CANCER

LETTERS
Cancer Letters 87 (1994) 55-63

Quercetin exerts a preferential cytotoxic effect on active

dividing colon carcinoma HT29 and Caco-2 cells

Georgine Agullo, Laurence Garnet *, Catherine Besson, Christian DemignC,

Christian RCmCsy
Laboratoire des Maladies Mktaboliques, INRA Theix. 63122 Ceyrat. France

Received 5 September 1994; accepted 20 September 1994

Abstract

The effect of the naturally occurring flavonol, quercetin, was investigated on cell growth and metabolism of two
human carcinoma cell lines, HT29 and Caco-2 cells, both during the exponentially growing phase and after confluence.
Our results show clearly that, after a 48-h period of treatment, quercetin (in the range of concentration from 15 pM
to 120 FM) exerted a preferential cytotoxic effect on active proliferating cells. This effect was dose dependent and was
accompanied by a simultaneous inhibition of lactate release and a dramatic decrease of total cellular ATP content.
In contrast, in confluent cells, quercetin failed to affect cell viability or lactate release, but led nevertheless to a deple-
tion of cellular ATP level. In conclusion, the cytotoxicity of quercetin is markedly higher in actively growing cells in
comparison with confluent cells.

Keywords: Flavonoid; Quercetin cytotoxic effects; Colon carcinoma cells; ATP content; Lactate release

1. Introduction or asiatic diets are more protective [2]. A variety

of micronutrients from plant products have been
Evidence from epidemiologic and experimental identified as compounds with preventive and anti-
studies suggest that the genesis of colon cancer carcinogenic properties [3]. Among these micro-
may be the result of complex interactions between nutrients, food polyphenols, and especially
environmental factors and genetic susceptibility flavonoids, may exert a protective role against
[ 11. Western diets with high intake of calories from colon cancer because of their high bioavaibility in
fats and low fiber supply has been linked to the large intestine [4]. Quercetin (5,7,3,4-
increase colon cancer incidence, while vegetarian flavonol) is one of the most widely distributed fla-
vonoids in the plant kingdom, and is a component
Abbreviafions: ATP, adenosine triphosphate; DMEM, of most edible fruits and vegetables. In humans,
Dulbeccos modified Eagles medium; FCS, fetal calf serum;
the average intake of all flavonoids has been
HEPES, N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic
acid\
__._,. estimated to be in the range of 0.5-l g/day but re-
* Corresponding author. cent investigations suggest that the actual daily in-

