cancer cachexia and gluconeogenesis

CANCER CACHEXIA A N D GLUCONEOGENESIS

Joseph Gold Syrucuse Cancer Research Institute Inc

Presidential Plaza Syracuse, New York 13202

Cachexia has long been recognized as a leading cause of death in cancer.’ Though this condition has been thought by some to be the result of a progressively inadequate dietary intake, it is obviously not so simple; many patients present initially with significant weight loss as their first and only symptom in the face of a completely adequate dietary intake. This suggests a specific mechanism apparently peculiar to the cancerous condition by which weight loss and body debilitation can proceed.

Such a thermodynamic mechanism for the production of cancer cachexia has been described by this author.2 It was shown that in cancer patients significant and progressive energy loss from host (i.e., noncancerous) tissues could occur by virtue of the establishment of a systemic energy-losing cycle dependent on an interplay of tumor glycolysis and host gluconeogenesis.

CARBOHYDRATE DEGRADATION IN CANCER TISSUES Before proceeding, it would be of value to review the fate of carbohydrate in

the body with respect to energy-production. Carbohydrate (glucose) is normally degraded via the Embden-Meyerhof pathway to yield a C3 fragment. pyruvate, that either proceeds to lactic acid (glycolysis) or enters the tricarboxylic acid cycle (TCA) (respiration) as acetate and is oxidized to carbon dioxide and water. The former pathway, anaerobic glycolysis, yields a net formation of two ATP molecules from every glucose molecule; the latter pathway, oxidative respiration, yields a net 15 ATP molecules from each molecule of pyruvate or a total of 30 additional A T P molecules from each original glucose molecule. Thus, oxidative respiration is an efficient energy production pathway, whereas glycolysis is a relatively inefficient pathway.

Cancers, in general, show high rates of anaerobic glycolysis. This increased capacity for glycolysis has been established from diverse3 experimental sources and is considered an outstanding metabolic characteristic of cancer cells. What is the significance of glycolysis in cancer? Research attention has generally adhered to the view that glycolysis is a primary means of energy production in tumors-or that it is the only means of energy production in tumors. This belief has been adhered to simply because of its “self-evidence,” with the implication that to interfere with glycolysis is to impair tumor growth. The problem is that clinically this does not work. Other vital normal tissues, such as brain, red blood cells, and skeletal muscles, apparently depend on glycolysis for a portion of their normal energy supply, and systemic interference with glycolysis is likely to result in more harm to these tissues than to the cancer. Thus. fluoroacetate. a glycolytic poison, causes serious central nervous system disorders before significantly inhibiting the tumor,“ and 2-deoxyglucose, though possessing the ability to promote tumor inhibition, has limiting side effects that rule out general clinical use.5 Virtually every effort that has attempted to exploit the finding of increased glycolysis in cancer has failed clinically. The problem of glycolysis in cancer has re- mained an enigma.

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104 Annals New York Academy of Sciences

Enigmas are often the product, however, of our inability to see beyond what is self-evident. If an unusual or outstanding feature of any disease cannot be clinically exploited, this usually indicates that either it is not a distinctive feature or that its complete significance has not been perceived. For glycolysis. therefore, we must ask: “What use, other than energy production, could glycolysis have to the tumor? Is there any other distinguishing feature of glycolysis? Indeed. glycolysis has a unique metabolic endpoint: namely, lactic acid. Although some of this lactate can be metabolized to COz and water locally, ample experimental evidence has shown that much of this lactate spills into the blood.6-10 Could this excess lactic acid production, a by-product of tumor glycolysis, be of importance to the cancerous process? Can the significance of glycolysis in cancer be related to the metabolic fate of this lactic acid production?

GLUCONE~GENBSIS AND CANCER A clarification of the above questions requires an understanding of gluconeo-

genesis. This is the process by which glucose and other hexose derivatives are synthesized in the liver and kidney cortex from noncarbohydrate sources that contain less than six carbon atoms. These noncarbohydrate precursors include lactic acid, amino acids (usually as breakdown products of protein), citrate, succinate, propionate, oxalacetate, and others. Gluconeogenesis is not normally a quantitatively important pathway but can become so under various physio- pathological Conditions, such as: I . a low carbohydrate diet, or fasting, which markedly stimulates gluconeogenesis; 2. heavy work or exercise, which results in increased lactic acid’in the muscles, most of which is reconverted to glucose; 3. increased secretion of glucocortiooids (hydrocortisone and corticosterone) from the adrenal cortex, which causes a rapid mobilization of protein for gluconeogenesis; 4. diabetes mellitus, which is accompanied by an abnormally high rate of conversion of protein to carbohydrates; other mechanisms may exist. Thus, under abnormal or unusual body conditions, gluconeogenesis can represent a major biosynthetic pathway capable of synthesizing up to 200 g or more of glucose per day in the adult,ll a figure in excess of the total minimum daily body requirements.

