Glucokinase) Activities in a Range of Tumor Cell Lines and Inhibition of Rates of Tumor Growth by the Specific Enzyme Inhibitor Man No Hep Tulose1

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[CANCER RESEARCH 55, 3278-3285. August 1, I"Â»5J High Km Glucose-phosphorylating (Glucokinase) Activities in a Range of Tumor Cell Lines and Inhibition of Rates of Tumor Growth by the Specific Enzyme Inhibitor Man no hep tulose1

Mary Board,2 Alison Colquhoun, and Eric A. Newsholme

Department of Biochemisriy. University of Oxford, South Parks Roail. Oxford OX l 3QU, Uniteti Kingdom

ABSTRACT activities are usually lower than those of phosphofructokinase in a range of tumor cell lines (10). Together with reports of the accumu Differences in modes of control of glycolysis in tumor cells, compared lation of fructose-l,6-bisphosphate, a product of the phosphofructoki with normal cells, have suggested that phosphofructokinase may not catalyse the rate-controlling step. Instead, hexokinase activity may assume nase reaction to high levels in untransformed proliferating cells (11-13) and to even higher levels in tumor cells (11, 14-16), evidence a more important regulatory role. Hexokinase activities are consistently lower than those of phosphofructokinase in tumor cells, and the former suggests that phosphofructokinase activity may not control rates of enzyme may be saturated with its substrate (M. Board et al, Biochem. J. glycolysis in tumor cells. This would mean that a different mode of 265: 503-509, 1990). The present work has focused on the glucose-phos- control is operating from that thought to regulate glycolysis in skeletal phorylation step in tumor cell glycolysis. A range of eight human tumor muscle, for example (17). It is considered that HK activity may play cell-lines, one human tumor tissue, and four rat tumor cell lines were an unusual regulatory role in tumor cells. The low Km of HK for its found to have an additional glucose-phosphorylating activity, with prop erties similar to hepatic glucokinase. Maximal activities range from 1.1-20 substrate suggests that it may be saturated at the intracellular glucose concentrations thought to be present in tumor cells (see nmol/min/mg cell protein, and the activity is consistently absent from any "Discussion"). If this is true, then HK would become the flux- untransformed cell line or tissue tested, except rat liver tissue (IS nmol/ min/mg cell protein). Tumor cell glucokinase activity has been character generating step for glycolysis from glucose. Moreover, lower ac ized by its high Am for glucose (8-11.8 m\n; inhibition by the specific tivities of HK, compared with phosphofructokinase, suggest that glucokinase inhibitor, mannoheptulose (Iso, 12.5 HIMI:and lack of inhibi the former enzyme may have a role to play in controlling rates of tion by 10 HIMglucose-6-phosphate. Mannoheptulose also causes inhibi flux through glycolysis. tion of glucose uptake by tumor cells (25-75% at 30 HIMmannoheptulose) Given the higher rates of glycolysis in tumor cells and the possible and inhibition of rates of growth of cultured tumor cell lines i Uâ€ž.21.4 HIM). Rates of growth of human tumors in experimental animals are regulatory role played by HK, it is expected that maximal HK activ dramatically reduced (by 65-79%) by a dose of 1.7 mg/g mannoheptulose ities would be dramatically increased compared with normal cells. daily for 5 days. The potential of the naturally occurring sugar, manno However, measurements of maximal HK activity in a range of tumor heptulose (which is purified from avocados and is assumed to be of low cells indicate that it is not elevated by comparison with untransformed toxicity), as a cancer treatment is discussed. cell lines and that the range of activities is very broad (10). During the present work, the glucose-phosphorylation step in INTRODUCTION tumor cell metabolism has been further studied. The discovery that all tumor cell lines tested thus far have a high Km glucose- Manifold reports of high rates of glucose import and glycolysis by phosphorylating activity, similar to hepatic GK, is reported. The rapidly proliferating cells imply important roles for these processes in activity is present in all transformed cells and tissues (including cell division. Both processes occur at high rates in proliferating metastatic, tumorigenic, and suppressed examples) and is absent untransformed cells (1-3) as well as in tumor cells [reported by from all untransformed cells and tissues (except rat liver tissue). Warburg (4), Broadfoot et al. (5), and Roos and Loos (6)]. However, Characterization of the activity has included measurements of A"m high rates may be constitutive only in the latter example and may be for glucose; inhibition by the specific inhibitor of hepatic GK, a response to mitogenic stimulation in the former. Attention has focused mannoheptulose; and the absence of inhibition by glucose-6-phos- on the possible benefits conferred on dividing cells by high glycolytic phate. The behavior of tumor cell GK, in all respects, suggests that rates. Energy extraction by this route is relatively inefficient (2 mol it has similar properties to hepatic GK. ATP/mol glucose), compared with complete oxidation (36 mol ATP/mol The inhibition of GK activity from various tumor cell lines by glucose), and observations that most consumed glucose is converted to mannoheptulose has been studied, and results are presented. Manno lÃ¡clateled to suggestions that substrates more readily oxidized by the heptulose inhibits glucose uptake by cultured cells, which show GK tumor cell may provide most of its energy. Glutamine has been proposed as the main energy-yielding substrate in many tumor cell lines (7-9). activity but not by those (untransformed) cells lacking the enzyme. This finding is discussed in the context of modes of control of However, glycolysis is known to fulfill another function in terms of the glycolysis in tumor cells. The effects of the inhibitor on proliferation provision of biosynthetic precursors for nucleotide and lipid synthesis, of tumor cells in culture are dramatic, and severe inhibition of the both of which would be necessary for dividing cells. Mechanisms underlying the maintenance of high rates of glycolysis rates of growth of a range of human cell lines was discovered. Growth by tumor cells are speculative. It has been reported that maximal HK' rate suppression can be mimicked by sugars of similar structure, which share the GK-inhibitory property. Again, the effect is selective

