Lack of Cell Proliferative and Tumorigenic Effects of4‑Hydroxyestradiol in the Anterior Pituitary of Rats: Role ofUltrarapid O‑Methylation Catalyzed by Pituitary Membrane-BoundCatechol‑O‑MethyltransferasePan Wang,†,§ Laura H. Mills,† Ji-Hoon Song,† Jina Yu,† and Bao-Ting Zhu†,‡,*†Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160,United States§State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences,Beijing 100101, China‡Kobilka Institute of Innovative Drug Discovery, The Chinese University of Hong Kong (Shenzhen), Shenzhen, Guangdong 518172,China
*S Supporting Information
ABSTRACT: In animal models, estrogens are complete carcinogens incertain target sites. 4-Hydroxyestradiol (4-OH-E2), an endogenousmetabolite of 17β-estradiol (E2), is known to have prominent estrogenicactivity plus potential genotoxicity and mutagenicity. We report here ourfinding that 4-OH-E2 does not induce pituitary tumors in ACI femalerats, whereas E2 produces 100% pituitary tumor incidence. To probe themechanism, we conducted a short-term animal experiment to comparethe proliferative effect of 4-OH-E2 in several organs. We found that,whereas 4-OH-E2 had little ability to stimulate pituitary cell proliferationin ovariectomized female rats, it strongly stimulates cell proliferation incertain brain regions of these animals. Further, when we used in vitrocultured rat pituitary tumor cells as models, we found that 4-OH-E2 hassimilar efficacy as E2 in stimulating cell proliferation, but its potency isapproximately 3 orders of magnitude lower than that of E2. Moreover, we found that the pituitary tumor cells have the ability toselectively metabolize 4-OH-E2 (but not E2) with ultrahigh efficiency. Additional analysis revealed that the rat pituitary expressesa membrane-bound catechol-O-methyltransferase that has an ultralow Km value (in nM range) for catechol estrogens. On thebasis of these observations, it is concluded that rapid metabolic disposition of 4-OH-E2 through enzymatic O-methylation in ratanterior pituitary cells largely contributes to its apparent lack of cell proliferative and tumorigenic effects in this target site.
■ INTRODUCTION
Estrogens have diverse physiological and pathophysiologicaleffects in humans and animals. The endogenous estrogen 17β-estradiol (E2) is among the most potent nonpeptidal mitogenin many target organs.1 Excessive stimulation of cellproliferation mediated by the estrogen receptors (ERs) hasbeen recognized as an important mechanism for estrogen-induced tumorigenesis in certain target organs (e.g., uterus,breast, and pituitary) in experimental animal models.2
However, estrogen may also exert antiproliferative or apoptoticactions depending on the types of estrogen and its doses usedas well as the cell/tissue types involved.3−5
In humans, catechol estrogens, such as 2- and 4-hydroxyestradiol (2-OH-E2 and 4-OH-E2), are major oxidativemetabolites of E2 formed by cytochrome P450 enzymes.6 Onthe basis of earlier studies, 4-OH-E2 has a relative bindingaffinities for ERα and ERβ ranging from 42−150% of E2 invitro,7−10 and it also has strong uterotropic activity in female
rats and mice.11−14 An earlier study showed that the pituitaryER has a dissociation constant of 0.15 ± 0.06 nM for 4-OH-E2,which is essentially the same as the value 0.13 ± 0.03 nMdetermined for the uterine ER.15 Another study showed thatthe in vivo translocating capacities of the various catecholestrogens (including 4-OH-E2) correlated well with theirbinding affinities for pituitary cytosol receptors determined invitro, indicating that 4-OH-E2 not only binds ER but alsoactivates ER in the pituitary in the same way as E2.
16 In additionto the significant estrogenic activity, catechol estrogens are alsocapable of undergoing redox cycling, which generates reactiveoxygen species that may cause DNA damage, mutagenesis, andultimately tumorigenesis.17,18
Earlier studies showed that 4-OH-E2 is complete carcino-genic in inducing tumorigenesis in the kidneys of male Syrian
hamsters.19,20 In recent years, the facile induction of pituitaryand breast tumors in intact female ACI rats by estrogens suchas E2 has been widely used as an animal model for studying themechanism of estrogen tumorigenesis.21−23 Surprisingly, anearlier study reported that 4-OH-E2 failed to induce mammarytumorigenesis in this animal model, whereas E2 under the sameexperimental conditions was a strong inducer of mammarytumorigenesis.24 The question as to whether 4-OH-E2 caninduce pituitary tumor formation, however, is not known,which is the focus of our present study.Metabolic O-methylation of catechol estrogens catalyzed by
catechol-O-methyltransferase (COMT) has received consid-erable attention in the past 30 years because it abolishes thepotential genotoxicity and mutagenicity of these chemicallyreactive estrogen intermediates and thus is considered to be animportant protective mechanism against estrogen tumori-genesis.25 COMT catalyzes the metabolic O-methylation ofcatechol substrates using S-adenosyl-L-methionine (AdoMet) asmethyl donor.26−28 COMT has low substrate specificity andcan catalyze the O-methylation of many substrates, includingendogenous catecholamines, endogenous catechol estrogens,and various catechol-containing xenobiotics.29,30
COMT is present in most mammals and exists in twodifferent forms, namely, the soluble form (S-COMT) and themembrane-bound form (MB-COMT).31,32 These two formsare encoded by a single gene using two separate promoters P1and P2.33 The rat S-COMT protein is composed of 221 aminoacids with a molecular weight of 24 kDa.34 The rat MB-COMTprotein contains 43 additional amino acid residues at the N-terminus, which contains a hydrophobic signal-anchor peptideanchoring the MB-COMT polypeptide to the endoplasmicreticulum membrane.35 Earlier studies showed that S-COMTand MB-COMT prepared from several tissues showed similaraffinity for the O-methylation of catechol estrogens.36 Similarly,the recombinant human S-COMT and MB-COMT havesimilar Km values for the O-methylation of catechol estrogens,30
although MB-COMT displays a 10-fold higher affinity than S-COMT for the O-methylation of catecholamines.30,37
In the present study, we found that 4-OH-E2 did not inducepituitary cell proliferation and tumorigenesis in female rats.Data are presented to suggest that the apparent lack of cellproliferative and tumorigenic activities of 4-OH-E2 in the ratpituitary may be partly due to its ultrarapid metabolicdisposition in this target site, likely catalyzed by the pituitaryMB-COMT, which has an ultralow Km value for the O-methylation of catechol estrogens.