0304-3835/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved

SSDI 0304-3835(94)03574-3
56 G. Agullo er al. /Cancer Lrtt. 87 11994) 55-63

take is frequently lower [5]. Only a part of the density (4 x lo4 cells per ml) in 35mm diameter
flavonoids is absorbed in the digestive tract, and Petri dishes in standard medium (2.5 ml per well).
their bioavaibility is still poorly known. Flavo- Maximum proliferative effect of FCS is obtained
noids have antioxidant and chelating properties in HT29 cells in the presence of 3% FCS, which in-
[6] and they have been shown to modulate various duces a doubling of cell population within a 30-h
cellular enzymes. Their potential use as anti- period of culture (data not shown). To check the
inflammatory, antiviral or antitumor agents has possible effect of various concentrations of querce-
also been examinated [7]. tin on HT29 and Caco-2 cell proliferation and me-
In this study we compare the effect of quercetin tabolism, quiescent cells were stimulated with
on cell growth and metabolism of two human col- sub-optimal doses of FCS (1%).
onic carcinoma cells, Caco-2 and HT29 cells both Concerning studies on exponentially growing
during the exponentially growing phase and after cells, 1 day after seeding, the cells were placed in
confluence. The results show that quercetin was serum-free DMEM in order to arrest cell growth.
cytotoxic to active dividing cells in contrast to con- After 24 h in serum-free medium, all experiments
fluent cells. were started by stimulating the cells with 1% FCS
in the presence of 10 mM sodium bicarbonate and
2. Materials and methods 10 mM HEPES. Simultaneously with the reinitia-
tion of proliferation, cells were treated by various
2.1. Drugs and chemicals doses of quercetin diluted in pure DMSO (2 pi/ml
Dulbeccos modified Eagle medium (DMEM) of medium). Medium was changed every day. At
and fecal calf serum (FCS) were obtained from the indicated time after the beginning of the exper-
Gibco BRL. Quercetin was from Sigma Chemical iment, the effect of quercetin on cellular metabo-
Co. (St. Louis, MO). Cellular toxicity kit, phenol lism was estimated by either ATP content analysis
red-free DMEM (for bioluminescence assays) and or lactate release; its effect on cell growth was
the ATP bioluminescence CLS kit were from determined by measurement of cellular protein.
Boehringer Mannheim Biochemica. All other
chemicals were purchased from Sigma (St. Louis, 2.3. Estimation of growth rate
MO) or from Merck and were of the highest purity Growth rate was estimated by measuring the
grade. total protein content per dish. This parameter has
been previously demonstrated to be linearly cor-
2.2. Cell culture related with the number of cells [lo]. Total protein
HT29 and Caco-2 cell lines have been establish- content per well in 6-well plates was determinated
ed in permanent culture from a human colon car- at the indicated time over a 6-day period of cul-
cinoma by Dr J. Fogh (Sloan Kettering Institute ture, using the method of Bradford [ll].
for Cancer Research, Rye, NY) [8]. These two cell-
lines differ by their capacity to differentiate at con- 2.4. Lactate release
fluence [9]. They were obtained from Unite IN- To measure lactate release, the medium was col-
SERM 317 in Toulouse (France). Routinely, lected 6 h after the beginning of the experiment
stock-cells were cultured in DMEM containing 25 and deproteinized with 0.6 M perchloric acid (1:2,
mM glucose, 43 mM bicarbonate, 60 PM/ml peni- v/v). L-Lactate concentration was measured in a
cillin, 100 pg/ml streptomycin at 37C under an 50-~1 aliquot of the deproteinized supernatant
air/CO;! (9/l) atmosphere. Caco-2 cells-medium using lactate dehydrogenase and NAD according
was supplemented with 10% heat-inactivated fetal to Hohorst and Bergmeyer [ 121. The change in op-
calf serum and 1% non-essential amino acids. tical density was read at 340 nm.
HT29 cells-medium was supplemented with 5%
FCS. For both cell lines, the medium was changed 2.5. Intracellular ATP content analysis
every 2 days. Cell layers from a six-well plates were scraped in
For the experiments, cells were seeded at low 0.6 M perchloric acid and cellular homogenates
 G. Agullo et al. /Cancer Lelt. 87 (1994) 55-63 57

were rapidly neutralized with 0.75 M K,COs. A We have avoided this phenomenon by lowering
200~~1 quantity of the neutralized supernatant bicarbonate level (10 mM) of culture medium and
were buffered with 400 ~1 HEPES 40 mM, pH by adding HEPES (10 mM).
7.75. ATP measurements were performed accor-
ding to the luciferin-luciferase method, using an 3.1. Effect of quercetin on cell growth as a function
LKB 1251 luminometer (LKB instruments, Orsay, of cellular proliferative activity and differentiation
France). status
Fig. 1 shows the dose-response study of querce-
2.6. Cell viabillty quantification tin on HT29 and Caco-2 proliferative activity dur-
The assay is based on the cleavage of the yellow ing their exponentially growing phase (48 h).
dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- Quercetin inhibition was dose dependent toward
tetrazolium bromide (MTT) to purple formazan the growth of the two cell lines. The ability of cells
crystals by dehydrogenase activity in mitochon- exposed to 240 PM of quercetin to proliferate was
dria, a conversion which occurs only in living cells completely suppressed (data not shown). The con-
[13]. For the MTT reduction assay, the cells are centration for 50% growth inhibition (IC& of
grown in 24-well multiplates. cells continuously treated with quercetin was
about 45-50 PM for the two cell lines.
2.7. Differentiation markers assays Microscopical examination at day 2 of HT29
Cells were homogenized in 50 mM TRIS buffer and Caco-2 cell-layers revealed a cytotoxic effect
(pH 7.5) by several passages through a 26 G needle of quercetin with a cell-layer desquamation which
fitted to a 2-ml syringe. Alkaline phosphatase ac- confirms our cell viability results (Table 1). In-
tivity was determined according to the method of deed, a decrease in the amount of purple formazan
Garen and Levinthal [14] as described previously crystal formed was observed in quercetin-treated
[ 151. Dipeptidyl peptidase IV activity was deter- cells, which directly correlates with an increase of
mined according to the method of Svensson et al. cell death since MTT reduction into formazan re-
[ 161. The enzyme activity is expressed as mU per quires viable cells with functional mitochondrial
mg of cellular protein (one unit being 1 pmol of dehydrogenases.
substrate hydrolysed per min). In order to test whether quercetin has the same
effect on confluent cells as on growing cells,
3. Results dose-response studies were performed on HT29
and Caco-2 confluent cells (Fig. 2). For this pur-
This study was performed under experimental pose, HT29 and Caco-2 cells, respectively, were
conditions that avoid quercetin instability in solu- grown in 5% and 10% FCS-containing medium for
tion and the observed effects are unlikely to be 10 days, after which, they reached confluence.
mediated through the degradation products of However, the effect of quercetin was tested with
quercetin as was reported previously [ 171. Indeed, the 1% FCS, since high concentrations of serum
during preliminary assays, we observed that alka- could bind this flavonol and affect its dose-
linization of the culture medium (containing 43 response. Postconfluent HT29 cells were still un-
mM bicarbonate) induced a rapid modification of differentiated whereas postconfluent Caco-2 cells
the quercetin spectra absorption, showing the exhibit a differentiated status as ascertained by the
transformation of the molecule, probably by auto- level of alkaline phosphatase and dipeptidyl pep-
oxidation. It is known that quercetin consumed tidase activity (40 + 1 mU and 118 f 5 mU
oxygen more rapidly when added to an aqueous respectivley in confluent Caco-2 cells; versus
pH 8 buffer and auto-oxidation of quercetin in- 10 f 1 mU and 30 f 2 mu, respectively, in ex-
creases considerably at the higher pH of 8-8.5 ponentially growing Caco-2 cells). As seen in Fig.
[18]. Alkalinization of the culture medium can 2, the inhibitory effect of quercetin was significant
occur rapidly during the starting of the experiment only at 120 PM on confluent HT29 cells. In Caco-2
when cells are not under an air/CO1 ratio control. confluent cells (Fig. 2) there was no significant
 58 G. Agullo
etal./Cancer
Lett.
87 (1994)55-63

