- Email: [email protected]
Inhibition of 6-phosphogluconate dehydrogenase by glucose 1,6-diphosphate in human normal and malignant colon extracts
Cancer Letters, 23 (1984) 193-199 Elsevier Scientific Publishers Ireland Ltd.
193
INHIBITION OF 6-PHOSPHOGLUCONATE DEHYDROGENASE BY GLUCOSE 1,6-DIPHOSPHATE IN HUMAN NORMAL AND MALIGNANT COLON EXTRACTS
JARDENA NORDENBERG*, RAM AVIRAM, EINAT BEERY, and ABRAHAM NOVOGRODSKY
KURT H. STENZEL* *
The Rogoff-Wellcome Medical Research Institute, Beilinson Medical Center, Petah-Tikva and Tel-Aviv University Sackler School of Medicine (Israel) (Received 18 January 1984) (Revised version received 21 March 1984) (Accepted 8 April 1984)
SUMMARY
Increased activity of 6-phosphogluconate dehydrogenase was found in human colon tumors as compared to the adjacent unaffected mucosa. Glucose 1,6diphosphate (Glc-1,6-P,), an endogenous potent regulator of glucose metabolism, markedly inhibited the activity of 6-phosphogluconate dehydrogenase (6-PGD) in extracts of the normal and malignant human colon. Glc-1 ,6-Pz also inhibited the activity of hexokinase in these extracts. The endogenous levels of Glc-1,6-P, in the colon and tumors were measured. Since the pentose cycle can be inhibited by Glc-1,6-P,, means to increase endogenous levels of Glc-1,6-Pz or to introduce it into cells, might result in antitumor effects.
INTRODUCTION
An increase in the activity of enzymes involved in carbohydrate metabolism is characteristic of many malignant tissues. Increased activity of glucose-6-phosphate dehydrogenase and 6-PGD, the regulatory enzymes of the pentose phosphate shunt, was found in colon carcinoma [8,20,21]. The pentose cycle plays an important role in generating NADPH and in providing ribose 5-phosphate, a precursor for the synthesis of nucleic acids. Glc-1 ,6-Pz, the cofactor of phosphoglucomutase [ 121, is a potent modulator of many key enzymes in glucose metabolism [4,7]. In animal tissues *Address all correspondence to: Dr. J. Nordenberg, Rogoff-Wellcome Medical Research Institute, Beilinson Medical Center, 49100, Petah-Tikva, Israel. **Permanent address: Rogosin Kidney Center, Cornell University Medical College, New York, U.S.A. 0304-3835/84/$03.00 0 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
194 it is an activator (de-inhibitor) of phosphofructokinase, the rate-limiting enzyme in glycolysis, and of pyruvate kinase [9,11] and it is a potent inhibitor of hexokinase [ 2,18,19]. In rodents this modulator has been shown to fluctuate under many physiological, hormonal and pathological conditions [1,3,4,6,7,14] and its levels can be modulated by several drugs [l&16]. We have recently shown that Glc-1,6-Pz inhibits the activity of 6-PGD in several rodent tissues [ 51. In the present study we found that Glc-1,6-PZ markedly inhibited 6-PGD and hexokinase activity in extracts of both, normal human colon and human colon carcinoma. The anti-tumor effect of Glc-1,6-P* should be evaluated since inhibition of the pentose cycle might lead to a decrease in ribose 5-phosphate levels and a consequent inhibition of nucleic acid synthesis. MATERIALS
AND METHODS