■ METHODSChemicals and Reagents. E2, 2-OH-E2, and 4-OH-E2 were
purchased from Steraloids (Newport, RI). 4-OH-E2 was purified withhigh-performance liquid chromatography (HPLC) to remove residualE2 contamination prior to use in the experiments. The purities of 4-OH-E2, E2, and 2-OH-E2 used in this study were higher than 99%based on HPLC analysis. Additional analysis of 4-OH-E2 by gaschromatography−mass spectrometry (GC/MS) found no detectableE2 as a contaminant. Dulbecco’s modified Eagle’s medium (DMEM),horse serum, fetal bovine serum, AdoMet, and 1,4-dithiothreitol wereobtained from Sigma-Aldrich (St. Louis, MO). [Methyl-3H]AdoMet(specific activity 11.2−13.5 Ci/mmol) was obtained from PerkinElmer(Waltham, MA). Monoclonal mouse anti-BrdU primary antibody,biotinylated antimouse IgG, Vectastain ABC kit, and DAB kit wereobtained from Vector Laboratories (Burlingame, CA). Anti-COMTantibody was obtained from Millipore (Billerica, MA). Sulfatase fromHelix pomatia was purchased from Sigma-Aldrich (St. Louis, MO). All
solvents used in this study were of HPLC grade or better and obtainedfrom Fisher Scientific (Springfield, NJ).
Animal Experiments. All experimental protocols involving the useof live animals were approved by the Institutional Animal Care andUse Committee of the University of Kansas Medical Center. Theanimal experiments were carried out in accordance with the NationalInstitutes of Health guidelines for humane treatment of animals. Afterarrival, the animals were allowed to acclimatize for a week before beingused in the experiments. The animals were housed under controlledconditions of temperature and photoperiod (12 h light/12 h darkcycle) and had free access to standard laboratory rodent chow andwater throughout the experimental period.
For the estrogen-induced tumorigenesis experiment, 62-day-oldintact female ACI rats were randomly divided into 3 groups (with 25−26 animals per group), and they were implanted s.c. with a small pelletcontaining a known amount of E2, 4-OH-E2, or vehicle alone (ascontrol). Each of the estrogen pellets, in 20 mg total weight, contained18 μmol of E2 (4.9 mg) or 4-OH-E2 (5.2 mg) plus cholesterol orcontained only cholesterol for the control group. The dose ofestrogens used in this study was comparable to the dose used in anearlier study, where a pellet containing 4 mg of E2 was used.
24 Thepelleting components were thoroughly mixed and manually com-pressed into a small cylindrical pellet using a Parr Pellet Press (ParrInstrument Company Moline, IL). These pellets were surgicallyimplanted under the back skin of the animals under halothaneanesthesia.
The animals were weighed weekly and also examined for thepresence of palpable mammary tumors. The decision to terminate ananimal during the course of the long-term tumorigenesis experimentwas made when apparent moribund signs were observed (usually dueto the presence of large pituitary tumors). At the end of theexperiment, the animals were sacrificed with CO2 overdose followedby decapitation. The trunk blood was collected in the Vacutainer testtubes (Fisher Scientific, Springfield, NJ) containing sodium heparin,and the plasma samples were prepared for storage at −80 °C for futureanalysis of hormone levels. At the time of death, the size and locationof the tumors in each animal were recorded, and the weights of thetumor(s) and of the pituitary were also determined. The pituitary wasremoved for histopathological analysis. The removed tissues werestored in 10% buffered neutral formalin overnight followed bydehydration through a sequential transfer through 80−100% ethanoland then 100% xylene. The tissues were embedded in paraffin blocks,cut into 7 μm sections, and placed on Superfrost microscope slides(Fisher Scientific, Springfield, NJ). The sections were stained withhematoxylin and eosin and evaluated under a light microscope by aboard-certified pathologist with expertise in cancer pathology.
For the short-term biological activity study, 6-week-old ovariec-tomized female Sprague−Dawley (SD) rats (from Harlan Sprague−Dawley Laboratory, Houston, TX) were used. Ovariectomized SD ratshave been widely used as the animal model for accessing the estrogenicactivity of chemicals.38,39 The animals were randomly divided intothree groups: control, E2 (20 μg/rat), and 4-OH-E2 (20 μg/rat). Thedose of the estrogens was selected according to earlier reports.40,41 Allsteroids were injected i.m. into the animals once every 2 days for 7days. To quantify the rate of cell proliferation in the pituitary and alsoother target organs, we performed immunohistochemical detection ofBrdU-stained cells as described in our earlier study.42 In brief, 2 hbefore sacrifice, the animals were injected i.p. with BrdU at 50 μg/gbody weight (dissolved in sterile saline). The pituitary and brain wereremoved, fixed, dehydrated, embedded in paraffin blocks, and cut into5 μm sections. The sections were rehydrated and blocked using 1%normal horse serum. The sections were incubated with anti-BrdUprimary antibody (Novocastra, Newcastle, UK) for 60 min followed bya biotinylated horse antimouse secondary antibody (Vector Labo-ratories, Burlingame, CA) for 45 min at room temperature. Detectionwas performed using ABC Elite reagent (Vector Laboratories), andcolor was developed using diaminobenzidine (Sigma Chemical Co.)for 5 min. Hematoxylin was used as a counterstain. The number ofcells that incorporated BrdU were determined using a lightmicroscope.
