Cancer Letters 105 (19%) 61-70

Comparative effects of flavonoids on the growth, viability and metabolism of a colonic adenocarcinoma cell line (HT29 cells)

Georgine Agulloa, Laurence Garnet-Payrastre b,*, Yvette Femandezb, Nathalie Anciauxa, Christian DernignCa, Christian RCmksya

“Luborutoire des M&dies M&uboliques, INRA de The-ix, 63122. Ceyrut, France bhborutoitz des X&obiotiyues, INRA, 180 Chemin de Tournefeuille, BP 3, 31931 Toulouse Cedex, Franc p

Received 5 March 1996; accepted 4 April 1996

Abstract

The aim of the present study was to compare the effect of five structural classes of flavonoids on the viability and me- tabolism of a colonic adenocarcinoma cell line (HT29 cells). The most prominent structural features of flavonoids favoring both their cytotoxic activity and their capacity to inhibit lactate release appear to be the desaturation of the 2, 3 bond and the position of attachment of the B ring. Indeed, flavonol and flavone are the most potent and, in both dasses, the order of po- tency can be modulated by hydroxyl or methoxyl substituents. On the other hand, in our model, we did not find any correla- tion between flavonoid structure and their capacity to modulate CAMP level. This last point is discussed.

Keyworcis: Flavonoids structure-activity; Colon carcinoma cells; Cytotoxic effects; Lactate release

1. Introduction

In recent years, there has been a growing body of evidence that a diet containing abundant vegetables, fruits and grains can reduce the risk of several can- cers, especially colon cancer [ 1,2]. Some compounds from plants products, including fibers, vitamins and micronutrients have been suggested to be capable of reducing cancer risk [3,4]. Among micronutrients, interest in food polyphenols and especially fla- vonoids has arisen. Some flavonoids have been clearly shown to inhibit in vitro and in vivo tumor cell growth [5], genistein and quercetin have been found to be the most potent [6,7]. The specific and --

* Corresponding author. Fax: +33 191 61285244.

exact mechanisms responsible for the antitumoral effect of these two different classes are not entirely elucidated but they are thought to be mediated through distinct mechanisms such as the inhibition of specific enzymes implicated in the transduction of mitogenic signals [5,8,9].

The other classes of flavonoids possess a large spectrum of biological activities and various investi- gations suggest that the specific activity of flavonoids on cell function is dependent on their structure [5,10,11]. Some authors have establish a model of minimal essential feature requited for their effect on some purified enzymes [5,12-141.

In a previously published work 1151, we have shown that quercetin is a potent cytotoxic agent on actively growing human adenocarcinoma cells (HT29

0304-3835/96/$12.00 0 1996 Elsevier Science Ireland Ltd. Ail rights reserved

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62 G. Agullo et al. /Cancer Letters 105 (1996) 61-70

cells).This effect was dose dependent and was ac- companied by an inhibition of lactate release. In the present study we have tried to see whether the prop- erties of quercetin on HT29 cell growth and metabo- lism could be extended to various flavonoids or are restricted to structurally related compounds.

2. Materials and methods

2.1. Drugs and chemicals

Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were obtained from Gibco BRL. Flavonoids were from Sigma (St Louis, MO) except for luteolin and diosmetin which were from Extrasynthese (Lyon, France). Cellular toxicity kit was from Boehringer (Meylan, France). Radioimmu- noassay kit for CAMP determination was from Im- munotech (Marseille, France). All other chemicals were purchased from Sigma (St Louis) or from Merck and were of the highest purity grade.

2.2. Cell culture

The HT29 cell line has been established in perma- nent culture from a human colon carcinoma by Dr. J. Fogh (Sloan Kettering Institute for Cancer Research, Rye, NY, USA) [ 161. It was obtained from Unite INSERM 317 (Toulouse, France). Routinely, stock cells were cultured in DMEM containing 25 mM glucose, 43 mM bicarbonate, 60@4/ml penicillin, lOOpg/ml streptomycin supplemented with 5% FCS at 37°C under an air/CO* (9:l) atmosphere; the me- dium was changed every 2 days.

