruvic acid is subsequently converted to CO2 and water in the mitochondria, or to lactic acid in the cytosol. HIF-1 (hypoxia inducible factor-1) – A transcription factor that can bind to hypoxia response elements and activate gene transcription. It has been implicated in, for example, the regulation of angiogenesis, glucose transport, glucose metabolism and nitric oxide metabolism. Pasteur effect – The inhibition of glycolysis by respiration. Phenotype – The observable characteristics of cells which are the outcome of the interaction between cellular genes (the genotype) and the environment. Xenograft – A graft of living tissue from an animal of one species (e.g. man) into that of a different species (e.g. mouse).
associated with tumourigenic transformation, chromosomal rearrangements, extracellular matrix breakdown, migration and invasion, induction of the expression of cell growth factors and proteases, and it is a prominent feature of a reaction–diffusion model of cancer invasion 18. Characteristics of the cancer phenotype might also reduce the viability of adjacent normal host cells.
Consequences of tumour acidity One consequence of metabolism in any tissue is the formation of H1, which must be removed from the cell if the internal milieu is to maintain its normal pH. In cancer cells, the H1 formed during glycolysis leaves the cell with lactate2, via the 17
Opinion
MOLECULAR MEDICINE TODAY, JANUARY 2000 (VOL. 6)
(5FU) is pH-dependent even though its pK lies outside the physiological range22.
Lower pHe
Higher pHe
5FU RB-6145 Camptothecin Chloramphenicol
Adriamycin Vinblastine Mitoxanthrone
Tumour cell
5FU Higher pHi
Molecular Medicine Today
Figure 3. Modulation of tumour pH to increase uptake of chemotherapeutic drugs. The figure indicates the modulation (decrease or increase) of pH necessary to increase the uptake of the named drugs, which are weak electrolytes21.
monocarboxylate/ H1 co-transporter. In addition, H1 is exported by the Na1/H1 antiporter, using the energy of the Na1 gradient. This antiporter, which is activated in tumour cells, elevates cytosolic Na1, which will subsequently be pumped out by the (ATP-driven) Na1/K1 ATPase. H1 might also be exported by vacuolar type H1 ATPases in the plasma membrane. A decreased Na1 gradient provides less energy for Na1/Ca21 exchange, which could lead to an accumulation of intracellular Ca21. Pi is also increased20 (tumour cells have a lower energy currency than normal tissues, caused in part by the elevated ion pumping), and disruption of both Ca21 and Pi metabolism might trigger tumour
The outstanding questions
• • • • • 18
Are the high rates of glycolysis in tumours determined by changes in gene expression? Does hypoxia-related gene expression determine the tumour metabolic phenotype? Is the extracellular acidity of tumours an intrinsic feature of their metabolic phenotype? Does changing the set point of pHi or pHe cause extracellular acidity? Could hypoxia be a feed-forward activator of invasiveness/metastasis?
calcification, especially if pHi remains slightly alkaline. Tumour calcification is a common feature of tumour pathology, forming the basis of diagnostic tests for cancer, most notably mammography. Dystrophic calcification is also seen in many other chronically injured tissues, such as scars from myocardial infarction, the common link being hypoxia leading to alterations in ion distribution.
What are the implications of tumour extracellular acidity for treatment? Tumour pH gradients have practical importance because most anticancer drugs must be transported either by active transport or by passive diffusion into cells, where they frequently undergo further metabolism. As all of these processes might be pH sensitive, the cytotoxic activity of anticancer drugs could depend on both pHi and pHe. In particular, drugs that are weak electrolytes enter cells by passive diffusion of the non-ionized form of the compound. Such drugs will tend to partition preferentially across the cell membrane into the compartment where their ionized form predominates. Thus, for example, primary amines tend to be excluded from, and carboxylic acids accumulated by, the more alkaline intracellular compartment21 (Fig. 3). In addition, the toxicity of a number of drugs is sensitive to variation in pH as a result of various mechanisms that are not dependent on ionization-dependent diffusion through the cell membrane. For example, melphalan toxicity is enhanced by pHe modification without increased uptake or accumulation, and the accumulation of weakly acidic 5-fluorouracil
Methods and consequences of modifying tumour pH in vivo Various strategies for altering pH i and pHe have been tried in the quest for new anticancer strategies for solid tumours. In this article, only tumours in the steady state have been considered. However, in an acute situation, changes in pH can be induced in vivo; for instance, hyperglycaemia alters tumour pHi and pHe, resulting in an increase in DpH, which increases retention of 5FU. As tumour retention of 5FU appears to depend on the size of the ∆pH, increases in pHi or decreases in pHe can also increase 5FU concentrations in cells in solid tumours. It is hoped that strategies such as these can be exploited to concentrate cytotoxic agents selectively in tumours. In addition, chronic lowering of tumour cell pHi, either by inhibition of the Na1/H1 exchanger with amiloride23, or by inhibition of the Na1-dependent HCO32/Cl2 exchanger with amiloride-analogues, is cytotoxic to isolated tumour cells and inhibits tumour growth24.
