Study of pharmacokinetics of prenylflavonoids and dynamics of estrogen action in sera following ingestion of epimedium using validated, ultra sensitive cell based bioassays 4

CHAPTER 4 DISCUSSION 4.1 Development of sensitive in-vitro estrogen-responsive bioassays as clinical tools to understand biological effects of estrogenic compounds, alone and in comb

CHAPTER 4

DISCUSSION

4.1 Development of sensitive in-vitro estrogen-responsive bioassays as

clinical tools to understand biological effects of estrogenic

compounds, alone and in combination

187

4.1.1 Ultra-sensitive bioassays expressing ERα and ERβ can be used

to detect ER-isoform selective activity and study of structure–

function relationships

187

4.1.2 MCF-7 breast cancer cell proliferation assay for quantification of

4.1.5 Application of in-vitro ER-responsive bioassays as clinical tools

to understand estrogenic effects of compounds in serum 197

4.2 Pharmacokinetics and pharmacodynamics of Epimedium

compounds

200

4.2.1 Pharmacokinetics of unconjugated prenylflavonoid aglycones

4.2.2 Pharmacokinetics and pharmacodynamics of unconjugated

prenylflavonoids after ingestion of an enriched Epimedium preparation by rats

204

4.2.3 Pharmacokinetics and pharmacodynamics of conjugated

prenylflavonoids after ingestion of an enriched Epimedium

preparation by rats

206

4.2.4 Accumulation of conjugated Epimedium compounds in-vivo 208

4.3 Gene expression profiling reveals partially overlapping but distinct

genomic actions of different estrogenic compounds in human breast

cancer cells

209

4.3.1 Convergence of global gene expression profiles MCF-7 breast

cancer cells treated with estrogenic Epimedium

prenylflavonoids, genistein and estradiol

209

4.3.2 Discovery of up-regulation of CYP1A1 transcription by

Epimedium compounds via global gene expression profiling 211

4.3.3 Epimedium prenylflavonoids are dual activators of the AhR and

ER

212

4.1 Development of sensitive in-vitro estrogen-responsive bioassays as clinical tools to

understand biological effects of estrogenic compounds, alone and in combination

4.1.1 Ultra- sensitive bioassays expressing ERα and ERβ can be used to detect isoform selective activity and study of structure–function relationships

ER-A panel of stable human cell lines derived from HeLa human cervical adenocarcinoma cells has been developed that specifically respond to estrogenic compounds that interact with human ERα and ERβ proteins thereby allowing the efficient screening of estrogenic activity of chemicals, alone or in combination, such as in a complex mixture encountered in botanical extracts and sera

A yeast-based reporter gene assay was not used as such it does not discriminate estrogenic and anti-estrogenic substances (Andersen et al., 1999; Jørgensen et al., 1998)

and may not accurately represent human systems, as the permeability of compounds through the yeast cell wall might be different relative to mammalian cell membranes (Scrimshaw & Lester, 2004)

HeLa cells were chosen, as in the untransfected form, they endogeneously express

undetectable amounts of ERα and ERβ proteins (Escande et al., 2006) The pERE4Luchygro reporter gene was first stably incorporated into HeLa cells consists of 4 tandem copies of consensus vitellogenin ERE cDNA cloned into a pGL3-basic plasmid upstream

-of the luciferase reporter gene Subsequently, cells with pERE4-Luchygro in their genome were stably transfected with coding sequences for either ERα or ERβ Two highly inducible clones that stably expressed ERα and ERβ proteins respectively were selected for the development of ERα and ERβ cell-based bioassays The production of stable cell lines eliminates the necessity of constantly producing DNA and variability associated

with transient transfection procedures (Paris et al., 2002)