For the experiments, cells were seeded at low density (4 X 104 cells per ml) in 35 or 120 mm di- ameter Petri dishes in standard medium. Maximum proliferative effect of FCS is obtained in I-IT29 cells in the presence of 3% FCS which induces a doubling of cell population within a 30 h-period of culture (data not shown). To check the possible effect of various concentrations of flavonoids on I-IT29 cell proliferation and metabolism, quiescent cells were stimulated with sub-optimal doses of FCS (1%).

One day after seeding, the cells were placed in se- rum-free DMEM in order to arrest cell growth. After 24 h in serum-free medium, all experiments were started by stimulating the cells with 1% FCS in the

presence of 10 mM sodium bicarbonate and 10 mM N-2-hydroxyethyl-piperazine-M-2-ethanesulfonic acid (HEPES). Simultaneously with the reinitiation of proliferation, cells were treated with various doses of flavonoids diluted in dimethylsulfoxide (DMSO) (2~1 per ml of medium). At the indicated time after the beginning of the experiment, the effect of fla- vonoids on cellular metabolism was estimated by lactate release and CAMP level; their effects on cell growth were determined by cell counting; cell viabil- ity was measured by MIT reduction.

2.3. Measurement ofjlavonoid autooxidation

Oxygen (02) consumption was measured polaro- graphically [17] with a YSI 5300 oxygen monitor (Yellow Springs Instrument Co, Yellow Springs, OH) equipped with a Clark electrode. Reactions were run in DMEM, containing 1% SVF and buffered with 10 mM HEPES (pH 7.45) at 37°C in a 2 ml reaction chamber. Flavonoids diluted in DMSO were added to the culture medium to give a final concentration of 60pM. After 1 hour, O2 consumed was quantified and 200 U/ml of catalase were added to the culture medium. O2 released under the action of catalase was used to calculate the amount of H202 (H202 + Hz0 + l/20*).

2.4. Estimation of growth rate

Growth rate was estimated by determining cell numbering which was performed 48 h after the be- ginning of the experiment. The cells were detached by treatment of the dishes with 0.25% trypsin 0.6 mM EDTA in phosphate buffered saline. An ali- quot of the cell suspension was diluted in Isoton II diluent and the cell number was determined using a Coulter counter. Determinations within experimental groups were run in triplicate.

2.5. Lactate release

To measure lactate release, the medium was col- lected 6 h after the beginning of the experiment and deproteinized with 0.6 M perchloric acid (1:2 v/v). L- Lactate concentration was measured in a 50~1 ali- quot of the deproteinized supernatant using lactate dehydrogenase and NAD according to Hohorst and

Bergmeyer [18]. The change in optical density was read at 340 nm.

2.6. Cell viability quantification

G. Agullo et al. /Cancer Letters 105 (I 996) 61-70 63

with flavonoids alone (basal values) or wrth forskolin ( low5 M) in the presence or absence of 3-isobutyl- I- methylxanthine (IBMX) (0.5 mM) for 4 h. Then, the culture medium was removed, the cell layer was ex- tracted using an ice-cold methanol/formic acid (19:l v/v) mixture. The cell lysate was spun down at 4000 X s for 10 min. An aliquot of the resulting su- pernatant was dried, diluted in the appropriate vol- ume of sodium acetate buffer (0.2 M) and CAMP was measured by radioimmunoassay. The protein concen- tration of the CAMP preparation was determined by the method of Bradford 1201.

The assay is based on the cleavage of the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide (MTT) to purple formazan crystals by dehydrogenase activity in mitochondria, a conversion which occurs only in living cells [ 191. For the MTT reduction assay, the cells were grown in 24- well multipiates.

2.7. Measurement of cyclic AMP by radioimmuno- assay

For CAMP determination, simultaneously with the reinitiation of proliferation, cells were treated

0” o

Hesperetin : R3’ = OH ; R4’ = OCH,

Flwanonol HO

6” ;; 0” o

Quercetin : R3’ = OH ; R4’ = OH Taxifolin : R3’ = OH ; R4’ = OH

Fiavan 3-01 HO

“’ R%f’4

Genistein : R4’ = OH R5

Fig. I. Chemical structures of the different classes of flavonoids used in the study.