Future research approaches Possible approaches to answering some of the questions raised in this article include: (1) determining the in vivo rates of glycolysis in tumour types of differing differentiation status, this could be achieved by using 13C MRS techniques; (2) investigating the location and mechanism of H1 movements between intra- and extracellular compartments – as the pH of the arterial supply and the venous drainage can, in principle, be measured directly, appropriate modelling could allow the present generation of (MR) pHe probes to be used to investigate this; (3) investigating the role of hypoxiarelated gene expression in determining the cancer metabolic phenotype, this information could be obtained from genetically-manipulated cells grown as solid tumours in vivo; (4) determining the control and regulation of the glycolytic pathway exerted by hypoxia-related gene expression, this information could be provided by metabolic control analysis10. By understanding how tumour pH is controlled, we should be able to exploit it to selectively concentrate cytotoxic agents in tumour cells. Acknowledgements. Our work is sponsored by the Cancer Research Campaign (CRC), UK.
References 1 Warburg, O. (1930) The Metabolism Of Tumours, Arnold Constable, London 2 Griffiths, J.R. et al. (1981) 31P-NMR investigation of solid tumours in the living rat. Biosci. Rep. 1, 319–325 3 Vaupel, P. et al. (1989) Blood flow, Oxygen and nutrient supply, the metabolic microenvironment of
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MOLECULAR MEDICINE TODAY, JANUARY 2000 (VOL. 6)
4
5
6
7
8
9
10
11
human tumours: a review. Cancer Res. 49, 6449–6465 Gillies, R.J. et al. (1992) Role of intracellular pH in mammalian cell proliferation, Cell. Physiol. Biochem. 2, 159–179 Stubbs, M. (1998) in Tumour pH, Blood perfusion and Microenvironment of Human tumours: Implications for Clinical Radio-Oncology. Diagnostic Imaging and Radiation Oncology (Molls, M. and Vaupel, P. eds), Vol. 11, pp. 113–120, SpringerVerlag, Berlin Gillies, R.J. et al. (1994) 31P-MRS measurements of extracellular pH of tumours using 3-aminopropylphosphonate. Am. J. Physiol. 267, C 195–203 Frenzel, T. et al. (1994) Non-invasive in vivo measurements using a fluorinated pH probe and fluorine19 magnetic resonance spectroscopy. Invest. Radiol. 29, S220–S222 Weber, G. (1968) Carbohydate metabolism in cancer cells and the molecular correlation concept. Naturwissenschaften 55, 418–429 Davies, S.E. and Brindle, K.M. (1992) Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae. Biochemistry 31, 4729–4735 Fell, D. (1996) in Understanding the control of metabolism. Frontiers in Metabolism (No. 2) (Snell, K. ed.), Portland Press Gleadle, J.M. and Ratcliffe, P.J. (1998) Hypoxia and the regulation of gene expression. Mol. Med. Today 4, 122–129
12 Semenza. G.L. et al. (1997) Structural and functional analysis of hypoxia-inducible factor 1. Kidney Int. 51, 553–555 13 Maxwell, P.H. et al., (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275 14 Stubbs, M. et al. (1999) Causes and consequences of acidic pH in tumours; a magnetic resonance study. Adv. Enzyme Regul. 39, 13–30 15 Newell, K. et al. (1993) Studies with glycolysis-deficient cells suggest that production of lactic acid is not the only cause of tumour acidity. Proc. Natl. Acad. Sci. U. S. A. 90, 1127–1131 16 Yamagata, M. et al. (1998) The contribution of lactic acid to acidification of tumours: studies of variant cells lacking lactate dehydrogenase. Br. J. Cancer 77, 1726–1731 17 Folkman, J. (1996) Tumour angiogenesis and tissue factor. Nat. Med. 2, 167–168 18 Gatenby, R.A. and Gawlinski, E.T. (1996) A reaction-diffusion model of cancer. Cancer Res. 56, 5745–5753 19 Ivanov, S.V. et al. (1998) Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel–Lindau transgenes. Proc. Natl. Acad. Sci. 95, 12596–12601 20 Stubbs, M. et al. (1994) Metabolic consequences of a reversed pH gradient in rat tumours. Cancer Res. 54, 4011–4016 21 Gerweck, L.E. (1998) Tumour pH: implications for