taco-2

Qumxlin Quercetin

Fig. 1. Dose dependent effect of quercetin on HT29 and Caco-2 cell growth during their exponentially growing phase. Quiescent cells
were stimulated with I/0 FCS alone (Control) or in the presence of quercetin diluted at the indicated concentrations in DMSO (0.2%
final). Cell growth was estimated by measuring the total protein content of the dishes after a 48-h period of culture as described in
Materials and methods. Results are the means f S.E.M. of 4 separate experiments. *P < 0.05, significant difference compared with
FCS stimulated cells (control).

effect of quercetin on cell density during a 48-h 3.2. Effect of quercetin on cellular metabolism
period of culture. However, after 6 days of expo- In these experiments, the effect of quercetin on
sure, a significant reduction of cell density (30%) HT29 and Caco-2 cellular metabolism was
with 60 PM quercetin was observed (results not estimated by measuring lactate release and ATP
shown). Cytotoxicity test confirms the low sensi- intracellular levels either during their exponential-
tivity of confluent cells to quercetin (Table 1); ly or stationary growing phase (Fig. 3a,b, respec-
however, a noticeable effect was observed in the tively).
presence of 120 PM quercetin. As seen in Fig. 3a, the cytotoxic effect of querce-

Table I
Effect of quercetin on HT29 and Caco-2 cells viability as a function of cellular density

Exponentially growing phase Stationary growing phase

HT29 Caco-2 HT29 Caco-2

Control 100 f I 100 f 2 100 f 3 100 f 2

DMSO (0.2%) 99 f 2 84 f 6 91 f 3 97 f 3
15 gM Quercetin 15 f 4* 61 f 5* 94 ?? I 96 * 4
30 gM Quercetin 60 f 3* 40 f 4* 90 f 4 97 f 7
60 (AM Quercetin 25 f 3* 34 f 6* 86 ?? 3 100 ?? 3

120 PM Quercetin 12 f 3* 24 f 5* 80 + 4* 80 f 6*

Cells were treated as described in Figs. 2 and 3. Results are the means of f S.E.M of 4 separate experiments and are expressed as
percent of control.
*P < 0.05, significant difference compared with FCS stimulated cells (control).
 G. Agullo et al. /Cancer Lerr. 87 (1994) 55-63 59

Chercetin Quercetin

Fig. 2. Dose response study of quercetin effect on HT29 and Caco-2 postconfluent cells. After a 24-h period of culture in serum-free
medium, postconfluent HT29 and Caco-2 cells were stimulated, during 48 h, with 1% FCS alone (Control) or in the presence of quer-
cetin diluted at the indicated concentrations. Cell growth was estimated as described in Fig. 2. Results are the means f S.E.M. of
4 separate experiments and are expressed as percent of control. *P < 0.05, significant difference compared with FCS stimulated cells
(control).