Cetechin : R3’ = OH ; R4’ = OH

3. Results

3. I. Cytotoxicity offlavonoids

Table I shows the dose response study of the dif-

Flavone HO

OH 0

Luteolin : R3’ = OH ; R4’ = OH

Flavonol HO

Isofkwone

64 G. Agullo et al. /Cancer Letters 105 (1996) 61-70

Table I

Dose response study of the effect of flavonoids on HT29 cells growth

lP”M 3,uM 7PM

15pM 30/4M 60pM

Quercetin Catechin

99-e4 100*5 93 k5 98 f 3 66*4* 105r9 46i4* 85 zt I 36 + 3* 98 f I 21 f I* 86 f I

Hesperetin

look3 10926 99 2 3 98 f 7 94 * 9 97 + 6

Taxifolin

92 k 8 105+5 99 + 6

lOOk7 96 + 9 95 +5

Genistein

lOOk3 92 f 6 93*4 90*9 89 k 9 60*5*

Luteolin

105*3 93 ” 7 95*4 71*9* 28*4* 3lt5+

Cells were stimulated with 1% FCS and treated simultaneously with either 0.2% DMSO (control) or indicated flavonoids diluted in DMSO (0.2%). Cell growth was estimated by cell counting after a 48 h period of culture as described in Section 2. Results are expressed as percent of control and are the mean + SEM of 4 separate experiments. *P < 0.05, significant difference compared with control cells.

ferent classes of flavonoids (shown in Fig. 1) on HT29 cell number (as percent of control) after a 48 h period of treatment. Whatever the concentration used, taxifolin, hesperetin or catechin did not alter serum-induced cell growth. When present at 60,~M, the isoflavone genistein displayed only a weak effect on cell number compared to DMSO treated cells. On the other hand, Table 1 shows that the flavone luteo- lin affects HT29 cell proliferation; its effect was sig- nificant at 15 ,uM and was proportional to the flavone concentration. However, luteolin effect was less pro- nounced than that of quercetin, since this latter af- fected the HT29 cells as soon as 7 ,uM.

According to the data from the M’IT reduction as- say shown in Table 2, the effect of luteolin is not due only to the cytostatic activity of the compound since cell death is found to increase dose dependently. However, luteolin affects HT29 cell viability to a lesser extent than quercetin since the concentrations

Table 2

Effect of flavonoids on HT29 cells viability

for a 50% inhibition of cell viability (ICsa) are about 30pM and 15pM respectively. Table 2 also shows that the reduction of cell number observed in 60pM- genistein treated cells is concomitant with a cytotoxic effect. Moreover, M’IT reduction assay confirms the lack of effect of taxifolin, hesperetin and catechin on cell growth and viability. Whatever the flavonoid concentration used, the percent of viable cells is not significantly different from control conditions.

In order to study the influence of polyhydroxyla- tion or methylation on the potency of flavonoids on HT29 cell growth and viability, the effect of two fla- vonols (myricetin and kaempferol) and two flavones (apigenin and diosmetin) were examined. As shown in Fig. 2, myricetin is more hydroxylated than quer- cetin and kaempferol lacks a hydroxyl group at the 3’ position. Apigenin differs from luteolin by the ab- sence of OH in 3’, and diosmetin is a methoxy- substituted flavone. All four flavonoids tested im-

I@’ 3pM 7W

l5pM 30yM 60/4M

Quercetin

lO2&4 93 + 4 8Oi5* 41 f 4* 32 i 4* 20 f 6*

Catechin

lOOi lO4*7 108+6 112k7 112*6 97 f I

Hesperetin Taxifolin Genistein Luteolin

loo*4 lOOi lOOi 105*5 105&l 102k3 105*6 96zt8 107+8 101 * 5 109*4 92+5

96 zt 7 99 f 5 102 f 9 8Oi6* 95 f 9 90*7 89+9 50*3* 99 i 8 92 i 5 65*3* 28 i 2*

Cells were treated as described in Table 1. Results are the mean + SEM of 4 separate experiments and are expressed as percent of control (DMSO) cells. *P < 0.05, significant difference compared with control cells.

