I9
SCREENING OF STEROIDS FROM PLANTS USING SACCHAROMYCES CEREVISI&E AS A HOST SYSTEM
THESIS
Presented to the Graduate Council of the
University of North Texas in Partial
Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
By
Camelia G. A. Maier, B. A., Denton, Texas
August, 1992 Maier, Camelia G. A., Screening of Steroids from Plants Usina Saccharomyces cerevisiae a a Host System. Master of Science (Biology), August, 1992, 60 pp., VII tables, 22 illustrations, bibliography, 50 titles. The reconstituted steroid transcription unit in Saccharomyces cerevisiae was used to screen for steroids in cell extracts prepared from 24 species of plants representing 16 families. Extracts from 7 species representing 5 families were able to activate both estrogen and progesterone receptors expressed in yeast. The transcriptional activity of the steroid reporter gene induced by plant extracts was dependent on the active compounds in extracts and increased proportionally with increasing amounts of extract used. The active compounds in Eustoma and Macluraextracts reduced the transcriptional activity induced by estradiol up to 32 and 55%, respectively. These phytoestrogens showed mixed agonism-antagonism characteristics, common among antisteroids such as nafoxidine and tamoxifen. Further studies could result in their possible functioning as tumor inhibitors. TABLE OF CONTENTS
Page
LIST OF TABLES ...... v LIST OF FIGURES ...... vi Chapter
1. INTRODUCTION ...... 1
General characteristics of steroids...... 1
Steroids and related compounds in plants ...... 5
Biological role of phytosteroids ...... 9
Phytoestrogens as antiestrogens for breast cancer therapy ...... 13
Saccharomyces cerevisiae as a host system for screening phytosteroids ...... 15 2. MATERIALS AND METHODS ...... 16
Chemicals and supplies...... 16
Yeast strains...... 16
Yeast media and buffers...... 17
Plant material...... 18
Callus culture ...... 20
Preparation of plant extracts...... 21
Screening for compounds with estrogenic and progesteronic activities from plant
extracts...... 22
Preparation of yeast extracts ...... 22
Beta-galactosidase assays ...... 23
iii 3. R ESULTS ...... 24 Transcriptional activity of steroid receptors in S. cerevisiae...... 24
Screening for steroids and related compounds in plant extracts ...... 26
4. DISCUSSION ...... 49 REFERENCES...... 56
iv LIST OF TABLES
Table Page I. Common, Scientific Names and Families of Plants Used to Screen for Steroid Compounds...... 18
II. Plant Organs and Tissues Used to Prepare the Cellular Extracts...... 22
III. Effect of Plant Extracts on the Steroid Receptor Activation...... 28
IV. Transcriptional Activity Induced by Polanisiaand Euphorbia bicolorExtractsin S. cerevisiae ER Strains...... 40
V. Transcriptional Activity of Plant Extracts in S. cerevisiae BJ3505-PR Strain...... 42
VI. Effect of Eustoma Extract and Maclura Fruit Extract on the Transcriptional
Activity Induced by Estradiol in S. cerevisiae BJ3505-ER Strains...... 44
VII. Effect of Increasing Amounts of MacluraFruit Extract on the Tanscriptional Activity Induced by Estradiol in S. cerevisiae BJ3505
Strain...... -......
V LIST OF FIGURES
Figure Page 1. The steroid nucleus and molecular structures of cholesterol and other sterols in mammals and plants...... 2
2. The sterol biosynthesis pathway...... 4
3. Steroids and related compounds in higher plants...... 6
4. The lanosterol-cycloartenol branch in the steroid biosynthesis...... 8 5. "Steroid-like" compounds...... 12
6. Non-steroidal antiestrogens synthetically produced ...... 14 7. The yeast expression system for steroid receptors...... 25
8. The transcriptional activity of human estrogen receptor reconstituted in yeast...... 27
9. Texas mulberry, Morus microphylla Buckl. (Moraceae)...... 30
10. Osage-orange, Maclurapomifera (Raf.) Schneid. (Moraceae)...... 31
11. Bluebell gentian, Eustoma grandiflorum (Raf.) Shinners (Gentianaceae)...... 32
12. Snow-on-the-prairie, Euphorbia bicolor Engelm. & Gray (Euphorbiaceae)...... 33 13. Clammy-weed, Polanisiadodecandra (L.) DC. (Cappariddaceae)...... 34
14. Tomato and tobacco calli...... 35
15. Transcriptional activity induced by the estrogen receptor expressed in S. cerevisiae BJ3505 strain in the presence of increasing amounts of Eustoma plantextract...... 36
vi 16. Transcriptional activity induced by the estrogen receptor expressed in S. cerevisiae BJ3505 strain in the presence of 100 gi Maclura fruit extract at increasing concentrations of copper sulfate...... 37
17. Transcriptional activity induced by Maclura plant extracts inS. cerevisiae BJ3505-ER strain...... 39
18. Transcriptional activity induced by increasing amounts of Euphorbia and
Polanisiaextracts in S. cerevisiae EU5A strain ...... 41
19. Transcriptional activity induced by increasing amounts of Eustoma amd Mactura extracts in S. cerevisiae BJ3505-PR strain...... 43
