Activity of enzymes related to carbohydrate metabolism in the HT 29 colon adenocarcinoma cell line and tumor

J. Biochm~. Vol. 16, No. I. pp. X7-91, 1984 Printed in Great BritGn All rights reserved

OO?O-7 I IX 84 $3.00+ O.I)O Copyright (’ 1984 Pergamon Pros\ I.td

l/u

ACTIVITY

OF ENZYMES METABOLISM i4DENOCARCTNOMA

C.

I~ENIS,

C.

CORTINOVIS,

Institut de Phyaiologie.

B.

Universite

RELATED TO CARBOHYDRATE IN THE HT 29 COLON CELL LINE AND TUMOR

TERRAIN,

V.

VIALLARD,

H.

PARIS

Paul Sabatier, 2 Rue F. Magendie. [Tei (61) 52-81671

and J. C.

MURAT

31400 Toulouse.

France

Abstract- I. Activity of several enzymes of the glycogen and carbohydrate metabolism IS studied m HT 29 colon adenocarcinoma cell line and in HT 29 tumors developed in nude mice, by reference to the normal human colon mucos’i.L 2. Activity of glycogen synthase, glycogen phosphorylase, pyruvate kinase, fructose- I .h-diphosphatase, glucose-6-phosphate dchydrogenase and lactate dehydrogenase is found to be increased in both the cultured cells and the tumors. 3. It indicates that the biochemical strategy of malignant cells. due to the neoplastic transformation process. involves specific changes in the carbohydrate metabolism of tumor as well as in ri/ro growing correspondent cell line.

INTRODUCTION

kinase (EC 2.7.1.1 I) and pyruvate kinase (EC 2.7.1.40) which are key enzymes for the glycolytic

The HT 29 cell line was established in permanent culture by Dr J. Fogh (Sloan Kettering Institute for Cancer Research, Rye, New York, U.S.A.; Fogh and Trempe, 1975) from a human colon cancer and the HT 29 tumors can be easily obtained in nude mice by subcutaneous injection of cultured cells. Up to now, HT 29 cells or tumors have been extensively studied in many directions: morphological studies have shown accumulation of polysaccharides (Rousset, 1982) and presence of a partially differentiated brush-border (Salomon, 1982); pattern of glycogen accumulation has been investigated in relationship to the growth rate (Rousset et al., 1980, 1981 a) and the control of glycogen metabolism was therefore much studied in this model (Rousset et al., 1981b; Castilla et cd., 1981; Paris et u/.. 1982, 1983); Laburthe et crl. (1980), studying the cell membrane receptors. have shown that the Vasoactive Intestinal Peptide (VIP) was able to induce a large increase of cyclic AMP level in the cells; effect of VIP on glycogenolysis was also reported (Rousset et d.. 1981b); very recently, we showed that an a+drenergic receptor was present on the cell membranes (Carp& it a/.. 1983); a specific binding for insulin has been reported by C&zard et ul. (1981) indicating that this hormone could act as a growth factor for the HT 29 cells. By contrast, scanty information is available concerning metabolic pathways and utilization rates of different energetic substrates. The aim of the present study was to report on the activity of some enzymes involved in carbohydrate metabolism. both in the HT 29 cultured cells and in the HT 29 tumors developed into nude mouse, by reference to the normal colon mucosa. The investigated enzymes were: glycogen synthase (EC 2.4.1 .ll) and glycogen phosphorylase (EC 2.4.1.1); hexokinase (EC 2.7. I. l), phosphofructoBCih,

F

pathway; glucose-&phosphate dehydrogenase (EC 1.I I .49) which controls the hexose monophosphate shunt; lactate dehydrogenase (EC I, 1.1.37) which deals with the high rate of lactate production in

cancer cells; fructose- I .6-diphosphatase (EC 3.1.3. I I ) and glucose-6-phosphatase (EC 3.1.3.9) which are involved in futile cycles and in gluconeogenesis: glycerokinase (EC 2.7.1.30) was investigated because HT 29 cells were found to contain lipids and because it was questionable whether the cultured cells could utilize glycerol which is sometimes present in the culture

medium. MATERIALS

AND METHODS

Cells were routinely cultured at 37 C in 25cm: plartlc flasks with Dulbecco’s modified Eagle medium supplemented with lo”, of fetal calf serum. Each cell culture was started by seeding 10” cells and was run for 18 day\. confluence being reached after I&-I 2 days. The medium wa\ changed each 48 hr. After 18 days, the number of cells in the monolayer of one flask was estimated to be 30 x IV. After medium removal, the monolayer was rapidly rinsed with 5 ml of ice-cold saline. immediately deep-frozen and kept at -WC until analysis. At the time of analysis. the frozen monolayer was scraped from the plastic surface into 2 ml of the appropriate ice-cold buffer, homogenized by multiple passages through a 26G needle fitted to a I ml plastic syringe, collected into 1.5 ml Eppendorf tubes, sonicated for 15 set (except for Glc-6-phosphatase determination) and spun down (30sec, 9OOOg). Enzyme activity was immediately determined in the supernatant. Protein content of an aliquot was estimated with the Brilliant Blue method of Bradford (1976).