For the validated ERα bioassay, the intra- and inter-assay variations near the EC50

concentration of the standard estrogen, estradiol, were about 6% and 14% respectively For ERβ, the intra- and inter-assay variations were determined to be 8.1% and 16%

respectively Detection limits for estradiol in ERα and ERβ bioassays were 8.4 and 13.1

pM, respectively This set of assays is simple and rapid to perform and offers throughput, as cells require only 24 h of incubation with test samples

high-The estrogen agonist properties of estradiol and its metabolites, which include estrone and estriol, were first examined The rank order in terms of decreasing estrogenic potency for this series of compounds in both ERα and ERβ bioassays is estradiol >

estriol > estrone This result is in good agreement with those reported by Paris et al.,

(2002) for their corresponding ERα bioassay Both assays are highly specific to estrogens and they displayed minimal cross reactivity with most steroidal hormones such as the androgens, progestogens and corticosteroids However, when testing samples that contain testosterone, such as sera from males, where testosterone levels can be high, an aromatase inhibitor, such as DL-aminoglutethimide, would have to be added

These two assays are also able to distinguish full agonists from partial agonists Estriol and estrone are found to be full agonists like estradiol Interestingly, some compounds elicited superagonist effects Flavonoids like apigenin and genistein exhibited superagonist effects in both assays Kaempferol and luteolin are superagonists in the ERα bioassay but are partial agonists in the ERβ bioassay

ligand, strongly activated our ERα bioassay in a dose-dependent manner but did not induce detectable luciferase activity in the ERβ bioassay in the range of concentrations tested Both genistein and DPN, ERβ-selective ligands, displayed ERβ selectivity when tested in both systems

The effect of hydroxylation on the estrogenic activities of four common

flavonoids - apigenin, kaempferol, luteolin, and quercetin was also studied Previous

studies indicated that the two hydroxyls in position 7 of ring A and 4’ of ring B represent the minimal hydroxylation pattern for estrogenic activity (Vaya & Tamir, 2004) and these four phytoestrogens have these two hydroxyl groups Above this number, an inverse relationship between the number of hydroxyl groups and ERα bioactivity was observed Increasing the number of hydroxyl groups decreased the molecule’s hydrophobicity, which reduces binding to the hydrophobic ligand-binding pocket

Addition of a hydroxyl group in the 3’ position in benzene ring B, which converts the flavone apigenin to luteolin, reduced estrogenic activity, reflected by higher EC50

values and lower relative peak activities This structure–bioactivity relationship was also observed among the flavonols—the presence of a hydroxyl in the same position changed kaempferol to the less estrogenic quercetin, suggesting a role for the 3’ position of ring B

in ERα activity Another active position was the hydroxyl in position 3 of ring C of the chromone backbone: its presence also reduced estrogenic activity (apigenin vs kaempferol; luteolin vs quercetin) This observation was consistent with previous reports

that hydroxyls at positions 3 and 5 decrease reporter gene activities (Le Bail et al., 1998)

Interestingly, the hydroxyl substitutions in position 3 of ring C, which define flavones and flavonols, suggest that the division of phytoestrogens into these categories also reflect intrinsic ERα activity A similar observation was also seen for ERβ, with the

exception of kaempferol, which suggests that the mechanisms of ERβ activation by kaempferol may differ from the rest of the phytoestrogens Positions 3’ of ring B and 3 of ring C may have important roles for estrogenic activity, and more detailed investigations

on these two positions may be warranted

The molecular basis of these structure–function differences is unlikely to be completely due to differential receptor affinity Cell culture media used in this study had

dextran-coated charcoal stripped serum added to it and ex-vivo samples from clinical trials

and animal studies use sera samples from volunteers and test animals In the absence of serum, binding of quercetin to recombinant ERα was comparable to that of genistein and

kaempferol (Maggiolini et al., 2004; Leung et al., 2004) The presence of serum reduced

ERα binding of quercetin in MCF-7 cells, with relative binding affinities for genistein, kaempferol, and quercetin being 0.1, 0.012, and 0.001, respectively (Zava & Duwe, 1997) This apparently low ERα affinity of quercetin in serum is not surprising, considering that

quercetin binds extensively to serum proteins (Boulton et al., 1999) Reflecting these

changes in relative affinity, transactivation effects of quercetin were comparatively higher

in serum-free media compared to experiments done with serum (Maggiolini et al., 2004; Harris et al., 2005)

