Minggu, 03 Juli 2016

Phytochemicals and Gene Expression

Source: www.blogs.oregonstate.edu


The practice of self-medicating with botanical products is probably as old as humankind
itself. Depicted in literature and film, we are well-familiar with the herbal remedies
prepared and administered by the healers of ancient Egypt, medieval physicians, and
American Indian cultures. Only in the past century has modern chemistry produced a
distinction: a choice between synthetically derived drugs vs. natural products. With the
great advances made by the pharmaceutical industry, our modern Western culture has
distanced itself from the notion that phytochemicals may have specific medicinal actions.
Most synthetically derived medicines are considered to have a specific target: as an
enzyme inhibitor (e.g., cholesterol-lowering statins), receptor agonists, or receptor
antagonists (the antidiabetic drug rosiglitazone is an agonist of the peroxisome
proliferator activated receptor). Synthetic drugs are generally considered to have high
specificity; hallmark examples may be action on specific serotonin receptor subtypes or
the selective estrogen receptor (ER) modulators (SERMs) that act more potently on ERα
or ERβ

Understanding the specific effects of phytochemicals represents distinct challenges.
Most often, commercially available products represent solvent-soluble extracts prepared
from a botanical. These products usually contain mixtures of a wide number of
compounds. The exact number and relative abundance of these products often varies
from preparation to preparation. Growing conditions and the geographical source of a
botanical may ultimately affect the potency of the extract. Evaluating the effect of a
mixture on a biological system—whether a cell or animal model—becomes a more
difficult task compared to evaluating the effect of a single compound. Different
compounds present in the extract may have distinct effects, and may even interact
negatively or positively. This interaction may vary in significance depending on the
relative abundance of the different compounds in the extract.
To evaluate the effect of phytochemicals on gene expression, one may use both cell
and animal models. Further, if a specific compound is presumed to be the bioactive
compound present in a botanical extract, that compound can be studied alone, as a
purified compound, in parallel with studies evaluating a mixture containing the putative
active compound. One would expect to see the same effect on the model system, if the
putative compound is indeed the active factor.

If a candidate phytochemical is being evaluated as a potential regulator of gene
expression, one might predict specific sites where gene regulation would be affected. For
example, a phytochemical acting as a ligand for a specific nuclear receptor might enhance
the trancription rate of a specific gene. Alternatively, a phytochemical might repress gene
transcription if it interferes with coactivator recruitment. Phytochemicals acting as
agonists or antagonists of kinases or phosphatases involved in a signal transduction
pathway may ultimately affect the expression of a gene or sets of genes regulated by that
specific pathway.

A wide number of research reports have detailed the interaction between various
phytochemicals. Rather than attempt to provide an encyclopedic collection of those
reports, we have chosen a number of examples where phytochemicals have been shown
to affect gene expression in different model systems.