Received 2/8/95; accepted 5/24/95. for those (transformed) cells that display GK activity. The inhibitory The costs of publication of this article were defrayed in part by the payment of page effects of mannoheptulose on cultured cells can be reproduced for charges. This article must therefore be hereby marked advertisement in accordance with tumors growing in experimental animals. Severe reduction in tumor 18 U.S.C. Section 1734 solely to indicate this fact. ' The work presented in this paper is the subject of patent application number PCT/GB burden with mannoheptulose administration is reported. Actions of 94/01676 (filed August 1, 1994). 2 To whom requests for reprints should be addressed. mannoheptulose in inhibiting proliferation of tumor cells in culture 3 The abbreviations used are: HK, hexokinase (B.C. 2.7.1.1); OK, glucokinase and in vivo allow discussion of the potential of this agent as a (E.G. 2.7.1.2); I5(1,concentration of inhibitor required to elicit 50% inhibition. treatment for cancer patients. 3278

MATERIALS AND METHODS activity bound to the discs was measured by adding 1 ml distilled water plus 10 mis aqueous scintillation fluid and by counting the dispersions per minute Materials. ATP, glucose-6-phosphate, creatine kinase, creatine phosphate, in a Beckman LSI701 scintillation counter. NADP, hexokinase, and glucose-6-phosphate dehydrogenase were purchased It is considered that the presence of 100 mM glucose will result in the GK from Boehringcr Mannheim (Lewes, United Kingdom). DTT, mannoheptu- enzyme approaching saturation but that the presence of 10 mM glucose-6- lose, mannoheptose, glucoheptose, N-acetylglucosamine, and mercaptoethanol phosphate will completely inhibit any low Km HKs that are present. Thus, the were purchased from Sigma Chemical Co. (Poole, United Kingdom). GIBCO radioactivity present in the discs at the end of the assay will be due to (Paisley. United Kingdom) supplied cell culture material, except PCS (Bio the production of l4C-glucose-6-phosphate as a result of GK activity only. logical Industries, Leicester, United Kingdom). Oxoid (Basingstoke, Hamp Measurements of Maximal HK Activity. HK activity was measured shire, United Kingdom) supplied tablets for PBS. All radiolabeled material was spectrophotometrically, principally according to the method of Bergmeyer (29) supplied by Amersham International (Buckinghamshire, United Kingdom), but incorporating creatine kinase and creatine phosphate to act as an ATP- and aqueous scintillation fluid (Optiphase Hisafe 3) and dry filter scintillation regenerating and ADP-removing system (30). Cells were homogenized in 5 fluid (Optiscint) came from Wallac Scintillation Products (Leicester, United volumes of a buffer (pH 7.5) containing 50 mM triethanolamine-HCl, 1 mM Kingdom). Whatman (Maidstone, United Kingdom) supplied DE81 ion ex EDTA, and 2 mM MgCU. Aliquots of the homogenate were added to a medium change discs. All other salts and buffers came from British Drug Houses (pH 7.5) containing UK)mM2-amino-2-hydroxymethyl-l,3-propandiol, 10 mM (Merck, Ltd., Poole, United Kingdom). Nude mice (nu/nu) were supplied by MgCl,, 1.1 mM EDTA, 1 mM KC1, 5 mM mercaptoethanol, 1 mM NADP, 2.5 HarÃan(Bicestcr, United Kingdom). mM ATP, 0.02 mg/ml glucose-6-phosphate dehydrogenase, 0.0125 mg/ml Human Cell Lines. H.Ep2 is a cell line of epithelial origin from a primary creatine kinase, 8 mM creatine phosphate, 0.1% Triton X-100, and 5 mM carcinoma of the larynx and is morphologically similar to the HeLa cell line glucose. The increase in absorbance at 340 nm is followed and is due to the (18). ESH TR1-2 (tumorigenic) and ESH p6 (suppressed) are a hybrid reduction of NADP. matched pair resulting from somatic cell hybridization of a HeLa cell deriva Measurements of N-Acetylglucosamine Kinase Activity. Activity of N- tive. D98, and a human keratinocyte (19). 2B1 TO (tumorigenic) and 2B1 Coll acetylglucosamine kinase was measured under the conditions of the GK assay. (suppressed) are a hybrid matched pair from hybridization of D98 and a human Thus, any activities obtained are not expected to be maximal. The 100 mM fibroblast (20). RVH 421 is a melanoma cell line (21). HT29 is a cell line glucose substrate (containing a trace of D-U-'4C-glucose) present in the GK seeded from a carcinoma of the colon (epithelial; Ref. 22). RT112 is a cell line assay was replaced by 100 mM /V-acetylglucosamine containing 4 fiCi/ml seeded from a carcinoma of the urinary epithelium (22), and T24/83 is a cell U-'4C-W-acetylglucosamine. All other details of the procedure are as outlined line seeded from a bladder carcinoma (23). MRC5 is an untransformed diploid for the assay of GK activity. fibroblast cell line from a fetal lung of 14 weeks gestation. MRC5 persists for Measurements of Cellular Protein Content. The protein content was 48 passages before the onset of senescence (24). determined using the method of Bradford (31). Bovine -y-globulin was used to Rat Cell Lines. The reel group of cell lines was established in the William make a standard protein solution. The standard and samples were treated Dunn School of Pathology, University of Oxford (25). Cells from a newborn similarly and dissolved in a solution comprising 100 mM triethanolamine-HCl rat heart were cultured, and three untransformed clones were isolated and and 400 mM