G. Agullo et al. /Cancer Letters 105 (1996) 61.-70

FlwmoSs Flavones

Off HO OH

OH 0 Kaempferol : R4’ = OH OH

O Apigenin : R4’ 5 OH

OH Quercaln : R3’ = OH ; R4’ = OH I - OH f Luteolin : R3’ q OH;R4’=OH

I II OH o

OH Dioemetin : R3” = OH ;R4’= OH 0

Myricetin : R3’ = OH R4’ q OH ; R5’ =

Fig. 2. Chemical st~chmx of different flavonols (kaempferol and myricetin) and flavones (apigenin and diosmetin).

OCH3

paired in a dose dependent manner the growth and survival of HT29 cells as ascertained by cell number (Fig. 3) and cell viability estimation (Fig. 4) after a 48 h period of treatment. The following order of de- creasing potency on NT 29 cell viability was found in the present model: quercetin > kaempferol > myri- cetin for flavonols and luteolin = apigenin > dios- metin for flavones.

Figs. 3 and 4 show the dose response study start- ing at 7,~uM because no significant effect could be observed at 1 or 3pM (shown for quercetin and luteolin in Tables 1 and 2).

3.2. Control of autooxidation ofjlavonoids

This study was performed under experimental conditions that greatly diminish flavonoid autooxida- tion in the culture medium [ 151. However in order to be sure that the observed cytotoxic effect of fla- vonoids is not mediated through their degradation

product, we measured the 02 consumed and Hz02 generated in the culture medium. In the presence of catechin, hesperetin or taxifolin in the culture there was no O2 consumed or H202 generated. Addition of apigenin, luteolin, genistein, kaempferol and dios- metin led to the consumption of less than 1OpM of oxygen and to the production of less than 5pM of H202. On the other hand, autooxidation of quercetin and myricetin is more marked since in their presence in the culture medium O2 consumed was 25 and 52 PM, respectively , and H202 produced was I5 and 43 PM, respectively.

3.3. Effects of cytotoxicflavonoids on k/T?9 ceil metabolism

In this second part, we investigated whether the distinct effects of flavonoids on cell growth and vi- ability could be linked with their effects on cellular metabolism, estimated by measuring lactate release in

G. Agullo et (11. /Cancer Letters 105 (19%) 61-70 66

8 g 60

8 6

c 40

P

2 20 5 0

7 15 30 60 (PM)

7 15 30 60 (PM)

Fig. 3. Effect of different doses of flavonols and tlavones on HT29 cell growth. Cells were stimulated by the addition of 1% FCS and treated by flavonols (a), i.e. myricetin (striped bar), kaempferol (gray bar) or quercetin (patterned bar), and by flavo- nes (b), i.e. diosmetin (close striped bar), apigenin (black bar) or luteolin (open striped bar) at the indicated doses. Growth rate was estimated by cell counting 48 h after the beginning of the experi- ment, and as described in Section 2. Results are expressed as percent of control (DMSO-treated cells) and are the mean + SEM of four separate experiments. “A significant difference compared to quercetin treated cells at P < 0.05. bA significant difference between kaempferol and myricetin treated cells at P < 0.05. ‘A significant difference compared to luteolin treated cells at P < 0.05. dA significant difference between apigenin diosmetin treated cells at P < 0.05.

the culture medium 4 h after the addition of the fla- vonoids (Fig. 5). The cytotoxic effect of quercetin, kaempferol, apigenin, luteolin and diosmetin was associated with an early and dose dependent lowering of lactate release in the culture medium. As for fla-

vonols, quercetin was more potent than kaempferol; it is noteworthy that the polyhydroxylated flavonol myricetin was without effect on lactate release. Both luteolin and apigenin exerted comparable effects, diosmetin being less potent. The other flavonoids