All materials were purchased from Sigma Chemical Co. Glucose lphosphate was essentially free of Glc-1,6-P*. Imidazole was the low fluorescence grade. Colon carcinomas and samples of the uninvolved adjacent colon were removed during therapeutic surgery at Beilinson Medical Center. The specimens were processed immediately for analysis. Tissues were debribed of fat and minced with scissors and separated from hemorrhagic areas. For the determination of Glc-1,6-P*, the minced pieces were frozen in liquid NZ. The histology of the tumors was confirmed in the clinical pathology laboratory. 6-PGD was extracted by homogenizing. 80-150 mg tissue in cold 0.05 Tris-HCI (pH 7.4) containing 0.1% Triton X-100 for 30 s. The homogenate was centrifuged in a microfuge for 4 min and the activity in the supematant was measured by a highly sensitive fluorometric method which was a modification of the spectrophotometric method of Glock and McLean, as described previously [5] : 1 munit 6-PGD represents the amount of enzyme activity which forms 1 nmol NADPH in 1 min at 25°C. Hexokinase activity was extracted and measured as described previously [6] at pH 7 and 0.25 mM Mg*+. Under these conditions the enzyme is sensitive to allosteric regulation: 1 munit of hexokinase activity represents the amount of enzyme activity which forms 1 nmol of NADPH at 25°C. Phosphofructokinase was extracted in Tris-HCl (pH 6.9) and its activity was measured fluorometrically in an assay mixture containing 0.05 M imidazole (pH 6.9), 2 mM Fru8-P, 0.2 mM ATP, 15 pM NADPH and excess of tr-glycerophosphate dehydrogenase, aldolase and triose phosphate isomerase. These auxilliary enzymes were dialyzed in 0.01 M Tris-HCl (pH 7.8) to remove ammonium sulfate: 1 munit of phosphofructokinase activity catalyses the formation of 1 nmol fructose 1,6-diphosphate per min at 25°C. Glc-1,6-P, was extracted and measured by the fluorometric method of Passomneau et al. [17]. Protein was measured by the method of Lowry etsl. [13].
195 TABLE 1 THE ACTIVITY OF 6-PGD IN COLON CARCINOMA AND IN THE SURROUNDING NORMAL MUCOSA MEASURED IN THE ABSENCE AND PRESENCE OF Glc-1,6-P, 6-PGD (munits/mg protein) (mean + S.E. )
Additions
None Glc-1,6-P,
(20 PM)
% inhibition by Glc-1,6-P,
Colon
Tumor
13.1 f 1.1 (9) 2.7 t 1.0 (8) 81 c7 (P < 0.001)
22.3*
t 1.8 (9) 6.9 f. 1.7 (8)
7po< o:oz,
The enzymes were extracted and measured as described in Methods. Numbers in parentheses represent the number of specimens. *Tumor vs. colon P < 0.001.
RESULTS
Activity of 6-PGD extracted from 9 human colon carcinomas and from adjacent unaffected colon was measured. A significant increase in the activity of 6-PGD was found in all carcinomas, as compared to the uninvolved colon tissues. Addition of Glc-1,6-P, resulted in a marked inhibition of 6-PGD activity in extracts from both tumor and normal colon. When enzyme activity was measured at various Glc-1,6-P, concentrations (Fig. l), enzyme in normal and tumor were similarly sensitive to inhibition of Glc-1,6-P*. Fifty percent inhibition was obtained by 10 PM Glc-1,6-P,. Since Glc-1,6-P? is an allosteric inhibitor of hexokinase, the enzyme that catalyzes formation of Glc-6-P, the substrate for the pentose cycle, we measured the effect of Glc-1,6-P, on human colon and colon carcinoma hexokinase activity. The activity of hexokinase was measured under suboptimal conditions sensitive to ahosteric regulation by Glc-1,6-P,. Similar activities of hexokinase were found in the tumors and in the normal colon (Table 2). Glc-1,6-P, at 20 PM inhibited hexokinase activity by 50% in extracts from either the colon or the tumor. In contrast to the pronounced inhibitory effect of Glc-1,6-P* on hexokinase and on 6-PGD, Glc-1,6-P, has been found to be a potent activator of muscle, brain and red blood cells phosphofructokinase [9,16,18]. We measured the activity of phosphofructokinase in extracts from 3 carcinomas and their surrounding uninvolved tissues. The activity of normal colon phosphofructokinase was 1.4, 2.0 and 2.0 munits, and in the corresponding tumor tissues was 1.2, 2.4 and 2.5 munits. Glc-1,6-P, was found to increase phosphofructokinase activity similarly in normal and tumor homogenates.