KOH. propagated (reel c2, reel c3, and reel c4), along with the heterogeneous parent Incubation of Cells for Measurements of Glucose Consumption. Cells cell line (reel). After 25 passages, clones with primary tumorigenic activity (5-10 X 10'' cells/ml) were incubated in PBS (gassed with 100% O: for 30 min could be isolated from reel, and these were cultured separately as rcclt. After prior to incubation), pH 7.4, containing 5 mM glucose and 2% BSA [previously 52 passages, clones with metastatic activity could be isolated from the tumor defatted by the method of Chen (32)] in sealed flasks (treated with 1,1,1- igenic culture, and these were further propagated as rcclm. A low level of dimethyldichlorosilane solution to confer water-repellent properties) at 37Â°C. tumorigenic activity is evinced by reel (heterogeneous) and reel c2 at all Flasks were gassed with 100% CK in the gas phase for 30 s before incubation. passages tested during the present work. WRC 256 is a carcinosarcoma A 120-min incubation was stopped by the addition of 0.2 volumes of 25% (Walker) cell line (26). perchloric acid and cooling on ice. Media in the flasks were quickly brought Culture and Preparation of Cells. Cells were grown in an adhesion- to neutral pH using 40% KOH. The glucose concentration was measured in the dependent manner in MEM in sealed 250-ml flasks with an atmosphere of 5% media (cells plus incubation medium) and compared with an appropriate blank CO2 in air at a constant temperature of 37Â°C.MEM contained 5 mM glucose to which perchloric acid was added before the cell suspension. Viability of and 2 mM glutamine plus 5% FCS. Harvesting of subconfluent populations of cells (measured by the method of trypan blue exclusion) was unaffected by the cells was accomplished by incubation of the flask with PBS containing 0.04% conditions of the incubation over 120 min. (w/v) EDTA for 10 min at 37Â°C,after which cells could be detached by Measurements of Glucose Concentration. A deproteinizcd, neutralized vigorous shaking. For growth rate measurements, cells were resuspcnded, to an sample from an incubation of cells outlined above was added to a medium (pH appropriate density, in fresh MEM. For other experiments, the cell pellet was 7.5) containing 254 mM triethanolamine-HCl, 2.5 mM MgCl,, 20 mM ATP, 1.6 washed three times with PBS only. For measurements of glucose consumption, mM NADP, 0.1 mg/ml HK, and glucose-6-phosphate dehydrogenase. The cells were resuspended in PBS, and for measurements of enzyme activity, HK, reaction mixture was incubated for 20 min at 25Â°C,after which there was no GK, or N-acetylglucosamine kinase, cells were resuspended in 5 volumes of an further increase in absorbance, indicating that the reaction was complete. The appropriate extraction buffer and homogenized using a Teflon homogenizer for increase in absorbance at 340 nm allows calculation of glucose concentration. 30 passes of the pestle. Measurements of Rates of Growth of Cultured Cells. Rates of growth of Measurements of Maximal Glucokinase Activity. GK activity was meas cells were measured in two different ways based on: (Â«)the increase in cell ured essentially according to the method of Stanley et al. (27) by means of numbers and (/?) the incorporation of 6-['H|thymidine. (a) Cells (1-8 X IO4/ incorporation of the label from '4C-labclcd glucose into 14C-labeled glucose- ml) were inoculated into 50-ml flasks with growth medium, as for long-term 6-phosphate and including some modifications of Van Schaftingen (28). Cells propagation, but which contained appropriate concentrations of mannoheptu- were resuspended and homogenized in 5 volumes of a buffer (pH 7.5) con lose or other GK inhibitor. Cells were gassed with 5% CO2 in air (in the gas taining 50 mM triethanoIamine-HCl, 300 mM sucrose, 1 mM EDTA, 100 mM phase) at time = 0 and propagated for 4 days (96 h). At intervals of 1 day (24 KC1, and 10 mM mercaptoethanol. Aliquots (30 /Â¿I)of homogenate were h), flasks were removed, and cells were detached and counted using a incubated in a medium comprising 70 mM 2-amino-2-hydroxymethyl-l,3- Neubauer improved cell counting chamber, (b) Cells (1 X 10s per ml) were propandiol (pH 8), 7 mM MgCl,, 5 mM ATP, 10 mM glucose-6-phosphate, 100 seeded into wells of a 96-well plate with MEM (containing 5 mM glucose, 2 mM glucose (containing 4 (Â¿Ci/mlD-U-l4C-glucose), 0.1% Triton X-100, 2.5 mM glutamine, 5% PCS, and 0.2 mCi/ml 6-['H]thymidine). Appropriate con mM DTT, and 100 mM KC1 in a volume of 120 Â¡i\.An 8-min incubation was centrations of mannoheptulose or other glucokinase inhibitors were included in stopped by addition of 100 jul ice-cold methanol and cooling on ice. Aliquots the medium. Plates were incubated for 72 h at 37Â°Cwith an atmosphere of 5% (40 /xl) of reaction mixture were spotted onto DEAE-cellulose ion exchange C02 in air, followed by harvesting using a Skatron cell harvester. Radioactivity discs (which bind glucose-6-phosphate, but not glucose) and allowed to dry at in the cells due to incorporation of 6-['H|thymidine into DNA was measured room temperature. Discs were washed with four changes of doubly distilled using 0.1 ml dry filter scintillation fluid/filter and by measuring the cpm in a water (about 1 liter/50 discs/wash) and allowed to dry overnight. The radio Beckman LS1701 scintillation counter. 3279