7 15 30 60 (PM)

b) l”), -

7 15 30 60 WV

Fig. 4. Comparative effects of flavonols and flavones on HT29 cell viability. Cells were stimulated as described in Fig. 3 and treated by flavonols (a), i.e. myricetin, kaempferol, or quercetin, and by flavones (b), i.e. diosmetin, apigenin or luteolin (key as in Fig. 3) at the indicated doses. Cell viability was estimated by the MTT test or trypan blue exclusion 48 h after the beginning of the experiment and as described in Section 2. Results am expressed as percent of control (DMSO-treated cells) and are the mean f SEM of four separate experiments. ‘A significant difference compared to quercetin treated cells at P < 0.05. bA significant difference between kaempferol and myricetin treated cells at P < 0.05. ‘A significant difference compared to luteolin treated cells at P < 0.05. dA signiticant difference between apigenin diosmetin treated cells at P c 0.05.

C. Agulio et nl. / Cuncer Letters 10.5 (I 996) 61-70 67

b) 'O"

1

7 15 30 60 WV

Fig. 5. Dose response study of flavonols and tlavones effects on the glycolytic activity of HT29 cells. Cells were stimulated ;I$ described in Fig. 3 and treated by flavonols (a), i.e. myricetin, kaempfcrol or quercetin, and by flavones (b), i.e. diosmetin, api- grnin or iuteolin (key as in Fig. 3) at the indicated doses. Lactate release was determined after a 4 h period of treatment and was quantified as described in Section 2. Results arc expressed as percent of control and are the mean + SEM of four separate ex- periments. “A significant difference compared to quercetin treated cells at P < 0.05. bA significant difference between kaempferol and myricctin treated cells at P < 0.05. ‘A significant difference compared to luteolin treated cells at P < 0.05. dA significant dif- ference between apigenin diosmetin treated cells nt P c 0.05.

tested were ineffective on lactate release in the me- dium, even at the highest dose used (60,~M) (i.e. taxifolin 97 4- 3; hesperetin 110 +- 5; catechin 101 + 3 and genistein 88 2 9).

3.4. Effects offavonoids on CAMP levels

Table 3 shows that at the concentration used (60pM), the flavonoids studied had no significant effect on the basal intracellular cAMP level. How- ever, the present results show that the isoftavone ge- nistein and the flavone luteolin significantiy elevated the CAMP accumulation induced by 10es M forskolin compared to control conditions. Their effects were amplified by the addition of 0.5 nM IBMX (i.e. CAMP levels in percent of basal value in g&stein and luteolin treated cells are: 5238 + 515 and 4242 + 379, respectively, in the presence of IBMX versus 2457 rt 528 and 1375 +- 133, respectively in the absence of IBMX). By contrast, the polyhydroxy- lated flavonol myricetin was the only one among the flavonoids tested which strongly depressed CAMP accumulation induced by forskolin in FIT29 cells.

4. Discussion

With regard to the flavonoid structure-activity re- lationship, and by comparing five different structural families, some structural features associated with cytotoxic activity of flavonoids on colon& adeno- carcinoma HT29 cells could be identified. It appears that flavones and flavonols are the most potent to affect cell viability in the present model, whereas

Table 3

Effects of flavonoids on basal and forskolin induced cAMP level of HT29 cells

Control Quercetin Kacmpferol Myricetin Catechin Hespcretin Taxifolin Genistein Luteolin Apigenin Diosmetin

Rasal

loo* I? 85% II 78 t 29 80* 17 93i31

1012 20 108 f 18 109 + 8 114+24 8532 I9 83* I2

Forskolin ( 10q5M) ~____

8?2*213 591 * I69 652 + 96 295 Tk 42* MXtt40

608 r 56 59b t 36

1457 * s2c(* 1375 f 133*

954 It 1% X3b 2 229

Cells were treated as described in Section 2. Values are the mean + SEM of 6 separate experiments and are expressed as percent of basal values. *P < 0 001. significant difference compllred with control cells.