Lactic acid occupies a fundamental position in gluconeogenesis. Even with the body in the resting basal state, lactate constitutes an important fraction of those precursors that act as substrates for gluconeogenesis. For instance, in the normal resting adult, 15% of glucose formed via gluconeogenesis originates from lactic acid, which is derived from the metabolism of brain and peripheral muscula- ture.12Ja In this “recycling” of lactic acid, the glucose formed from lactic acid is not really “new glucose,” because the lactate originally derived from glucose. Thus, lactic acid is recycled to glucose, as described originally by Meyerhof and by Cori and Cori in 1928, and this specific recycling is referred to as the “Cori cycle.”14 The Cori cycle assumes increased significance in cancer, because sub- stantial amounts of lactate formed as a result of tumor glycolysis find their way into the blood; there is also evidence that lactate may exert a facilitory action on gluconeogenesis, as inhibiting concentrations of intermediates along the gluconeogenitic pathway do not occur when lactate is a substrate. Thus, Reichard and colleagues15 demonstrated that the value of the Cori cycle (recycled lactate) in less advanced cancer patients was two to four times greater than in patients who suffer from other diseases. In one case of metastatic carcinoma of the cervix, the absolute value of this recycling of lactate to glucose was 68 mg glucose/kg body weight/hr (or approximately 114/day, based on a 70-kg person); in a

Gold: Cachexia & Gluconeogenesis 105

patient with a diffuse lymphoma, the absolute value was 89 mg/kg/hr (or approximately 150 grams of glucose from lactate per day). These results are corroborated by earlier work with tumor-bearing animals.16 It was concluded by Reichard and coworkers that “essentially all or most of the lactate that enters the blood is resynthesized to glucose.” This presents a most unusual metabolic situation in cancer patients: namely, there is evidently an obligatory conversion of lactate to glucose, with little, if any, of the lactate finding its way into the urine’’ or undergoing oxidation in the tricarboxylic acid (TCA) cycle.

The above figures represent recycled lactate only. No figures are yet available on the quantity of new glucose formed via gluconeogenesis from amino acid precursors. It is estimated, however, that these figures must be equally as high or higher than lactate, because it is known in cancer patients that the rate of protein mobilization to amino acid, under the influence of increased glucocorticoids, is high.

MECHANISM OF CACHEXIA

In cancer patients therefore a situation exists in which high rates of glycolysis and protein mobilization yield increased amounts of lactic acid and other precursors, and a high rate of gluconeogenesis converts these substances back to glucose. The unique significance of the cycle, however, is that these two processes do no? occur in the same tissues to any extent. Although both processes share many of the same enzyme systems, conditions for their optimal reaction rates in each direction are quite different.” Thus, liver and kidney cortex, which display high gluconeogenesis, display low glycolysis, and malignancies that commonly display a high rate of glycolysis exhibit a low rate of gluconeogenesis. The metabolic implications of this situation are sufficient to account for a cachexic mechanism; in the anaerobic breakdown of glucose to lactic acid in tumors, a net two ATP molecules per glucose molecule is yielded to the tumor, but synthesis of glucose from the resulting lactic acid (or from any other precursor) via gluconeogenesis requires the utilization of the equivalent of at least six ATP molecules derived from normal host sources, In the Cori cycle per se, the equivalent of at least 14 ATP molecules are therefore “lost” to the body economy with each specific recycling (based on two equivalents of “lactate” being recycled to glucose), tw.0 ATP molecules to the cancer cell and 12 ATP molecules from normal host tissues. As the tumor enlarges, consuming ever-increasing amounts of glucose, vast energy reserves from the host can be depleted in maintaining the circuit of glucose presentation to the malignant cell, reconversion of lactic acid and other precursors to glucose in host cells, and re-presentation of the resulting glucose to the cancer cell. This systemic “metabolic circuit” (FIGURE 1 ) , which is character- ized by a malignancy’s utilization of glucose to produce relatively small amounts of energy for its own needs at the expense of relatively large amounts of energy from the host, gives every indication of being an operational biochemical mechanism for the production of cancer cachexia. The only thermodynamic prerequi- site for this metabolic circuit to actually function is that lactic acid from the glycolyzing tumor enter the blood.