rumors Tumors were obtained from HT 29 cells injected subcutaneously into nude mice reared in our laboratory ( IO” cells 87

in ii total ~~olumc of 0.2ml culture medium per mouse). After 3 uccks. the average waght of tumors was I g.: nucr were killed by decapitation and excised tumors were kept at X0 (‘ until analysis. For enrymc activity determination. tumor samples were homogenized mto the appropriate Ice-cold huller using a glass Potter. Homogenates were sonicatcd and spun down as for the cells.

SampIca of colon mucosa were taken from several adults \utTering an irreversible coma due to cranial trauma: these patients were also kidney donors. Clean samples of scraped muco\a wcrc hcpt in liquid nitrogen. then treated as tumor snmpla.

Chcmlcala wcrc ohtaincd from Scrco (Heidelberg. W. Cicrmany) or from Sigma (St. Louis. Missouri. IJ.S.A.): analytical en/-ymes were from Boehringer (Mannheim. W. German)): [‘JC]glucose-l-phosphate and UDP-[Vlglucose were from New England Nuclear (Boston. Massachusetts. U.S.A.). Glycogcn synthasc activity was determined according to the method of Sate VI tri. (1973): 0. I ml of assaymixture contained: 50 mM Tria, 4 mM EDTA. X mM NaF, 0.25 M \ucI-o\c (pH 7.4). I’),, oyster glycogen. IO mM UDP[“C‘]glucnsc: total (a + b) form of the enzyme was measured in the prcscncc of 6.5 mM glucose-h-phosphate and R form (Saugmann and Esmann. 1977) of the enzyme was estimated in the prc\encc of 0.65 mM,g~ucosc-h-phosphate. Incubation was run at 30 C‘ after addltlon of supernatant from cell or tissue homogenate; at 0, 20 and 40 min. 0.05 ml ahquots wcrc spotted on filter paper and rinsed in 66”,, ethanol: tilter papers were then dried and radioacti\itj was measured in a hcta scintillation counter. Glycogcn phosphorylase activity was determined according, to Wang and Eamann (I 972) by following the incorporatlon of [lJC]glucose-l-phosphate into glycogen. ().I-ml of assay mixture contained: SO mM PIPES. SO mM NaF. 4mM EDT:2 (pH 6.X). I”,, oyster glycogen. 0.1 M [“C]plucosc-l-phosphate, I mM call’elnc, I .5 mM AMP: 0. I ml of supernatant was added to start the incubation at 30 C. The procedure was then the same as for the glycogen \ynthasc assa>. HcxokinaTc activity was measured according to Joshi and Jaganmithan ( I%h): 0.I ml of supernatant wa? mixed with 0.9 ml of 75 mM Tria (pH 7.6) containing 35 mM MgCl?. 0. I mM EDTA. 0.5 mM ATP. I .SmM NADP. I5 mM glucobc and 7 li ml glucose6-phosphate dehydrogenase. The reaction M’IICrun at 25 C‘ for IO min and change in optlcal dcnslt) wa recol-ded at 340 nm. Phosphofructokinasc activity was estimated according to Mandercau and Boicin ( 1973) by mixing 0.05 ml of supernatant with I ml of 0. I M Tris (pH 7.S) containing 0.1 mM AMP. 5 mM KII,PO,. 5 mM MgCI:. 6 mM dithiothreitol. I .5 mM ATP. I mM fructose-h-phosphate. 0. I5 mM NADH?. I U.mI of aldolase. IO !J;ml of trlosephosphate i\onicrasc, 7 li:ml of glyccraldehyde-phosphate dehydrogcnasc. The oxidation of NADH: \vas followed at 25 C. for IO min. iit 340 nm. P)I-u\atc kinase activity was estimated according to Gutmann and Bcrnt (1974): the incubation mixture contamed 0.05 ml of supcrnatant and 0.65 ml of l60mM tricthanolammc (pH 7.5). I20 mM KCI, 21 mM MgSO,. I.3 mM I:DTA. 3 mM NADH,. 33 mM phosphocnolpyruvatc. IO0 mM ADP. 2X0 Ljsrnl of lactate dehydrogcnase. Reaction uas run at 25 C for IO min and NADFI, oxqdatlon was read at 240 nm. C;luco\c-6.phosphat~se activity uas measured according to H uson c/ (I/. ( I97 I ). Samples were homogenized in 0. I M cacodylate bufl’cr (~11 6.5); 0.1 ml of homogenate was incubated with 0.1 ml of0. I M glucose-h-phosphate at 25 C for 30 men: at this time. 0.5 ml of trichloroacetlc acld-

