(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 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.
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) ()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.
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
() 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.
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 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.
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 () 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.
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 () 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.
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. , transfected CHO cells expressing the glucose
transporter GLUT1.
, transfected CHO cells overexpressing the
human placental insulin receptor.
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 () 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.
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 () 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.
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 [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) .
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.