The Effect of 2-Desoxy-D-glucose on Glycolysis and Respiration of Tumor and Normal Tissues
GLADYSE. WOODWARDANDMARIET. HUDSON
(Biochemical Research Foundation, Newark, Delaware)
Inhibition of metabolism by structural analogs end of each experiment. Reaction rates are expressed of metabolites is one of the newer concepts of of dry tissue/hour, and are based on the initial steady rate. chemotherapy. It seems possible that this concept The symbols, QCOJ.Qco2>an
Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1954 American Association for Cancer Research. TABLE 1 INHIBITION OF GLUCOLYSISOFRAT TUMORSLICESBY2DG (Glucose concentration, 0.015 M) GLDCOLTSISRATE GlucoseTonos»FJFJW256FJFJFJW256FJW256FJFJW256W256IDGCONC.U0.0150.00750.003750.0150.00750.003750.0150.00750.003750.00750.00750.003750.0020.0010.0010.0150.0150.0150.0075+2DG Glucose2DG:GLDCO8EBATIÓ1:1.5:1.25:11:1.5:1.25:11:1.5:1.25:1.5:1.5:1.25:1.133:1.067:1.067:11:11:11:1.5:1addrd added<&10.017.624.67.412.417.87.418.225.010.312.823.432.223.728.6Qco,6.74.89.815.2o1*'Qco,(anaerobic)31.031.023.627.132.432.042.727.629.3(aerobic)20.512.124.622.7NoadditionQNl2.05.25.44.03.53.84.52.53.8Qco,-1.3-1.5-3.00INHIBITIONorOLOCOLTSIS(per cent)724«i*ill7251893007808SO2715363545433
* FJ —Flexner-Joblingcarcinoma. W256—Walker 256 carcinoma.
TABLE 2 REVERSALOF2DG INHIBITIONOFANAEROBICGLUCOLYSISOFRATTUMOR SLICESBYINCREASEDGLUCOSE
<; r I . i .1.1.-IM BATI
TUMOB*FJW256W256FJFJW256GLUCOSECONC.M0.0150.030.060.0150.030.060.0150.030.0450.060.0150.030.060.0150.030.060.0150.030.06Glucose+2DGaddedONzyco¡8.418.018.411.cent)7100556007447372578482064412838110 2DG)17.220.133.2(with0.015 H
S10.421.411.819.822.626.08.618.625.210.215.618.627.638.448.6GlucoseaddedQN««cot(with
0.0075 H2DG)30.825.042.9NoadditionQ&4.84.04.42.42.03.0INHIBITIONorGLOCOLTSIS(per
*For abbreviations, see footnote to Table 1.
Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1954 American Association for Cancer Research. WOODWARDANDHUDSON—2-Desoxy-D-glucose and Tissue Metabolism 601 inhibited by 2DG (Table 3). A concentration of Brain.—Brain tissue, which can glycolyze glu 0.06 Mfructose was used in these studies, since this cose at a considerable rate anaerobically, although concentration was found necessary for maximum not aerobically (7), was chosen as a normal tissue fructolysis. In this medium, 2 DG in concentra upon which to test the effect of 2DG on glucolysis. tions of 0.0005 M or higher produced inhibition. The data in Table 4 show that the anaerobic glu On the basis of the 2DG ¡fructoseand 2DG ¡glucose colysis of brain slices was very susceptible to in ratios required for equal inhibition, it is apparent hibition by 2DG. With brain tissue, as with tu that fructolysis is at least 50 times more sensitive mor tissue, the extent of the inhibition produced to 2DG than is glucolysis. by 2DG at any one concentration varied from
TABLE 3 INHIBITIONOFANAEROBICFRUCTOLYSISOFRATTUMORSLICESBY2DG (Fructose concentration, 0.06 M) FBDCTOLTSIBHATE
FRUCTOSERATIO0. TUMOR*FJFJFJW256FJ«DGCONC.M0.0150.00750.003750.003750.0018750.0010.0010.00050.0010.00050.000250.0010.000250.000062DG:cent)10010010082878679488083207520 25:10.125:10.062:10.062:10.031:10.016:10.016:10.008:10.016:10.008:10.004:10.016:10.004:10.001:1Fructose+ÕDGaddedQCÔ4.84.87.26.05.25.48.010.87.67.215.97.819.420.6Fructoseadded«S,18.818.815.318.619.8NoadditionOS,7.23.26.04.88.7INHIBITIONOFFRUCTOLYSII(per
* For abbreviations, see footnote to Table 1.
