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(Received for publication, November 28, 1995; and in revised form, January 26, 1996) From the
Genistein is a dietary-derived plant product that inhibits the
activity of protein-tyrosine kinases. We show here that it is a potent
inhibitor of the mammalian facilitative hexose transporter GLUT1. In
human HL-60 cells, which express GLUT1, genistein inhibited the
transport of dehydroascorbic acid, deoxyglucose, and methylglucose in a
dose-dependent manner. Transport was not affected by daidzein, an
inactive genistein analog that does not inhibit protein-tyrosine kinase
activity, or by the general protein kinase inhibitor staurosporine.
Genistein inhibited the uptake of deoxyglucose and dehydroascorbic acid
in Chinese hamster ovary (CHO) cells overexpressing GLUT1 in a similar
dose-dependent manner. Genistein also inhibited the uptake of
deoxyglucose in human erythrocytes indicating that its effect on
glucose transporter function is cell-independent. The inhibitory action
of genistein on transport was instantaneous, with no additional effect
observed in cells preincubated with it for various periods of time.
Genistein did not alter the uptake of leucine by HL-60 cells,
indicating that its inhibitory effect was specific for the glucose
transporters. The inhibitory effect of genistein was of the competitive
type, with a K
In mammalian cells, there are two discrete hexose transport
systems whose function is to provide the cells with the basic cellular
fuel, glucose. These two systems are the family of facilitative hexose
transporters (glucose transporters, GLUTs) (1, 2) that
transport glucose down a concentration gradient and are widely
expressed in all cells and tissues, and the sodium-glucose
cotransporters that transport glucose against a concentration gradient
and are mainly expressed in small intestine and kidney(3) . Six
genes encoding different glucose transporter isoforms have been
molecularly
cloned(4, 5, 6, 7, 8, 9) ,
and more than one isoform is usually expressed in a given cell or
tissue. Five isoforms, GLUT1-GLUT5 are expressed on the cell
membrane (4, 5, 6, 7, 8) and one,
GLUT7, is restricted in expression to the internal membranes of the
endoplasmic reticulum(9) . Glucose transporters of the
facilitative type are the universal transporters of glucose in
mammalian cells(1, 2) . One isoform, GLUT5, is not a
glucose transporter but instead is involved in the transport of
fructose in specialized tissues and cells such as
spermatozoa(10) . GLUT2, the isoform abundantly expressed in
liver, has the ability to transport both glucose and fructose but with
different affinities(11) . Evidence has accumulated indicating
that the glucose transporters participate in the cellular accumulation
of substrates other than
glucose(12, 13, 14, 15, 16, 17) .
We demonstrated that the glucose transporters GLUT1, GLUT2, and GLUT4
are efficient transporters of the oxidized form of vitamin C,
dehydroascorbic acid(15) . We found that glucose transporters
are also the main pathway mediating the transport of dehydroascorbic
acid in normal human neutrophils (15) and HL-60 myeloid
leukemia cells(16, 17) . The data suggest that the
facilitative glucose transporters may be the universal transporters of
both glucose and dehydroascorbic acid in mammalian cells. Many
cellular functions are regulated at the levels of gene expression and
of protein function by discrete phosphorylation-dephosphorylation
events. Tyrosine phosphorylation plays a major role in the transduction
of cellular signals induced by the binding of insulin to its cognate
receptor(18) , and the effect of insulin on GLUT4 is an
important point of regulation for glucose transport in mammalian cells.
Insulin induces the translocation of GLUT4 from intracellular membrane
pools to the cell surface(19, 20) , and there is also
evidence suggesting that insulin may modulate the intrinsic functional
activity of the glucose transporters(21) . The role of
phosphorylation in regulating the activity of the glucose transporters
is, however, controversial (22, 23, 24, 25, 26) . The isoflavone genistein (4`,5,7-trihydroxyisoflavone) is a
dietary-derived natural product (27) present in a variety of
plant foods that selectively inhibit the activity of protein-tyrosine
kinases, as opposed to protein-serine/threonine kinases(28) .