COLON
CARClNOMA
Glc-1,6-P,
(ykl)
Fig. 1. The effect of Glc-1,6-P, at different concentrations on the activity of 6-PGD extracted from colon and colon carcinoma. The enzyme was extracted and measured in the presence of various Glc-1,6-P, concentrations as described in Methods. The values are the mean of 2 specimens of colon and 2 of carcinoma. TABLE 2 THE INHIBITORY EFFECT OF Glc-1,6-P, ON HEXOKINASE NORMAL COLON AND FROM COLON CARCINOMAS Additions
None Glc-1,6-P, (20 PM) % inhibition by Glc-1,6-P,
ACTIVITY
FROM
Hexokinase activity (munits/mg protein) Colon
Tumor
3.5
2.9
f 0.6 (5) 1.5* t 0.2 (5) 57 + 11
* 0.4 (5) 1.5* + 0.2 (5) 49 f 12
Enzyme extraction and measurement was as described in Methods. Numbers in parentheses represent on the number of samples. *+Glc-1,6-P, vs. -Glc-1,6-P, P < 0.001.
197 TABLE 3 THE LEVELS OF Glc-1,6-P, IN COLON AND CARCINOMA Colon Carcinoma
Glc-1,6-P, mmol/kg wet wt 1.80 of 0.57 (5) 1.25 * 0.31 (5)
Glc-1,6-P, was extracted and measured as described in Methods.
Glc-1 ,6-Pz (16 PM) increased phosphofructokinase activity by 50% in one tumor homogenate and in its corresponding normal mucosa. In the second specimen, the enhancement by Glc-1,6-Pz was 150%. To evaluate the possibility that Glc-1,6-P, regulates the level of 6-PGD in the colon, we measured the levels of Glc-1,6-Pz in the colon and in the carcinomas. A small decrease in the levels of Glc-1,6-Pz was found in the carcinomas as compared to the colon tissue (Table 3). DISCUSSION
In the present study we measured the activities of rate-limiting enzymes of glucose metabolism in human colon carcinoma and compared them with those found in adjacent uninvolved colon mucosa. There was a consistent increase in the activity of 6-PGD in the human colon carcinomas. Similar results were recently obtained by Denton et al. [ 81. Glc-1,6-Pz, the potent modulator of many key enzymes in glucose metabolism, was found to be a potent inhibitor of human colon 6-PGD. Although the activity of 6-PGD in carcinomas is elevated, it maintains its sensitivity to inhibition by Glc-1,6-P*. Physiologic concentrations of Glc-1 ,6-Pz are inhibitory. Levels of Glc-1,6-P, measured in the colon tissue are low compared to human red blood cells [ 181, muscle (Beitner and Nordenberg, unpublished results) and many rodent tissues [ 5 ] . It might be possible that the levels measured do not reflect the real tissue concentration, since the tissues could not be frozen immediately after removal from the patients. The activity of hexokinase and of phosphofructokinase were similar in normal and colon tumors when measured under regulatory conditions [S] . The inhibition of hexokinase by Glc-1,6-P, and the concomitant activation of phosphofructokinase may lead to a decrease in the level of Glc-1 ,6-P2, the substrate for pentose biosynthesis. The effects of Glc-1,6-P* may lead to inhibition of ribose &phosphate biosynthesis in 2 ways: (1) inhibition of 6-PGD, one of the rate-limiting enzymes in the pentose-cycle; (2) reducing the level of Glc-6-P, due to inhibition of hexokinase and thus reducing the substrate availability for both the oxidative pentose cycle and the non-oxidative L. type pentose
198