ascorbic acid solution was added and released PO, was measured according to Chen clr (I/. (1956). Non specilic phosphatases were checked by incubating 0.1 ml ol homogenate with 0.1 ml of 0.1 M beta-glycerophohphate. Fructose-1.6.diphosphatase activity was estimated according to Latzko and Gibbs (1974): 0. I ml of supcrnatant was mixed with 0.9ml of 0.2 M Tris (pH 7.5). SO mM MgC12, 80 mM 2-mercaptoethanol. 0.5 mM NADP. 0.3 M fructose-l.h-diphosphate. 7 U/ml of phosphoglucosc isomerase. 10 IJ/ml of glucose-h-phosphate dehydrogcnaae. The reduction of NADP was followed at 25 C. for IO min. at 340 nm. Glycerokinase activity was measured accordmg to Wieland (1957): 0. I ml of supernatant was mixed with I ml of 0.2 M glycine buffer (pH 9.X). 0.25 M hydrazine. 2 mM M&Cl,, I.6 mM ATP. 0.7 mM NAD. I7 U/ml of glycero-iphosphate dehydrogenase and 0. I ml of 0. I M glycerol was added to start the reaction at 25 C for IO min: NAD reduction was read at 340nm. Lactate dehydrogenasc activity was cstimatcd according to Bcrgmeyer and Bcrnt (I 974): 0.02 ml of supcrnatant was mixed with I ml of 0. I M phosphate bulTer (pH 7.5) containing 22.7 mM sodium pyruvate and 1.2 mM NADH. The reaction was followed at 340nm. at 25 C for lOmu. Cilucose-h-phosphate dehydrogenasc activity was cstimated according to Liihr and Wailer (1974): 0.0.5 ml 01 supernatant was mixed with I ml of 50 mM Tris buffer (pH 7.6) containing 5 mM EDTA, 0.5 mM NADP. I .35 mM glucose-h-phosphate. The reduction of NADP was rccordcd at 340 nm, at 25 C for IO min.

RESlJLTS AND DISCUSSION

All the results

are shown in Table I.

Activity of both total (a + b) and R forms of glycogen synthase is strongly increased in the HT 2Y cells as well as in the HT 29 tumors. It is tempting to correlate this fact with the difference in glycogen contents which was reported by Rousset (1982), viz: 2.5 pg/mg protein in the normal colon mucosa, 15-45 (according to the growth phase) pg/mg protein in the cultured HT 29 cells and 15.5pg!mg protein in the HT 29 tumors grown into nude mice. It is of interest to find a R form of glycogen synthase in the normal mucosa and in the malignant cells: this form of the enzyme, activated by low glucose-6-phosphate concentration and non inhibited by PO: or ATP (Saugmann. 1977), was reported to be present only in leucocytes (Saugmann, 1977: Saugmann and Esmann, 1977), adipose tissue (Kaslow et (II., 1979) and liver (Tan. 1982). It is likely that increase of R form of glycogen synthase in HT 29 cells is an important factor for explaining the special pattern of glycogen metabolism described in adenocarcinoma cells (Rousset rf u/., 1983; Paris et al., 1983). The R form of glycogen synthase is also reported to be present in the Ehrlich ascite malignant cells (Granzow. personal communication, 1983). Glycogen phosphorylase activity is also found to be about 5 times increased in the malignant cells when compared with the normal mucosa. It must be pointed out that the a form activity is very low. as a rule, in standard conditions; however, we recently reported that glycogen phosphoryiase was transiently converted into a form when glycogenolysis was in-

Carbohydrate Table

1. Activity

of

enzymes

of

metabolism glycogen

adenocarcinoma

and

cultured

enzymes carbohydrate

cells and

HT

of HT 29 colon cancer cells metabolism 29 tumors

in crown

human into

Normal

EtllymeS

EC

Glycogen

synthaae

(total

Glycogen

synthase

(R

Glycogen

phosphorylase

form)

No.