Measurements of Rates of Tumor Growth in Vivo. Nude mice (female that the activity is not a feature of the mode of propagation of the MF1 nu/nu) were maintained in isolation from other animals and in accordance tumor cells. The present study established that the activity is also with the United Kingdom Coordinating Committee on Cancer Research guide absent from cultured rat lymphocytes, whether these are quiescent or lines (33) with a 12-h light/12-h dark cycle and fed ad libitum. Tumor cells have been mitogenically stimulated with concanavalin A, lipopolysac- (2â€”5x IO6 cells/200-jxl injection) in MEM were implanted s.c. in the lumbar charide, or phytohemagluttinin. This suggests that the presence of GK region. Mice developed visible tumors within 3-5 days, and these increased in diameter over the subsequent 15-20 days. Tumor burden did not exceed 5% of activity is not a universal feature of rapidly dividing populations of the animal's weight in any case. cells, nor is it a nonspecific adaptation to growth in culture. Indica Mannoheptulose (100 (nl of a 1.7 mg/g body weight solution) was admin tions are that manifestation of GK activity is restricted to transformed istered by i.p. injection daily for a 5-day period, starting at day 15 of tumor- cells whether these produce tumors in experimental animals (meta bearing. Control animals received 100 fil of 0.9% saline by a similar route. The static and tumorigenic) or not (suppressed). increase in tumor diameter was measured daily, and animals ate and drank The range of maximal activities of HK present in the tumor cell normally, exhibiting normal behavior patterns over the tumor-bearing period. lines listed in Table 1 is also broad (11 to 109 nmol/min/mg protein) At the end of the experiment, mice were sacrificed, and the tumors were and shows no correlation with the malignancy of the cell lines dissected and weighed. (assessed as rates of growth). However, capacities for glucose phosphorylation by these cell types can be seen as the sum of the maximal RESULTS activities of GK and of HK. Ranges of this value, although overlap ping, tend to be higher for the tumorigenic group of cell lines (14-117 GK activity can be detected in a range of human and rat cell lines nmol/min/mg protein) than for the suppressed (29-31 nmol/min/mg (Table 1). Activity is present in tumorigenic cell lines (1.1 to 19.6 protein) or untransformed (11-40 nmol/min/mg protein) cell lines and nmol/min/mg protein) and suppressed cell lines (9.4 to 16.5 nmol/ are generally much higher than for rat liver tissue (27 nmol/min/mg min/mg protein). There is no detectable activity in the untransformed protein). cell lines, MRC5, reel c3, and reel c4. Although the range of GK Hepatic GK is generally considered to have a much higher Km for activities is broad, its distribution is marked by its presence in all the glucose substrate than isoenzymes I, II, or III of HK. GK activity tumor cell lines tested and its absence from all untransformed cell from a range of cell lines of the present study gave Kms for glucose in lines or tissues (except rat liver tissue). It is interesting to note that the region 8-11.8 mM. These values agree well with previously whereas the assay of rat heart tissue produced the expected absence of reported Kms for glucose of 6-16 mM (34, 35) for the hepatic enzyme. detectable GK activity, the activity is present in the transformed cell /kms of HK I, II, and III for glucose are generally thought to be within lines cultured from newborn rat heart (reel, reel c2, rcclt, and rcclm; the range 30 /IM to 0.25 mM (35). Differences in Km between GK and Table 1). Ranges of maximal activity for metastatic tumor cells HK suggest that the activity present in the tumor cells of the present (1.1 to 8.1 nmol/min/mg protein) and for primary tumorigenic cells study is likely to resemble hepatic GK. However, GK activity is (3.8 to 19.6 nmol/min/mg protein) illustrate that there is no correlation usually considered to be confined to hepatic parenchymal cells (36, with malignancy, and an attempt to correlate tumor cell growth rate 37) and pancreatic ÃŸcellsof the islets of Langerhans (38). Allen et al. with maximal GK activity revealed no direct relationship between (39) have suggested that reports of high Km glucose-phosphorylating these two variables. activities in extrahepatic and extrapancreatic tissues are due to the In addition to its presence in all cultured tumor cell lines tested, GK presence of A'-acetylglucosamine kinase, which can phosphorylate activity can also be detected in human melanoma tissue, indicating glucose at low rates and has a Km for this substrate of about 210 mM. However, under the conditions of the GK assay performed in the present work, none of the cell lines tested showed any detectable rate Table 1 Maximal activities of glucokinase and hexokinase in human tumor celi lines of A'-acetylglucosamine phosphorylation. Thus, the GK activity pre and human melanoma tissue sent in tumor cell lines is unlikely to be due to A'-acetylglucosamine Means (Â±SEM)are calculated for at least eight separate batches of each cell line. kinase activity. activity activity (nmol/min/mg cell (nmol/min/mg cell Unusually high activities of the mitochondrially bound form of HK MalignantstateMetastaticTumorigenicSuppressedUntransformedGlucokinaseprotein)H.EP2rcclmESHTR1-22B1Cell line protein)12.8 have been reported for tumor cells compared with untransformed +0.24"109 cells. This activity shows reduced susceptibility to inhibition by Â±0.691 Â±7.4Â°11 glucose-6-phosphate compared with cytosolic HK, giving it some 1 Â±0.9220+ Â±1.3"17 similar characteristics to those of GK. However, we consider the 1.15.3 Â±0.64"85 TGrccltRVH421HT29RT1 activity reported here to be more similar to GK than to mitochondria! Â±0.3317 Â±4.5"36 Â±0.857.1+0.783.8 Â±2.362 HK for the following reasons. The Km for glucose of mitochondrial HK is in the range 56-83 /U.Mfor the hepatoma cell line, H-91, 12/84reelreel Â±0.369.4 Â±14"51 whether the enzyme is bound or solubilized (40). This contrasts with Â±4.94.2 A"msof 8-11.8 mM for the activity of the present study. Additionally, c2Human Â±0.186.1 Â±1.8"20 melanomatissue''ESH +1.19.4 Bustamente and Pedersen (40) report that the mitochondrial HK from their H-91 cells is inhibited by 53% by 2 mM glucose-6-phosphate. p62B1 Â±0.6316 Â±0.49"15 Collreel Â±1.5ND'ND'ND'18 +0.49"35 We find no apparent inhibition when activity is measured by the +0.81"40 radiochemical method, which includes 10 mM glucose-6-phosphate c3reel (see "Materials and Methods"), compared with the spectrophoto- c4MRC5Rat +2.9"11 Â±0.63"1.4 metric method of Dipietro and Weinhouse (41) in which there is no exogenous glucose-6-phosphate. livertissueRat +0.9726ND'Hexokinase livertissue'Rat The 7-carbon sugar, mannoheptulose, is considered to be a specific heart tissue1.1+0.118.1 inhibitor of hepatic GK (42) and does not inhibit HK. Mannoheptu " Values for maximal HK activity taken from Board el al. (10). ' Mean calculated for seven different samples of human melanoma tissue. lose, at a concentration of 20 mM, produces pronounced inhibition of ' ND, not detectable. tumor cell GK activity (Table 2). The percentage of inhibition ranges rfCalculated from data given by Stanley et al. (27). from 3 to 100%, compared with 69% for rat liver GK, and is not 3280