Radioimmunoassay (RIA) of the Plasma Levels of Prolactin(PRL), Luteinizing Hormone (LH), and Follicle StimulatingHormone (FSH). To measure the plasma levels of E2, PRL, LH, andFSH, whole blood samples were collected from each animal afterdecapitation and placed in Vacutainer test tubes containing heparinsodium (Fisher Scientific, Springfield, NJ). The collected whole bloodsamples were centrifuged for 20 min at 1000g. The plasma sampleswere transferred precisely to another set of small vials with sealed capsand stored at −80 °C. The plasma levels of rat LH, FSH, and PRL(ng/mL) were determined by highly sensitive, specific, andquantitative RIAs using the RIA reagent sets prepared and distributedby the National Hormone and Peptide Program of the NationalInstitute of Diabetes and Digestive and Kidney Diseases.Tumor Cell Culture. Rat pituitary tumor cell lines GH3 and RC-
4B/C and human breast cancer cell line MCF-7 were obtained fromthe American Type Culture Collection (Manassas, VA). The culturemedium for GH3 is DMEM supplemented with 12.5% horse serumand 2.5% fetal bovine serum (FBS). The culture medium for RC-4B/Ccells is DMEM supplemented with 10% FBS, 0.2 mg/mL of bovineserum albumin, and 2.5 ng/mL of epidermal growth factor. MCF-7cells are maintained in EMEM containing 10% FBS supplementedwith 100 units/ml of penicillin, 100 μg/mL of streptomycin, 2 μg/mLof insulin, 0.5 mM sodium pyruvate, 10 mM nonessential amino acids,and 2 mM L-glutamine. To study the proliferative effect of E2 and 4-OH-E2 in GH3, RC-4B/C, and MCF-7 cells, we used phenol red-freemedium supplemented with dextran-coated charcoal-stripped FBS andhorse serum. The charcoal-stripping procedure was carried out asdescribed earlier,43 which was employed to remove the endogenousestrogens present in the serum.The cells were first propagated in the 75 cm2
flasks to approximately80% confluence under 37 °C air with 5% CO2 and 95% humidity.They were then detached from the flask by treatment with 3 mL of thetrypsin-EDTA solution for 5 min. Cell suspensions were centrifuged.and the cell sediments were resuspended in the culture medium at thedesired 105 cells/mL density. A 100 μL aliquot of the cell suspensionwas then added to each well of the 96-well microplate usually at a finaldensity of 104 cells per well. After the cells were allowed to attach andgrow for 24 h, the cell culture medium was changed, and different drugtreatments were introduced at that time. In most experiments, thedrug treatment lasted for 6 days with one medium change on thefourth day following the initial drug treatment.The cell viability in the 96-well microplates was determined using
the MTT assay. Briefly, 22 μL of 5 mg/mL MTT was added to eachwell of the 96-well microplate, and the plate was incubated at 37 °C for1 h. The medium was removed at the end of the incubation, and 100μL of DMSO was added to dissolve the formazan crystals. Theabsorbance value of each well was measured at 560 nm with a UVmaxmicroplate reader (Molecular Device, Palo Alto, CA).Analysis of Estrogen Levels in Cell Culture Medium. GH3
cells were seeded in 6-well plates in regular culture medium, and 100nM of 4-OH-E2 or E2 in DMEM (without serum) was introduced. Themedium was collected after 60 min of incubation. Aliquots (1 mL) ofthe culture medium were transferred to a 1.5 mL microcentrifuge tubecontaining 200 μL of Na2CO3 buffer (2 M, pH 5.0). The mixture wascentrifuged at 10, 000g for 5 min, and 1 mL of supernatant wastransferred to a small glass tube. Ten microliters of ascorbic acid (0.15mg/μL) was added as antioxidant; 20 μL of 0.5 ng/μL deuterated 17β-estradiol-d5 (E2-d5) (in 200 proof ethanol) was added as internalstandard, and 10 μL of H-2 sulfatase (2,000 units/mL) was added asthe enzyme for hydrolysis of sulfated estrogens. The reaction mixturewas incubated at 37 °C for 4 h. After incubation, the tubes werecentrifuged at 1500g for 10 min, and the supernatant was transferredto another set of test tubes and extracted with 5 mL of ethyl acetate.The organic extracts (4.5 mL) were precisely transferred and driedunder a stream of nitrogen gas. For derivatization, bis(trimethylsilyl)-trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS)(50 μL) was added to the dried sample in a small glass vial, and themixture was incubated at 65 °C for 0.5 h.The GC/MS was carried out as described earlier.44,45 Briefly, the
GC/MS apparatus consisted of an Agilent 6890N gas chromatogra-
pher with a 7683 autosampler and the HP-5MS capillary column (30m × 0.25 mm × 0.25 μm) coupled with an Agilent-5973 massspectrometer. Helium was used as the carrier gas. The injector anddetector temperatures were 260 and 280 °C, respectively. Duringanalysis, the column temperature was increased from 180−260 °C at arate of 4 °C/min and then maintained isothermally at 260 °C for 5min. Then, the temperature was increased to 300 °C at a rate of 5 °C/min and then maintained isothermally at 300 °C for 5 min. The massspectrometer was operated in the electron impact mode (70 eV). Massabundance was determined by the SIM mode. The E2 and 4-OH-E2were quantified according to the area of the peak and normalized withinternal standard E2-d5.
Preparation of GH3 Cell Lysates and Rat Pituitary and LiverHomogenates. GH3 cells were propagated in 75 cm2
flasks toapproximately 80% confluence. They were then detached from theflasks by treatment with 3 mL of a trypsin-EDTA solution for 5 min.The cell pellets were washed in PBS once and then kept at −80 °Cuntil they were used for preparation of cellular lysates. The pituitaryglands from female Sprague−Dawley rats were purchased from Sierrafor Medical Science (Whittier, CA).
Cell pellets, rat pituitaries, and livers were defrosted on ice andimmersed in 50 mM Tris-HCl buffer (pH 7.5) containing a proteaseinhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Cell pellets werelysed with ultrasound sonication on ice for 5 min. Rat pituitary andliver tissues were homogenized on ice using a Wheaton homogenizer(Millville, NJ) for 10 min. The whole homogenates were thencentrifuged at 4 °C at 10,000g for 15 min to remove intact cells andnuclei. The supernatant was used as the cell lysates or crude tissuehomogenates in the COMT-mediated methylation assays.
In some of the experiments, the microsomal and cytosolic fractionswere further isolated from the cell lysates or crude tissue homogenatesas described earlier.46 The GH3 cell lysates or crude tissuehomogenates were subjected to centrifugation at 4 °C at 100,000gfor 2 h, and the supernatant fraction (cytosolic fraction) was used asthe source of S-COMT. The pellet (microsomal fraction) wassuspended in Tris-HCl buffer (pH 7.5) containing a protease inhibitorcocktail and it was used as the source of MB-COMT.