20. Transcriptioanl activity induced by estradiol in S. cerevisiae BJ3505-ER
strain in the presence of incresing amounts of Maclura fruit extract...... 45
21. Transcriptional activity induced by different dilutions of estradiol in S.
cerevisiae BJ3505-ER strain in the presence of Eustoma and Maclura extracts...... 46
22. Transcriptional activities induced by tomato and tobacco extracts in S. cerevisiae EU5A-ER strain...... 48
vii CHAPTER 1
INTRODUCTION
General Characteristics of Steroids
Steroids represent a class of chemical compounds found in animals and plants, as
well as in many prokaryotes (1). They occur in a variety of derivatives such as hormones
and vitamins in mammals and insects, saponins, alkaloids and glycosides in plants and marine organisms, and steroid antibiotics in fungi (2).
Steroids are organic compounds biosynthetically derived from 6-isopentenyl
pyrophosphate units and contain the perhydro-cyclopentane-phenanthrene nucleus. This
steroid nucleus is composed of three six-membered rings (A, B and C) and one five membered ring (D) (Fig. 1A). A side chain at C17 of the D ring is present in the majority of
steroids (2-3). Nes and McKean (4) define a steroid as a compound with the 1,2
cyclopentane-phenanthrene skeleton which in its biosynthetic pathway has passed through the "steroid transition", a state possessing stereochemistry similar to the trans-syn-trans
anti-trans-and configuration.
Eukaryotic steroids are considered to be derived from one key compound, common
to all eukaryotes: sterol, the 3 B-alcohols of about 27-30 carbons (5). Cholesterol is the
classic chemically defined sterol. The structures of the most relevant mammalian and plant
sterols are shown in Fig. 1B.
Sterols are present in the eukaryotic cell at a constant level of about 1 mg per g of dry weight and are integral parts of the cell membranes (1, 3, 5, 6). Some eukaryotes, such as plants, fungi and vertebrates are capable of endogenous sterol biosynthesis. Members of the sterol group differ from each other in the number and location of double bonds and in
1 2
12 18 17
1 1 oo 13 16
9 14 15 210 8 A B 3 7 5 4 6
B
Cholesterol 7-Dehydrocholesterol
HOig s r Stigmasterol Sitosterol
Ergosterol
FIGURE 1 - A. Steroid nucleus. The perhydro-cyclopentane-phenanthrene nucleus is composed of three six membered rings, A, B and C, and one five-membered ring , D. The numbering of the carbon skeleton is shown. B. Molecular structures of cholesterol and other sterols in mammals and plants. Sterols are the 3 8 alcohols of 27-30 carbons (5). Cholesterol is the classic chemically defined sterol. All natural sterols have an oxygen function at C3 and a side chain of at least seven carbons at C17 (7). The most abundant sterols of the higher plants are sitosterol and stigmasterol (2). 3 the juncture between rings A and B. All natural sterols have an oxygen function at C3 and a side chain of at least seven carbons at C17 (7) (Fig. 1B).
The biosynthesis of sterols goes through the well-known isopentenoid pathway of acetyl-CoA, mevalonate, isopentenyl pyrophosphate, geranyl pyrophosphate, farsenyl pyrophosphate, and squalene (Fig 2). Squalene-2,3-oxide or epoxide, formed by the aerobic cyclization of squalene, is a key intermediate in the formation of cholesterol and all related sterols of living systems (8-10).
In mammals, cholesterol is the intermediate for vitamin D, bile salts and steroid hormones (glucocorticoids, mineralocorticoids, and reproductive hormones) (9). The action of the steroid hormone on target cells has been studied intensively as the steroid hormones coordinate complex events involved in development and differentiation, as well as in the physiological response to stimuli (11). They exert their effects on target cells through two different mechanisms: a genomic one, by means of intracellular receptor proteins, acting on target genes, and a non-genomic one by increasing the level of cAMP and affecting the permeability of the cell (12).