The functional significance of glycolysis in cancer appears therefore to be twofold: as a source of energy production (growth) for the tumor and as a source of lactate that initiates a progressive energy loss (cachexia) in the host through marked stimulation of gluconeogenesis. Inhibition of gluconeogenesis thus suggests itself not only as a means of inhibiting this energy loss and the resulting cachexia, but also, if tumor energy gain and host energy loss are functionally

106 Annals New York Academy of Sciences

T G L U C O S t 2 ATP (Malignant tissue) (L iver kidney -6 ATP

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/p r(

Amino acids, /‘ LACTATE TCA cycle

intermediates

Ficuw, 1. Schematic representation of proposed systemic metabolic circuit responsible for energy loss in cancer.

interrelated, as seems probable, as a possible means of inhibiting tumor growth itself.

INHIBITION OF GLUCONEOGENESIS Gluconeogenesis is an approximate reversal of the process of glycolysis (FIGURE

2) . Important differences in their biochemical pathways do exist, though. Thus, in glycolysis, phosphoenolpyruvate (PEP) proceeds directly to pyruvate, whereas in gluconeogenesis, pyruvate must first go through oxalacetate before proceeding to PEP. This is an important difference, because it yields the possibility of imposing a selective metabolic block against gluconeogenesis-a block that has no effect on glycolysis, an important therapeutic consideration, as has already been discussed. For instance, the gluconeogenitic enzymes that convert pyruvate to oxalacetate (pyruvate carboxylase) or oxalacetate to PEP (phosphoenolpyruvate carboxykinase) can be inhibited without inhibiting the glycolytic enzyme that converts PEP to pyruvate (pyruvate kinase). A selective inhibition of gluconeogenesis from lactate alone can be achieved by blocking either pyruvate carboxylase or phosphoenolpyruvate carboxykinase (PEP CK) ; however, because substances other than lactate enter the gluconeogenitic pathway, and thus impose their energy drain on the host, it would be advantageous to be able to block these, too. FIGURE 2 depicts the point of entry of most of these latter substances at the oxalacetate stage. Therefore, by instead inhibiting the conversion of oxalacetate to PEP, that is, by inhibiting the enzyme PEP CK, it is possible to inhibit virtually all substances from becoming glucose via gluconeogenesis and thus to inhibit the attendant host energy loss caused by this process.

SUMMARY OF EXPERIMENTS Known inhibitors of the enzyme PEP CK were tested against the in vivo

growth of several tumor types in an attempt to elicit a beneficial response in the host. Procedural techniques have been described elsewhere.18 L-Tryptophan, hydrazine sulfate, and pyridine-2,3-dihydrazide, which represent three distinct classes of PEP CK inhibitors, were found to produce significant tumor inhibition and extension of survival time in these animals; experimental data with the Walker carcinosarcoma are presented in TABLES 1 and 2 for illustrative purposes. Antitumor response ranged from moderate (L-tryptophan) to extremely potent (hydrazine sulfate and pyridine-2,3-dihydrazide), and toxicity was manifested in weight loss’alone (no mortality at indicated dosages). Similar results have been obtained** with B-16 melanoma (solid), Murphy-Sturm lymphosarcoma, and, to a limited degree, with L-1210 leukemia (solid). (Further tumor types, in


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108 Annals New York Academy of Sciences Lactate

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TABLE 2 EFFECT OF HYDRAZINE SULFATE ON SURVIVAL OF WALKER 256 ASCITES CARCINOSARCOMA IN RATS

Survival Res onse (days) MST (MJTPT') T/C

Dosage (mg/ks)

60 21.0 233 40 21.0 233 25 14.0 155

Controls 9.0 -

* Mean survival time.