treatment and novel drug design. Semin. Radiat. Oncol. 8, 176–182 22 Ojugo, A.S.E. et al. (1998) Influence of pH on the uptake of 5-Fluorouracil into Lettre ascites tumour cells. Br. J. Cancer 77, 873–879 23 Yamagata, M. and Tannock, I.F. (1996) The chronic administration of drugs that inhibit the regulation of intracellular pH: in vitro and anti-tumour effects. Br. J. Cancer 73, 1328–1334 24 Vukovic, V. and Tannock, I.F. (1997) Influence of low pH on cytotoxicity of paclitaxel, mitoxanthrone and topotecan. Br. J. Cancer, 75, 1167–1172
Marion Stubbs DPhil* Deputy Director Paul M.J. McSheehy PhD Senior Research Fellow John R. Griffiths MB, BS, DPhil Director C. Lindsay Bashford DPhil. Reader in Biochemistry CRC Biomedical MR Research Group, Department of Biochemistry, St. George’s Hospital Medical School, London, UK SW17 0RE. Tel: 144 181 725 5852 Fax: 144 181 725 2992 e-mail: [email protected]
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19
Opinion
Causes and consequences of tumour acidity and implications for treatment Marion Stubbs, Paul M.J. McSheehy, John R. Griffiths and C. Lindsay Bashford
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. In the 1930s, Warburg noted ‘the remarkable extent to which living tumour cells are able to convert carbohydrate into lactic acid’1. Subsequently, for about 50 years, it was assumed that tumour cells would have an acidic intracellular pH (pHi). Microelectrode measurements of tumour pH appeared to confirm that the pH of tumour cells was low. However, when 31P magnetic resonance spectroscopy (MRS) was introduced for the study of tumours in situ2, it provided a simple, noninvasive means of estimating pHi, and tumour cells turned out to have neutral or slightly alkaline pHi values3. Like normal cells, tumour cells regulate their cytoplasmic pHi within a narrow range to provide a favourable environment for various intracellular activities. Indeed, many tumour cells have a high pHi, which is considered to be permissive for cell growth4. In tumours, it is the extracellular fluid that is relatively acid. It was mainly this compartment that the microelectrodes had been sampling, and such measurements are now acknowledged largely to reflect extracellular pH (pHe ). The result is a reverse or negative pH gradient (pHi . pHe) across the tumour-cell plasma membrane in situ compared with normal tissues where pHi (~7.2) is lower than pHe (~7.4) (Ref 5).
pH measurement by MRS pHi The MRS measurement of pHi is based on a pHdependent chemical shift difference between the 31P inorganic phosphate (Pi) signal and an
endogenous reference signal. At physiological pH, the position of the Pi signal reflects the relative concentrations of the two phosphate species (H2PO42 and HPO422) present. There is phosphate in both the intra- and extracellular compartments, so unless two Pi peaks can be resolved in the MRS spectrum, the MRS measurement of tissue pH is a weighted average of pHi and pHe. In normal tissue, the concentration of intra- and extracellular Pi is approximately the same. It is generally considered that the pH measured by MRS is intracellular because the extracellular volume, for example of liver, is less than 25% of the total cell volume and thus extracellular Pi is only a minor component. For tumours, however, this assumption might not apply because, in addition to having higher-than-normal Pi signals, their extracellular volume could also be high, owing to necrotic areas and cysts for example. Estimates of the proportion of Pi signal coming from the intracellular volume can be made if the total tumour volume and the fractional volume of extracellular water are known. Calculations show that if the extracellular volume does not exceed 55% then pH measured by MRS largely represents pHi (Ref. 5).