TABLE 4 INHIBITIONOFANAEROBICGLUCOLYSISOFRATBRAINSLICESBY2DG (Glucose concentration, 0.015 M) GLUCOLYBIBBATE
RATUSEDAS SOURCEOF :OLUCOSERATIO0.25:10.25:10.125:10.125:10.067:10.125:10.067:10.125:10.067:10.033:10.017:10.067:10.067:10.033:10.017:1Glucose+*DGadded0N,^COJ1.72.04.113.213.85.45.73.35.810.610.87.66.46.312.0GlucoseaddedQNl12.111.99.911.710.810.013.49.49.79.511.6NoadditionQCO.1.41.68.84.22.71.53.21.21.91.52.7INHIBITIONorGLUCOLYBIS(per TISSUE*W256-bearingNormalFJ-bearingW256-bearingW256-bearingNormalFJ-bearingFJ-bearingNormalFJ-bearingFJ-bearing«DGCONC.If0.003750.003750.0018750.0018750.0010.0018750.0010.0018750.0010.00050.000250.0010.0010.00050.000252DGcent)979588006763797528252242400
For abbreviations, see footnote to Table 1.
Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1954 American Association for Cancer Research. 602 Cancer Research
animal to animal. The variation, however, did not therefore, at least 100 times less sensitive to 2DG appear to depend upon whether the brain tissue than is tumor fructolysis. was from a normal or tumor-bearing rat. It should be noted that the brain slices from one Walker 256- EFFECTOF2DG ONRESPIRATION bearing rat (fourth rat, Table 4) were entirely re 2DG did not significantly affect the endogenous sistant to 2DG in concentrations at which a high respiration of rat tissues, as shown in Table 8. The degree of inhibition was produced with the brain respiration of tumor, liver, and diaphragm is de slices from other rats. pendent mainly upon endogenous material. With 2DG at a concentration of 0.001 M in the pres these tissues, Qo, in the presence of added glucose ence of 0.015 M glucose produced an average in was no greater than in the absence of glucose. In hibition of about 50 per cent with brain tissue, fact, the addition of glucose, as had been observed whereas with tumor tissue 2DG at a concentra by many workers, at times brought about a slight tion of 0.0075 M was required for a similar extent lowering of Qo,. The slight lowering of Qo, in the of inhibition. The anaerobic glucolysis of brain presence of added 2DG is of a magnitude similar tissue appears, therefore, to be about 7.5 times to that produced by glucose, and is therefore not more sensitive to 2DG than that of tumor tissue. to be attributed to inhibition of respiration by Reversal of the inhibition was produced by in 2DG per se. creasing the glucose concentration (Table 5). The With brain tissue, the endogenous respiration inhibition with brain tissue, therefore, appears to is very low, but there is considerable oxidation of be of the competitive type as it was with tumor added glucose or fructose. 2DG in a 1:1 ratio to tissue. glucose did not appreciably inhibit the oxidation Diaphragm.—Since glucolysis of diaphragm of added glucose by brain tissue, but when the can be measured without slicing the tissue, the ratio was increased to 4:1, inhibition of about 50 effect of 2DG on this tissue was studied. It was per cent was produced. Since a 2DG ¡glucoseratio found that the anaerobic glucolysis of diaphragm of only about 0.067:1 was required for 50 per cent was also inhibited by 2DG (Table 6). Although inhibition of anaerobic glucolysis (Table 4), brain the results varied more with different samples of respiration in the presence of added glucose is diaphragm than with different samples of tumor about 60 times less sensitive to 2DG than is brain or brain, the susceptibility of diaphragm to inhibi glucolysis. Fructose oxidation by brain tissue, on tion appeared to be intermediate between that of the other hand, was inhibited by 2DG in a very tumor and brain. small proportion to fructose (Table 8). Since a Liver.