Genistein affects oncogene-induced tumorigenesis(29) , cell
proliferation (30, 31, 32) , cell
differentiation(33, 34) , angiogenesis(35) ,
and signal transduction mechanisms activated by growth
factors(36, 37, 38, 39) . The
multiple effects of genistein in cellular systems are presumed to
reflect its ability to inhibit the activity of protein-tyrosine
kinases. We show here that genistein is a potent inhibitor of the
functional activity of the glucose transporters present in human
myeloid HL-60 cells, Chinese hamster ovary (CHO) (
Figure 1:
Genistein blocks the uptake
of deoxyglucose and dehydroascorbic acid in HL-60 cells. A,
HL-60 cells were incubated in the presence of the indicated
concentrations of genistein for 30 min, and uptake of deoxyglucose
(
The above assays were carried out using
cells preincubated with genistein for 30 min, a typical protocol used
in studies analyzing the effect of genistein on the activity of
cellular protein-tyrosine kinases. As uptake under these conditions is
a complex function of transport and intracellular trapping of the
transported substrate, genistein could be inhibiting uptake at either
of these steps. Total inhibition of deoxyglucose (Fig. 1B) and dehydroascorbic acid (data not shown)
transport was observed in cells preincubated with 100 µM genistein from 0 to 60 min, indicating that the full inhibitory
effect of genistein on uptake develops instantaneously and no
preincubation is required. Genistein affects multiple cellular
activities as a result of its ability to inhibit the activity of
protein-tyrosine
kinases(29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) .
The observed inhibition of deoxyglucose and dehydroascorbate uptake by
genistein therefore could be linked to the inhibition of
phosphorylation events involved in the cellular uptake of these
compounds. While protein phosphorylation can modulate the activity of
GLUT4(23, 24, 25, 26, 43) ,
there is no evidence indicating that GLUT1 is phosphorylated on
tyrosine residues. Therefore, we hypothesized that the inhibitory
effects of genistein on transport reflect a direct action of genistein
on the glucose transporters rather than inhibition of cellular
protein-tyrosine kinases. Support for this hypothesis was obtained
from experiments analyzing the effect of genistein under experimental
conditions tailored to measure transport as distinct to
accumulation(17) . We determined the dose dependence of
genistein's effect on the transport of deoxyglucose,
dehydroascorbic acid, methylglucose, and leucine in HL-60 cells using a
30-s uptake assay. Methylglucose is not trapped or metabolized, and,
therefore, its kinetics of transport can define the time frame at which
transport is still occurring under initial velocity conditions. We
measured the transport of leucine to control for the specificity of the
effect of genistein on the activity of the glucose transporters, as
opposed to possible nonspecific effects on membrane transport. Leucine
is transported into cells by transport systems functionally unrelated
to the glucose transporters(44) . The initial rate of transport
of dehydroascorbic acid, deoxyglucose, methylglucose, and leucine by
the HL-60 cells was linear for the first 60 s of uptake (data not
shown), validating the assay for the determination of the kinetic
constants of transport. When genistein was added at the beginning of
the uptake assays, it inhibited the transport of dehydroascorbic acid,
deoxyglucose, and methylglucose, with 50% inhibition observed at
10-15 µM genistein (Fig. 2, A-C). Complete inhibition of transport was
observed at 100 µM genistein, but some transport was
observed at concentrations of genistein greater than 100
µM. No effect of genistein on the transport of leucine by
the HL-60 cells was observed in these studies, with less than 20%
inhibition at 100 µM genistein (Fig. 2D),
indicating the specificity of the effect of genistein on the activity
of the glucose transporters and militating against the possibility that
it could be caused by a general biological effect on the cell membrane.