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. HIGH Kâ€žGLUa>Si:-PIIOSPHORYLATING ACTIVITIES IN TUMOR CELLS directly proportional to maximal activity. Estimations of GK inhibi Table 3 Values for /5(ts for inhibition of tumor cell glucokinase activity and rates of tion of four cultured human cell lines, RVH 421, H.Ep2, ESH TR1-2, growth by mannoheplulose and analogues Means were calculated for inhibition of GK activity or rates of growth of a range of and HT29, with mannoheptulose (and other sugars of similar struc four different human tumor cell lines. Values represent the means of at least five separate ture) were used to arrive at mean values for the I5()s.I5()represents the batches of cells in each case (Â±SEM). Fifty r/c inhibition of GK activity could not be attained with W-acetylglucosaminc up to 50 mM. Maximal effects were attained by about concentration of inhibitor required to elicit 50% inhibition of maximal 10 mM /V-acetylglucosamine. enzyme activity. The mean I5(, for mannoheptulose over the four tumor types is 12.5 mM(Table 3). This is similar to the I5()for rat liver (m"!Inhibition GK, which is about 15 mM[calculated from data given by Stanley et of of al. (27)]. The combination of evidence of A"mforsubstrate, absence of CompoundMannoheptulose glucokinaseactivity12.5 growthrate21.4 inhibition by glucose-6-phosphate, and inhibition by mannoheptulose Â±2.1 Â±3.2 suggests that the glucose-phosphorylating activity present in the tu Mannohcptose 84 Â±3.3 Glucoheptose 34Â±1Inhibition 85 + 10 mor cell lines of the present study has similar characteristics to those W-Acetylglucosarnine'so 85 + 12 of hepatic GK activity and is not due to contamination with N- acetylglucosamine kinase activity nor to confusion with low KmHK activity. this value is somewhat higher than the mean value for inhibition of The presence of 30 mM mannoheptulose also inhibited rates of GK activity (12.5 mM).Three other sugars (mannohcptose, glucohep- uptake of glucose by tumor cell lines during incubations of cell tose, and Af-acetylglucosamine)were found to inhibit tumor cell GK suspensions where glucose was the only substrate present (Table 2). activity, and mean values for I5()are shown in Table 3. All other Glucose consumption is inhibited by 25-75% by 30 mMmannohep sugars were less potent inhibitors than mannoheptulose, but all tulose for tumor cell lines, and this inhibition correlates with that of showed a similar property of inhibiting growth rates of tumor cell GK activity by 20 mMmannoheptulose (r = 0.992). Glucose uptake lines (mean values of I5(),84-85 mM;Table 3). This indicates that by the untransformed fibroblast cell line, MRC5 (which does not inhibition of tumor cell growth rate is a collective property of agents manifest GK activity), is not inhibited by mannoheptulose. This that will inhibit GK activity, illustrating the crucial importance of this indicates that the activity of GK closely reflects and may control rates activity for proliferation of tumor cells. Results presented in Table 3 of glucose uptake by the tumor cell lines of the present study. The also illustrate that, of commercially available sugars showing GK- presence of GK in addition to HK activity may confer on these cells inhibitory action, mannoheptulose is the most potent thus far tested. an increased capacity for glucose uptake and phosphorylation (see I50sfor the inhibition of growth rate are higher in all cases than for the below for discussion). inhibition of GK activity. This may indicate that the specificity of the Rates of growth of seven human tumor cell lines, measured as the tumor cell glucose transporter, although broad enough to allow car rate of incorporation of 6-[3H]thymidine (rate of DNA synthesis) by riage of mannoheptulose into the cell, does not extend to transporting the cells, are inhibited by the presence of 30 mMmannoheptulose. The this sugar as efficiently as glucose. Thus, higher external concentra percentage inhibition of rates of growth ranges from 14-50% tions of mannoheptulose may be necessary in order to elevate internal (Table 2). In a similar pattern to that of glucose consumption, rates of concentrations to a sufficient degree to inhibit the enzyme. growth of MRC5 in culture are unaffected by the presence of man Time courses of inhibition of growth rates by mannoheptulose over noheptulose. When rates of growth of the same range of cell lines a 96-h period are shown in Fig. 1. Growth rates were measured as the were measured in the presence of galactose (which does not inhibit increase in numbers of cells/flask with time. Of the three cell lines, the GK), it was discovered that galactose, up to a concentration of 150 rates of growth of which were affected by mannoheptulose, inhibition mM, does not affect rates of growth of any cell line tested. Table 3 became apparent by day 3 (ESH TR1-2 and ESH p6) or day 4 shows a mean value for I5(lfor mannoheptulose inhibition of growth (H.Ep2). The rates of growth of the untransformed MRC5 cell line are rate for a range of four different human tumor cell lines. At 21.4 mM, unaffected by the presence of mannoheptulose over the 4-day period (Fig. Id). In each of the three affected cell lines, mannoheptulose inhibits rates of growth in a dose-dependent manner, with 100 mM Table 2 Inhibition of tumor cell glucokinase activity unti glucose uptake by mannoheptulose producing a more severe suppression of cell numbers mannoheptulose than 20 mM.The H.Ep2 cell line shows a delayed response compared Means (Â±SEM)are calculated from at least five different batches of cells or tissue samples in each case. Rates of growth were measured by means of incorporation of with ESH TR1-2 or ESH p6, and this may be related to the lower 6[3H]thymidine by cells. Rates of growth of a similar range of cell lines were unaffected maximal activity of GK in the former, compared with the latter two by the presence of 30 mM galactose. cell lines (Table 1). The persistence of the mannoheptulose effect % inhibition of inhibition of % inhibition of suggests that it is still active after 4 days (Fig. 1, a-c). This may glucokinase activity glucose uptake growth rate by 20 mM by 30 mM by 30 mM indicate either that the sugar itself has not been metabolized by the mannoheptuloseH.Ep2Cell line/Tissue mannoheptulosemannoheptulose25 cells or that some other more long-term effect than competitive Â±2 0.575+ 1 28 Â± inhibition has been evoked by its presence. It is considered unlikely rcc 1mESH 1UOÂ±1861 Â±547 that mannoheplulose will be phosphorylutcd by tumor cell GK or HK, TR1-22B1 Â±520 Â±3 33 Â±931+614 TGRT112HT29T24/83RVH Â±220 since the sugar is not metabolized by rat brain or liver extracts (43). This would suggest a long half-life for mannoheptulose in the culture Â±350 medium of growing cells and would account for the persistence of Â±925 421reelreel 471+5 + growth rate-suppressing effects up to day 4 after administration of the Â±1100Â±1785 inhibitor. c2ESH In addition to inhibiting rates of growth of tumor cells in culture, p6 Â±15 Â±60 23 2B1CollMRC5Human 3Â±0.5NAÂ°21 mannoheptulose administration also causes inhibition of rates of 0 growth in vivo(Table 4). Two human tumor cell lines were inoculated tissueRatmelanoma Â±569 into nude mice, and mannoheptulose administration resulted in 65 and liver tissue31 Â±4% 79% inhibition of tumor growth, assessed as eventual tumor weight. " NA. not applicable. Mannoheptulose was administered for only 5 days of a 20-day tumor- 3281