Fragmentation of GH3 Cell Lysates with Gel Filtration. TheGH3 whole cell lysates containing 100 mg of total protein wereapplied on a column (20 cm × 3 cm) packed with Bio-Rad P60 gel.Elution was performed on an FPLC with 50 mM Tris-HCl as themobile phase and a UV detector for protein monitoring. The fractionwas collected at a flow rate of 0.1 mL/min and a volume of 1 mL/tube.The whole procedure was carried out at 4 °C.
Assay of the COMT-Mediated O-Methylation of CatecholSubstrates. The COMT-mediated O-methylation of catechol estro-gens (4-OH-E2 or 2-OH-E2) was carried out as described earlier.30
The reaction mixture consisted of 0.5 mg/mL of total protein, differentconcentrations of 4-OH-E2 or 2-OH-E2 as substrate, 250 μM AdoMet(containing 0.5 mCi [methyl-3H]AdoMet), 1.2 mM MgCl2, and 1 mMdithiothreitol in a final volume of 200 μL of 50 mM Tris-HCl buffer(pH 7.5). The reaction was initiated by adding the enzyme preparationand was carried out at 37 °C for 30 min. After incubation, the reactionwas arrested by immediately placing the tubes on ice and followed byaddition of 500 μL of ice-cold saline and then extracted with 2 mL ofn-heptane. After a brief centrifugation at 1000g, 1 mL of the organicextracts was measured for radioactivity content in a liquid scintillationcounter. The enzyme kinetic parameter Km values for the O-methylation of catechol estrogens were calculated using thecommercial enzyme kinetics curve regression program of theSigmaPlot software (version 9; Systat Software, Inc., San Jose, CA),which is based on the Michaelis−Menten equation and its curvepattern.
Western Blotting Analysis for COMT. Cytosolic and microsomalfractions that were prepared from the whole homogenates of GH3cells and rat pituitary tissues were boiled in the SDS sample buffer(containing 2% SDS, 10% glycerol, 1.2% 2-mercaptoethanol, and0.02% bromophenol blue in 50 mM Tris-HCl, pH 6.8) for 5 min at100 °C. Equal amounts of total proteins (approximately 30 μg/lane)were electrophoresed in 12% polyacrylamide gel and then transferred
to PVDF membranes. The membranes were treated with 5% nonfatmilk for 1 h to block nonspecific binding and then rinsed andincubated with the COMT antibody (at 1:5000 dilution). Themembranes were then treated with horseradish peroxidase-conjugatedantirabbit IgG (at 1:5000 dilution) for 1 h. The protein bands weredetected on X-ray films with the aid of a chemiluminescence substrate.Statistical Analysis. The quantitative data are expressed as means
± SEM. The Student’s t-tests were used to compare paired data andthe one-way analysis of variance followed by Dunnett’s tests was usedfor multiple comparisons. For the BrdU staining data, the Chi-squaretest was used. P < 0.05 was considered statistically significant.
■ RESULTS
4-OH-E2 Fails to Induce Pituitary Tumorigenesis inFemale ACI Rats. First, we compared the tumorigenic activityof 4-OH-E2 and E2 in the pituitary of intact female ACI rats. Allanimals implanted with an E2 pellet developed a large pituitarytumor with an average weight of 254 ± 19 mg (Figure 1A). Allanimals in this group became severely moribund at 5−6.5months after E2 implantation, most likely due to the presenceof a large pituitary tumor, and they had to be euthanized. Incomparison, all the control animals (implanted with a vehiclepellet) and those implanted with a 4-OH-E2 pellet remainedhealthy during the entire course of the experiment. Macro-
scopic examination of the pituitary revealed that not a singleanimal in these two groups developed a pituitary tumor. Theaverage pituitary weights in 4-OH-E2-treated animals were 17.3± 1.3 mg, which were slightly increased over the control group(11.7 ± 0.8 mg) (P < 0.05) (Figure 1A).The plasma levels of prolactin (PRL) in control animals and
in animals chronically treated with 4-OH-E2 were 70.0 ± 20.6and 71.1 ± 11.0 ng/mL, respectively, whereas the plasma levelsof prolactin in E2-treated animals were 6050 ± 467 ng/mL(Figure 1B). Histopathological examination showed thepresence of adenomas (prolactinoma) with focal hemorrhage,cystic change, and apoplectic necrosis in E2-treated animals, butno appreciable histological abnormalities were observed in thepituitary for 4-OH-E2-treated animals (data not shown). Thedrastic change in plasma prolactin levels is consistent with thehistological observation of the presence of prolactinoma in theanterior pituitary. Moreover, chronic E2 administrationmarkedly reduced the plasma levels of FSH and LH (Figure1B), and this effect is known to be an estrogen receptor-mediated action (commonly referred to as estrogen’s feedbackregulation) on the anterior pituitary cells that secrete FSH andLH.47 In comparison, 4-OH-E2 had a detectable but weakereffect than E2 in regulating FSH and LH release (Figure 1B),
Figure 1. Effect of chronic administration of E2 or 4-OH-E2 on pituitary tumor induction in female ACI rats. (A) Weights of rat pituitary glandsfollowing chronic treatment with E2 or 4-OH-E2. The y-axis values represent the mean ± SEM of the pituitary weights of the animals, and the x-axisvalues represent the mean ± SD of the treatment days for all animals. The filled circle, filled triangle, and empty circle indicate the treatment groupsthat received vehicle only (control), E2, or 4-OH-E2, respectively. (B) Plasma levels of PRL, FSH, and LH at the end of the long-term experiment,which were analyzed at the National Hormone and Pituitary Program using the 125I-labeled RIAs. Each value is the mean ± SEM (N = 25). *P <0.05 compared to the control, and #P < 0.05 compared to the E2 group.
which is consistent with the lower estrogenic activity of 4-OH-E2 in vivo.Chronic administration of E2 is known to decrease an
animal’s body weight gain in a dose-dependent manner, andthis effect was reported to be associated with the activity ofERα.48 Although 4-OH-E2 had no tumorigenic activity in thepituitary, it surprisingly produced a slightly stronger reductionin body weight during the first 150 days of estrogen treatment(Figure 2). We observed in this study that 4-OH-E2 has a
stronger effect than E2 for reducing the daily food intake by theanimals (data not shown). The body weight reduction in E2-treated animals accelerated after the first 150 days following E2treatment, likely due to the rapid development of pituitarytumors.4-OH-E2 Fails to Stimulate Pituitary Cell Proliferation.