The specificity of the effect of genistein was further confirmed in
experiments which showed that daidzein (4`,7-dihydroxyisoflavone), an
analog of genistein that lacks a hydroxyl group at position 4 (Fig. 2, E and F) did not affect the transport
of dehydroascorbic acid, deoxyglucose, methylglucose, or leucine in the
HL-60 cells (Fig. 2, A-D). Similarly,
staurosporine, a general inhibitor of protein phosphorylation, did not
affect the transport of dehydroascorbic acid, deoxyglucose,
methylglucose, or leucine (data not shown) at concentrations
(1-10 µM) known to completely block protein
phosphorylation in HL-60 cells(45) . Genistein competitively
inhibited the transport of both deoxyglucose and methylglucose in HL-60
cells with a K
Figure 2:
Dose dependence of the effect of the
isoflavones genistein and daidzein on the transport of hexoses and
amino acids by HL-60 cells. Transport of dehydroascorbic acid (A), deoxyglucose (B), methylglucose (C),
and leucine (D) was measured using a 30-s uptake assay in the
presence of the indicated concentrations of genistein (
Figure 3:
Genistein inhibits in a competitive manner
the transport of deoxyglucose and methylglucose in HL-60 cells. A, double-reciprocal plot of the effect of genistein on the
substrate dependence for deoxyglucose transport. Transport of
deoxyglucose at 2, 3, 5, and 10 mM was measured for 30 s in
the absence (
Figure 4:
Genistein inhibits the uptake of
dehydroascorbic acid and deoxyglucose in CHO cells expressing the
glucose transporter GLUT1. A, time course of the uptake of
dehydroascorbic acid. B, time course of the uptake of
deoxyglucose. C, dose dependence of the effect of genistein on
the uptake of dehydroascorbic acid using a 1-min uptake assay. D, dose dependence of the effect of genistein on the uptake of
deoxyglucose using a 1-min uptake assay. Genistein was present during
the uptake assay. Data represent the mean ± S.D. of four
samples.
Figure 5:
Genistein inhibits in a competitive manner
the uptake of deoxyglucose in human erythrocytes. A, dose
dependence of the effect of genistein on the uptake of deoxyglucose. B, double reciprocal plot of the effect of genistein on the
substrate dependence for deoxyglucose uptake in the absence (
Figure 6:
Genistein inhibits in a competitive manner
the binding of cytochalasin B to the glucose transporter GLUT1 present
in human erythrocyte membranes. A, dose dependence of the
effect of genistein on the binding of cytochalasin B to human
erythrocyte membranes. B, Scatchard analysis of the binding of
different concentrations of cytochalasin B to human erythrocyte
membranes in the absence (
We show here that genistein is a potent inhibitor of the
cellular uptake of dehydroascorbic acid, deoxyglucose, and
methylglucose, all substrates that enter cells through hexose
transporters of the facilitative type. We used as experimental systems
HL-60 cells and human erythrocytes, cells that express the facilitative
hexose transporter GLUT1. Additionally, we used stably transfected CHO
cells expressing GLUT1. It is possible that genistein could be
inhibiting the activity of protein-tyrosine kinases whose
phosphorylating activity is fundamental in maintaining the activity of
GLUT1. There is, however, no available evidence indicating that GLUT1
is phosphorylated on tyrosine residues. Our data are compatible with
the concept that genistein inhibits the cellular uptake of hexoses and
dehydroascorbic acid by a mechanism unrelated to its ability to inhibit
the activity of protein-tyrosine kinases. Rather, genistein interacts
directly with GLUT1 and competitively inhibits the transport of hexoses
and dehydroascorbic acid across the cell membrane. The data from
time course experiments were consistent with the hypothesis that the
effect of genistein was not mediated through inhibition of
protein-tyrosine kinases. No genistein preincubation step was necessary
to observe its effect on the uptake of methylglucose, deoxyglucose, and
dehydroascorbic acid. The effect of genistein was instantaneous and
maximum when genistein was added to the uptake assay simultaneously
with the test substrate at time 0. On the other hand, the available
evidence indicates that the effect of genistein on protein tyrosine
phosphorylation is a time-dependent phenomenon that in some cases
requires long incubation times to fully
develop(28, 34, 36, 37, 39) .