pHe Recently, several MR-specific extracellular markers for pHe have become available6,7 that allow the simultaneous measurement of pHi and pHe in tumours and normal tissue. Studies using these
1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1357-4310(99)01615-9
probes confirm that tumour pHe in many (but not all) animal models is lower than pHi. However, it should be noted that pHe comprises both the interstitial and vascular compartments, and the latter (pHb) usually has a pH of about 7.4.
Why do tumours have high rates of glycolysis? Many tumours have high rates of glycolysis, regardless of whether their supply of oxygen is good (Warburg’s original observation) or poor. Most tumours in vivo synthesize some ATP by oxidative metabolism, and some by glycolytic metabolism to lactate (aerobic glycolysis). Clearly, if the oxygen supply is removed (acute hypoxia), the tumour cells switch to anaerobic glycolysis, just as would normal tissue. Research on hepatomas has shown that the rate of tumour glycolysis appears to be associated with the differentiation status and growth rate of the tumour8. But the question as to why some of the energy demand is satisfied by aerobic glycolysis, rather than by mitochondrial oxidative phosphorylation, remains unanswered. Several normal cell-types that are not necessarily hypoxic (for example, leukocytes and enterocytes) also have high rates of glycolysis and produce lactic acid. Leukocytes, after many rounds of cell division, have fewer mitochondria than their stem-cell precursors, and some tumour cells contain about half the mitochondria of comparable, untransformed tissue. If there are not enough mitochondria to replace ATP 15
Opinion
MOLECULAR MEDICINE TODAY, JANUARY 2000 (VOL. 6)
pH Extracellular H+ store
H+ pump
6
pHe ~6.8 Intracellular H+ source 7
pHi ~7.2
Tumour Normal
f1
Blood H+ sink
(a) pHe ~7.3
f2
pHb ~7.4
(b) 8 Molecular Medicine Today
Figure 1. Factors affecting pHi and pHe in normal and tumour tissue. H1 produced by metabolism is pumped from the intracellular compartment into the interstitial compartment and subsequently flows into the blood. The concentration of H1 in the interstitial compartment is relatively high in tumours (low pHe ) compared with normal tissue. At steady state, the flow (f) of H1 between the compartments is equal (f1 5 f2). The increased interstitial acidity of tumour cells could be caused by (a) an increase in H1 pumping by, for example, expression in the plasma membrane of vacuolar-type H1 pumps or (b) an increase in resistance induced either by altered gene expression or by the action of cytokines on cells at the interstitial/vascular interface.
at the required rate, enhanced glycolysis would be an obvious metabolic solution, and many tumour cells appear to adopt a mixed metabolic economy of simultaneous oxidative phosphorylation and glycolysis. In most normal cells, the levels of ADP and Pi needed to sustain a high glycolytic rate would be sufficient to maximally activate oxidative phosphorylation. The changes in metabolic control that allow both pathways to be active, but not saturated, in tumours (and mitochondrially deficient normal cells) remain uncertain. The observation that overexpression of key glycolytic enzymes in yeast abolishes the Pasteur effect9 might suggest that the oncogenic events that switch on uncontrolled DNA replication in the nucleus fail to cause an equivalent increase in replication of mitochondrial DNA, and permit overexpression of glycolytic enzymes.