The genomic mechanism of steroid hormones follows an entirely different pattern from that of the hormones that act through plasma membrane receptors (13). Being relatively small hydrophobic molecules, the steroid hormones cross the plasma membrane by simple diffusion and bind specifically to intracellular receptors. The binding of the hormone causes the receptor protein to undergo a conformational change that increases its binding affinity for DNA. Thus, the hormone-receptor complex can activate the expression of target genes which ultimately alter the functions and the phenotype of the cell (11). 4
3 Acetyl-CoA 4
coo Squalene epoxide 0H2 0I H3C- C -OH
CHZ Mevalonate I H CH2 CH33 CHCR2CH2CH =C OH CR3
Squalene
I HO Lanosterol CH2 mcmcH
C - CH3 d3-Isopentenyl pyrophosphate Cu, CR3 CH2 0 0 II H2C- O-P-0-P CHCR2CHZC2CH
4 0 0 CH3 C H3 OH HO Cholesterol CH3 CH2 0H2 CH3 C - CH3 C CH II CH2 O2
CH2 CH3 CH2 CH3 Farsenyl SyGeranylhae N / pyrophosphate c pyrophosphate -~ C 11 CH CH o 0 o 0 - O H2C - 0- P -0 - P -o H2C- 0 -p-O-P o 0 O 0
FIGURE 2 - Sterol biosynthetic pathway 5
Steroids and Related Compounds in Plants
name 'sterol' applies specifically All plants contain steroids of some kind (14). The with a hydroxyl group to steroid alcohols. Since, practically, all plant steroids are alcohols (15). at C3, as shown in Fig. 1B, they are usually called sterols C5 building block, Plants produce a great variety of products based on a branched primary metabolites, the isoprenoid unit, isopentenyl pyrophosphate. Some of these are synthesized by animals. Various such as sterols, and are similar or identical to compounds 1,25-dihydroxyvitamin D have animal steroids such as cholanic acid, cholecalciferol, and and ecdysterone have also been also been found in plants. The insect hormones ecdysone particularly progestogens and found in higher plants (15). Mammalian steroid hormones, (6). the corticosteroid deoxycorticosterone exist in plants as well by plants are However, the majority of the 'terpenoid' compounds synthesized (14, 16). Such plant secondary metabolites, and are considered unique plant products derivatives (15): sapogenins, steroids frequently occur not free but as more complex of some of the known plant sterols and glycosides, steroid alkaloids, etc. The structures abundant sterols of higher plants derivatives are shown in Fig. 3. In Fig. lB the most
namely B-sitosterol and stigmasterol are shown (2). the past 50 years, but plant sterol Interest in the biosynthesis of animal sterols spans only since about 1965. The investigations biosynthesis has been extensively investigated similar, there still are some differences in demonstrated that, although the pathways are and plants, but also between particular steroid biosynthesis not only between animals role appears to be corelated to the biological classes of plants (17). The biosynthetic scheme and plants, which, in turn, is linked to of steroids in different classes of animals
phylogenesis (1). and plants proceed along the The initial stages of sterol production in both animals 6
OH
oH
OH TpCO HO H 0 Thyphasterol
OH
OH H 0 Bufalin H Digitoxigen 0
O
HOH HO
HO Diosgenn H Digitogenin
HOa H Tomatidine Solanidine
that FIGURE 3 - Steroids and related compounds in higher plants. Different classifications of steroids exist are not clearly chemical or biological. Important classes of steroids are shown: phytoecdysones (ecdysone), and brassinosteroids (thyphasterol), cardiac-active steroids (digitoxigenin and bufalin), sapogenins (diosgenin
digitogenin), and steroidal alkaloids (solanidine and tomatidine). 7 well-established isoprenoid route (mentioned above) from acetyl-CoA through hydroxymethylglutaryl-CoA to mevalonic acid and thence via phosphorylated intermediates to farsenyl pyrophosphate and squalene (Fig. 2).
The nature of the sterol precursor produced by squalene-2,3-oxide cyclization shows a striking dichotomy between photosynthetic and non-photosynthetic organisms.
Fungi and animals produce lanosterol, which then is converted to cholesterol. By contrast, photosynthetic plants produce the 9 8, 19-cyclopropane compound called cycloartenol, which then undergoes modifications to yield the characteristic phytosterols (17) (Fig. 4).
Cycloartenol has consistently been identified among the labelled products of the sterol pathway, which is in accord with its proposed role as a phytosterol precursor (2, 17). It differs from lanosterol in the presence of a three-membered ring (9,19-cyclo grouping) in place of a A8 bond (10) (Fig. 4).
There are very few reports of lanosterol in green plants, and careful reinvestigations have resulted in the identifying lanosterol in only a few higher plants, notably in the latex of the Euphorbiaceae(17).
Cholesterol (24-desalkyl sterol) has been considered for many years as the typical animal sterol and the 24-alkyl sterols as typical for plants, thus giving rise to the terms zoosterol and phytosterol (1). It is now recognized that cholesterol is widespread in the plant kingdom also. Moreover, the 24-alkyl sterols occur in lower animals as well (1). By weight, cholesterol accounts for only 3% of the sterol mixture in plants since it appears to be rapidly formed and metabolized. Its function in plants, as that of all the sterols, is to serve as the starting material for the biosynthesis of all other steroids (8, 14). 8
SQUALINE EPOXIDE
HO I - n"4 'H CYCLOARTENOL LANOSTEROL
ANIMALS LOWER AND HIGHER FUNGI PHOTOSYN. PLANTS 0 0
z
FUNCTIONAL STEROLS (CHOLESTEROL, SITOSTEROL, ETC.)