Thus, the instantaneous effect of genistein on uptake argues against
the involvement of inhibition of protein tyrosine phosphorylation in
the mechanism of action of genistein. Further support for the lack of
involvement of protein-tyrosine kinases in the effect of genistein on
transport was provided by data from the uptake experiments in human
erythrocytes. The similar dose-effect curves for the effect of
genistein on uptake in the HL-60 cells, transfected CHO cells, and
human erythrocytes indicated that the effect of genistein was
cell-independent and likely occurred through the same mechanism in the
three cell types analyzed. Although HL-60 and CHO cells are sensitive
to the effect of tyrosine kinase inhibitors, human erythrocytes do not
express tyrosine kinase activities inhibited by genistein. Our data
indicate that genistein affects the facilitated transport of hexoses
and dehydroascorbic acid as distinct from the trapping/accumulation of
the transported substrates. The similar dose-response curves for
inhibition of uptake in both short uptake studies measuring transport
and in extended uptake assays measuring accumulation of the transported
substrate suggest that genistein is inhibiting uptake at the common
transport step previous to the intracellular trapping of the
transported substrates. Trapping of deoxyglucose depends on its
intracellular phosphorylation to deoxyglucose 6-phosphate which is not
further metabolized and cannot leave the cell because is not
transported by GLUT1(1, 2) . On the other hand,
trapping of dehydroascorbic acid depends on its reduction to ascorbic
acid that is not a substrate for GLUT1 and therefore accumulates
intracellularly(15, 16) . These are two very different
biochemical processes that depend on different enzyme activities
unlikely to be similarly affected by genistein. The effect of genistein
on uptake of the nonmetabolizable methylglucose is confirmative. Thus,
our data indicate a direct action of genistein on transport as opposed
to accumulation. The data showing that genistein inhibited the
transport of methylglucose and deoxyglucose in HL-60 cells and
deoxyglucose in human erythrocytes in a competitive manner strongly
support the concept that genistein exerts its effect on transport by
directly interacting with GLUT1. This notion is consistent with the
binding data indicating that genistein blocked the glucose-sensitive
binding of cytochalasin B to GLUT1 present in human erythrocytes.
Although the precise nature of the interaction between genistein and
GLUT1 cannot be deduced from these experiments, the data indicate that
genistein interacts with sites in the transporter involved in the
binding or transport of dehydroascorbic acid and hexoses and that this
interaction is also responsible for interfering with the binding of
cytochalasin B. The multiple effects of genistein on cell physiology
are believed to represent its ability to inhibit protein tyrosine
phosphorylation. Genistein has been shown, however, to have cellular
targets other than the protein-tyrosine kinases such as
protein-histidine kinase (50) and DNA topoisomerase
II(51) . Here, we provide evidence indicating that genistein is
a potent inhibitor of the facilitative glucose transporter GLUT1. Our
findings have implications for the interpretation of experiments
revealing a major effect of genistein on a general cellular function.
The literature contains reports of genistein's effects in
different cellular systems where the concentrations used are well
within the range that causes strong inhibition of the functional
activity of the glucose transporters. These include inhibition of
endothelial cell proliferation and in vitro angiogenesis (35) and cell cycle and apoptotic events induced in HL-60 and
Molt 4 cells(30) . Furthermore, genistein inhibits the growth
of the human prostatic carcinoma cell lines LNCaP and PC-3 without
inhibiting the tyrosine kinase activity of the epidermal growth factor
receptor in these cells(52) . Genistein also caused apoptosis
in thymocytes without altering protein tyrosine
phosphorylation(53) . Given the presence of glucose
transporters in all cells and tissues(1, 2) , and the
importance of these proteins for the provision of cellular nutrients
essential for normal cell function, it seems reasonable to consider
that some of the effects of genistein on cell proliferation and
differentiation may be related to its capacity to inhibit the activity
of the glucose transporters. GLUT1 is expressed in all cells and
tissues, is especially abundant in erythrocytes and brain, and is
responsible for the basal cellular uptake of
glucose(1, 2) . The finding that genistein inhibits
the functional activity of GLUT1 may be particularly relevant to the
physiology of cancer cells. It has been known for a long time that one
of the primary characteristics of cancer cells is an increased
metabolism of glucose(54) . Since cancer cells do not
accumulate intracellular stores of glucose in the form of glycogen or
fat as does liver and adipose tissue, glucose must be obtained
continuously from external sources and transported intracellularly. The
increased ability of cancer cells to transport glucose is used
clinically to locate tumors in patients and to assess their metabolism
and response to therapy in a noninvasive manner by positron emission
tomography scanning using [ The molecular details of the
interaction of GLUT1 with genistein, a compound functionally defined by
its specific interaction with protein-tyrosine kinases, are unknown. It
has been suggested that the glucose transporters possess a nucleotide
binding site and experimental evidence has been presented showing that
they can be photolabeled with azido-ATP (62) and
azidoadenosine(63) . Furthermore, there is controversy
regarding the ability of ATP and ADP to modulate the functional
activity of the glucose
transporters(42, 64, 65, 66) . Data
are available indicating that genistein, while unable to inhibit the
insulin receptor kinase activity, markedly decreased basal and
insulin-stimulated glucose uptake in rat adipocytes(67) .