phosphate cycle that also utilizes hexose g-phosphate as a precursor [lo] . Since the pentose cycle can be inhibited by Glc-1,6-P2, means to increase endogenous levels of Glc-1,6-P* or to introduce it into cells, might result in anti-tumor effects. REFERENCES 1 Beery, E., Klein, S., Nordenberg, J. and Beitner, R. (1980) The influence of Ca”ionophore A23187 on glucose-l ,6-diphosphate and ATP levels, and on the activities of phosphofructokinase and phosphoglucomutase in isolated rat diaphragm muscle. Biochem. Int., 1, 526-531. 2 Beitner, R., Haberman, S. and Livni L. (1975) Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase: the role of glucose-1,6diphosphate. Biochim. Biophys. Acta, 397,355-369. 3 Beitner, R., Haberman, S. and Nordenberg, J. (1978) The effect of epinephrine and dibutyryl cyclic AMP on glucose-1,6-diphosphate levels and the activities of hexokinaae, phosphofructokinase and phosphoglucomutase in the isolated rat diaphragm. Mol. Cell. Endocrinol., 10, 135-147. 4 Beitner, R. (1979) The role of glucose-l ,6diphosphate in the regulation of carbohydrate metabolism in muscle. Trends. Biochem. Sci., 4, 228-230. 5 Beitner, R. and Nordenberg, J. (1979) Inhibition of 6-phosphogluconate dehydrogenase(decarboxylating) by glucose-1,6_bisphosphate. Biochim. Biophys. Acta, 583, 266-269. 6 Beitner, R., Klein, S. and Nordenberg, J. (1982) The participation of glucose-1,6diphosphate in the regulation of hexokinase and phosphoglucomutase activities in brains of young and adult rats. Int. J. Biochem., 14, 195-199. 7 Beitner, R. (1984) Control of levels of glucose-1,6-bisphosphate. Int. J. Biochem., in press. 8 Denton, J.E., Lui, M.S., Aoki, T., Sebolt, J., Takeda, E., Eble, J.N., Glover, J.L. and Weber, G. (1982) Enzymology of pyrimidine and carbohydrate metabolism in human colon carcinomas. Cancer Res., 42,1176-1183. 9 Hofer, H.W. and Pette, D. (1968) Wirkungen und wechselwirkungen von Substraten und Effectoren an der phosphofructokinase des Kaninchen-Skeletmuskels. Hoppe Seyler’s Z. Physiol. Chem., 349, 1378-1392. 10 Kim, S., Klein, J. and Graham, O.L. (1957) Pathways of ribonucleic acid pentose biosynthesis by lymphatic tissues and tumors. J. Biol. Chem., 229, 853-863. 11 Koster, J.F., Slee, R.G., Staal, G.E. and Van Berkel, Th.J.C!. (1972) The influence of glucose-1,6diphosphate on the enzymatic activity of pyruvate kinase. Biochim. Biophys. Acta, 258,763-768. 12 Leloir, L.F., Trucco, R.E., Cardini, C.E., Paladini, A.C. and Caputto, R. (1948) The coenzyme of phosphoglucomutase. Arch. Biochem., 19,339-340. 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measure ment with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. 14 Nordenberg, J., Heffetz, D., Cohen, T.J. and Beitner, R. (1981) Glucose-1,6diphosphate and carbohydrate metabolism in skeletal muscle of old rats. Int. J. Biochem:, 13,317-321. 15 Nordenberg, J., Klein, S., Beery, E., Kaplansky, M. and Beitner, R. (1981) Changes in the levels of glucose-l ,6diphosphate and ATP and in the activities of phosphofructokinase and phosphoglucomutase induced by local anesthetics in the isolated rat diaphragm muscle. Int. J. Biochem., 13, 1005-1109. 16 Nordenberg, J., Kaplansky, M., Beery, E., Klein, S. and Beitner, R. (1982) Effects of lithium on the activities of phosphofructokinase and phosphoglucomutase levels in rat muscles, brain and liver. Biochem. Pharmacol., 31, 1025-1031.
199 17 Passonneau, J.V., Lowry, O.H., Schulz, D.W. and Brown, J.G. (1969) Glucose-1,6diphosphate formation by phosphoglucomutase in mammalian tissues. J. Biol. Chem., 244,902-909. 18 Rapaport, S. (1974) Control mechanism of red cell glycolysis. In: The Human Red Cell in Vitro, pp. 153-178. Editors: T.J. Greenwalt and G.A. Jamieson. Grune and Stratton, New York. 19 Rijksen, G. and Staal, G.E.J. (1977) Regulation of human erythrocyte hexokinase by glucose-l ,6diphosphate and inorganic phosphate. FEBS Lett., 80, 61-65. 20 Shonk, C.E., Arison, R.N., Koven, B.J., Masima, H. and Boxer, G.E. (1964) Enzyme patterns in human tissues III Glycolytic enzymes in normal and malignant tissues of the colon and rectum. Cancer Res., 25, 206-213. 21 Weber, G., Kizaki, H., Tzeng, D., Shiotani, T. and Olah, E. (1978) Colon tumor: Enzymology of the neoplastic program. Life Sci., 23,729-736.