How might the activities of the enzymes participating in glycolysis be raised? Modern metabolic control analysis has shown that the control of metabolic pathways is distributed over many enzymes; if glycolytic flux is increased, many of the glycolytic enzymes are likely to have raised activities10. Recently, a widespread system of oxygen-related gene expression, based on the activation of the transcription factor hypoxia inducible factor-1 (HIF-1), has been defined11. Intriguingly, these molecular genetic approaches 16
have revealed that sequences that are 59 to the coding region of genes for glycolytic enzymes contain a common motif12, and this could provide the basis for coordinated upregulation of the pathway. Chronic hypoxia also upregulates the expression of a number of ‘stress’ proteins, several of which are glycolytic enzymes. For example, the major hypoxic stress protein p34 is an isozyme of lactate dehydrogenase. These findings suggest that hypoxia is a trigger that coordinates induction of gene expression and may well be one of the factors that determines the metabolic phenotype of tumours. Thus, it is possible to imagine that the genetic disruptions that produce tumour cells might also stimulate the HIF-1 system without the need for the hypoxic stimulus. Indeed, HIF-1 is activated constitutively in cells that are defective in an important tumour suppressor, the von Hippel–Lindau gene product13. The modified cells would have the metabolic phenotype associated with tumours and could induce the chaotic vasculature typically found in tumours. Subsequent hypoxia would reinforce this pattern by super-induction of the HIF-1 system12.
Why is pHe of tumours acidic? Does excess lactate production cause extracellular acidity? Following glycolysis, the major pathway of lactate export from cells is the H1-monocarboxylate cotransporter. The steady-state intracellular lactate
concentration of tumour cells tends to be at least twofold higher than the extracellular concentration. This follows from the intimate association between lactate and H1 gradients and the direction of the H1 gradient across the tumour-cell plasma membrane14. One obvious hypothesis to explain the low pHe in solid tumours is that metabolic acids (lactate and/or CO2) exported from the cancer cells into the interstitial fluid cannot be exported to the blood rapidly enough. This poor H1 clearance could be due to the disorganized vasculature of tumours, poor lymphatic drainage and elevated interstitial pressure. However, when a cancer cell line that produced large amounts of lactic acid was compared with a mutant line that had a defective glycolytic pathway and produced very small amounts of lactic acid, solid tumours grown up from both the wild-type and mutant lines still had an acidic interstitial pH (Ref. 15). One possible explanation for this is that the mutant cells had produced large amounts of CO2 by oxidative metabolism, which would also acidify the extracellular compartment. However, other mutants defective in the glycolytic pathway produced similar amounts of CO2 to the normal cells. This implies that the acidity of tumours is not caused simply by excessive production of lactate and CO2 (Ref. 16).
Does changing the set point of pHi regulation cause extracellular acidity? Tumour cells might raise their set point for pHi by increasing the export of H1 from the intracellular compartment4. Increased H1 export could permit faster overall production of acid by tumour metabolism (‘H1 source’ in Fig.1) and lead indirectly to increased extracellular acidity. The increased H1 export could be achieved via activation of the mitogen-sensitive Na1/H1exchanger, or via increased functional expression in the plasma membrane of H1-pumping ATPases. Functional expression of H1-pumping ATPases has been measured on the cell surface of some tumour cells, particularly those with a higher pHi, and it is associated with greater ATP turnover, increased glycolysis and decreased protein degradation4, all features of the tumour metabolic phenotype.
Is pHe regulated and does changing the set point of pHe cause extracellular acidity? The pH of any compartment at steady-state is determined by the balance between H1 entering and leaving that compartment (Fig.1) and the nature of the internal buffers. pHi and pHe correlate strongly in cell culture experiments in which the pH of the medium has been manipulated. However in four rodent models of solid tumours (including one human xenograft) in which pHi
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Glossary
Tumour cell plasma membrane
(a)
Calcification – The deposition of calcium salts in tissue.
pHe ~6.8 H+
Chemical shift – A shift in resonating frequency between molecules that depends on the electron shielding of their chemical bonds. For example, HPO422 resonates at a slightly different frequency to that of HPO42.
(f) Ca2+
pHi ~7.2
Na+
Na+ ADP+Pi ATP
H+
ATP ADP+Pi K+
(e)
(b)
Lactate−
HCO3− Glycolysis Cl−
H+
(c)
Na+ ?