FIGURE 4 - The lanosterol-cycloartenol branch in the sterol biosynthesis. There is a clear dichotomy in the biosynthesis pathway of sterol relative to the presence or absence of photosynthesis which is associated to the cyclization of squalene epoxide to cycloartenol in the former case and to lanosterol in the latter.
Cycloartenol differs from lanosterol in the presence of a three-membered ring (9, 19-cyclo grouping) in place of a A 8 bond (10). (Adapted from Nes W.R. (1)). 9
Biological Role of Phytosteroids
In nature, the primary role for sterols is a non-metabolic one as architectural components of membranes. Much attention has been given to their metabolic role as intermediates in the biosynthesis of hormones and other substances such as vitamins D, bile alcohols and acids (1, 18).
The function of steroids in plants is a controversial issue. In only a few cases, definite ecological roles are known. They are either protective as bitter or toxic substances or behave as feeding attractants (5). It is thought that glycoalkaloids (nitrogenous steroidal glycosides found in most Solanum species) have a role as general, nonspecific protective agents against microbial and insect invasions (2, 3, 19). The cucurbitacins of Cucurbitaceae are not only repellents for some insects (20) but also attractants for others (15). Phytoecdysones also protect the plant from insect attack (15).
Ecdysones or molting hormones have achieved a special attention since they are considered growth and differentiation hormones in insects similar to the estrogens and androgens of mammals (2). Moreover these molting hormones have been found to be widely distributed among the plant kingdom (21). It is not known if they are end products of sterol metabolism, the plant analogues of bile acids, or if they have a physiological or biochemical function in plant growth and development (2). Apart from the stabilizing effect of free sterols on phospholipid-containing membranes, a nongenomic effect, other functions have been suggested such as transport forms, auxin synergists, and precursors for hormones (22). Frequently encountered steroid esters represent either storage forms or metabolic pools. In case of sterol glycosides there is especially good evidence for a metabolic role as carriers for glucose oxidation (1).
It seems that animals and plants have utilized sterols for their growth and reproduction during evolution since the appearance of sterol some 1.5 billion years ago 10
(23). Progesterone in plants is synthesized from either cholesterol or alkylated plant sterols by a pathway similar to that in animals. Traces of estrone, testosterone, deoxycorticosterone and closely related compounds have been found in plants but nothing is known of their biosynthesis (6). Individual observations exist as to the possible roles of
steryl glycosides as hormones or precursors. For instance, stigmasteryl-B-D-glucoside enhances the growth-promoting effect of auxin in Avena coleoptiles (18). Brassinosteroids (named from Brassica napa L., Cruciferae), have been identified
as plant growth hormones of steroid nature. Such brassinosteroids show several growth promoting effects on plants such as stimulation of cell elongation and cell division, and are regarded as native hormones (1, 15, 24).
It might be possible that steroid hormones in plants induce growth and
differentiation by a mechanism similar to that described for mammalian steroid hormones.
It might also be possible that they exert their genomic effect in plant cells through receptor
proteins which have not yet been identified (14). Experiments showed that steroids as well as non-steroidal or "steroid-like"
compounds from plants are able to complex with receptor proteins and activate target genes in animal cells (25-27). The steroidal structure is not exclusive to substances having
estrogenic activity. It was shown that estrogenic substances occurring in some species of plants had a non-steroidal structure (25-27).
Plants containing compounds with estrogenic activity were believed to contribute to
infertility (called "clover disease" in Australia) and diseases of the urogenital system in farm
animals (26, 27). Isolation and structural elucidation experiments showed that the
compounds responsible for these problems are nonsteroidal substances of the three major
chemical types of phytoestrogens: coumestans (coumesterol), flavones and isoflavones
(genistein and formononetin) (28). 11
It is also possible that phytoestrogens in the diet can contribute to the growth of hormone-dependent breast tumors in postmenopausal women (28), since estrogens are potentially capable both of inducing tumors and enhancing growth of established tumors
(29), and since the low levels of circulating estrogens in postmenopausal women may not be sufficient to provide optimal growth in hormone-dependent tumors (12). Martin et al.
(29) showed that coumestrol, genistein and formononetin (Fig. 5) could bind to the estrogen receptor in the human breast cancer cell line MCF-7. The nuclear receptor phytoestrogen complex is processed in a manner analogous to the estradiol-bound receptor, and an appropriate biological response, that is growth stimulation, is achieved. Thus, these nonsteroidal compounds commonly present in food may function in the same manner as estradiol and are potentially capable of having profound effects on estrogen target cells (29
30).