Although the authors did not address the mechanism of action of
genistein in their experimental system, they observed that treatment
with genistein decreased the labeling of GLUT4 present in the plasma
membrane to the same extent that it inhibited glucose uptake without
inhibiting the insulin-induced recruitment of GLUT4 to the plasma
membrane. These data are consistent with the evidence presented here
indicating a direct interaction of genistein with GLUT1 and suggest
that genistein has the capacity to interact with other members of the
family of facilitative glucose transporters. Our data show that the
interaction of genistein with GLUT1 is quite specific, as exemplified
by the fact that daidzein, an isoflavone that differs from genistein in
lacking one hydroxyl group, did not affect the transport of
dehydroascorbic acid, deoxyglucose, or methylglucose in HL-60 cells.
Although daidzein does not inhibit the activity of the epidermal growth
factor receptor, a receptor whose activity is inhibited by genistein,
it inhibits thromboxane A2-stimulated protein tyrosine
phosphorylation(28, 68) . Overall, these observations
suggest the existence of a variety of compounds able to interact with
the glucose transporters in a highly specific manner that are
potentially capable of modulating their functional activity. We show
here that genistein directly inhibits the transport function of GLUT1.
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 8719-8724
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of approximately 12
µM for inhibition of the transport of both methylglucose
and deoxyglucose. Binding studies showed that genistein inhibited
glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte
ghosts in a competitive manner, with a K of 7 µM. These data indicate that genistein
inhibits the transport of dehydroascorbic acid and hexoses by directly
interacting with the hexose transporter GLUT1 and interfering with its
transport activity, rather than as a consequence of its known ability
to inhibit protein-tyrosine kinases. These observations indicate that
some of the many effects of genistein on cellular physiology may be
related to its ability to disrupt the normal cellular flux of
substrates through GLUT1, a hexose transporter universally expressed in
cells, and is responsible for the basal uptake of glucose.
)cells
overexpressing the glucose transporter GLUT1, and human erythrocytes.
The characteristics and the specificity of the inhibition, and the
results of studies showing that genistein affects the
glucose-displaceable binding of cytochalasin B to GLUT1 in erythrocyte
membranes, indicate that the effect of genistein is related to its
direct interaction with GLUT1. These results emphasize the ability of
GLUT1 to interact with molecules structurally unrelated to glucose and
have important implications for our understanding of the effects of
genistein on cellular physiology in normal and malignant cells.
Cell Culture
HL-60 cells were cultured in
Iscove's-modified Dulbecco's medium supplemented with 10%
fetal bovine serum and antibiotics. Cell viability was greater than
95%, as determined by trypan blue exclusion. Chinese hamster ovary
cells were cultured in Iscove's-modified Dulbecco's medium
supplemented with 10% fetal bovine serum and 0.25 mg/ml G418. CHO cells
expressing GLUT1 were a gift from Dr. Michael Czech (Program in
Molecular Medicine, University of Massachusetts Medical
Center)(40) . CHO cells expressing the human placental insulin
receptor were constructed in the laboratory of the late Dr. Ora M.
Rosen at Sloan Kettering Institute(41) . Human erythrocytes
were purified from outdated blood samples obtained by the Hematology
Service of the Regional Hospital in Valdivia.Uptake Assays
For uptake assays(17) ,
cells were incubated at room temperature in incubation buffer (17) containing 0.1-1 µCi of L-[
C]ascorbic acid (specific activity,
4.74 mCi/mmol, DuPont NEN),
2-[1,2-
H]deoxy-D-glucose (specific
activity 26.2 Ci/mmol, DuPont NEN), or
[H]methylglucose (specific activity 86.7 Ci/mmol,
DuPont NEN) and adequate concentrations of the respective unlabeled
compounds for the times indicated in the figures. Solutions of ascorbic
acid were prepared daily before use and kept at 4 °C in incubation
buffer containing 0.1 mM dithiothreitol. For dehydroascorbate
uptake experiments, ascorbate oxidase (0.1 to 10 units) was added to
the incubation mixture to generate dehydroascorbic acid(16) .