(d)
Endothelial cells Glucose
? (g)
pHb ~7.4 Molecular Medicine Today
Figure 2. Mechanisms involved in maintenance of intracellular neutrality in tumours. When lactic acid is produced from glucose (a non-electrolyte), the lactate ion and H1 can pass into the extracellular fluid via the monocarboxylate carrier (a). Some lactate ions accumulate in the cell and contribute to the high lactate levels commonly observed in tumours. The Na1/H1 antiport (b), which is activated in transformed cells, exports H1 and imports Na1 contributing to the high intracellular Na1, another common tumour-cell feature. There is also some buffering by HCO32, which enters the cell by the Na1 dependent HCO32 /Cl2exchanger (c). There are many additional mechanisms which may also play a role in the regulation of maintenance of neutral pHi including the ATP-dependent Na1/K1 antiport (d) and vacuolar H1-pump (e). The low energy of the Na1 gradient causes Ca21 to accumulate (f) and together with high Pi may initiate tumour calcification. Also shown is a hypothetical exchanger at the tumour–endothelial cell interface (g) which might regulate pHe.
and pHe were measured simultaneously in vivo (using 3-aminopropylphosphonate6 or ZK150471 (Ref. 7) as extracellular pH markers), only one model (a xenograft of human HT29 cells) showed a correlation between pHi and pHe, whereas the other three models (transplanted rodent tumours) showed no correlation between these two parameters14. Does this simply reflect a higher output of hydrogen ions by the HT29 cells or is there usually regulation of pH in the extracellular space that is somehow subverted by the tumour cell? If, like pHi, tumour pHe is regulated, any correlation between these two parameters would depend on factors other than pHi, as was observed in the three rodent tumour models. Under these circumstances, the acidic pHe of tumours could be a consequence of some interaction between the tumour cell and its host-cell matrix. For example, the set point for pHe might be altered at the intersti-
tial–vascular interface (Figs 1 and 2); tumour endothelial cells are known to release growth factors and cytokines that regulate tumour cell function and vice versa17. Human cytokines released by the HT29 xenograft might have been unable to modulate the host (rodent) stromal cells.
Is low pH an intrinsic feature of the cancer phenotype? e
These considerations imply that acidic pHe, a feature of tumours that has been proposed to facilitate tumour progression18, might not be just a consequence of tumour metabolism, but an intrinsic tumour property. For example, tumour cells might change their pHe set point either as a result of tumour gene-expression, for example by overexpression of carbonic anhydrase isozymes caused by inactivation of the VHL tumour suppressor gene19, or as a result of a tumour cell–host-matrix interaction. Low pHe has been
Cytokines – Locally produced proteins that regulate the differentiation, proliferation and activities of cells. Glycolysis – The sequence of metabolic reactions that transforms glucose into pyruvic acid with the concomitant formation of two molecules of ATP from ADP and Pi. Pyruvic acid is subsequently converted to CO2 and water in the mitochondria, or to lactic acid in the cytosol. HIF-1 (hypoxia inducible factor-1) – A transcription factor that can bind to hypoxia response elements and activate gene transcription. It has been implicated in, for example, the regulation of angiogenesis, glucose transport, glucose metabolism and nitric oxide metabolism. Pasteur effect – The inhibition of glycolysis by respiration. Phenotype – The observable characteristics of cells which are the outcome of the interaction between cellular genes (the genotype) and the environment. Xenograft – A graft of living tissue from an animal of one species (e.g. man) into that of a different species (e.g. mouse).
associated with tumourigenic transformation, chromosomal rearrangements, extracellular matrix breakdown, migration and invasion, induction of the expression of cell growth factors and proteases, and it is a prominent feature of a reaction–diffusion model of cancer invasion 18. Characteristics of the cancer phenotype might also reduce the viability of adjacent normal host cells.
Consequences of tumour acidity One consequence of metabolism in any tissue is the formation of H1, which must be removed from the cell if the internal milieu is to maintain its normal pH. In cancer cells, the H1 formed during glycolysis leaves the cell with lactate2, via the 17
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(5FU) is pH-dependent even though its pK lies outside the physiological range22.