Nes (1) defines these "steroid-like" compounds as molecules which, to a greater or lesser degree, can play the particular biological roles which classical sterols can play. In some cases these molecules assume a conformation approximating the steroidal structure (Figure 5). One might expect to find them instead of, or along with, sterols in some organisms. They do, in fact, accompany sterols in many plants and perhaps play membraneous roles, too (1). Farnsworth et al. (30) classified the plant compounds possessing estrogenic activity as sterols, such as B-sitosterol, and non-steroidal compounds, such as the isoflavones and coumestans. There is a striking similarity of the skeletal structure of these compounds with the structure of the synthetic estrogen diethylstilbestrol (Fig. 5). 12
OH
090 HOO HO Estradiol trans-Diethyistilbestrol
0H 0. H H OH 0
OHI1 0 HO 0 HO 0 CH30 Coumestrol Genistein Formononetin
OH
H
HO
alpha-Onocerin HO Carotenol
FIGURE 5 - "Steroid-like" compounds. Non-steroidal or "steroid-like" compounds can play particular
biological roles which classical sterols play (1). Coumestrol, genistein and formononetin can bind to the
estrogen receptor in the human brerast cancer cell line MCF-7 (29). In some cases, "steroid-like"
compounds assume a conformation approximating the steroidal structure, e.g., alpha-onocerinand carotenol
structure (1). 13
Phytoestrogens as Antiestrogens for Breast Cancer Therapy
As Martin et al. (29) demonstrated, phytoestrogens can compete with estradiol for
binding to the mammalian estrogen receptor and attenuate the action of estradiol. In the
presence of phytoestrogen, estradiol acted in a manner similar to that of weak or impeded estrogens.
A possible role for phytoestrogens in breast cancer has been considered in that their
effect may lie in both their ability to antagonize the natural steroid hormones and in their own intrinsic estrogenic activity. It is known that estrogens have a dual, dose-dependent
effect on mammary tumor induction and growth: large doses inhibit tumor development and
supress growth of established tumors, but they also can induce mammary tumor growth
even at physiological level. Phytoestrogens may have similar dual potentials (29).
Several antiestrogens have been tested clinically as therapeutic agents for breast
cancer. In the 1960s, clomiphene, nafoxidine, and tamoxifen (Fig. 6) were clinically tested
as potential tumor-inhibitors, but only tamoxifen is now available for general use. The
ability of these compounds to antagonize estrogen-stimulated cell growth and to suppress the growth of some (60%) of the estrogen receptor-positive human breast cancer cells explains the clinical utility of these compounds (28, 31-32). Recently, the compound trioxifene (Fig. 6), which has antiestrogenic and antitumor actions in laboratory animals, has been tested but it is not yet available for therapy (28). All these compounds are synthetically produced chemicals. Because of their low potency, partialy agonistic activity, and the occurrence of side effects and resistance to them in patients, current interest focuses upon the development of natural compounds. Such natural compounds should have a high affinity for the estrogen receptor and, as a result, increased antiestrogenic activity. They should also show minimum side effects if none. 14
OCH2CH2N
CH30 Nafoxidine (U l1,100A)
/ CH3
OCH2CH2N CR3
Tamoxifen (ICI 46,474)
NCH2CH20
Trioxifene
FIGURE 6 - Synthetically produced non-steroidal antiestrogens. Nafoxidine, tamoxifen and trioxifene are triphenylethylene derivatives. Trioxifene has been clinically tested but is not yet available for therapy (28). 15
Saccharomyces cerevisiae as a Host System for Screening Phytosteroids
An intensive search for plant products having antitumor activity began in 1957, shortly after establishment of the Cancer Chemotherapy National Service Center (CCNSC) within the National Cancer Institute (NCI).
The search for tumor-inhibitory compounds from higher plants is enhanced considerably by a newly developed estrogen responsive transcriptional system in
Saccharomyces cerevisiae (33). In the classical approach of studying these naturally occurring compounds, only those compounds which were most easily separated from a plant extract and most easily crystallized were studied. In the program established by CCNSC, biological assays were done at each stage of the fractionation of the extracts (34) without knowing from the beginning which plant extract contained active compounds. The recently reconstituted steroid regulated transcription unit in yeast (35) and used in this research project, significantly simplifies the search for possible tumor-inhibitory compounds in that it tests first for the total plant extract that can stimulate or inhibit steroid responsive reporter genes through the steroid receptors. Those extracts that are shown to be positive are then fractionated and the active fractions further characterized. CHAPTER 2
MATERIALS AND METHODS
Chemicals and Supplies
Estradiol-17-8, progesterone, adenine sulfate, o-nitrophenyl B-D-galactopyranoside
(ONPG) and other general reagents were purchased from Sigma Chemical Co., (St. Louis,
MO). Casamino acids, yeast nitrogen base without amino acids, dextrose and other yeast medium components as well as the agar for the plant tissue culture were obtained from
Difco-BRL (Bethesda, MD). The glass beads were obtained from Thomas Scientific
(Swedesdbore, NJ). BioRad protein assay dye reagent and bovine serum albumin were from BioRad (Richmond, CA). The chemicals for Murashige and Skoog medium were of
analytical grade and were purchased from Fisher Scientific Co., (Fairlawn, NJ). EDTA and
the plant hormones: indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and 6
benzylaminopurine (BA) were obtained from U.S. Biochemicals Co., (Cleveland, OH).