20 10 9 OmM Mannoheptulose OmM Mannohoptuloss a 20mM Mannoheptulose B 20mM Mannoheptulose â€¢100mM Mannoheptulose Â«-tOOmM Mannoheptulose

5 10 -

1 2 3 1 1 234S Time (days) Time (days)

50 11

-D-OmM Mannoheptulose 10 OmM Mannoheptulose â€¢n 40-1 a 20mM Mannoheptulose 9 â€¢100mM Mannoheptulose a 20mM Mannoheptulose 8 - 100mM Mannoheptulose

30 â€” 7 6 5 20 - 4 3 10 2 1 0 2 3 2 3 Time (days) Time (days)

Fig. 1. Rales of growth of ESH TR1-2 (a), ESH p6 (i>), H.Ep 2 (r), and MRC5 ( P > 0.001: ", P < 0.001.

bearing period and at a dose giving a total tissue concentration animals, since the animals ate and drank normally and exhibited 2.7-fold lower than the I5() for inhibition of growth in culture. normal behavior patterns during the tumor-bearing period. However, growth of human tumors in vivo was still severely inhibited. Thus, there may be a more potent effect of the inhibitor DISCUSSION in vivo than is observed in vitro. Growth of the rat tumor, WRC 256, in nude mice was not significantly inhibited by mannoheptu The presence of GK activity has implications for the understanding lose, although there was a trend towards inhibition that may of tumor cell glucose metabolism. High rates of glucose import (5, 44, indicate that a higher dose would be more effective. There were no 45) and lÃ¡clateproductionwere among the first metabolic character apparent toxic side-effects of mannoheptulose administration to istics reported for tumor cells and tissues. Subsequent work on altered properties of the glucose transporter showed that this may be a contributory factor. The tumor cell glucose transporter displays in Table 4 Rates of tumor growth in vivo with mannoheptulose creased activity (46, 47), perhaps due to a decreased Kmfor glucose Means are presented (Â±SEM)with numbers of animals in parentheses. Doses of 100 Â¿il (48) and an increased density of transporter proteins in the membrane of 1.7 mg/g mannoheptulose solution were administered by daily i.p. injection for a 5-day of the transformed cell (49, 50). Available evidence suggests that period. Control animals received 100 /il of 0.9% saline daily by the same route. glucose transport into the tumorigenic cell is a near-equilibrium Weight of tumor (mg)" process, where concentrations of intracellular glucose approach those inhibition Cell line used for inoculum Control Mannoheptulose of growth in the extracellular medium at physiologically relevant glucose con RT112/84 (human colon carcinoma) 152 Â±39 (17) 32Â°Â±15 (13) 79 centrations (51). These factors may result in a similar mode of transport to that into the liver cell. Transport may also have a greater T24/83 (human bladder carcinoma) 4.6 + 0.4(14) 1.6* Â±0.3(15) 65 simple diffusion component in tumor cells (52) than that for untrans-

WRC 256 (rat carcinoma) 435 Â±83 (15) 366 Â±58 (14) 16 formed cells. If the process is near-equilibrium, the presence of GK " Differences between tumor weight in the presence and absence of mannoheptulose. enzyme will confer on the tumor cell a vastly increased capacity for * P < 0.001. Other differences are not significant. uptake of glucose by displacement of the glucose equilibrium across 3282