Uptake was stopped by washing the cells in cold (4 °C) stopping
solution (phosphate-buffered saline without Ca
and
Mg). The cells were solubilized in Tris-HCl, pH 8.0,
containing 0.2% sodium dodecyl sulfate (SDS) and processed for liquid
scintillation counting. When testing the effects of different
competitors and inhibitors on uptake, they were added at the beginning
of the experiment from concentrated stock solutions freshly prepared,
or, alternatively, the cells were preincubated with them.Cytochalasin B Binding
Pink erythrocyte ghosts
were prepared from washed red cells by hypotonic lysis in 5 mM Na
HPO
buffer, pH 7.4, containing 2 mM EDTA, followed by four successive centrifugation/wash cycles in
the same buffer, and were resealed in 10 mM NaHPO buffer, pH 7.4(42) .
Specific binding of cytochalasin B to functional glucose carriers was
estimated from the difference between cytochalasin B bound in the
presence of 500 mML-glucose and 500 mMD-glucose. Equilibrium cytochalasin B binding was
initiated by the addition of the membrane preparation (suspended in 10
mM NaHPO buffer, pH 7.4) to the
binding assay solution. The latter contains 10 mM NaHPO buffer, pH 7.4, cytochalasin E,
[4-H]cytochalasin B, and D- or Lglucose. The total volume of the reaction mixture was 150
µl. The final composition was 0.06 to 0.1 mg/ml erythrocyte
membrane protein (equivalent to 1-1.6 
10
cells), 10 µM cytochalasin E, 500 mMD- or L-glucose, 0.02-0.04 µCi of
[4-H]cytochalasin B (11.9 Ci/mmol, DuPont NEN),
and cold cytochalasin B for final concentrations of 0.01 to 5
µM. The mixture was incubated at room temperature for 10
min before collecting the membranes by centrifugation at 15,000 g for 10 min. The amount of specifically bound cytochalasin B
was estimated by determining the amount of radioactive ligand
associated with the membrane pellet and from the difference in the
amount of soluble radioactivity before and after centrifugation. Both
determinations gave similar results.
Effect of Genistein in HL-60 Cells
Genistein
inhibited the uptake of deoxyglucose and dehydroascorbic acid by HL-60
cells in a dose-dependent manner, with 50% inhibition observed at
12-15 µM genistein (Fig. 1A). 100
µM genistein completely blocked the uptake of
dehydroascorbic acid and deoxyglucose. The HL-60 cells express the
glucose transporter GLUT1, and the functional characteristics of this
transporter are similar to those described for GLUT1 present in other
cell types, including its ability to transport dehydroascorbic acid and D- but not L-hexoses, and its sensitivity to
inhibition by cytochalasin B but not cytochalasin
E(16, 17) .
) and dehydroascorbic acid (
) was measured in a 10-min
assay. Genistein was also present during the uptake assay. Data are
presented as percentage of control (samples not treated with genistein)
and represent the mean of four samples. B, HL-60 cells were
incubated in the absence () or in the presence () of 100
µM genistein for the indicated times (0-60 min)
before measuring uptake of deoxyglucose in a 10-min assay in the
presence of genistein. Data represent the mean of four
samples.
of approximately 15 µM (Fig. 3, A-D). These observations
indicate a direct interaction of genistein with the glucose transporter
expressed by HL-60 cells.
) or
daidzein (). Data are expressed as percentage of control
(transport in the absence of genistein) and represent the mean ±
S.D. of four samples. The structures of genistein and daidzein are
shown in E and F.
) or in the presence of 5 (), 10 (
), and
30 µM (
) genistein. Data represent the mean of four
samples. B, secondary plot of the effect of genistein on the
substrate dependence for deoxyglucose transport. C, double
reciprocal plot of the effect of genistein on the substrate dependence
for methylglucose transport. Transport of methylglucose at 3, 4, 8, and
15 mM was measured for 30 s in the absence () or in the
presence of 10 (), 20 () and 40 µM ()
genistein. Data represent the mean of four samples. D,
secondary plot of the effect of genistein on the substrate dependence
for methylglucose transport.