Sucrose was from Baker Inc., (Phillipsburg, NJ). Inositol and pyridoxine hydrochloride
were purchased from Eastman Kodak Co., (Rochester, NY). Nicotinic acid was obtained
from Matheson Co., (Norwood, OH) and thiamine was from Sigma Chemical Co. (St. Louis, MO).
Yeast Strains
Screening for estrogenic compounds in plant extracts was carried out in two
Saccharomyces cerevisiae strains: BJ3505 (MAT a, pep 4::His 3, prb 1-A 1.6R, his 3-A
200, lys 2-801, trp 1-A 101 (gal 3), ura 3-52 (gal 2), can 1) carrying the estrogen receptor
expression plasmid YEPE10 and the estrogen responsive reporter plasmid YRPE2, and
16 17
EU5A (MAT a, pep 4::His 3, prb 1-A 1.6R, his 3-A 200, lys 2-801, trp 1-A 101 (gal 3),
ura 3-52 (gal 2), can 1, ssn 6) containing the estrogen receptor plasmid YEPKB1 and the estrogen responsive reporter plasmid YRPE2.
Screening for progesteronic compounds was carried out in the Saccharomyces
cerevisiae BJ3505 strain (MAT a, pep 4::His 3, prb 1-A 1.6R, his 3-A 200, lys 2-801, trp 1-A 101 (gal 3), ura 3-52 (gal 2), can 1) carrying the progesterone receptor expression
plasmid YEPhPR1 and the progesterone responsive reporter plasmid PC3G2. These
Saccharomyces cerevisiae strains were kind gifts from D. McDonnell and Z. Nawaz,
Baylor College of Medicine, Houston, Texas.
Yeast Media and Buffers
The yeast growth medium used in this study is a minimal medium containing 1%
casamino acids, 2% dextrose, 1% yeast nitrogen base without amino acids, and 0.0012% adenine sulfate.
Yeast strains containing the receptor expression plasmids (estrogen receptor and progesterone receptor) and reporter plasmids (estrogen responsive and progesterone
responsive) were grown at 300 C overnight in a medium without uracil and tryptophan, in
the presence of either hormone (17-B-estradiol or progesterone) or plant extract and copper
sulfate (100 gM). Since the estrogen and progesterone receptor genes are under the control of the yeast metallothionein (CUP 1) promoter, the addition of copper sulfate in the medium will induce the production of estrogen and progesterone receptors.
The transcriptional buffer for the 0-galactosidase assays contains 16.1 g Na2HPO4.7H20, 5.5 g NaH2PO4.H20, 0.75 g KCl, 0.246 g MgSO4.7H20, and 2.7 ml mercaptoethanol, pH 7.0. 18
Plant Material
The plants were collected when completely mature. Nineteen species of plants from
14 families were collected from Thomsen Foundation property and neighborhood, in
Southeastern Montague County, Texas, in August 1991. Oranges, Citrus aurantium L.,
Rutaceae, were purchased from a grocery-store, as were the seeds for Texas bluebonnet,
Lupinus texensis Hook, Papillionaceae.Tobacco plants, Nicotiana tabacum L., and tomato plants, Lycopersicon esculentum Mill., Solanaceae, were obtained under greenhouse conditions.
The determination of their common and scientific names and families was done (36
38) and confirmed by Drs. D.W. Smith and F. Schafer (Table 1).
TABLE I
Common, scientific names and families of plants used to screen for steroid compounds
Family Scientific name Common name
ANACARDIACEAE Toxicodendron poison-ivy, climbing radicans (L.) Kuntze. climath
CAPPARIDACEAE Polanisia clammy-weed dodecandra(L.) DC.
COMPOSITAE Helianthus silverleaf sunflower argophyllus T. & G. Ambrosia short rag-weed
artemisiffolia L.
CONVOLVULACEAE Ipomoea lacunosaL. morning-glory CUCURBITACEAE Melothriapendula L. meloncito 19
TABLE I - Continued I Family Scientific name Common name
EUPHORBIACEAE Croton capitatusMichx. hogwort, wooly croton
Euphorbia bicolor snow-on-the-prairie
Engelm. & Gray
Euphorbia/Pointsettia warty spurge
dentata var. cuphosperma
(Engelm.) Small.