St. John’s wort is used to treat mild to moderate depression and anxiety (1). It is
composed of numerous constituents, although its active component remains speculative.
One component, hyperforin, inhibits synaptic uptake of various neurotransmitters such as
serotonin and dopamine in vitro, although in vivo studies suggest other potential modes
of action not fully dependent on hyperforin (1). St. John’s wort serves a wide array of
therapeutic uses, not only historically, but also currently in both the United States and
Europe (2). Recent reports have suggested that St. John’s wort decreases the efficacy of
medications metabolized by cytochrome P450 3A4 (CYP3A4), a member of the
cytochrome P450 monooxygenase family (CYP) (Table 1) (3). The CYP family of
enzymes is responsible for clearing the majority of prescription drugs and ingested
pollutants (4). These enzymes are located in hepatocytes and intestinal cells and are
capable of metabolizing many endogenous compounds along with exogenous drugs and
pollutants (5). Several CYP families exist, although CYP1, CYP2, and CYP3 metabolize
most xenobiotic substrates (5). Moore et al. investigated the effect of St. John’s wort on
the human pregnane X receptor (PXR), a nuclear receptor responsible for regulating
CYP3A4 transcription (Figure 1) (3). They found that three different commercial brands
of St. John’s wort extract activated PXR. Of the components tested, hyperforin induced
PXR at half-maximal effective concentration (EC50) of 23 nM. Importantly, this
concentration is well below the 200 nM level observed in plasma of individuals using St.
John’s wort on a regular basis. Hyperforin was also found to directly bind PXR, as
confirmed by competition binding assays. Finally, Moore et al. found that the St. John’s
wort extract and hyperforin induced the expression of CYP3A4, validating the premise
that this botanical could interfere with and increase the metabolism of drugs metabolized
by CYP3A4 (5). Wentworth et al. also found that St. John’s wort and hyperforin
activated the ligand-binding domain of the steroid X receptor (SXR), known as the
human PXR (6). This occurs via the activation function site 2 (AF-2) of S×R. Steroid
receptor coactivator-1 is a coactivator recruited by various nuclear receptors including
S×R. St. John’s wort and hyperforin mediated the SXR-SRC1 association, further
confirming of activation of CYP3A transcription by this phytochemical (6). Hyperforin
has recently been confirmed by crystal structure analysis to bind to the ligand-binding
domain of PXR (7). St. John’s wort has also been implicated to increase the expression of
CYP1A2, the second most abundant CYP accounting for over 10% of human hepatic
CYP content (4). However, a human in vivo study did not find evidence of increased
CYP1A2 activity after two weeks of St. John’s wort intake (300 mg three times per day),
although CYP3A4 activity was significantly induced in the intestinal wall (8). These
studies concluded that St. John’s wort increases the metabolism of certain medications by
increasing the expression of CYP3A4 via hyperforin binding and activating human PXR.


Guggulsterone is the active, lipid-lowering fraction of gugulipid, a gum resin extract from
the Commiphora mukul tree used in India for thousands of years to treat hyperlipidemia
(9). Numerous animal studies and clinical studies have been conducted since the 1960s
when the initial scientific studies were begun on the hypolipidemic effect of the gum
resin and its extracts. Most of the clinical studies found that gugulipid or guggulsterone
reduced serum cholesterol levels by an average of 30% (9). Upon further investigation,
Urizar et al. proposed that the lipid-lowering mechanism of this gum resin occurs via the
antagonistic activity of guggulsterone for the farnesoid X receptor (FXR) (10). FXR
heterodimerizes with the retinoid X receptor (RXR) upon ligand binding (11) and is
known as the “bile acid sensor” because it is responsible for repressing bile acid synthesis
via transcription of ileal bile acid-binding protein (I-BABP). Ligands of the nuclear
receptor FXR include bile acids such as chenodeocycholic acid (CDCA). Activation of
FXR also increases bile acid recirculation due to elevated bile acid concentrations within
the cell (11). In the recent study by Urizar et al., the Z-guggulsterone isomer had no
effect on FXR alone, although the E- and Z-guggulsterone isomers were able to inhibit
CDCA activation of FXR along with FXR-regulated genes (10). The isomers were also
able to inhibit CDCA activation of small heterodimeric partner (SHP). Small heterodimeric
partner is a nuclear receptor that heterodimerizes with other nuclear receptor
complexes such as the active FXR-RXR complex, although it does not have a DNAbinding
motif, as do most other nuclear receptors (11). Guggulsterone inhibited
transactivation of FXR-RXR and not DNA-binding of these complexes, indicating that
the isomers were exerting an inhibitory effect via the ligand-binding domain of the FXR (10). This effect was confirmed by using a fluorescence resonance energy transfer (FRET)-based coactivator
binding assay, in which guggulsterone was found to directly compete with CDCA for the
ligand-binding domain, inhibiting the recruitment of a necessary coactivator, SRC-1.
Finally, FXR-null mice did not respond to the cholesterol-lowering effect of
guggulsterone seen in the wild-type mice, indicating that FXR is necessary for the
hypolipidemic effect of guggulsterone (10). Wu et al. also found that guggulsterone had
an antagonistic activity of FXR in the presence of FXR activators and was able to
decrease gene expression of FXR-regulated genes (12). More recently, a study found that
guggulsterone induced the expression of the FXR-regulated bile salt export pump gene
(BSEP) in vitro in the presence of two different FXR ligands, CDCA and GW4064 (13).
This induction was also evident in rats fed a diet containing either 2.8 or 5.6%
guggulsterone, in which both BSEP and SHP mRNA were elevated compared to the
control-fed rats. However, mRNA levels of other FXR-regulated genes tested—such as
cholesterol 7α-hydroxylase (CYP7α1), sterol 12α-hydroxylase (CYP8b1), and I-BABP—
were unaffected. In this study, guggulsterone blocked coactivator recruitment of p120
and PBP as well as SRC-1, consistent with the prior report (13). These results indicate
that guggulsterone may have selective antagonistic activity on required coactivator
recruitment for FXR-mediated transcription, but also agonist-enhancing activity on selective FXR-regulated genes.