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. HIGH AT-,GLUCOSh-I'MOSFHORYLATING ACTIVITIES IN TUMOR CELLS the plasma membrane. This would give the GK activity a similar role their resources to the provision of precursors for nucleotide synthesis. to that suggested in liver, where it allows an extra capacity to take up In addition, if cytosolic pyruvate carboxylase is present, oxaloacetate, and phosphorylate glucose (which can then be stored as glycogen) at and hence aspartate (for nucleotide synthesis), may be produced from higher blood glucose concentrations. This differs from the glucose the pyruvate intermediate of glycolysis. Glycolytic rates are high sensor role played by GK in the pancreatic ÃŸ-cell,a cell type which among many untransformed cell types that have the ability to prolif does not store appreciable quantities of glycogen. The presence of erate on stimulation, and these cell types also tend to show aerobic tumor cell GK activity would thus allow preferential access to plasma glycolysis (56-59) with a diminished or absent Pasteur effect. How glucose over host tissues and may explain earlier observations of high ever, it may be that tumor cells alone show constitutively high rates of rates of glucose import. glycolysis (55), and this pathway is only up-regulated on mitogenic GK activity augments the role of HK by catalyzing the same stimulation of untransformed cell types. Indications from the present reaction while having a higher Km for the common glucose substrate. work are that for tumor cells, inhibition of glycolysis, due to a It is considered that HK (Km = 30 Â¡J.Mto0.25 ITIM;Ref. 35) will be decrease in the availability of the glucose-6-phosphate substrate when saturated at the intracellular glucose concentrations thought to GK is inhibited, will suppress rates of growth of tumor cells by be present in the tumor cell. This would normally make HK activity starving the cells of energy, biosynthetic precursors, or both. Which of a contender for control by factors other than the glucose concentra these two factors may be the more important is difficult to assess. tion. However, the presence of GK activity complicates the situation However, for tumor cells growing in MEM, where both glucose (at since the capacity for glucose phosphorylation is likely to be GK plus 5 mM) and glutamine (at 2 mM) are available, rates of glucose HK activity, rather than HK alone. Tumor cell GK is likely to be consumption are 41 to 52 jumol/day/mg protein (for H.Ep.2 and ESH TR1-2 cell lines), and rates of glutamine consumption are 1.3 to 1.4 substantially active at most concentrations of plasma glucose, since Kms fall within the range 8-11.8 mM, and plasma glucose is likely to jumol/day/mg protein. A lower limit of ATP production from glucose be 5-10 mM. Any control of HK activity alone could only control rates can be estimated by assuming that all consumed glucose is converted to lÃ¡clate.This gives 82-104 /xmol ATP produced/day/mg protein. of glucose uptake when the intracellular glucose concentration falls to levels that would render the activity of GK very low. The fact that Estimated rates of ATP production from glulamine (assuming that all total glucose-phosphorylating capacity (HK + GK; Table 1) is still consumed glutamine lhal is noi converted to glutamale is fully oxi dized) are 11-12 /xmol/day/mg protein. Thus, mean ATP production lower than maximal phosphofructokinase activity (10) for many tu from glucose is approximalely 7-9 times that from glulamine, despite mor cell lines does leave the possibility that rates of phosphorylation the more efficient oxidative metabolism of the latter substrate. Partial, of glucose and hence, perhaps, rates of glycolysis may be strongly rather then full, oxidation of glutamine (7, 60, 61) further limits ATP influenced by plasma glucose concentration. When the plasma glu production. Under similar conditions, the ratio of ATP production cose (and hence intracellular glucose) concentration rises, for exam from glucose to that from glutamine is 3 for the untransformed MRC5 ple, GK will respond with an increase in activity, and phosphofruc tokinase may respond to the resulting increase in fructose-6-phosphate cell line (which does noi display GK aclivity). This suggests that ATP production from anaerobic glucose metabolism may far oulslrip lhat concentration. Thus, it may be that the presence of GK activity is an from the oxidative glutamine metabolism for tumor cells under culture adaptation by the tumor cell to ensure that rates of glucose phospho conditions. Manifold reports of hypoxia in a rapidly growing tumor rylation respond with increased sensitivity to plasma glucose concen mass may increase the differential by limiting rates of oxygen-requir tration and also that high rates are maintained even under conditions ing glulamine melabolism further and increasing Ihe reliance of Ihe where HK activity is substantially inhibited. HK is inhibited by a high cells on the anaerobic glucose substrale. This situation has been ATP:ADP concentration ratio and by high concentrations of its prod addressed by Kallinowski et al. (62), who suggest that the fully uct, glucose-6-phosphate. Neither of these inhibitory factors applies oxygenaled incubation conditions used by those who suggest gluta toGK. mine as the main energy-yielding substrate for tumors (7-9, 63) may Since mannoheptulose inhibits glucose uptake by the tumor cell, be unrealistic for the tumor in vivo. indications are that the role of GK eclipses that of HK and may be the The evidence outlined above suggests lhal the limit on entry of single most important factor in controlling this process. The impor glucose into glycolysis imposed by the GK inhibitor, mannoheptulose, tance of glucose as a tumor cell substrate has been inferred previously may reduce the availability of energy to Ihe cells beyond the critical from its high rates of import and metabolism. Tumor cells and tissues point necessary to maintain proliferation rales. It may be that this convert most consumed glucose into lÃ¡clate(8, 53, 54), suggesting a energy-starving role of mannoheplulose is more important lhan its relatively inactive oxidative metabolism with glucose. Experimental role in limiting availability of biosynthetic precursors. Results pre- observations seem to confirm this (8). With a glucose substrate, senled above suggesl lhal ATP produclion from glucose will be therefore, the cells may rely on their anaerobic glycolytic metabolism inhibited by 25-75% in the presence of 30 mM mannoheptulose. A to generate ATP via substrate-level phosphorylation. Rates of lÃ¡clate similar constraint is not placed on proliferation of unlransformed cells in production are high (so high for the cells of the present study that lÃ¡clateis released into the growth medium),4 and this suggests simi culture, since growth rates of MRC5 are unaffected by mannoheptulose. The presence of GK aclivily may represent an adaptation by the larities to the anaerobic metabolism of vigorously exercising muscle, lumor cell/tissue to allow it lo compete more effectively with other where limiting rates of O-, diffusion dictate that most ATP for glucose-consuming organs via Ihe presence of a larger capacily for contraction must come from glycolysis. glucose uplake. Certainly, the tumor which shows GK activity will However, the glycolytic pathway serves as a pool of metabolite have preferential access to the glucose subslrale. However, a GK precursors for biosynthesis, as well as a source of ATP-generating inhibitor may reduce Ihe exlra glucose uplake capacity so advanta reactions. Glucose-6-phosphate can produce ribose units (for nucleo- geous to the tumor cell, and thus, there is a potential role for such tide synthesis) and NADPH (for lipid synthesis) when it is metabo agents in the trealment of cancer patienls. The dramalic reduction in lized through the pentose phosphate pathway. Eigenbrodt et al. (55) growth rates of tumors growing in vivo by mannoheplulose adminis- have described tumor cells as nucleigenic, cells which devote much of tralion lends support to Ihis idea. Mannoheptulose is a naturally occurring sugar, its usual route of 4 Unpublished observations. purification being from avocados (Persea gratissima; Ref. 64), and is 3283