Lower pHe
Higher pHe
5FU RB-6145 Camptothecin Chloramphenicol
Adriamycin Vinblastine Mitoxanthrone
Tumour cell
5FU Higher pHi
Molecular Medicine Today
Figure 3. Modulation of tumour pH to increase uptake of chemotherapeutic drugs. The figure indicates the modulation (decrease or increase) of pH necessary to increase the uptake of the named drugs, which are weak electrolytes21.
monocarboxylate/ H1 co-transporter. In addition, H1 is exported by the Na1/H1 antiporter, using the energy of the Na1 gradient. This antiporter, which is activated in tumour cells, elevates cytosolic Na1, which will subsequently be pumped out by the (ATP-driven) Na1/K1 ATPase. H1 might also be exported by vacuolar type H1 ATPases in the plasma membrane. A decreased Na1 gradient provides less energy for Na1/Ca21 exchange, which could lead to an accumulation of intracellular Ca21. Pi is also increased20 (tumour cells have a lower energy currency than normal tissues, caused in part by the elevated ion pumping), and disruption of both Ca21 and Pi metabolism might trigger tumour
The outstanding questions
• • • • • 18
Are the high rates of glycolysis in tumours determined by changes in gene expression? Does hypoxia-related gene expression determine the tumour metabolic phenotype? Is the extracellular acidity of tumours an intrinsic feature of their metabolic phenotype? Does changing the set point of pHi or pHe cause extracellular acidity? Could hypoxia be a feed-forward activator of invasiveness/metastasis?
calcification, especially if pHi remains slightly alkaline. Tumour calcification is a common feature of tumour pathology, forming the basis of diagnostic tests for cancer, most notably mammography. Dystrophic calcification is also seen in many other chronically injured tissues, such as scars from myocardial infarction, the common link being hypoxia leading to alterations in ion distribution.
What are the implications of tumour extracellular acidity for treatment? Tumour pH gradients have practical importance because most anticancer drugs must be transported either by active transport or by passive diffusion into cells, where they frequently undergo further metabolism. As all of these processes might be pH sensitive, the cytotoxic activity of anticancer drugs could depend on both pHi and pHe. In particular, drugs that are weak electrolytes enter cells by passive diffusion of the non-ionized form of the compound. Such drugs will tend to partition preferentially across the cell membrane into the compartment where their ionized form predominates. Thus, for example, primary amines tend to be excluded from, and carboxylic acids accumulated by, the more alkaline intracellular compartment21 (Fig. 3). In addition, the toxicity of a number of drugs is sensitive to variation in pH as a result of various mechanisms that are not dependent on ionization-dependent diffusion through the cell membrane. For example, melphalan toxicity is enhanced by pHe modification without increased uptake or accumulation, and the accumulation of weakly acidic 5-fluorouracil
Methods and consequences of modifying tumour pH in vivo Various strategies for altering pH i and pHe have been tried in the quest for new anticancer strategies for solid tumours. In this article, only tumours in the steady state have been considered. However, in an acute situation, changes in pH can be induced in vivo; for instance, hyperglycaemia alters tumour pHi and pHe, resulting in an increase in DpH, which increases retention of 5FU. As tumour retention of 5FU appears to depend on the size of the ∆pH, increases in pHi or decreases in pHe can also increase 5FU concentrations in cells in solid tumours. It is hoped that strategies such as these can be exploited to concentrate cytotoxic agents selectively in tumours. In addition, chronic lowering of tumour cell pHi, either by inhibition of the Na1/H1 exchanger with amiloride23, or by inhibition of the Na1-dependent HCO32/Cl2 exchanger with amiloride-analogues, is cytotoxic to isolated tumour cells and inhibits tumour growth24.
Future research approaches Possible approaches to answering some of the questions raised in this article include: (1) determining the in vivo rates of glycolysis in tumour types of differing differentiation status, this could be achieved by using 13C MRS techniques; (2) investigating the location and mechanism of H1 movements between intra- and extracellular compartments – as the pH of the arterial supply and the venous drainage can, in principle, be measured directly, appropriate modelling could allow the present generation of (MR) pHe probes to be used to investigate this; (3) investigating the role of hypoxiarelated gene expression in determining the cancer metabolic phenotype, this information could be obtained from genetically-manipulated cells grown as solid tumours in vivo; (4) determining the control and regulation of the glycolytic pathway exerted by hypoxia-related gene expression, this information could be provided by metabolic control analysis10. By understanding how tumour pH is controlled, we should be able to exploit it to selectively concentrate cytotoxic agents in tumour cells. Acknowledgements. Our work is sponsored by the Cancer Research Campaign (CRC), UK.