4.1. Genistein and PPARs
Soy isoflavones are phytochemicals often termed “phytoestrogens” due to the estrogenic
properties of these botanically derived products (14). Soy isoflavones have been credited
to have antiatherosclerotic, antidiabetic, and anticarginogenic properties, although the
specific physiological and cellular mechanisms affected by isoflavones are an area of
controversy and debate (15–17). Recent studies found that soy isoflavones were able to
activate two isoforms of the peroxisome-proliferator-activated receptors (PPARα and
PPARγ), proposing a novel way in which the isoflavones may be exerting their
antiatherosclerotic and antidiabetic properties (Figure 2) (18,19). The PPARs are nuclear
receptors involved in cellular lipid homeostasis (20). They have a promiscuous ligandbinding
domain able to bind a variety of lipophilic ligands, resulting in receptor
activation. Activation of PPARα results in increased expression of genes involved in fatty
acid catabolism, whereas activation of PPARγ results in increased expression of genes involved in cellular differentiation and insulin sensitization (20). Dang et al. found that
the soy isoflavone genistein was able to activate PPARγ in a dose-dependent manner, and
genistein also increased the expression of PPARγ-regulated genes and adipogenesis in
KS483 cells at a dose of 25μM (19). Genistein interacts directly with the nuclear
receptor, as verified by a membrane-bound PPARγ binding assay. However, a lower dose
of genistein (1 μM) actually had an inhibitory effect on PPARγ-regulated genes as well as
on adipogenesis. This is most likely due to the ability of low concentrations of genistein
to activate estrogen receptor-mediated activity, resulting in a decrease of PPARγ
activation (19). Mezei et al. found that both genistein and the soy isoflavone daidzein
were able to activate PPARγ-mediated transcription (18). Furthermore, female obese
Zucker rats fed a highisoflavone-containing soy diet had significantly improved glucose
tolerance compared to casein and low-isoflavone-containing diets, consistent with effects
of PPARγ activation. Genistein and daidzein were also able to activate PPARα-mediated
transcription. Both male and female obese Zucker rats fed a high-isoflavone-containing
diet had reduced liver cholesterol, liver triglycerides, and total liver weight, consistent
with effects of PPARα activation (18). Harmon and Harp found an opposing effect of
genistein on PPARγ (21). In this study, genistein was found to inhibit PPARγ expression
as well as adipogenesis in adipocytes, a well-characterized consequence of PPARγ
activation. However, these inconsistent effects may be due to the elevated concentration
used in this study. Other studies found that a genistein concentration of 50 μM was
enough to induce apoptosis in certain culture models such as colon carcinoma cell lines,
whereas Harmon et al. used a genistein concentration of 100 μM (22, 23). Genistein and
daidzein are not the only phytochemicals with PPAR-activating ability. Takahashi et al.
discovered that farnesol and geranylgeraniol, two common fruit and herb isoprenols, are
able to activate both PPARα and PPARγ along with several PPAR-regulated genes (24).
Therefore, these studies give new insight on the mechanism by which soy isoflavones and
other botanicals exert their favorable consequences.