2.4.1.1

colon I

24.1.11

form) (total

form)

mucosa

colon nude

HT

mucosa.

HT

29 colon

HT

29

mice

29

cultured

cells

tumors

I .89 2 0.30

9.44 f 0.36t

7.49 + 0.5bt

1.09~0.15

5.51 i0.12.t

2.72&0.2lt

2.4.1.1

52.34 + 3.65

253.16i832t

Hexokmaae

2.7.1.1

21.59 + 1.60

20.42 i

Phosphofructokmase

2.7.1.11

3.22 + 0.30

Pyrwate

2.7. I .40

kinase

x9

270.63

+ 36.2

123.67 + l5.U 2.33

2.68 & 0. I9 I 266.49

+ 42 It

27 96 & 2.74 3.20 + 0.28 X33.82 + 46 it

Glucose-6.phosphatase

3.1.3.9

0.77 * 0.20

0.70 & 0.22

I .05 -i_ 0.23

Fructose-1.6.diphosph~t~s~

3.1.3.11

2.99 * 0.55

5.02 5 o.a*

4.30 & 0.22*

Glucose-h-phosphate

I. I. I .49

Lactate

dehydrogenase

I

dehydrogenase

2.7.1.30

Clvcerokinase Activities

are expressed

synthase Values

and

tn nmol

dlffcrent

of five determmations from

duced by glucose starvation (Paris et al., 1983). Hesokinuse.

of substrate

II

+ 2.37

110.42 k 5.8Ot

121.50~20.71

961.30*34.It

X07 25 i

4.06 + 0.82

converted.

per mm.

the normal

5.25 f 0.09

per mg of protein,

at 25 C (at

56.0+

4.1 I * 0.27 30 C for

glycogen

in cultured

iSEM.

colon

mucosa:

'P i 0.05: tP < 0.001.

HT 29 cells

pil~)sphofructokina.~e and pywate

and

in cancer cells, which could indicate an activation the fructose-6-phosphate-fructose-l,6-diphosphate futile cycle.

of

kinase

Table I shows that only pyruvate kinase is significantly modified in the HT 29 cells or tumor. It IS a general feature that, in cancer cells, pyruvate kinase is highly active (Knox, 1976; Weber, 1977). In the present study, the method for measuring the whole hexokinase activity (see “Materials and Methods”) does not allow to distinguish between different lsoenzymes or different subcellular localizations. It is possible that a significant difference could exist from this point of view, as suggested by Bustamante ct al. (1981) for the malignant cells. Further studies are necessary to elucidate this point. By contrast to what was reported by Weber (1982) who studied several colon carcinomas, we find no Ggnificant difference in phosphofructokinase activity between normal colon mucosa and HT 29 cells or tumors. It is somewhat puzzling to see that in highly glycolytic HT 29 cancer cells, activity of key enzymes of the glycolytic pathway are not shifted to a similar extent; however. it is a common observation and it was also reported by Weber (1977), when studying the glycolytic enzymes of cancer cells, that the enlymes activity ‘of one pathway were modified in different orders of magnitude. (;luco.se-h-phosp,hatase /~hutuse

22.

155.01 2 17.3

phosphorylase)

are the mean

Signilicantly

1.1.27

jiuctose-1,6-diphos-

As seen in Table I, a slight activity of glucose-6phosphatase is observed in the normal mucosa as well xs in the HT 29 cells or tumor. It is already known rhat this enzyme., mainly present in liver and kidney, is also found in the digestive mucosa (Hugon et a/., 1971). The trace activity found in the HT 29 cells is not able to produce significant amount of free glucose from glucose-6-phosphate since tracing the formation of labelled glucose from gluconeogenic precursors was negative (unpublished result). In the case of liver neoplastic transformation, a great decrease of glucose-6-phosphatase activity is considered as an index of malignancy (Bannasch et a/., 1980). In HT 29 cells, the fructose-1,6-diphosphatase activity is increased by reference to normal mucosa. It is the first report dealing with the activity of this enzyme