193
INHIBITION OF 6-PHOSPHOGLUCONATE DEHYDROGENASE BY GLUCOSE 1,6-DIPHOSPHATE IN HUMAN NORMAL AND MALIGNANT COLON EXTRACTS
JARDENA NORDENBERG*, RAM AVIRAM, EINAT BEERY, and ABRAHAM NOVOGRODSKY
KURT H. STENZEL* *
The Rogoff-Wellcome Medical Research Institute, Beilinson Medical Center, Petah-Tikva and Tel-Aviv University Sackler School of Medicine (Israel) (Received 18 January 1984) (Revised version received 21 March 1984) (Accepted 8 April 1984)
SUMMARY
Increased activity of 6-phosphogluconate dehydrogenase was found in human colon tumors as compared to the adjacent unaffected mucosa. Glucose 1,6diphosphate (Glc-1,6-P,), an endogenous potent regulator of glucose metabolism, markedly inhibited the activity of 6-phosphogluconate dehydrogenase (6-PGD) in extracts of the normal and malignant human colon. Glc-1 ,6-Pz also inhibited the activity of hexokinase in these extracts. The endogenous levels of Glc-1,6-P, in the colon and tumors were measured. Since the pentose cycle can be inhibited by Glc-1,6-P,, means to increase endogenous levels of Glc-1,6-Pz or to introduce it into cells, might result in antitumor effects.
INTRODUCTION
An increase in the activity of enzymes involved in carbohydrate metabolism is characteristic of many malignant tissues. Increased activity of glucose-6-phosphate dehydrogenase and 6-PGD, the regulatory enzymes of the pentose phosphate shunt, was found in colon carcinoma [8,20,21]. The pentose cycle plays an important role in generating NADPH and in providing ribose 5-phosphate, a precursor for the synthesis of nucleic acids. Glc-1 ,6-Pz, the cofactor of phosphoglucomutase [ 121, is a potent modulator of many key enzymes in glucose metabolism [4,7]. In animal tissues *Address all correspondence to: Dr. J. Nordenberg, Rogoff-Wellcome Medical Research Institute, Beilinson Medical Center, 49100, Petah-Tikva, Israel. **Permanent address: Rogosin Kidney Center, Cornell University Medical College, New York, U.S.A. 0304-3835/84/$03.00 0 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
194 it is an activator (de-inhibitor) of phosphofructokinase, the rate-limiting enzyme in glycolysis, and of pyruvate kinase [9,11] and it is a potent inhibitor of hexokinase [ 2,18,19]. In rodents this modulator has been shown to fluctuate under many physiological, hormonal and pathological conditions [1,3,4,6,7,14] and its levels can be modulated by several drugs [l&16]. We have recently shown that Glc-1,6-Pz inhibits the activity of 6-PGD in several rodent tissues [ 51. In the present study we found that Glc-1,6-PZ markedly inhibited 6-PGD and hexokinase activity in extracts of both, normal human colon and human colon carcinoma. The anti-tumor effect of Glc-1,6-P* should be evaluated since inhibition of the pentose cycle might lead to a decrease in ribose 5-phosphate levels and a consequent inhibition of nucleic acid synthesis. MATERIALS
AND METHODS
All materials were purchased from Sigma Chemical Co. Glucose lphosphate was essentially free of Glc-1,6-P*. Imidazole was the low fluorescence grade. Colon carcinomas and samples of the uninvolved adjacent colon were removed during therapeutic surgery at Beilinson Medical Center. The specimens were processed immediately for analysis. Tissues were debribed of fat and minced with scissors and separated from hemorrhagic areas. For the determination of Glc-1,6-P*, the minced pieces were frozen in liquid NZ. The histology of the tumors was confirmed in the clinical pathology laboratory. 6-PGD was extracted by homogenizing. 80-150 mg tissue in cold 0.05 Tris-HCI (pH 7.4) containing 0.1% Triton X-100 for 30 s. The homogenate was centrifuged in a microfuge for 4 min and the activity in the supematant was measured by a highly sensitive fluorometric method which was a modification of the spectrophotometric method of Glock and McLean, as described previously [5] : 1 munit 6-PGD represents the amount of enzyme activity which forms 1 nmol NADPH in 1 min at 25°C. Hexokinase activity was extracted and measured as described previously [6] at pH 7 and 0.25 mM Mg*+. Under these conditions the enzyme is sensitive to allosteric regulation: 1 munit of hexokinase activity represents the amount of enzyme activity which forms 1 nmol of NADPH at 25°C. Phosphofructokinase was extracted in Tris-HCl (pH 6.9) and its activity was measured fluorometrically in an assay mixture containing 0.05 M imidazole (pH 6.9), 2 mM Fru8-P, 0.2 mM ATP, 15 pM NADPH and excess of tr-glycerophosphate dehydrogenase, aldolase and triose phosphate isomerase. These auxilliary enzymes were dialyzed in 0.01 M Tris-HCl (pH 7.8) to remove ammonium sulfate: 1 munit of phosphofructokinase activity catalyses the formation of 1 nmol fructose 1,6-diphosphate per min at 25°C. Glc-1,6-P, was extracted and measured by the fluorometric method of Passomneau et al. [17]. Protein was measured by the method of Lowry etsl. [13].