To determine whether 4-OH-E2 can stimulate cell proliferationin the pituitary or other estrogen target organs or tissues, weused the BrdU-labeling method and compared the proliferativeeffect of E2 and 4-OH-E2 in several target tissues of femaleovariectomized rats. Previous studies have reported that E2 canstrongly stimulate cell proliferation in both the pituitary andsubventricular zone (SVZ) of the brain.1,49−51 Administrationof 4-OH-E2 for 7 days did not show a significant stimulation ofanterior pituitary cell proliferation (Figure 3A, Figure S1), but itstrongly stimulated cell proliferation in the SVZ of the brain. 4-OH-E2 also increased uterine wet weight gain (Figure 3A). Theeffect of 4-OH-E2 in SVZ cell proliferation and uterus wetweight gain was slightly weaker than those of E2, which is in linewith their known ERα-binding affinities.7 On the basis of thesein vivo observations, it is apparent that the lack of proliferativeeffect of 4-OH-E2 in the rat pituitary is an organ-selectivephenomenon.4-OH-E2 has High Efficacy but Low Potency in
Stimulating Rat Pituitary Tumor Cell Proliferation inCulture. To probe whether the selective inability of 4-OH-E2
to stimulate rat pituitary cell proliferation is due to an intrinsiclack of sensitivity of pituitary cells to this estrogen metabolite ordue to the low bioavailability of 4-OH-E2 inside these cells, weconducted in vitro experiments to compare the proliferativeeffect of 4-OH-E2 and E2 using two ER-positive pituitary tumorcell lines (GH3 and RC-4B/C) as models. Our basicassumption is that, if the pituitary cells indeed cannot respondto 4-OH-E2’s proliferative action, then we should not be able tosee significant stimulation of cell proliferation in these cellseven when very high concentrations of 4-OH-E2 are present inthe culture medium. However, if it is simply due to the lowbioavailability of 4-OH-E2 inside the pituitary cells, then weshould still be able to see significant cell proliferation whensufficiently high concentrations of 4-OH-E2 are present in theculture medium.As shown in Figure 3B, E2 strongly stimulated the
proliferation of GH3 and RC-4B/C cells in vitro with EC50values of 0.02 and 0.11 nM, respectively. The EC50 values of E2in these two cell lines are consistent with the knownmechanism of ERα-associated stimulation of cell proliferation.In comparison, 4-OH-E2 did not have an appreciablestimulatory effect on cell proliferation in these cells until itsconcentration reached >10 nM. As such, the potency of 4-OH-E2’s proliferative effect in GH3 and RC-4B/C cells isapproximately 3 orders of magnitude lower than that of E2.Notably, despite the very low potency of 4-OH-E2, its dose−response curve patterns in stimulating the proliferation of GH3and RC-4B/C cells closely resemble those of E2, which suggeststhat the proliferative effects of both estrogens are mediated bythe same ERs and that 4-OH-E2 has a similar efficacy to that ofE2. Together, these results suggest that the very lowbioavailability of 4-OH-E2 in rat pituitary cells is the majorunderlying cause for its apparent lack of a cell proliferativeeffect in vivo.
Rat Pituitary Cells Metabolize 4-OH-E2 Much Fasterthan E2. To provide experimental evidence for the suggestionthat the apparent lack of a cell proliferative effect by 4-OH-E2 inthe rat pituitary is due to its rapid local metabolic disposition,we used cultured GH3 tumor cells as an in vitro model andexamined the rate of metabolic disposition of E2 and 4-OH-E2.We found that the disappearance of 4-OH-E2 in the GH3 cellculture medium was far faster than the disappearance of E2under the same cell culture conditions (Figure 4). These datasuggest that the rapid metabolism of 4-OH-E2 may beresponsible for the abrogation of the proliferative andtumorigenic activities of this highly estrogenic E2 metabolitein rat pituitary cells.
Rat Pituitary COMT Displays Two-Component Ki-netics for the O-Methylation of Catechol Estrogens. Itis known that the COMT-mediated O-methylation is a selectivepathway for the metabolic inactivation of catechol estrogens butnot for the inactivation of E2.
28 Therefore, we hypothesized thatthe pituitary may be able to very rapidly methylate catecholestrogens. To test this hypothesis, we first sought tocharacterize the enzyme kinetics for the O-methylation of 4-OH-E2 when the crude lysates prepared from GH3 cells wereused as the enzyme source (Figure 5A). We found that theenzyme kinetics had two components as revealed by theEadie−Hofstee plot: one with a very low Km at 51 nM and theother with a high Km at 7.7 μM. Similarly, the COMT presentin rat pituitary homogenates also showed similar two-component kinetics with Km values of 40 nM and 6.7 μM,respectively, for the O-methylation of 4-OH-E2 (Figure 5B).
Figure 2. Body weight changes after chronic administration of E2 or 4-OH-E2 in female ACI rats. Each value is the mean ± SEM (N = 7).
Figure 3. Effect of E2 or 4-OH-E2 on pituitary cell proliferation in vitro and in vivo. (A) Effect of E2 and 4-OH-E2 on the uterine wet weight (left)and cell proliferation in the anterior pituitary (middle) and subventricular zone (SVZ) (right) in ovariectomized female rats. Each value is the mean± SEM (N = 7). (B) Effect of E2 and 4-OH-E2 on the proliferation of GH3 and RC-4B/C cells in culture. Cells were treated with differentconcentrations of E2 or 4-OH-E2 for 6 days with one medium change on the third day. Each value is the mean ± SEM (N = 6). *P < 0.05 comparedto the control, and #P < 0.05 compared to the E2 group (Chi-square test).