TABLE 3 INHIBITION OF GLUCONEOGENESIS IN THE ISOLATED PERFUSED RAT LIVER B Y L-TRYPTOPHAN,

HYDRAZINE SULFATE, AND PYRIDINE-2,3-DIHYDRAZlDE

Test Compound Micromoles of Glucose/lOO g Body Weight/hr

L-Tryptophan (2.4 mM) 0 Hydrazine sulfate (2.4 mM) 0 Pyridine-2,3-dihydrazide (2.4 mM) 0 Control (lactate, 10 mh4) 120

TABLE 4 EFFECT OF HYDRAZINE SULFATE, PYRIDINE-2,3-DIHYDRAZIDE, A N D L-TRYPTOPHAN ON

HELA CELLS IN CULTURE

Test Compound TDm (pglml)

Hydrazine sulfate Pyridine-2.3-dihydrazide L-Tryptophan

51 133

> 1000


addition to other anti-PEP CK or antipyruvate carboxylase agents, are currently undergoing testing.)

These same three inhibitors of the enzyme PEP CK were next shown to be inhibitors of gluconeogenesis in organ systems, in this instance in the isolated, perfused rat liver. The results are presented in TABLE 3; previously performed crossover-type studies have established that the enzymic site of inhibition is at PEP CK.19.20 Thus, an initial experimental link can be made between inhibition of gluconeogenesis at the PEP CK reaction and beneficial host response in tumor- bearing animals.

In validating this approach, it was next necessary to demonstrate that the three above substances did not achieve their results by direct cytotoxicity against cancer cells. These substances were therefore tested against HeLa cell growth in culture; the HeLa dilution protein assay was employed.21 The results are given in TABLE 4. In this assay system, the TD50 of a direct cytotoxic agent might be expected to be of the order of 0.1 pg/ml, so that hydrazine sulfate (TD50 = 51), pyridine-2,3- dihydrazide (TD50 = 133), and L-tryptophan (TDS0 > 1000) would have to he considered 500, 1300, and over 10,000 times less active, respectively, than a direct cytotoxic agent. Based on these data, it would be extremely difficult to ascribe the potent antitumor effects of these compounds to direct cellular cytotoxicity. Although the body might convert one of these compounds to an actively cytotoxic form, as in the case of cyclophosphamide, the chances that this occurs in all three compounds are remote. Thus, TABLE 4 demonstrates that hydrazine sulfate, pyridine-2,3-dihydraide and L-tryptophan have virtually no direct cytotoxicity on cancer cells, which therefore reinforces their proposed mechanism of action as inhibitors of gluconeogenesis at the PEP CK reaction.

TABLE 5 represents a summary of the above experimental findings, which cor- relates beneficial host response or antitumor activity with PEP CK inhibition and inhibition of gluconeogenesis at the PEP CK reaction in the face of no direct cytotoxic action on cancer cells by the agents used.

TABLE 5 CORRELATION OF in V i m AND in Vivo PROPERTIES OF PEP CK INHIBITORS

W I ~ H THEIR EFFECTS ON in Vivo TUMOR GROWTH AM) DIRECT CYTOTOXICITY

Test Compound

Inhibition of Gluconeo enesis in In Vtvo Direct

Inhibition Rat Liver Inhibition toxicity In Vitro PEP CK Isolated kerfused Tumor cyto-

Hydrazine sulfate yes yes yes no Pyridine-2.3-dihydrazide yes yes yes no L-Tuptophm yes (metabolite) yes yes no

CONCLUSIONS L-Tryptophan, hydrazine sulfate, and pyridine-2,3-dihydrazide serve to illus-

trate that there does indeed appear to be a causal relationship between gluconeogenesis inhibition at the PEP CK reaction and beneficial host response in tumor- bearing animals. The data are further consistent with the overall projected mechanism of action being an inhibition of host energy loss (cachexia) caused by a greatly augmented pathway of gluconeogenesis in cancer. Although the actual

110 Annals New York Academy of Sciences mechanism of tumor inhibition per se is not yet known, a new approach to cancer chemotherapy is nonetheless indicated, based on inhibition of gluconeogenesis at the PEP CK reaction.

ACKNOWLEDGMENTS I wish to thank Dr. Henry A. Lardy, Enzyme Institute, University of Wiscon-

sin, Madison, Wisconsin, for his valuable help in the performance of the isolated, perfused rat liver studies presented here. Thanks are also due to Dr. William T. Bradner, Bristol Laboratories, Syracuse, New York, for performance of the HeLa cell culture studies.

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cancer cachexia and gluconeogenesis

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