195 TABLE 1 THE ACTIVITY OF 6-PGD IN COLON CARCINOMA AND IN THE SURROUNDING NORMAL MUCOSA MEASURED IN THE ABSENCE AND PRESENCE OF Glc-1,6-P, 6-PGD (munits/mg protein) (mean + S.E. )
Additions
None Glc-1,6-P,
(20 PM)
% inhibition by Glc-1,6-P,
Colon
Tumor
13.1 f 1.1 (9) 2.7 t 1.0 (8) 81 c7 (P < 0.001)
22.3*
t 1.8 (9) 6.9 f. 1.7 (8)
7po< o:oz,
The enzymes were extracted and measured as described in Methods. Numbers in parentheses represent the number of specimens. *Tumor vs. colon P < 0.001.
RESULTS
Activity of 6-PGD extracted from 9 human colon carcinomas and from adjacent unaffected colon was measured. A significant increase in the activity of 6-PGD was found in all carcinomas, as compared to the uninvolved colon tissues. Addition of Glc-1,6-P, resulted in a marked inhibition of 6-PGD activity in extracts from both tumor and normal colon. When enzyme activity was measured at various Glc-1,6-P, concentrations (Fig. l), enzyme in normal and tumor were similarly sensitive to inhibition of Glc-1,6-P*. Fifty percent inhibition was obtained by 10 PM Glc-1,6-P,. Since Glc-1,6-P? is an allosteric inhibitor of hexokinase, the enzyme that catalyzes formation of Glc-6-P, the substrate for the pentose cycle, we measured the effect of Glc-1,6-P, on human colon and colon carcinoma hexokinase activity. The activity of hexokinase was measured under suboptimal conditions sensitive to ahosteric regulation by Glc-1,6-P,. Similar activities of hexokinase were found in the tumors and in the normal colon (Table 2). Glc-1,6-P, at 20 PM inhibited hexokinase activity by 50% in extracts from either the colon or the tumor. In contrast to the pronounced inhibitory effect of Glc-1,6-P* on hexokinase and on 6-PGD, Glc-1,6-P, has been found to be a potent activator of muscle, brain and red blood cells phosphofructokinase [9,16,18]. We measured the activity of phosphofructokinase in extracts from 3 carcinomas and their surrounding uninvolved tissues. The activity of normal colon phosphofructokinase was 1.4, 2.0 and 2.0 munits, and in the corresponding tumor tissues was 1.2, 2.4 and 2.5 munits. Glc-1,6-P, was found to increase phosphofructokinase activity similarly in normal and tumor homogenates.