4.1.2 MCF-7 breast cancer cell proliferation assay for quantification of estrogenic activity

Unlike HeLa cells, MCF-7 cells are breast cancer cells which express ERα and ERβ to no relevant extent They require estrogenic stimulation for transformation from dormant into proliferating cells MCF-7 cells have been used widely as a highly sensitive

tool for the detection of even small agonistic effects at ERα An in-vitro assay based on

the proliferation of MCF-7 cells commonly called the ‘E-screen’ is performed by incubating cells with test samples over a period of six days The number of cells is quantified and comparisons can be made between cells treated with test samples with those incubated with the vehicle control

Although the assay procedure is more laborious than ERα and ERβ stable cells, the MCF-7 cell proliferation assay is more sensitive towards estrogens as it has a lower detection limit (ERα: 8.45 pM; ERβ: 13.1 pM & MCF-7: 0.112 pM) The rank order in terms of estrogenicity of estradiol, estrone and estriol were similar to that obtained via the stable ERα and ERβ HeLa cell lines where estradiol is the most potent, followed by estriol and estrone

To develop the MCF-7 cell proliferation assay for quantification of estrogenic

activity of ex-vivo samples, we incubated large doses of steroids such as

dihydrotestosterone, progesterone, and cortisol in the presence of estradiol and found that these do not significantly affect the bioassay However, there is a need to include the aromatase inhibitor DL-aminoglutethimide to prevent conversion of testosterone to estradiol by aromatase in MCF-7 cells

Like ERα and ERβ cell-based bioassays, the MCF-7 cell proliferation assay also possesses the ability of being able to distinguish full agonists from partial agonists

Superagonism in cell proliferation was not observed in the range of compounds and concentrations tested The difference in maximal stimulatory levels in ERα and ERβ bioassays; and MCF-7 cell proliferation assay could be a reflection of differences in endogenous versus transfected genes Superagonistic ER activity exhibited by cells that have been stably transfected with a reporter gene had also been observed for genistein and

reservatrol (Legler et al., 1999; Harris et al., 2005; Gehm et al., 2004) One possible

mechanism of this superagonist effect may be related to the function of the ER activation function-1 domain, since its removal prevented enhanced reporter gene activity with

resveratrol (Gehm et al., 2004) Because the ERα activation function-1 domain is

subjected to phosphorylation, superagonist effects could be contributed by the actions of

the AP-1 or the mitogen-activated protein kinase-responsive pathway (Frigo et al., 2002)

4.1.3 Estrogenic activities of Epimedium compounds

The estrogenicities of prenylated flavonoid compounds found in the Traditional

Chinese Medicine herb, Epimedium, namely, icariin, icariside I, icariside II, icaritin and

desmethylicaritin, were examined using ERα and ERβ; and MCF-7 cell proliferation assays in this study

Out of the five Epimedium compounds, icariin was not estrogenic and this is in

agreement with results reported in an earlier study by Liu et al., 2005 Icariside I, icariside

II, icaritin and desmethylicaritin, were found to be estrogenic in all three assays but were less potent compared to estradiol and the well-known soy isoflavone, genistein In terms

of the magnitude of maximal inducible responses, those elicited by genistein, desmethylicaritin and icaritin were similar to that given by estradiol, albeit at varying concentrations, indicating these three phytoestrogens are full estrogen agonists in the MCF-7 cell proliferation assay This contrasts with results obtained from experiments involving stable ERα and ERβ HeLa cell lines where genistein was found to be a superagonist where the maximal luciferase induction of genistein exceeded that achieved

by estradiol In both stable ERα and ERβ HeLa cell lines, desmethylicaritin and icaritin were partial agonists where their maximal luciferase inductions were lower than estadiol Icariside I and icarside II were also both found to be partial estrogen agonists and less potent than icaritin and desmethylicaritin in all three assays

In a paper involving protein–ligand docking simulations published by Wang et al.,