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. HIGH Kâ€žGLUCOSE-PHOSPHORYLATING ACTIVITIES IN TUMOR CELLS anticipated to be of low toxicity. These aspects make it suitable for of normal and virus-transformed chick cells in culture. Proc. Nati. Acad. Sci. USA, 70: 2951-2955, 1973. consideration as a potential therapeutic agent. However, the I50 of TildÃ³n, J. T. An alteration in glucose metabolism associated with a defect in ketone GK for mannoheptulose is relatively high at about 12.5 mM. This body metabolism. Proc. Nat. Acad. Sci. USA, 70: 210-214, 1973. implies that, were this sugar considered as a potential treatment, Culvenor, J. G., and Wcidemann, M. J. Phytohaemagluttinin stimulation of rat thymus lymphocyte glycolysis. Biochim. Biophys. Acta, 437: 354-363, 1976. high doses would be required. Equivalents of the daily doses given Gumaa, K. A., and McLean, P. The kinetic quantitation of ATP:D-glucose-6-phos- to experimental animals during the present study would be 1.7 g/kg photransferases. Biochem. J., //5: 1009-1029, 1969. for human patients (119 g for an average-sized person of 70 kg). Diamond, 1., Lcgg. A., Schneider, J. A., and Rozengurt, E. Glycolysis in quiescent cultures of 3T3 cells. J. Biol. Chem., 253: 866-871, 1978. This is a high dose, and no information is available concerning a Reitzer, L. J., Wice, B. M., and Kennel, D. J. The pentose cycle. J. Biol. Chem., 253: minimum effective dose. On the positive side, the sugar is very 5616-5626, 1980. Newsholme, E. A., and RÃ¤ndle. P. J. Regulation of glucose uptake by muscle. soluble and stable, and administration in the form of a drink might Biochem. J., 80: 655-662, 1961. be possible. Moore, A. E.. Sabachowsky, L., and Toolan, H. W. Culture characteristics of four Few toxic side-effects are anticipated from mannoheptulose admin permanent lines of human cancer cells. Cancer Res.. 15: 598-602, 1955. Peehl, D. H., and Stanbridge, E. J. Characterisation of human keratinocytes X HeLa istration. Previous studies (65, 66) failed to uncover toxicity towards somatic cell hybrids. Int. J. Cancer, 27: 625-635, 1981. experimental animals beyond reversible suppression of insulin secre Klinger, H. P. Suppression of tumorigenicity in somatic cell hybrids. Cytogenet. Cell. tion and transient hyperglycemia. There is an automatic selectivity of Genet., 27: 254-266, 1980. McCormick, D., Wallace, I., Kirk, J., Dinsmore, S., and Allen, I. The establishment mannoheptulose for tumor tissue. Its target enzyme, GK, is only and characterisation of a cell-line and mouse xenografts from a human malignant present in the liver and pancreas among normal tissues. Even in these melanoma. Br. J. Exp. Pathol., 64: 103-115, 1983. Marshall, C. J., Franks, L. M., and Carbonell, A. W. Markers of neoplastic transfor two tissues, GK protein is not thought to be constantly present at the mation in epithelial cell-lines derived from human carcinomas. J. Nail. Cancer Inst., same high levels but is produced in response to blood glucose and 58: 1743-1747, 1977. insulin levels in order to fulfill its roles in storage (liver) and glucose Bubenik, J., Baresova, M., Viklicky, V., Jakovbkova, J., Sainerova, H.. and Donner, J. Established cell-line of urinary bladder carcinoma containing tumour-specific sensing (pancreas). antigen. Im. J. Cancer, //: 765-773, 1973. These factors suggest that mannoheptulose may be a potential Jacobs, J. P., Jones, C. M., and Bailie, J. P. Characteristics of a human diploid cell designated MRC5. Nature (Lond.), 227: 168-170, 1970. treatment for cancer patients. Actions in reducing tumor burden and Moraes. O. Tumour progression: in vitro and in vivo investigations of "spontaneous ameliorating host tissue wastage by increasing availability of glucose ly" transformed rat cell-lines, Ph.D. thesis. University of Oxford, 1989. for nontumor tissues are anticipated. This agent may be especially Earle, W. R. A study of the Walker rat mammary carcinoma 256 in vivo and in vitro. effective in conjunction with conventional treatments. The severe toxic- Am. J. Cancer, 24: 566-612, 1935. Stanley, J. C., Dohm, G. L. McManus, B. S., and Newsholme, E. A. Activities of hies of chemotherapy and radiotherapy tend to limit the frequency with glucokinase and hcxokinase in mammalian and avian livers. Biochem. J., 224: which sessions can be given to patients. Tumor regrowth is a hazard 667-671, 1984. Van Schaftingcn, E. Protein confers on glucokinase the property of regulation by fructose- during the intervening periods, and a nontoxic, noninvasive agent would 6-phosphate and fructose-1-phosphate. Eur. J. Biochem., 779: 179-184, 1989. seem a suitable supplementary treatment. Bergmeyer, H-U. Hexokinase. Methods Enz. Anal., /: 473-474, 1974. High ^Â¡sof tumor cell GKs for mannoheptulose are seen as a Crabtree, B., and Newsholme, E. A. The activities of phosphorylase, hexokinase, phosphofructokinase, lÃ¡clatedehydrogenase and the glycerol-3-phosphate dehydro- disadvantage in the further development of this particular compound. genases in muscles from vertebrates and invertebrates. Biochem. J., 726: 49-58, Studies are under way on structural analogues of the sugar in order to 1972. identify compounds of increased potency. Bradford, M. M. A rapid and sensitive method for the quanlitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem., 72: 248-254, 1976. Chen, R. F. Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. ACKNOWLEDGMENTS Chem., 242: 173-181, 1967. United Kingdom Coordinating Committee on Cancer Research. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. High Km Glucose-phosphorylating (Glucokinase) Activities in a Range of Tumor Cell Lines and Inhibition of Rates of Tumor Growth by the Specific Enzyme Inhibitor Mannoheptulose

Mary Board, Alison Colquhoun and Eric A. Newsholme

Cancer Res 1995;55:3278-3285.

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