Figure 4.Metabolism of 4-OH-E2 and E2 in GH3 cell culture medium. Cells were treated with 100 nM 4-OH-E2 or E2 for 60 min and, then 1 mL ofcell culture medium was retrieved and processed for GC/MS analysis. The quantity (in ng) of each estrogen was normalized to the internal standardE2-d5. The control value was derived from the respective cell culture medium containing 100 nM of 4-OH-E2 or E2 right before it was added to thecell culture. Each value is the mean ± SEM (N = 4).
For comparison, we also determined the COMT in rat liverhomogenates and found that the liver COMT had single-component kinetics with an apparent Km value of 10.3 μM(Figure 5C).Similarly, when 2-OH-E2 was used as substrate, the GH3 cell
lysates and the rat pituitary tissue homogenates also displayedtwo-component kinetics with low Km values of 135 and 68 nM,respectively, and high Km values of 7.1 and 4.2 μM, respectively(Figure 6A and B). In comparison, the rat liver crudehomogenates showed similar single-component kinetics withan apparent Km value of 13.3 μM (Figure 6C).MB-COMT Shows Ultrahigh Activity for Catechol
Estrogens. COMT exists in two different forms, namely, S-COMT and MB-COMT.31,32 To determine which protein wasresponsible for the low and high Km COMT activity, we used agel filtration column (Bio-Rad P60 gel) to separate the wholecellular protein preparation from GH3 cells according to theprotein size. Then, we assayed the COMT activity of theseparated fractions (data shown in Figure 7A). It is apparent
that there are two different proteins (labeled COMT I andCOMT II) that contain the catalytic activity for O-methylation.As revealed by Western blotting analysis (Figure 7B), theCOMT I fractions contained the MB-COMT, whereas theCOMT II fractions contained S-COMT. The MB-COMT waseluted much earlier through the gel filtration column, indicatingthat its gross molecular weight is much larger than that of S-COMT, probably due to its association with the membranelipids.Next, we separately analyzed the enzyme kinetics of the
COMT I and COMT II fractions using 4-OH-E2 as substrate.As shown in Figure 7C, COMT I displayed the single-component kinetics with a low Km value of 76 nM for 4-OH-E2,whereas COMT II displayed the single-component kineticswith a high Km value of 15 μM for 4-OH-E2.To further characterize the kinetics of MB-COMT and S-
COMT, we separated the microsomal fraction (containingmostly MB-COMT) from the cytosolic fraction (containingmostly S-COMT) using the standard high-speed centrifugation
Figure 5. GH3 pituitary tumor cells and rat pituitary tissue display two-component kinetics for the O-methylation of 4-OH-E2. Enzyme kinetics forthe O-methylation of 4-OH-E2 when (A) GH3 cell, (B) rat pituitary, or (C) rat liver crude homogenate was used as the enzyme source. The rightpanels are the Eadie−Hofstee plots that are converted from the data in the corresponding left panels. Each value is the mean ± SEM (N = 4).
for both GH3 cells and rat pituitary tissues. As shown byWestern blotting analysis (Figure 8A), MB-COMT wasconcentrated in the microsomal fraction, whereas S-COMTwas concentrated in the cytosolic fraction prepared from bothGH3 cells and the rat pituitary tissue. In addition, GH3 cellsand rat pituitary tissues contained roughly equal levels of MB-COMT and S-COMT (Figure 8A). Next, we separatelydetermined the kinetics of MB-COMT and S-COMT using4-OH-E2 and 2-OH-E2 as substrates (Figures 8B and C and 9Aand B). The Km values are summarized in Table 1. Both MB-COMT and S-COMT displayed single-component kineticswhen 4-OH-E2 or 2-OH-E2 was used as substrate. MB-COMThad a much lower Km value compared to that of S-COMT forboth 4-OH-E2 and 2-OH-E2. MB-COMT had no preference for4-OH-E2 over 2-OH-E2, but S-COMT displayed a slightdifference (approximately 1-fold higher preference for 2-OH-E2
over 4-OH-E2).
■ DISCUSSION
It has been known for many years that estrogen can inducepituitary cell proliferation and tumorigenesis in some of the ratstrains in both male and female rats.1,2,52,53 Pathologicalanalysis revealed that the pituitary tumors induced by chronictreatment with an estrogen are usually of the prolactinomatype,54 which is also confirmed in the present study. 4-OH-E2 isan endogenous estrogen metabolite with strong estrogenicactivity and high binding affinity for the ERs.6,7 Moreover, thisestrogen metabolite is chemically reactive, capable of causinggenotoxicity and mutagenicity.17,18,20 Earlier studies haveshown that 4-OH-E2, like E2, could induce tumorigenesis inmale Syrian hamsters with a high tumor incidence.19 Therefore,it is rather unexpected that this highly tumorigenic estrogenderivative fails to induce tumorigenesis in the pituitary offemale ACI rats, whereas E2 serves as a strong tumor inducer.In line with the observation of a lack of tumorigenic activity, we
Figure 6. GH3 pituitary tumor cells and rat pituitary tissue display two-component kinetics for the O-methylation of 2-OH-E2. Enzyme kinetics forthe O-methylation of 2-OH-E2 when (A) GH3 cell, (B) rat pituitary, or (C) rat liver crude homogenates were used as the enzyme source. The rightpanels are the Eadie−Hofstee plots that are converted from the data in the corresponding left panels. Each value is the mean ± SEM (N = 4).
also found that 4-OH-E2 fails to stimulate cell proliferation inthe pituitary of female ACI rats. It is apparent that this lack ofproliferative effect in rat pituitary is organ-selective, because 4-OH-E2 is able to induce cell proliferation in the SVZ brainregion in female rats.Because it is known that rat pituitary expresses ERα and that
both E2 and 4-OH-E2 have high binding affinities for ERα,7,55 it
is puzzling that even though E2 is a strong mitogen and a strongtumor inducer in rat pituitary, 4-OH-E2 fails to induce cell
proliferation and tumorigenesis in this organ. A more plausibleexplanation for this discrepancy is because 4-OH-E2 may havevery low bioavailability in pituitary cells. Another less likelypossibility may be that there is a unique modification inpituitary ERs that renders the cells selectively insensitive to theproliferative action of 4-OH-E2. To experimentally probe thesetwo possibilities, we designed an in vitro experiment tocompare the proliferative effect of 4-OH-E2 and E2 in two ER-positive pituitary prolactinoma cell lines (GH3 and RC-4B/C).