Effect of Genistein in CHO Cells
We next analyzed
the effect of genistein on the uptake of dehydroascorbic acid and
deoxyglucose in CHO cells transfected with a plasmid carrying the cDNA
for GLUT1. Immunoblotting experiments using anti-GLUT1 antibodies
revealed that the transfected cells expressed an increased number of
glucose transporters as compared to control cells stably transfected
with a plasmid carrying the cDNA for the human placental insulin
receptor (data not shown). CHO cells overexpressing GLUT1 had an
increased capacity to take up deoxyglucose and dehydroascorbic acid as
compared to the control cells, and uptake was linear for at least 10
min (Fig. 4, A and B). Genistein caused a
dose-dependent inhibition of uptake and, at 100 µM,
completely inhibited uptake of both dehydroascorbic acid and
deoxyglucose in the GLUT1-expressing as well as the control cells (Fig. 4, C and D). In both cell lines, 50%
inhibition of uptake was observed at approximately 12 µM genistein. These results show that the effect of genistein on the
activity of GLUT1 is independent of the cell context in which the
transporters are expressed. The simplest interpretation of the above
results is that genistein interferes with the transport of
dehydroascorbic acid, deoxyglucose, and methylglucose by directly
interacting with GLUT1.
, transfected CHO cells expressing the glucose
transporter GLUT1. , transfected CHO cells overexpressing the
human placental insulin receptor.
Effect of Genistein in Human
Erythrocytes
Genistein inhibited the uptake of deoxyglucose in
human erythrocytes in a dose-dependent manner, with 50% inhibition
observed at approximately 15 µM genistein (Fig. 5A). Kinetic analyses indicated that genistein
competitively inhibited the uptake of deoxyglucose with a K of about 12 µM (Fig. 5, B and C). The data, therefore, are consistent with
the direct interaction of genistein with the erythrocyte glucose
transporter. We further tested this hypothesis by analyzing the effect
of genistein on the binding of radiolabeled cytochalasin B to the
hexose transporters present in purified human erythrocyte ghosts.
Affinity labeling experiments show that cytochalasin B binds covalently
to the glucose transporter in a D-glucose-displaceable
manner(46, 47) . We reasoned that if genistein and
cytochalasin B compete for the glucose binding or transport sites in
the transporter, they may also compete with each other. The pink ghosts
used in these experiments bound 0.49-0.55 nmol of cytochalasin B
per mg of protein in a D-glucose-sensitive fashion, with a
dissociation constant of 0.25 µM (data not shown). From
these data, we calculated that there are 1.8-2.1 10
cytochalasin B binding sites related to the glucose transporter
per cell, based on estimating the amount of membrane protein per cell
at 0.61 pg. These sites are competed by high concentrations of D- but not L-glucose. The calculated number of sites
per cell agrees with previous estimates of 2-3 10 sites/cell derived from cytochalasin B binding studies (48) and chemical labeling of the GLUT1 protein(49) .
Increasing concentrations of genistein efficiently competed for the
glucose-sensitive cytochalasin B binding sites present in the
erythrocyte membranes (Fig. 6A). Approximately 15
µM genistein inhibited the binding of 0.1 µM cytochalasin B by 50%, while total inhibition of binding was
observed at 100 µM genistein. A small increase in binding
was observed at concentrations of genistein greater than 100
µM, a result similar to the increase in transport observed
in HL-60 cells treated with similar concentrations of genistein (Fig. 6A and Fig. 2, B and C).
Genistein was a linear competitive inhibitor of the binding of
cytochalasin B to the erythrocyte ghosts (Fig. 6B). A
secondary plot of the apparent extent of cytochalasin B binding versus genistein concentration was linear and gave an
inhibition constant for genistein of approximately 8.0 µM (Fig. 6C). The above observations are consistent
with the concept that genistein interacts directly with the glucose
transporters.
) or
in the presence of 10 (
), 15 (), 20 (), and 40
µM (
) genistein. C, secondary plot of the
effect of genistein on the substrate dependence for deoxyglucose
uptake.
) or in the presence of 5 (), 10
(), 15 (), and 30 µM () genistein. C, secondary plot of the effect of genistein on the binding of
cytochalasin B to the erythrocyte
membranes.
F]fluorodeoxyglucose,
a molecule transported by facilitative hexose
transporters(55, 56) . The mechanism whereby cancer
cells increase their ability to take up glucose involves the selective
overexpression of
GLUT1(57, 58, 59, 60) , a
transporter we show here is inhibited by genistein. In this regard,
there is now a growing interest in the possible use of genistein or
genistein-containing soy matrices in chemoprevention trials for breast
and prostate cancer(61) .
)
We thank Dr. Michael Czech for providing the
transfected CHO cell line overexpressing GLUT1.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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