68 G. Agullo et al. /Cancer Letters 105 (1996) 61-70

flavan-3-01, flavanone and isoflavone were inactive. From these data it seems that the absence of the C2- C3 double bond and/or attachment of ring B to the chromone structure results in a loss of their cytotoxic potency on HT29 cells. Since the effect of quercetin is more pronounced than that of luteolin, it seems also that hydroxylation at the 3 position confers a more potent cytotoxic effect. Furthermore, these properties can vary as a function of the number and the position of hydroxyl or methoxyl groups on the B ring of the flavone or flavonol nucleus. The lack of the hydroxyl group in the 3’ position seems to corre- spond to a lower effect of flavonol on HT29 cells and from our results it is clear that the cytotoxicity of flavonoids seems to inversely correlate with the number of hydroxyls. Substitution at the C4’ position with a methoxyl group significantly alters the cyto- toxic activity of flavones on HT29 cells. In summary, the order of potency on cellular toxicity is quer- cetin > kaempferol > myricetin in the flavonol fam- ily, and luteolin = apigenin > diosmetin in the fla- vone class.

Flavonoids are known to possess many biological activities at the cellular level and various hypotheses have been made to explain their potential anticancer- ous properties (for a review see Ref. [5]). Our results clearly show that cytotoxic activity of flavones and flavonols is linked in the same order of potency to an inhibition of glycolytic activity of HT29 cells and/or a diminished lactate release. This last effect has been described in other cell lines and seems to be mediated through their action on the lactate transporter or on the Na+/K+ ATPase enzymes as well as on glycolysis associated enzymes [21-231. As reported by Laugh- ton et al. [24], in spite of their well known anti- oxidant properties, flavonoids can also have pro- oxidant effects under some reaction conditions which lead to cellular damage. However, our results clearly show that there was no correlation between the cyto- toxicity of flavonols and flavons and their various degrees of autooxidation in the culture medium. Thus a possible or important effect of H202 generated on HT29 cell metabolism and viability could be rule out. As myricetin is still the one flavonol which displays cytotoxic activity without affecting lactate release, one can suggest that its effect on cell viability is linked at least in part to a substrate-independent res- piratory burst in mitochondria, due to its ability to

produce cytotoxic oxygen radical as described previ- ously [25].

In the present work, it was not possible to distin- guish any systematic relationships between the ability of flavonoid to modulate CAMP-induced forskolin accumulation and a particular structural feature. Only three structurally unrelated flavonoids (i.e. luteolin, genistein and myricetin) affected this process in a significant manner. Since the addition of IBMX had an additive effect in luteolin and genistein treated cells, one can suggest that these flavonoids do not function in the present model as a phosphodiesterase inhibitor. Their effect on CAMP level could be asso- ciated with a modulation of adenylate cyclase activ- ity. Furthermore, the elevation of cyclic AMP level induced by luteolin and genistein in forskolin treated cells may result indirectly from the inhibition of Na+/K+ ATPase. Further work is needed to confirm these hypotheses. Surprisingly, incubation of HT29 cells with myricetin produces a noticeable inhibition of forskolin-induced CAMP accumulation. Fla- vonoids have generally been found to be potent CAMP protein kinase and phosphodiesterase inhibi- tors [26-291. This property seems to vary as a func- tion of their structure and confers on flavonoids their potency as inhibitors of platelet aggregation [30,31]. On the other hand, Di Carlo et al. [32] have demon- strated that some flavonols could also interact with the a-2-adrenergic receptor. In this view, it could be interesting to test whether the inhibitory effect of myricetin on CAMP accumulation in HT29 cells is mediated through the a-2 adrenergic systems. Intra- cellular CAMP levels are often linked in various cell systems to the cellular proliferative activity [33]. However, in the present model, it was not possible to find any correlation between the effect of luteolin, myricetin and genistein on cellular AMPc level and their properties on HT29 cell growth or viability.

Further studies are in progress in our laboratory to elucidate the cellular mechanism underlying these effects and in order to assess more precisely the beneficial effects of flavonoids in the human diet; total flavonoids consumption is likely to be as high as 1 g per day [34].

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