4.2. Isoflavones and Estrogen Receptors
It is estimated that 80% of women over the age of 45 use some type of non-prescription
therapy to manage menopause symptoms, ranging from the consumption of soy or
Genomics and proteomics in nutrition 252
evening primrose oil to acupuncture (25). Of these therapies, the use of isoflavones as an
alternative to hormone replacement therapy may be an attractive alternative to classical
“hormone replacement therapy” (HRT) for many postmenopausal women, although their
potential side effects and long-term health implications are still not fully understood (26).
One of the soy isoflavones, genistein, has been shown in studies to have estrogenic
activity (27, 28). Because some postmenopausal women with estrogen-dependent breast
tumors may be consuming genistein as an alternative to HRT, Ju et al. studied the effect
of genistein on estrogen-dependent breast cancer growth (29). In this study, mice with
estrogen-dependent tumors had a significant, dose-dependent increase in tumor
presenelin-2 (pS2) mRNA levels when provided dietary genistein. The level of induction
seen with the higher genistein doses was similar to the induction produced by the
subcutaneous 17β-estradiol pellet. The pS2 gene is an estrogen-responsive gene and
indicative of estrogen-dependent growth. The ability of genistein to induce estrogendependent
growth in these tumors was also observed in tumor size and proliferation; both
were significantly increased with genistein ingestion or 17β-estradiol supplementation
(29). Therefore, the results of this study indicate that genistein consumption may promote
the growth of certain estrogen-dependent breast tumors. Another phytochemical with
known estrogenic activity is resveratrol, a polyphenolic compound in grapes and wine
(30). Resveratrol has also been attributed to have cancer-preventative properties in colon
cancer cell lines (31, 32). However, the effect of resveratrol on breast cancer growth is
controversial, especially with respect to estrogen-dependent tumors (33). Levenson et al.
found that resveratrol was able to induce gene expression of the estrogen-responsive gene
tumor growth factor a (TGFα) in a dose-dependent manner in breast cancer cells
expressing wild-type estrogen receptor (33). Higher doses of resveratrol were needed to
mimic this effect in breast cancer cells expressing a mutant form of the estrogen receptor.
However, resveratrol did not further stimulate TGFα expression when 17β-estradiol was
present in its optimal concentration. Resveratrol inhibited the growth of the breast cancer
cells regardless of the presence of the estrogen receptor or the antiestrogen ICI, indicating
that growth inhibition by resveratrol is, at least in part, estrogen receptor-independent.
Estrogen receptor protein levels were analyzed in both wild-type and mutant estrogen
receptor-expressing cells. Both 17 β-estradiol and resveratrol decreased the wild-type
estrogen receptor levels. Finally, resveratrol and 17(β-estradiol both increased the protein
levels of p21cip/WAF1, a cyclin-dependent kinase inhibitor, although this increase
appears to be an estrogen-mediated effect (33). Both resveratrol and genistein have
estrogenic effects and are able to regulate many estrogen receptor-mediated genes. This
activity may explain some of the beneficial effects of these phytochemicals, but also
warrants further investigation due to possible harmful side effects.

4.3. Genistein and Gene Expression Patterns
Recent studies utilizing microarray technology reveal that genistein affects the regulation
of many genes, including those involved in reproductive development and prostate cancer
(34–36). Naciff et al. found that genistein had a gene expression profile similar to an
estrogen (17 α-Ethynyl estradiol) and a weak estrogenic chemical (bisphenol A) in the
developing uterus and ovary of the rat (34). Genes involved in cell growth (growth
hormone receptor), differentiation (progesterone receptor), stress response (glutathione transferase M5), and apoptosis (interleukin 4 receptor) were regulated similarly by all
three compounds. RT-PCR confirmed some of these results, such as increased expression
of the progesterone receptor by 17 α-Ethynyl estradiol, bisphenol A, and genistein (34). It
is important to note that although the three compounds have a similar gene expression
profile in the developing reproductive system, the gene expression profiles of 17 α-
Ethynyl estradiol and bisphenol A were more similar to each other than to genistein. This
may be due to the mainly “estrogenic” activity of these compounds, whereas genistein
has other known activities, such as tyrosine kinase and topoisomerase-II inhibition along
with activity as a PPAR agonist profiled earlier (34). Two recent studies also analyzed
the gene expression profile of genistein using a human prostate cancer cell line (35, 36).
Li and Sarkar found that genistein downregulated genes involved in angiogenesis, such as
vascular endothelial growth factor and its receptor, and upregulated genes inhibiting
angiogenesis, such as connective tissue growth factor and connective tissue activation
peptide (35, 36). Furthermore, genistein also downregulated genes necessary for tumor
cell invasion and metastasis (MMP-9/type IV collagenase, urokinase plasminogen
activator, and urokinase plasminogen activator receptor). These results indicate that
genistein may inhibit tumor metastasis and growth. Another study by Li and Sarkar found
that genistein caused a difference in expression profiles of genes involved in cell cycle
control, apoptosis, and cell signaling (36). Genistein downregulated cell cycle promoter
genes such as cyclin A and cyclin B and induced genes that inhibit cell cycle progression,
such as cyclin G2 in human prostate cancer cultured cells. Genes involved in the
inhibition of apoptosis (survivin) and genes involved in cell growth (pescadillo) were also
downregulated in genistein-treated cells (36, 37). Genistein also downregulated signaling
genes such as NF-κB-inducing kinase and MAP kinase kinase potentially resulting in
decreased cell proliferation (36). Therefore, microarray analysis revealed that genistein is
able to affect many genes involved in biological processes such as cell growth, cell cycle
control, differentiation, stress response, angiogenesis, tumor cell invasion, metastasis,
signaling, and apoptosis.