Figure 7. Separation of COMT proteins in GH3 cell lysates for analysis of the catalytic activity. (A) COMT activity for each fraction eluted from thegel filtration column. The estimated protein size of each fraction was labeled under the elution volume. (B) Western blotting analysis of the COMTproteins contained in each fraction eluted from the gel filtration column. “T” represents the cell lysate. (C) Enzyme kinetics for O-methylation with4-OH-E2 catalyzed by the COMT I and COMT II fractions.
Our basic premise is that, if the pituitary cells indeed cannotrespond to 4-OH-E2’s proliferative action due to the presenceof a unique modification in the ERs, then we should not be ableto see significant stimulation of proliferation in these cells evenwhen very high concentrations of 4-OH-E2 are present.However, if it is simply due to the low bioavailability of 4-OH-E2 in pituitary cells in vivo, then we should still be able tosee a full-range of growth stimulation when higher concen-trations of 4-OH-E2 are used under controlled in vitroconditions.Our results showed that E2 can strongly stimulate the
proliferation of GH3 and RC-4B/C cells in vitro with EC50
values at 0.02 and 0.11 nM, respectively. The effects of E2 inthese cells are as expected, and the EC50 values are consistent
with the known mechanism of ERα-mediated stimulation ofcell proliferation.43 In comparison, 4-OH-E2 does not have anappreciable growth-stimulatory effect in these cells until itsconcentrations reaches >10 nM. Notably, the overall dose−response curve patterns of 4-OH-E2’s proliferative effect in bothGH3 and RC-4B/C cells are very similar to those of E2,although the estimated potency of 4-OH-E2 is approximately 3orders of magnitude lower than that of E2. Considering the factthat the difference in the ER-binding affinity of E2 and 4-OH-E2
are very small in vitro,7 these results suggest that the very lowbioavailability of 4-OH-E2 in rat pituitary cells likely is the mainunderlying cause for its apparent lack of proliferative effect invivo. Providing experimental support for this suggestion, wefound that approximately 99% of 4-OH-E2 disappeared from
Figure 8. Enzyme kinetics for the O-methylation of 4-OH-E2 catalyzed by microsomal and cytosolic fractions prepared from GH3 cells and ratpituitary tissues. (A) Western blotting analysis of the COMT proteins in the microsomal (M) and cytosolic (C) fractions prepared from GH3 cellsand rat pituitary tissues. (B and C) Enzyme kinetics for the O-methylation of 4-OH-E2 catalyzed by the microsomal and cytosolic fractions preparedfrom GH3 cells (B) and rat pituitary tissues (C). The fractionation of cell lysates and crude tissue homogenates was carried out using the differentialcentrifugation method as described in the Methods section.
the culture medium of rat pituitary tumor cells within the firsthour of addition, whereas ∼90% of E2 remained in the culturemedium. However, it is also possible that an estrogen may exertsome of its pituitary effects in vivo via its initial action in thehypothalamus, and this possibility cannot be ruled out.Considering the fact that the metabolic machinery present in
pituitary cells can only rapidly dispose of 4-OH-E2 but not E2, itnaturally prompted us to probe the COMT-mediated O-methylation in pituitary cells because COMT can only catalyzethe metabolic disposition of catechol estrogens (including 4-
OH-E2), but not E2. We found, for the first time, that ratpituitary MB-COMT displays an ultrahigh affinity for the O-methylation of catechol estrogens with Km values around 40−51 nM, whereas the pituitary S-COMT has an affinityapproximately 150-times lower with a Km of approximately 7μM.Notably, there were several earlier studies that had also
determined the Km values for the COMT-mediated O-methylation of catechol estrogens in selected tissues(summarized in Table 2).30,36,56,57 They all reported quite
Figure 9. Enzyme kinetics for the O-methylation of 2-OH-E2 catalyzed by microsomal (M) and cytosolic (C) fractions prepared from (A) GH3 cellsand (B) rat pituitary tissues. The fractionation of the cell lysates and crude tissue homogenates was carried out using the differential centrifugationmethod as described in the Methods section.
high Km values, which range from 0.77 to 10 μM. Although anearlier study reported relatively low Km values for both MB-COMT and S-COMT in rabbit aorta for 2-OH-E2 (0.15 and0.27 μM, respectively), no drastic differences were noted for theKm values of these two COMTs.36 Therefore, the results of ourpresent study are the very first report of an ultralow Km valuefor pituitary MB-COMT with catechol estrogens as substrates.In fact, to our knowledge, this is the lowest Km value everreported for COMT-mediated O-methylation of all knownsubstrates.Most of the known metabolizing enzymes have rather low
specificity with relatively high Km values.27,58 It is very rare tohave a general-purpose metabolizing enzyme with a substrate’sKm in the nM range. Usually, only highly specialized enzymessuch as those involved in the biosynthesis of endogenoushormones (such as steroid-synthesizing cytochrome P450enzymes) have very low Km values, i.e., very high affinity forthe substrates.6 The possible mechanisms underlying theultrahigh activity of pituitary MB-COMT may include uniqueprotein structures and/or post-translational protein modifica-tions of MB-COMT in the pituitary gland. However, in breastcancer cells, the single-component kinetics indicates that MB-and S-COMT had the same Km values (Figure S2).Nevertheless, the low Km value of COMT in breast cancercells may also lead to rapid metabolic inactivation of 4-OH-E2,resulting in drastically reduced cell proliferative activity inbreast tissues compared to that of E2 (Figure S3).