COLON
CARClNOMA
Glc-1,6-P,
(ykl)
Fig. 1. The effect of Glc-1,6-P, at different concentrations on the activity of 6-PGD extracted from colon and colon carcinoma. The enzyme was extracted and measured in the presence of various Glc-1,6-P, concentrations as described in Methods. The values are the mean of 2 specimens of colon and 2 of carcinoma. TABLE 2 THE INHIBITORY EFFECT OF Glc-1,6-P, ON HEXOKINASE NORMAL COLON AND FROM COLON CARCINOMAS Additions
None Glc-1,6-P, (20 PM) % inhibition by Glc-1,6-P,
ACTIVITY
FROM
Hexokinase activity (munits/mg protein) Colon
Tumor
3.5
2.9
f 0.6 (5) 1.5* t 0.2 (5) 57 + 11
* 0.4 (5) 1.5* + 0.2 (5) 49 f 12
Enzyme extraction and measurement was as described in Methods. Numbers in parentheses represent on the number of samples. *+Glc-1,6-P, vs. -Glc-1,6-P, P < 0.001.
197 TABLE 3 THE LEVELS OF Glc-1,6-P, IN COLON AND CARCINOMA Colon Carcinoma
Glc-1,6-P, mmol/kg wet wt 1.80 of 0.57 (5) 1.25 * 0.31 (5)
Glc-1,6-P, was extracted and measured as described in Methods.
Glc-1 ,6-Pz (16 PM) increased phosphofructokinase activity by 50% in one tumor homogenate and in its corresponding normal mucosa. In the second specimen, the enhancement by Glc-1,6-Pz was 150%. To evaluate the possibility that Glc-1,6-P, regulates the level of 6-PGD in the colon, we measured the levels of Glc-1,6-Pz in the colon and in the carcinomas. A small decrease in the levels of Glc-1,6-Pz was found in the carcinomas as compared to the colon tissue (Table 3). DISCUSSION
In the present study we measured the activities of rate-limiting enzymes of glucose metabolism in human colon carcinoma and compared them with those found in adjacent uninvolved colon mucosa. There was a consistent increase in the activity of 6-PGD in the human colon carcinomas. Similar results were recently obtained by Denton et al. [ 81. Glc-1,6-Pz, the potent modulator of many key enzymes in glucose metabolism, was found to be a potent inhibitor of human colon 6-PGD. Although the activity of 6-PGD in carcinomas is elevated, it maintains its sensitivity to inhibition by Glc-1,6-P*. Physiologic concentrations of Glc-1 ,6-Pz are inhibitory. Levels of Glc-1,6-P, measured in the colon tissue are low compared to human red blood cells [ 181, muscle (Beitner and Nordenberg, unpublished results) and many rodent tissues [ 5 ] . It might be possible that the levels measured do not reflect the real tissue concentration, since the tissues could not be frozen immediately after removal from the patients. The activity of hexokinase and of phosphofructokinase were similar in normal and colon tumors when measured under regulatory conditions [S] . The inhibition of hexokinase by Glc-1,6-P, and the concomitant activation of phosphofructokinase may lead to a decrease in the level of Glc-1 ,6-P2, the substrate for pentose biosynthesis. The effects of Glc-1,6-P* may lead to inhibition of ribose &phosphate biosynthesis in 2 ways: (1) inhibition of 6-PGD, one of the rate-limiting enzymes in the pentose-cycle; (2) reducing the level of Glc-6-P, due to inhibition of hexokinase and thus reducing the substrate availability for both the oxidative pentose cycle and the non-oxidative L. type pentose
198