Soy consumption may have many advantageous outcomes, such as an improved
management of blood lipids and a decreased risk of cancer (38–40). In one rodent study,
Tovar-Palacio et al. found that soy-fed gerbils had significantly reduced levels of
circulating apolipoprotein B and significantly increased circulating levels of
apolipoprotein A-I after a 28-day feeding study (41). However, apolipoprotein A-I gene
expression was significantly reduced in gerbils fed a soy diet containing various amounts
of isoflavones. This reduction was not reflected in the circulating protein content. This
discrepancy may be a result of decreased circulating lipoprotein turnover or a
downregulation of apolipoprotein A-I synthesis due to its elevated level in circulation in
the soy-fed animals. It is important to note that the mRNA levels of apolipoprotein E an
apolipoprotein also synthesized in the liver and similar in abundance to apolipoprotein A,
remained unchanged (41). Two other genes also affected by soy consumption in gerbils
are phosphoribosylpyrophosphate synthetase-associated protein (PAP) and a member of
the cytochrome P450 2A family (CYP2A) (42). In a study by Mezei et al., gerbils fed a soy diet with increasing levels of isoflavones had a dose-dependent increase in both PAP
and CYP2A gene expression. PAP is a protein that negatively regulates
phosphoribosylpyrophosphate synthetase (PRPP-synthetase) activity, an enzyme
involved in nucleotide synthesis. Therefore, soy may be able to decrease nucleotide
synthesis and cell proliferation via PAP regulation. CYP2A belongs to a family of
enzymes used to metabolize endogenous and exogenous toxins and other xenobiotics
(43). Therefore, upregulation of this CYP might decrease mutagenic threat to the cell.
Ronis et al. found an induction in CYP3A protein levels in dexamethasone-treated (DEXtreated),
soy-fed rats relative to DEX-treated, casein-fed rats (44). This induction in
protein level appears to be due in part to the increased expression of CYP3A2 mRNA.
CYP3Al, CYP3A9, and CYP3A18 did not have increased mRNA levels in DEX-treated, soy-fed rats compared to the DEX-treated, casein-fed rats. Neither
CYP2B1 protein levels nor mRNA levels were different between these soy-fed and
casein-fed rats. However, the enzymatic activity of CYP2B1 was greater in the DEXtreated,
soy-fed rats. This effect was also seen in CYP3A enzymatic activity except when
CYP3A18-specific lithocholic acid was used as substrate (44). Therefore, soy had other
stimulatory effects on CYP enzymes besides increased gene and subsequent protein
expression. A second study by Ronis et al. focused on CYP1 A induction and activity
resulting from a casein, whey, or soy protein source in 3methylcholanthrene- (3-MC) or
isosafrole- (ISO) induced rats (45). 3-MC is an environmental carcinogen that induces
CYP1A expression via the aryl-hydrocarbon receptor (AhR) located in the promoter of
CYP1A genes, and ISO is a common phytochemical component of foodstuffs that
induces CYP1A in an AhR-independent manner (45). 3-MC induced CYP1A1 gene
expression while protein levels were significantly reduced in soy-fed rats compared to
casein-fed rats. CYP1A2 mRNA levels were also significantly reduced in soy-fed rats
compared to casein-fed rats, although protein levels were comparable. Finally, the
enzymatic activities of both CYP1A1 and 1A2 were lower in the soy-fed group (45).
Consistent with the results just noted, AhR expression was 50% lower in soy-fed rats, and
AhR expression was highly correlated to 3-MC-induced CYP1A1 expression (45). The
selective regulation of soy on CYP expression and activity may account for some of the
anticargenogenic activities attributed to soy.