Glucose-6-phosphate

dehydrogenase

The activity of this enzyme is five times higher in cultured HT 29 cells and tumor than in normal colon. The increased activity for pentose-phosphates synthesizing enzymes throws light on the mechanism of the increased ability of cancer cells in culture as well as in tumors to produce pentoses for nucleic acids synthesis. A fairly wide range of concentrations of this enzyme is found among normal tissues. In slow or fast growing hepatomas, it was shown that the glucose-6phosphate dehydrogenase activity was not significantly changed by reference to the normal liver (Knox, 1976); this is in contrast with the present data and what is generally reported in malignant tissues. When studying 5 other cell lines derived from human adenocarcinoma, we found, as a rule, an increased activity of the glucose-6-phosphate dehydrogenase (unpublished data). Lactute dehydrogenase This enzyme is known to exist as a sum of several isoenzymes. The values reported in Table 1, which represent the total activity, show that lactate dehydrogenase is much more active in HT 29 cells and tumor than in normal mucosa. Such high activity is consistent with the increased production of lactate which was earlier reported to be the rule in cancer cells (Warburg, 1956); it could also allow the HT 29 cells to convert back the lactate into pyruvate when other fuel sources are exhausted in the culture medium, as we have observed in cultures left for 5 days without renewing the medium (unpublished data). The lactate dehydrogenase activity is already quite high in the normal gut mucosa; this is related to the ability of this tissue to convert easily glucose into lactate (Roediger, 1982); it seems, thus, that high rate of lactate production is a functional characteristic of the whole gut epithelium which is just enhanced in the transformed neoplastic tissue.

The presence of this enzyme in colon mucosa indicates the ability of this tissue to utilize glycerol as energetic substrate and as precursor for glycerides synthesis. The activity of the enzyme is found to be in the same order of magnitude in FIT 39 cells, which indicates that these cells can use glycerol when present in the culture medium.

Bradford M. M. (1976) A rapid sensitive method for the qu~lltit~tion of protein utilizing the principle oiprotein dye binding. Anr+t. Hiciihr~~)1.72, 248 -754. Bustamante E., Morris 1. P. and Pcder\cn P. L. (19x1) Energy metabolism for tumor cells: requirement for u form of hexokinase with a propensity for mmirochondri;tl binding. J. hiol. C’ho,l. 256. X699 X704. Carpene C.. Paris H.. Cortinovis C., Vlallnrd V. and Miirar J. C. (1983) Characterlration of alpha,-adronergic rcceptars in the human colon adenocarclnoma cell lint FlT ?9 in culture by (‘H)-yohimbinc blnding. (;L’II. /‘lrirrr!rtrc,. 14. 701 70.3.

(‘ONC’I,& IISIONS < The data presented above are a contribution to the question of a specific bi~~chelni~~~l pattern in colon cancer which distin~uislles it from normal mucosd. The most striking moditications of enzyme activities by reference to normal colon epithelium are observed both in the HT 29 cultured cells and in the HT 29 tumors grown into nude mice: in other words, the biochemical variations seen in the cultured cells are also present in the tumors. These modifications mainly concern: I Err~~m~.cO/ ~/JYY~P~mttrho/i.vnz. It confirms that glycogen metabolism plays a very peculiar role in malignant tissue as it probably does in fetal tissues. Further studies are necessary to understand to what extent glycogen itself or glycogen metabolizing enzymes arc linked to the process of cell division as proposed by Rousset ti ui. ( l%O. 1% la. b). 3. pI.rtftafe Xincrsf. High rate of glycolysis is a common feature in cancer cells. However, no difference in malignancy, as tested by tumorigenicity in nude mouse, was found in a low glycolytic strain derived from highly piycolytic malignant tibroblasts (Pouyssegur ct IL/., 1980; Franchi CI 4.. 1981) or in BALB 3T3 derived cell lines (Peterkofsky and Prather, 1982). Similarly, we found no difference in tumorigenicity of a low glycolytic cell line obtained by heat-shock treatment of the H7‘ 29 cells (unpublished results). Therefore, the relationship between high pyruvate kinase activity and rna~i~nan~y is still an open question. 3. Girccc~sc-~-~?~l~~~spl~fft~l ri~~li~drofPnci.sr. Increased activity of the pentose phosphate pathway is supposed to supply more ribose needed when cells divide actively. It also allows many synthetic activities which need reduced NADP. The question of whether a high activity of the pentose phosphate pathway controlling enzymes is essential for expressing malignancy is very important but not yet solved. ~~I\riolt,l~,~~~~,,n~t?t.v~ -This work was supported by grants t’rom 1.F4.S.E.R.M. and from Llniversiti- P;lul Sabatier. The skilful technical assistance of Colette Pirtan and JcanCiaude

Coulomiers

was greatly appreciated.

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