phosphate cycle that also utilizes hexose g-phosphate as a precursor [lo] . Since the pentose cycle can be inhibited by Glc-1,6-P2, means to increase endogenous levels of Glc-1,6-P* or to introduce it into cells, might result in anti-tumor effects. REFERENCES 1 Beery, E., Klein, S., Nordenberg, J. and Beitner, R. (1980) The influence of Ca”ionophore A23187 on glucose-l ,6-diphosphate and ATP levels, and on the activities of phosphofructokinase and phosphoglucomutase in isolated rat diaphragm muscle. Biochem. Int., 1, 526-531. 2 Beitner, R., Haberman, S. and Livni L. (1975) Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase: the role of glucose-1,6diphosphate. Biochim. Biophys. Acta, 397,355-369. 3 Beitner, R., Haberman, S. and Nordenberg, J. (1978) The effect of epinephrine and dibutyryl cyclic AMP on glucose-1,6-diphosphate levels and the activities of hexokinaae, phosphofructokinase and phosphoglucomutase in the isolated rat diaphragm. Mol. Cell. Endocrinol., 10, 135-147. 4 Beitner, R. (1979) The role of glucose-l ,6diphosphate in the regulation of carbohydrate metabolism in muscle. Trends. Biochem. Sci., 4, 228-230. 5 Beitner, R. and Nordenberg, J. (1979) Inhibition of 6-phosphogluconate dehydrogenase(decarboxylating) by glucose-1,6_bisphosphate. Biochim. Biophys. Acta, 583, 266-269. 6 Beitner, R., Klein, S. and Nordenberg, J. (1982) The participation of glucose-1,6diphosphate in the regulation of hexokinase and phosphoglucomutase activities in brains of young and adult rats. Int. J. Biochem., 14, 195-199. 7 Beitner, R. (1984) Control of levels of glucose-1,6-bisphosphate. Int. J. Biochem., in press. 8 Denton, J.E., Lui, M.S., Aoki, T., Sebolt, J., Takeda, E., Eble, J.N., Glover, J.L. and Weber, G. (1982) Enzymology of pyrimidine and carbohydrate metabolism in human colon carcinomas. Cancer Res., 42,1176-1183. 9 Hofer, H.W. and Pette, D. (1968) Wirkungen und wechselwirkungen von Substraten und Effectoren an der phosphofructokinase des Kaninchen-Skeletmuskels. Hoppe Seyler’s Z. Physiol. Chem., 349, 1378-1392. 10 Kim, S., Klein, J. and Graham, O.L. (1957) Pathways of ribonucleic acid pentose biosynthesis by lymphatic tissues and tumors. J. Biol. Chem., 229, 853-863. 11 Koster, J.F., Slee, R.G., Staal, G.E. and Van Berkel, Th.J.C!. (1972) The influence of glucose-1,6diphosphate on the enzymatic activity of pyruvate kinase. Biochim. Biophys. Acta, 258,763-768. 12 Leloir, L.F., Trucco, R.E., Cardini, C.E., Paladini, A.C. and Caputto, R. (1948) The coenzyme of phosphoglucomutase. Arch. Biochem., 19,339-340. 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measure ment with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. 14 Nordenberg, J., Heffetz, D., Cohen, T.J. and Beitner, R. (1981) Glucose-1,6diphosphate and carbohydrate metabolism in skeletal muscle of old rats. Int. J. Biochem:, 13,317-321. 15 Nordenberg, J., Klein, S., Beery, E., Kaplansky, M. and Beitner, R. (1981) Changes in the levels of glucose-l ,6diphosphate and ATP and in the activities of phosphofructokinase and phosphoglucomutase induced by local anesthetics in the isolated rat diaphragm muscle. Int. J. Biochem., 13, 1005-1109. 16 Nordenberg, J., Kaplansky, M., Beery, E., Klein, S. and Beitner, R. (1982) Effects of lithium on the activities of phosphofructokinase and phosphoglucomutase levels in rat muscles, brain and liver. Biochem. Pharmacol., 31, 1025-1031.
199 17 Passonneau, J.V., Lowry, O.H., Schulz, D.W. and Brown, J.G. (1969) Glucose-1,6diphosphate formation by phosphoglucomutase in mammalian tissues. J. Biol. Chem., 244,902-909. 18 Rapaport, S. (1974) Control mechanism of red cell glycolysis. In: The Human Red Cell in Vitro, pp. 153-178. Editors: T.J. Greenwalt and G.A. Jamieson. Grune and Stratton, New York. 19 Rijksen, G. and Staal, G.E.J. (1977) Regulation of human erythrocyte hexokinase by glucose-l ,6diphosphate and inorganic phosphate. FEBS Lett., 80, 61-65. 20 Shonk, C.E., Arison, R.N., Koven, B.J., Masima, H. and Boxer, G.E. (1964) Enzyme patterns in human tissues III Glycolytic enzymes in normal and malignant tissues of the colon and rectum. Cancer Res., 25, 206-213. 21 Weber, G., Kizaki, H., Tzeng, D., Shiotani, T. and Olah, E. (1978) Colon tumor: Enzymology of the neoplastic program. Life Sci., 23,729-736.