Flavonoids are polyphenolic phytochemicals with antioxidative, anticarginogenic, and
estrogenic activity that exert their effects through various biological processes including
signaling cascades (46–48). Frigo et al. found that the flavonoids apigenin, flavone, and
chalcone induced activator protein-1 (AP-1) activation in two estrogen-unresponsive cell
lines (46). AP-1 is a transcription factor that is a target for multiple signaling cascades.
Chalcone was the only flavonoid that induced all of the transcription factors tested (AP-1,
Elk-1, c-Jun, and CHOP). The flavonoids kaempferide and apigenin inhibited PMAinduced
Elk-1 and c-Jun activity, decreasing cellular proliferation signaling important in
tumor prevention (46). Genistein is another flavonoid that can moderate its biological
effects such as cell-cycle arrest through intracellular signaling pathways (47). Frey and
Singletary showed that genistein inhibited the growth of immortalized human breast
cancer cells, as seen by DNA synthesis arrest (47). In this study, genistein was able to
cause phosphorylation of the p38 mitogen-activated protein kinase (p38 MAPK) in a
dose- and time-dependent manner and increase its activity. This ultimately caused the
downregulation of Cdc25C, a cell-cycle promoter protein. Genistein also inactivated
ERK1/ERK/2 and had no effect on SAPK/JNK activity, indicating that this isoflavone
has a selective action on MAPK signaling pathways that may be dependent on cell type
(47). Another flavonoid that affects signaling cascades is naringenin, a flavonoid found in
grapefruit (48). Harmon and Patel found that this flavonoid inhibited insulin-mediated
glucose uptake in adipocytes (48). Naringenin arrested Akt activation, but had no effect
on the insulin receptor (IRβ), insulin receptor substrate-1 and -2 (IRS-1, IRS-2), or
phosphoinositide 3-kinase (PI3K) phosphorylation status. Although naringenin did not
affect the phosphorylation state of PI3K, it did inhibit the activity of PI3K, resulting in
the observed decrease in Akt phosphorylation (48). In conclusion, flavonoids such as
chalcone, genistein, and naringenin are able to mediate biological processes such as cellcycle
arrest or altered gene transcription via intracellular signaling cascades.


One of the most exciting advances in the field of regulation of gene expression by dietary
constituents is the explosion of information becoming available regarding nuclear
receptors such as PPAR, FXR, PXR, AhR, and many others. A particularly important
aspect that must be considered is that many nuclear receptors are “promiscuous”
receptors, having the ability to bind many different endogenous and exogenous ligands.
Thus, is becomes immediately apparent that many phytochemicals have such lipophilic
Genomics and proteomics in nutrition 258
properties that they may make excellent ligands for one or more of these nuclear
receptors. In previous sections, we have discussed some of the recent examples just
beginning to show how phytochemicals interact with these promiscuous receptors. One of
the exciting challenges of future research will be to identify further phytochemical
ligands for these receptors and to study phytochemical/phytochemical and
drug/phytochemical interactions. The ability to point out both negative interactions and
potentially promising positive interactions between drugs and phytochemicals may
provide great practical health benefits to the consumer.

Author: Orsolya Mezei and Neil F.Shay
Department of Biological Sciences, University of Notre Dame,
Notre Dame, Indiana, U.S.A.

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