GENTIANACEAE Eustoma grandiflorum bluebell gentian (Raf.) Shinners
JUGLANDACEAE Carya illinoinensis pecan (Wang.) K. Koch
LABI TA E Salvia azurea Lam. giant blue sage LILIACEAE Nothoscordum crow-poison bivalve (L.) Britt.
MORACEAE Maclurapomifera osage-orange, (Raf.) Scheind. bow-wood
Morus microphilla Texas mulberry Buckl.
ONAGRACEAE Oenothera fluttermill, missouriensisi Missouri primrose
macrocarpaSims.
PAPILLIONACEAE Lupinus texensis Hook Texas bluebonnet PLANTAGINACEAE Plantagorhodosperma Dcne. red-seeded plantain 20
TABLE I - Continued
Family Scientific name Common name
RUTACEAE Citrus aurantiumL. orange SOLANACEAE Solanum silver-leaf eleagnifolium Cav. nightshade Solanum rostratum buffalo-bur Dun. Solanum Carolina carolinense L. horse-nettle Nicotiana tabacum L. tobacco Lycopersicon tomato esculentum Mill.
Callus Culture
Tomato seeds, Lycopersicon esculentum Mill. cv. Improved Summertime, and tobacco seeds, Nicotiana tabacum L. cv. Wisconsin 38, were obtained from Texas A & M University Experimental Research Station, College Station, TX. Tomato plants were grown under greenhouse conditions. The youngest fully expanded leaves of 3- to 4-week-old plants were removed, rinsed for 10 seconds in 95% ethanol, washed with distilled water, and then soaked for 10 minutes in 15% commercial Clorox with three drops of Tween 20 (polyoxyethylene-sorbitan monolaurate) per 100 ml of solution. The leaves were then rinsed with three changes of sterile distilled water. Uniform squares of leaf were cut and placed on the medium in plastic petri plates which were sealed with parafilm and incubated in darkness. 21
Tobacco plantlets were obtained by aseptically germinating seeds in Murashige and
Skoog medium (MS medium) (39) without growth regulators, supplemented with 3% sucrose, and solidified with 0.8% agar. The leaves were uniformly cut in squares which were placed on the medium.
The nutrient medium used to initiate and maintain the callus was MS medium
suplimented with 3% sucrose and IAA (2 x 10-3 g) and BA (2 x 10-4 g) for tobacco, or
NAA (5 x 10-7 M) and BA (1 x 10-5 M) for tomato, and solidified with 0.8% agar. The MS medium contains: nitrates (1.65 g NH4NO3, 1.9 g KNO3), sulfates (0.37 g
MgSO4.7H20, 1.69 x 10- 2 g MnSO4.H20, 8.6 x 10-3 g ZnSO4.7H20, 2.5 x 10-5 g CuSO4.5H20), halides (0.44 g CaCl2.2H20, 8.3 x 10-4 g KI, 2.5 x 10-5 COC12.6H20),
0.17 g KH2PO4, 6.2 x10-3 g H3BO3, 2.5 x 10-4 g Na2MoO4.2H20, 3.7 x 10- 2 g
Na2EDTA, 2.7 x 10-2 g FeSO4.7H20, and vitamins (5 x 10-5 g nicotinic acid, 5 x 10-5 g
pyridoxine, 2 x 10- 4 g thiamine), and 0.1 g inositol.
The pH of the medium was adjusted to 5.7 with 0.1 N NaOH or 0.1 N HCl prior
sterilization (1210C, 12-15 psi, 25 minutes).
Preparation of Plant Extracts
Fresh samples of whole plants or of their appropriate organs or tissues were
extracted with 95% ethanol (1:4 w/v) while grinding for 3-5 minutes in a Waring blender (34, 40-42). Call were ground using porcelain mortars and pestles (Table II). The homogenates were left standing at the room temperature for 24 hours and then
centrifuged at 3,700 X g for 15-20 minutes. The supernatants were collected after filtering
through MFS 1 (Micro Filtration System, Dublin, CA) filter paper and stored at -200 C
until used. Before using, small amounts of plant extracts were recentrifuged in
microcentrifuge tubes at 10,000 X g for 10 minutes to remove the residual debris. 22
TABLE II
Plant organs and tissues used to prepare the cellular extracts
Part used Plant
Whole plant clammy-weed, ragweed, morning
glory, hogwort, snow-on-the-prairie,
warty spurge, bluebell gentian, giant blue sage, red-seeded plantain, silver leaf nightshade, buffalo bur, horse
nettle
Aerial part silverleaf sunflower, meloncito, crow
poison, fluttermill, osage orange, mulberry
Fruit osage-orange, tomato
Exocarp orange
Leaves poison-ivy, pecan, tobacco, bluebell
gentian
Seedlings Texas bluebonnet
Callus tobacco, tomato
Screening for Compounds with Estrogenic and Progesteronic Activities from Plant Extracts
Preparation of yeast extracts
Yeast cells containing the receptor expression plasmid and the reporter plasmid were grown overnight in selective medium with CuSO4 at a final concentration of 100 pM.