(2006), the prototypical estradiol molecule docked in the ERα ligand-binding domain was found to be characterized by three hydrogen bonds formed between amino acid residues Arg-394, Glu-353, and His-524 with estradiol, which are known to be essential for the molecule’s agonistic activity Using the same approach on the two aglycones, namely,

icaritin and desmethylicaritin, the former compound’s higher estrogenicity could be attributed to the ability of the hydroxyl group on ring B to interact with Glu-353 and Arg-

394 and another similar interaction involving the hydroxyl group on ring A with His-524

In contrast, for the case of icaritin, the interaction with Glu-353 and Arg-394 in the ERα ligand-binding domain was blocked due to the presence of the hydrophobic methyl group

in ring C The lower estrogenicities of icariside I, icariside II and icariin could be explained by the steric hindrance produced by the presence of bulky sugar groups which prevents the required docking to the receptor site and increases the molecules’ hydrophilicity

Prenylation at the 8-position of the flavonoid backbone was observed to lead to an enhancement of estrogenicity Desmethylicaritin is the prenylated version of kaempferol and the estrogenic potency of the desmethylicaritin is ~ 55 times higher than that of kaempferol A similar observation has been reported in studies comparing the estrogenicities of naringenin and its prenylated derivative, 8-prenylnaringenin, which is

found in hops and beer (Milligan et al., 2000; Schaefer et al., 2003; Kretzschmar et al.,

2010)

Icaritin and icariside I were discovered to be ERα-selective ligands in this study This is quite unlike common flavonoids, such as apigenin, kaempferol, luteolin, quercetin and genistein, which are mainly ERβ-selective 8-Prenylnaringenin, the prenylated

version of naringenin, was also found to be ERα-selective (Schaefer et al., 2003)

4.1.4 Estrogenic activities of binary mixtures

The ability of ERα and ERβ stable cell lines and MCF-7 breast cancer cells to detect estrogenic agonistic and antagonistic activities of mixtures was studied ERα and ERβ stable cells; and MCF-7 breast cancer cells were first exposed to binary mixtures containing sub-maximal dose of estradiol and well known ER-antagonists such as 4-hydroxytamoxifen, raloxifene and and ICI 182,780 All three compounds exhibited

antagonistic activity towards estrogen-induced luciferase activity in a dose-dependent

manner as reported in previous studies (Sonneveld et al., 2005; Escande et al., 2006)

MCF-7 breast cancer cells also showed reduced cell proliferation when incubated with estradiol in the presence of increasing doses of 4-hydroxytamoxifen in a dose-dependent manner

Phytoestrogens have been reported in some studies to possess both additive and antagonistic properties First, the effects of the well known soy phytoestrogen, genistein,

on estrogen-induced luciferase transactivation were examined using ERα and ERβ cell lines and were found to exhibit superagonistic activity in the presence of estradiol in a dose-dependent manner In contrast, there was insignificant enhancement of cell proliferative effects when MCF-7 breast cancer cells were exposed to low doses of genistein and estradiol When doses of genistein were increased to 100 nM and greater, inhibitory effects on estrogen-induced cell proliferation were observed In all three assays,

low doses of compounds derived from Epimedium did not show any significant additive

estrogenic activity in the presence of estradiol Above 1 µM in the MCF-7 cell

proliferation assay and 10 µM in ERα and ERβ cell lines, Epimedium compounds were

observed to suppress estrogen-induced bioactivity

Any antagonistic effects against estrogen stimulation exerted by test compounds need to be examined more closely as these may be brought about by non-specific inhibition of reporter gene activity or overall cytotoxicity, or more specific effects such as

inhibition of protein synthesis or mRNA transcription (Sonneveld et al., 2005) Genistein

and other flavonoids have also been demonstrated to influence effects on other signaling pathways, such as tyrosine kinases, mitogen-activated protein kinases, and protein kinase

C inhibition (Akiyama et al., 1987; Kuo & Yang, 1995; Knight & Eden, 1996; Kurzer &

Xu, 1997)