References 1 Warburg, O. (1930) The Metabolism Of Tumours, Arnold Constable, London 2 Griffiths, J.R. et al. (1981) 31P-NMR investigation of solid tumours in the living rat. Biosci. Rep. 1, 319–325 3 Vaupel, P. et al. (1989) Blood flow, Oxygen and nutrient supply, the metabolic microenvironment of
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human tumours: a review. Cancer Res. 49, 6449–6465 Gillies, R.J. et al. (1992) Role of intracellular pH in mammalian cell proliferation, Cell. Physiol. Biochem. 2, 159–179 Stubbs, M. (1998) in Tumour pH, Blood perfusion and Microenvironment of Human tumours: Implications for Clinical Radio-Oncology. Diagnostic Imaging and Radiation Oncology (Molls, M. and Vaupel, P. eds), Vol. 11, pp. 113–120, SpringerVerlag, Berlin Gillies, R.J. et al. (1994) 31P-MRS measurements of extracellular pH of tumours using 3-aminopropylphosphonate. Am. J. Physiol. 267, C 195–203 Frenzel, T. et al. (1994) Non-invasive in vivo measurements using a fluorinated pH probe and fluorine19 magnetic resonance spectroscopy. Invest. Radiol. 29, S220–S222 Weber, G. (1968) Carbohydate metabolism in cancer cells and the molecular correlation concept. Naturwissenschaften 55, 418–429 Davies, S.E. and Brindle, K.M. (1992) Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae. Biochemistry 31, 4729–4735 Fell, D. (1996) in Understanding the control of metabolism. Frontiers in Metabolism (No. 2) (Snell, K. ed.), Portland Press Gleadle, J.M. and Ratcliffe, P.J. (1998) Hypoxia and the regulation of gene expression. Mol. Med. Today 4, 122–129
12 Semenza. G.L. et al. (1997) Structural and functional analysis of hypoxia-inducible factor 1. Kidney Int. 51, 553–555 13 Maxwell, P.H. et al., (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275 14 Stubbs, M. et al. (1999) Causes and consequences of acidic pH in tumours; a magnetic resonance study. Adv. Enzyme Regul. 39, 13–30 15 Newell, K. et al. (1993) Studies with glycolysis-deficient cells suggest that production of lactic acid is not the only cause of tumour acidity. Proc. Natl. Acad. Sci. U. S. A. 90, 1127–1131 16 Yamagata, M. et al. (1998) The contribution of lactic acid to acidification of tumours: studies of variant cells lacking lactate dehydrogenase. Br. J. Cancer 77, 1726–1731 17 Folkman, J. (1996) Tumour angiogenesis and tissue factor. Nat. Med. 2, 167–168 18 Gatenby, R.A. and Gawlinski, E.T. (1996) A reaction-diffusion model of cancer. Cancer Res. 56, 5745–5753 19 Ivanov, S.V. et al. (1998) Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel–Lindau transgenes. Proc. Natl. Acad. Sci. 95, 12596–12601 20 Stubbs, M. et al. (1994) Metabolic consequences of a reversed pH gradient in rat tumours. Cancer Res. 54, 4011–4016 21 Gerweck, L.E. (1998) Tumour pH: implications for
treatment and novel drug design. Semin. Radiat. Oncol. 8, 176–182 22 Ojugo, A.S.E. et al. (1998) Influence of pH on the uptake of 5-Fluorouracil into Lettre ascites tumour cells. Br. J. Cancer 77, 873–879 23 Yamagata, M. and Tannock, I.F. (1996) The chronic administration of drugs that inhibit the regulation of intracellular pH: in vitro and anti-tumour effects. Br. J. Cancer 73, 1328–1334 24 Vukovic, V. and Tannock, I.F. (1997) Influence of low pH on cytotoxicity of paclitaxel, mitoxanthrone and topotecan. Br. J. Cancer, 75, 1167–1172
Marion Stubbs DPhil* Deputy Director Paul M.J. McSheehy PhD Senior Research Fellow John R. Griffiths MB, BS, DPhil Director C. Lindsay Bashford DPhil. Reader in Biochemistry CRC Biomedical MR Research Group, Department of Biochemistry, St. George’s Hospital Medical School, London, UK SW17 0RE. Tel: 144 181 725 5852 Fax: 144 181 725 2992 e-mail: [email protected]
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