—The anaerobic utilization of added glu 2DG¡fructose ratio of 0.033:1 was required for cose by liver slices is so small (4, 7) that measure about 50 per cent inhibition of fructose oxidation, ment of the effect of 2DG on liver glucolysis would the inhibitory effect of 2DG on the oxidative fruc be practically impossible. However, anaerobic tose metabolism of brain is about 100 times great utilization of added fructose is appreciable (4). er than on the oxidative glucose metabolism. The anaerobic fructolysis system was used, there fore, to test the effect of 2DG on liver metabolism. DISCUSSION Since 0.06 M fructose was required to give a maxi The experimental results show that the anaero mum rate of fructolysis with tumor, this concen bic glycolysis of both tumor and normal tissues is tration of fructose was used as well as a concentra inhibited by the glucose analog, 2DG. Since the tion of 0.015 M which was sufficient for maximum 2DG inhibition with both tumor and brain was activity with liver. reversed by increased glucose, the inhibition ap Liver fructolysis was found to be relatively pears to be due to competition between 2DG and insensitive to 2DG (Table 7). With 0.06 M fruc glucose for an enzyme of the glycolytic system. tose, 2DG at a concentration of 0.015 M failed to The variation of results with different tissue sam produce inhibition of liver fructolysis, whereas ples, due to the nonhomogeneity of the tissue 2DG in this proportion to fructose (0.25:1) had material, makes difficult absolute proof that the produced practically complete inhibition of tumor inhibition is entirely of the competitive type. How fructolysis. At both substrate levels, 0.06 M and ever, with yeast, which metabolizes glucose anaer- 0.015 M, 2DG in a 1:1 ratio with fructose was re obically through an enzymatic pathway identical quired to produce consistently extensive inhibition except at the last stage to the enzyinatic pathway with liver, while with tumor 2DG in a ratio of only in animal tissues, it has been shown (2) that the about 0.008:1 was required (Table 3) to produce inhibition of glucose fermentation by 2DG was a similar extent of inhibition. Liver fructolysis is, purely of the competitive type.
Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1954 American Association for Cancer Research. TABLE 5 REVERSALOF2DG INHIBITIONOFANAEROBICGLUCOLYSISOFRATBRAIN- SLICESBYINCREASEDGLUCOSE GLUCOLYSIS RATE INHIBITION RAT i -M>AS GLUCOSECONC.1C0.0150.030.0450.060.0150.030.060.0150.030.060.0150.030.0450.060.0150.030.0450Glucose No or SOURCE OF added addition GLUCOLYSI8 TISSUE* N2 (per cent) (with 0.002 M 2DG) FJ-bearing 8.6 2.0 76 7<> 88 S W2S6-bearing 0.015 6.0 11.1 2.4 59 50 0 Normal 0.015 2.6 8.8 1.6 86 (¡it 22 (with 0.001 M 2DG) FJ-bearing 14.8 1.8 78 65 21 ¿1 Normal 0.015 5.6 11.8 1.6 61 40 0 060.0150.030.0450.06Glucose+«DGaddedQco,3.63.68.48.46.06.311.42.63.87.24.66.412.012.05.66.813.812.27.67.610.013.60 Normal 0.015 7.6 15.4 1.8 37 0.03 7.6 57 0.045 10.0 40 0.06 13.6 13 •Forabbreviations, see footnote to Table 1.
TABLE 6 INHIBITIONOFANAEROBICGLUCOLYSISOFRATDIAPHRAGMBY2DG (Glucose concentration, 0.015 M) GLUCOLYSISRATE
+SDG orOLUCOLYSIS(per AS2DGCONC.SOUBCERATUSED :OLUCOSEBATIÓ0.5:10.5:10.25:10.25:10.133:10.133:10.067:10.067:10.067:10.067:10.033:1Glucoseadded0N¡ addedQN! additionQNl"co,3.45.72.62.93.24.21.5S.«2.73.82.9INHIBITION MFJ-bearingFJ-bearingFJ-bearingFJ-bearingNormalNormalFJ-bearingFJ-bearingNormalNormalFJ-bearing0.00750.00750.003750.003750.0020.0020.0010.0010.0010.0010.00052DGOF TISSUE* QC.OZ4.95.94.93.3566.64.43.64.67.26.0Glucose«COj7.27.77.05.67.69.64.86.5i.a6.96.1Nocent)609048854550121006003
* For abbreviations, see footnote to Table 1.
Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1954 American Association for Cancer Research. 604 Cancer Research
Since hexokinase is generally considered to be Rather surprising is the observation that oxida the enzyme which initiates glycolysis, it might be tion of glucose by brain tissue is about 60 times presumed that 2DG inhibits at this stage of the less sensitive to 2DG than is the glycolysis of glycolytic pathway. It is known that 2DG is phos- brain tissue. Since it is generally accepted that phorylated by the hexokinase of yeast (2), of brain both the glycolytic and oxidative metabolism of (6), and of tumor1 but that it is not fermented or glucose require primary phosphorylation of glu glycolyaed. It is possible, therefore, that 2DG cose by hexokinase, it would be expected that interferes in the glycolytic system by removing oxidation as well as glycolysis would be inhibited some of the adenosinetriphosphate (ATP) which if the hexokinase reaction is the site of 2DG in is necessary for phosphorylation of glucose by hibition. The relative insensitivity of respiration hexokinase. Proof of the actual site of inhibition by to 2DG may be due to the presence of higher lev 2DG, however, must await further study. els of ATP under aerobic than under anaerobic
TABLE 7 INHIBITION OF ANAEROBICFRUCTOLYSISOFRAT LIVERSLICESBY2DG FRUCTOLYSISRATE
RATUSEDAS +2DGCONC. No or • SOURCEOFTISSUE*FructoseeoeaddedM 2DG: FRUCTOSE addedINHIBITIONadditionFHUCTOLYSISQCÓI BATIÓ QÇQ(with cent>fructose)914913911124036860466238440204683440filSI5850M(P*r 11FJ-bearingW256-bearingNormalNormalW256-bearingNormalFJ-bearingFJ-bearingW256-bearingW256-bearingNormalNormalFJ-bearingNormal0000000000000006060606OS03015OS015015015015007500751:10.06 71:1 101:1 71:.5:.5:.25:2:1:1:109812(with78101:1
fructose)98141011988605208456545s86020765220483444120
81:1 8.5:1 8.5:1 978ft0S840.0152047060Fructose * For abbreviations, see footnote to Table 1.
TABLE 8 EFFECT OF 2DG ON THE RESPIRATIONOF RAT TISSUESLICES BATETISSUE*Tumor OJCTQENUPTAKE
addedQo,6.14.24.311.07.42.42.2
u6.68.4 as4.162 (FJ) Tumor (FJ) 0.015 6.04.7 Liver from FJ-bearing rat 0.015 4.67.75.3Õ.74.7 LiverratDiaphragm from normal 0.0150.015 7.64.4 6.95.25.211.9IS from FJ-bearing rat Diaphragm from normal rat 0.015 4.6 BrainratBrain from normal 0.0150.060.0150.060.0150.0152.82.22.3(0.0153.12.2fructose ratBrainfrom normal ratBrainfrom normal 0substrate)11.210.5 H1.81.3 asSubstrateaddedQo,substrate)5.0 ratBrainfrom normal from W256-bearing rat Brain from normal rat*DGCONC.H0.0150.0005NoadditionQoi(0.0152.62DGaddedQo,glucose11.2Substrate+2DG6.3 * For abbreviations, see footnote to Table 1.
1G. E. Woodward, unpublished data.
Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1954 American Association for Cancer Research. WOODWARDANDHUDSON—g-Desoxy-o-glucoseandTissue Metabolism 605 conditions (3). The higher level of ATP under of slices of rat brain and sections of rat diaphragm oxidative conditions may also explain why the was also inhibited by 2DG. Brain was about 7.5 aerobic glycolysis of tumor tissue was slightly less times more sensitive to 2DG than was tumor. sensitive to 2DG than was the anaerobic glycoly Diaphragm was intermediate between brain and sis. tumor in sensitivity toward 2DG. The fact that fructose metabolism, both gly- The type of inhibition appeared to be com colytic and oxidative, is inhibited at a much lower petitive, since inhibition was reversed by in concentration of 2DG than is glucose metabolism creased glucose in the case of tumor and brain. is evidence in favor of the hexokinase reaction as 2DG also inhibited the anaerobic glycolysis of the site of 2DG inhibition. It has been shown that fructose (fructolysis) by tumor tissue slices. Tu the affinity of glucose for brain (6) and yeast (5) mor fructolysis was at least 50 times more sen hexokinase is greater than the affinity of fructose sitive to 2DG than was tumor glucolysis. The for these hexokinases. Therefore, it would be ex anaerobic fructolysis of liver was also inhibited by pected that the hexokinase reaction with fructose 2DG, but was about 100 times less sensitive to would be inhibited by 2DG more than the reac 2DG than was tumor fructolysis. tion with glucose. 2DG did not significantly inhibit the endoge In considering the possible usefulness of 2DG nous respiration in any of the tissues studied. Oxi to inhibit tumor growth in vivo by inhibiting gly dation of glucose by brain tissue slices was in colysis, certain differences in the metabolism of hibited only by a very high proportion of 2DG to turnor and normal tissues should be borne in mind. glucose, while oxidation of fructose was about 100 Tumor tissue, as shown by Warburg (7) and con times more sensitive to 2DG than was oxidation firmed by many workers, utilizes glucose mainly of glucose. by glycolysis, aerobically as well as anaerobically. The possibility is discussed that 2DG in vivo A few normal tissues, of which brain is an example, might have a selective inhibitory effect on tumor can glycolyze glucose to a considerable extent metabolism. anaerobically but can do so but slightly aerobical ly. Most normal tissues depend mainly upon res REFERENCES 1. CHAMES,F. B. A Procedure for the Preparation of 2- piration for their energy, while tumors depend to Desoxy-D-glucose. J. Franklin Inst., 253:277-80, 1952. a large extent upon glycolysis. Since respiration is 2. CBAMER,F. B., and WOODWARD,G.E. 2-Desoxy-D-glucose not inhibited by 2DG in concentrations at which as an Antagonist of Glucose in Yeast Fermentation. J. glycolysis is inhibited, it seems possible that in Franklin Inst., 253:354-60, 1952. vivo, where aerobic conditions prevail, tumor me 3. OCHOA,S.Efficiency of Aerobic Phosphorylation in Cell-free Heart Extracts. J. Biol. Chem., 161:493-505, 1943. tabolism might be selectively inhibited by 2DG. 4. ROSENTHAL,O. Die Vergärbarkeit verschiedener Zucker 2DG, in tests against a few mouse tumors in arten durch Rattenleber. Ihre Abhängigkeitvom Ernäh vivo, carried out in this laboratory by Ely,2 has rungszustand des Versuchstiers und von jahreszeitlichen indeed shown some beneficial effects; survival Einflüssen.Biochem. Ztschr., 227:354-84, 1930. time was increased 19-49 per cent, and tumor 5. SLEIN, M. W.; Com, G. T.; and COBI, C. F. A Compara tive Study of Hexokinase from Yeast and Animal Tissues. growth was retarded. J. Biol. Chem., 186:763-80, 1950. 6. SOLS, A., and CRANE,R. K. Some Properties of Brain SUMMARY Hexokinase. Fed. Proc., 12:271, 1953. The glucose analog, 2-desoxy-D-glucose (2DG), 7. WABBUBO,O.The Metabolism of Tumours. London: Con stable & Co., 1930. has been shown to inhibit the anaerobic and aero 8. WOODWARD,G.E., and CRAMER,F. B. 2-Desoxy-D-glucose bic glycolysis of glucose (glucolysis) by slices of as an Inhibitor of Anaerobic Glycolysis in Tumor Tissue. the rat tumors, Flexner-Jobling carcinoma and J. Franklin Inst., 254:259-60, 1952. Walker 256 carcinoma. The anaerobic glucolysis 9. UMBBEIT,W. W.; BUBRIS, R. H.; and STAUFFEH,F. J. Manometric Techniques and Tissue Metabolism. Minne 1J. O. Ely, J. Franklin Inst., 268:157-60, 1954. apolis: Burgess Publishing Co., 1949.
Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1954 American Association for Cancer Research. The Effect of 2-Desoxy-d-glucose on Glycolysis and Respiration of Tumor and Normal Tissues
Gladys E. Woodward and Marie T. Hudson
Cancer Res 1954;14:599-605.
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