An experiment in the form of an ‘estrogen rescue’ can be used to verify receptor-specific inhibitive effects of the test compound on the reporter gene activation or MCF-7 cell proliferation The ‘estrogen rescue’ experiment is easily performed by increasing the concentration of estradiol so that receptor-specific inhibitive effects can be reversed This approach worked well with a range of androgen receptor antagonists tested

non-in a study non-involvnon-ing of an androgen receptor luciferase reporter assay by Sonneveld et al.,

(2005) where all inhibitory responses were reversed by co-incubation with excess dihydrotestosterone, demonstrating the specificity of the response In contrast, the inhibitory effects of high levels of a number of individual brominated flame retardants congeners could not be reversed by excess dihydrotestosterone, which coincided with cytotoxicity of these ligands, as assessed through inhibition of expression of a constitutively expressed reporter gene and a positive response in the MTT assay (Hamers

4.1.5 Application of in-vitro ER-responsive bioassays as clinical tools to understand

estrogenic effects of compounds in serum

In this study, eight healthy male subjects were enrolled and administered

separately of estradiol valerate and Epimedium pubescens decoction Serum samples were obtained and assayed ex-vivo for levels of estrone and estradiol by tandem mass

spectrometry, for ERα and ERβ bioactivity and MCF-7 breast cancer cell proliferative effects This study provides new validated assays, which measure global activity in serum,

to evaluate the effects of ligands on ERα, compared to ERβ activity and breast cancer cell growth

Regression modelling that was performed indicated that ERα and ERβ bioassays correlated better with summated estrone and estradiol compared to either estrogen alone, demonstrating their ability to reflect the global activity of estrogens In contrast, MCF-7 cell proliferation appeared to be driven mainly by estradiol This is not surprising considering that MCF-7 cells express predominantly ERα

Estradiol is a more potent estrogen than estrone and estrone is quantitatively the predominant estrogen both endogenously and after ingestion of estradiol valerate Estrone

is produced by the rapid metabolism of estradiol in the liver (Vree & Timmer, 1998) However, estrone levels are not measured routinely in clinical practice, and its

contribution to estrogenicity is regarded as negligible (Kuiper et al., 1997) In our study,

coefficient ratios for estrone/estradiol were two-fold higher for ERβ compared to ERα, suggesting that estrone contributed twice as much to ERβ activity in subjects’ sera Estrone can induce steroid receptor coactivator-1 recruitment to ERβ with much higher

efficiency than for ERα (Margeat et al., 2003), and estrone–ERβ complexes can bind coactivator LXXLL fragments with higher affinity than estrone–ERα complex (Ozers et

al., 2005) Our data indicated that consumption of estradiol valerate induced 600% higher incremental mean estrone AUC compared to the 72% increase observed with estradiol The higher concentrations of estrone, plus the distinct affinity of the estrone–ERβ complex for coactivator peptides, may mean that estrone can exert significant estrogenic effects following ingestion of estradiol valerate The associations between high serum

estrone levels with breast cancer risk in postmenopausal women (Miyoshi et al., 2003; Missmer et al., 2004) and with bone health in elderly men (van den Beld et al., 2000)

underline the need for further evaluation of the relative significance of this estrogen to health in aging populations

There was a consistent pattern of diurnal variation of estrogenicity in the subjects, consistent with previous studies that reported night time fall in testosterone and estradiol

levels in pubertal males (Albertsson-Wikland et al., 1997) This intrinsic rhythm with a

clear nocturnal and early morning rise is driven by changes in GnRH pulse generator and

LH secretion from the pituitary gland After adjustment for diurnal variation, the effects

of estradiol valerate on ERα, ERβ, and MCF-7 AUC profiles paralleled that of estrone and estradiol, confirming that these were indeed the main bioactive products causing estrogenicity in sera Ingestion of estradiol valerate resulted in a 67% increase in adjusted AUC over baseline for both ERα and ERβ, comparable to the 72% observed for estradiol The AUC increase for MCF-7 was 23%, reflecting a saturation effect limiting the proliferation of breast cancer cells The data indicated that ERα, ERβ, and MCF-7 cell proliferation bioassays are useful instruments to measure changes in estrogenicity in sera