One of the biological functions of the ultralow Km pituitaryCOMT may serve as a built-in mechanism to effectively protectthis organ against highly tumorigenic catechol estrogens. It isalso possible that the ultralow Km pituitary COMT may rapidlymetabolize dopamine, which is an important neurotransmitterin the anterior pituitary and can inhibit prolactin release andlactotroph proliferation.59 On the other hand, it should benoted that the rat pituitary expresses high levels of catecholestrogen-forming enzymes,60 which can convert endogenousestrogens to catechol estrogens. Moreover, the pituitary alsohas the unique ability to rapidly convert catechol estrogens tomethoxyestrogens as shown in this study. It is possible thatmethoxyestrogens may be endogenously formed estrogenderivatives with some unique biological functions in certaintarget sites (such as the pituitary). In line with this speculation,it is of note that methoxyestrogens were reported to have aunique ability to activate the nongenomic pathways in certainsystems61,62 despite the fact that they are devoid of significantbinding affinity for the ERs.7
It is worth noting that an earlier report also showed that 4-OH-E2 failed to induce mammary tumor formation in femaleACI rats, whereas E2 under the same experimental conditionsstrongly induced mammary tumorigenesis.24 The lack of amammary tumorigenic effect of 4-OH-E2 is likely due to acombination of two contributing factors. One is the lack ofelevated prolactin release in animals treated with 4-OH-E2(Figure 1); epidemiological studies revealed that circulatingPRL is an important risk factor for breast cancer.63,64 In rodentanimal models, overexpression of prolactin induces breastcancer development.65 Another important factor (data shownin Figure S2) is that 4-OH-E2 is also very rapidly O-methylatedby COMT in mammary glandular cells, which abrogates itsestrogenicity and cell proliferative activity. We found that theCOMT proteins contained in the crude homogenates fromMCF-7 human breast cancer cells have high affinity for the O-methylation of 4-OH-E2 with an apparent Km of 310 nM withsingle-component kinetics (Figure S2). Moreover, the EC50 of4-OH-E2 for stimulating MCF-7 cell proliferation is approx-imately 1 nM, which is 50-fold higher than the EC50 of E2 inthese cells (approximately 0.02 nM) (Figure S3), whereas theERα-binding affinity of 4-OH-E2 is approximately 70% of E2.
7
Together, these data likely suggest that the rapid COMT-mediated O-methylation of 4-OH-E2 may also be a contributingfactor for its apparent lack of tumorigenicity in the breast.The carcinogenesis of estrogens mainly involves two
mechanisms. First, excessive stimulation of cell proliferationmediated by the estrogen receptors (ERs) has been recognizedas an important mechanism for estrogen-induced tumori-genesis.66 Second, numerous studies have shown that thecytochrome P450-catalyzed formation of chemically reactivemetabolites of endogenous estrogens, i.e., catechol estrogens, iscapable of undergoing redox cycling between o-quinones andsemiquinone radicals.2,17,18,67 This redox cycling leads to theformation of reactive oxygen species (ROS) including super-oxide, hydrogen peroxide, and ultimately the hydroxylradical.2,68,69 These free radicals cause oxidation of thenucleotide residues of DNA and oxidative cleavage of theDNA backbone, which results in the formation of DNAadducts, DNA strand breaks, and generated DNA mutationsthat can lead to cancer initiation.67,70−75 Thus, the damage ofDNA by estrogens is mostly dependent on the conversion ofestrogens to catechol estrogens, whereas the cell proliferativeeffect is not. Our results suggest that, in the pituitary where 4-
Table 1. Km Values for the MB- and S-COMTs Present in theGH3 Cells, Rat Pituitary Tissue, and Rat Liver Tissue When2-OH-E2 and 4-OH-E2 Were Used as Substrates
OH-E2 fails to induce tumor formation due to its rapid O-methylation, E2’s carcinogenicity is probably not dependent onits conversion to 4-OH-E2. This indicates that the cellproliferative effect of estrogen may play a relatively moredominant role in estrogen-induced tumorigenesis in the ratpituitary. Somewhat in line with this suggestion, it is of notehere that estrogen-induced tumorigenesis in the rat pituitary isknown to be mostly benign in nature (partly due to the lack ofhigh estrogen-induced mutagenicity), whereas estrogen-in-duced mammary tumors in rats are mostly malignant in nature,which is consistent with the presence of mutagenic catecholestrogens and the accumulation of oncogenic DNA mutations.
■ CONCLUSIONSThe results of this study show that, unlike E2, 4-OH-E2 doesnot stimulate cell proliferation and tumorigenesis in thepituitary of ACI female rats. However, using cultured ratpituitary tumor cells as in vitro models, we found that 4-OH-E2can still stimulate cell proliferation just like E2, but its potency isapproximately 3 orders of magnitude lower than that of E2.Moreover, we find that the rat pituitary tumor cells in culturehave a unique ability to selectively metabolize 4-OH-E2 (butnot E2) with exceptionally high efficiency. Additional studiesshow that the rat pituitary MB-COMT has an ultralow Km valuefor the O-methylation of catechol estrogens with Km values of40−51 nM. This finding may suggest that the rat pituitary hasan important defense mechanism for protection against 4-OH-E2-induced tumorigenesis. On the basis of these findings, it issuggested that the apparent lack of proliferative andtumorigenic activity of 4-OH-E2 in the pituitary is due to theultrarapid metabolic inactivation of this endogenous estrogenmetabolite. The results of this study also raise the possibilitythat some of the estrogen derivatives, in particular the catecholestrogens and methoxyestrogens, may serve unique biologicalfunctions in the pituitary. This possibility is intriguing andmerits further investigation.
■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.chemres-tox.7b00096.
Figure S1, BrdU staining of anterior pituitary cells inovariectomized female Sprague−Dawley rats after treat-ment with E2 or 4-OH-E2 for 7 days; Figure S2, enzymekinetics for the O-methylation of 4-OH-E2 catalyzed bywhole cell lysates (containing both cytosol and micro-somes) prepared from MCF-7 breast cancer cells; andFigure S3, effect of E2 or 4-OH-E2 on MCF-7 breastcancer cell proliferation in culture (PDF)
■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. Phone: +86-755-84273851.ORCIDBao-Ting Zhu: 0000-0001-8791-8460Author ContributionsB.T.Z. conceived the initial ideas. P.W. and B.T.Z. designed theexperiments. P.W., L.H.M., J.-H.S., and J.Y. performed theexperiments, and P.W. and B.T.Z. analyzed the data and wrotethe paper.
FundingThis research was supported by grants RO1-CA97109 andRO1-CA92391 from the National Cancer Institute of theNational Institutes of Health, grants from the Natural ScienceFoundation of China (No. 81473224), from Shenzhen CityPeacock Team Project (No. 201619854), and from a Shenzhencity municipality grant (No. JCYJ20140714151402768).NotesThe authors declare no competing financial interest.
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