tin previously observed on HT29 and Caco-2 ac- 4. Discussion

tively growing cells is preceeded by an early
decrease of lactate release (measured during the This study shows that quercetin is a potent cyto-
first 6 h of incubation). In agreement with the less toxic molecule on colon cancer cells in vitro. It
cytotoxic effect of quercetin on stationary growing also appears that this cytotoxic effect is dependent
cells, results in Fig. 3b show that lactate release of on cellular proliferative activity. Indeed, in spite of
postconfluent HT29 cells is not significantly af- the marked effect of quercetin on the proliferation
fected by the different doses of quercetin com- of HT29 and Caco-2 cells, this flavonol does not
pared with control DMSO-treated cells. On the seem to impair the growth and viability of con-
other hand, Caco-2 differentiated cells presented a fluent cells. The effects observed on the two types
dramatic stimulation of lactate release for the of cells were relatively comparable; however, the
highest dose of quercetin (120 PM). Measurement well differentiated Caco-2 cells seem to be more re-
of ATP by bioluminescence revealed that querce- sistant to quercetin.
tin, at most concentrations studied, caused a Quercetin is known to display a variety of bio-
dramatic depletion of ATP in Caco-2 and HT29 logical actions and numerous studies have
actively growing cells and also in confluent cul- reported its powerful growth inhibitory activity in
tures (Fig. 4a,b). However, in Caco-2 confluent vitro on various tumor cells [7]. In a range of con-
cells, the lowest quercetin concentration was inef- centrations between 30 and 70 PM, this flavonol
ficient to deplete cellular ATP in contrast to HT29 has been shown to arrest cell cycle at the Gt/S
cells. transition [ 19,201. Lower concentrations seem also
60 G. Agullo et al. /Cancer Lelt. 87 (1994) 55-63

Fig. 3. Dose response effect of quercetin on the glycolytic activity of HT29 and Caco-2 cells either during their exponentially growing
phase (a) or during their stationary growing phase (b). Cells were treated as described in Figs, 2 and 3 for (a) and (b), respectively.
Lactate release was determinated after a 6-h period of incubation in the deproteinized medium as described in materials and methods.
Results are the means f S.E.M. of 4 separate experiments. *P < 0.05, significant difference compared with FCS stimulated cells
(control).

effective since authors have demonstrated that might alter the typical cellular metabolism of ac-
concentrations between 10 nM and 10 PM tively proliferative cells, i.e. the glycolytic pathway
modulate cell growth in some tumor cells [21]. and energetic metabolism. Indeed, in these cells
This study also showed that quercetin could quercetin induces a rapid decrease in lactate re-
exert a preferential cytotoxic effect on actively lease and a fall in the ATP level. Inhibition of lac-
proliferating cells, as compared with slowly grow- tate release by flavonoids has been described in
ing cells. These results suggest that this flavonol other cell lines and seems to be mediated through
 G. Agullo et al. / Cancer Let!. 87 (1994) 55-63 61

a Cam-2
=1

I J

Quercelin Quacetin

Fig. 4. Dose response effect of quercetin on ATP intracellular levels of HT29 and Caco-2 cells either during their exponentially grow-
ing phase (a) or during their stationary growing phase (b). ATP intracellular levels were measured on deproteinized cell layers as
described in Materials and methods. Results are the means of 4 separate experiments. *P < 0.05, significant difference compared
with FCS stimulated cells (control).

their action on both the lactate transporter and the deshydrogenase and pyruvate kinase) [24]. In
Na/K ATPases [22]. Hirano et al. [23] suggested growing cells, ATP depletion could arise from the
that the cytoxic effect of flavonoids may be in pro- inhibition of glycolysis; however, the fact that
portion to their ability to inhibit the Na/K AT- cellular ATP was also diminished in confluent cells
Pases and could disturb the maintenance of is consistent with a direct effect of quercetin on
cellular ionic gradients, leading to cell death. ATP release or ATP wastage [25]. It has also been
Moreover, flavonoids have been reported to inhi- shown that quercetin induces DNA strands break
bit some glycolysis-associated enzymes (lactate (reviewed by Middleton and Kandaswami [7]) and
62 G. Agullo et al. /Cancer Lett. 87 (1994) 5.5-63

that activation of the DNA repairing enzyme these micronutrients could exert a protective effect
(polyADPribose polymerase) consumes nicotin- on large intestine in addition to the well-known
amide adenine dinucleotide sufficiently to interfere effect of dietary fibers.
with the ATP metabolism [26]. The observed ATP
fall in quercetin-treated cells could also be linked Acknowledgments
to the stimulation of the DNA repairing system.
The great cytotoxic effect of quercetin on growing We wish to thank A. Talmant and F. Esclapez
cells could be also explained by the extra supply of for their successful technical assistance.
energy needed for cellular division.
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