When the cells reached an absorbance of 0.7 at 600 nm, the appropriate hormone and plant extract were added to the culture, and the cells were allowed to grow for additional 6-7 23 hours. At an absorbance of 1.0 at 600 nm, the cells were harvested by centrifugation. The homogenates were centrifuged at 10,000 X g for 10 minutes and the supernatants (crude cytosols) were collected.
Total protein concentrations in these cytosols were measured according to the method described by Bradford (43), using bovine serum albumin as standard.
Beta-galactosidase assays
The level of B-galactosidase produced was then measured according to the method of Miller (44). Each assay point was done in triplicate using 5-15 gg of protein. The protein diluted in lml of transcriptional buffer was incubated at room temperature for 10 minutes and then 200 gl of ONPG solution (4 mg/ml) was added. After a 30-minute incubation, the reaction was stopped by lowering the pH of the reaction with 500 ml of 1M Na2CO3 and the absorbance (A) at 420 nm was measured. The results are expressed in
Miller Units per mg of protein. The Miller units were calculated as follows:
Specific Activity = A420 X 1000 T X P
where T is the time of the assay, in minutes, and P is the amount of protein assayed, in mg. CHAPTER 3
RESULTS
Transcriptional activity of steroid receptors in Saccharomyces cerevisiae
The search for tumor-inhibitory compounds from higher plants is facilitated considerably by a newly developed steroid responsive transcriptional system in
Saccharomyces cerevisiae. The classical approach of studying these naturally occurring compounds in plants is very laborious. The yeast system significantly simplifies the search for possible tumor-inhibitory compounds, as it tests first for the total plant extract that can stimulate or inhibit a steroid responsive reporter gene through the specific steroid receptor. The S. cerevisiae strains were transformed with both a steroid receptor expression plasmid (YEpSR) and a reporter plasmid (YRpSR) and the transformants were selected for tryptophan and uracil prototrophy (33). The expression plasmid contains the copper responsive yeast metallothionein (CUP 1) promoter for the transcription of the human steroid receptors such as estrogen receptor (ER) and progesterone receptor (PR) fused to the carboxyl-terminus of the ubiquitin gene (Fig. 7). The CUPI promoter is inducible by copper ions.
The translation of mRNA begins from the ubiquitin AUG, producing a fused
ubiquitin-receptor protein. Ubiquitin is removed shortly after synthesis by ubiquinase, a
yeast processing enzyme, leaving an intact receptor. This ubiquitin-fusion technology allows high levels of receptor-protein production by enhancing stability and solubility of
the receptor proteins, and by protecting them from endogenous proteolysis (33, 35, 45).
To test the transcriptional activity of the steroid receptor expressed as described
above, a cis-trans transcription assay was developed by cotransforming the receptor
24 25
r amp
2u YEpSR CUP1 ub trpl term
receptor cDNA
CuInduction
Ub Receptor
Processing ura3 2
SUbi Receptor YRpSR r amp
+ Ligand
Transactivation cycI lacZ
B-galactosidase
Figure 7 - Yeast expression system for steroid receptors.The expression plasmid YEpSR was produced by using a ubiquitin gene fused to the receptor cDNA in the yeast 2p natural plasmid. Location of CUP promoter, ubiquitin-hSR, cyc terminator, tryptophan selection marker, and the 2 sequences for replication and segregation are indicated. The reporter plasmid YRpSR contains the cyci promoter fused to the E. coli
8-galactosidase gene. The synthetic SREs, uracil selection marker and the 2 sequences for replication and
segregation are indicated (adapted from Nawaz, Z., Ph. D. dissertation (33)). 26 expression plasmid, YEpSR, and the reporter plasmid, YRpSR (Fig. 7), into the yeast cell, and determining the ability of the receptor to activate the transcription of the reporter gene. The reporter plasmid contains the yeast iso-1-cytochrome c proximal promoter elements fused to the E.coli f-galactosidase gene (lac Z). It has two synthetic steroid response elements upstream of the cyc promoter elements. The induction of B-galactosidase activity by steroid receptor is solely dependent on the specific ligand and on the presence of the steroid response elements (33, 35, 45). The transcriptional activity of human steroid receptors was determined by measuring the B-galactosidase activity. To test the transcriptional activity of hER, yeast cells containing an estrogen receptor expression plasmid (YEPE10) and an estrogen responsive reporter plasmid (YRPE2) were grown overnight in the presence of varying concentrations of estradiol and with 100 gM copper sulfate. Fig. 8 shows that the estrogen receptor expressed in yeast activates the transcription of the target gene in the presence of
hormone. The half-maximal activity of transcription occurred at about 1 nM estradiol. This
is similar to the activity of the estrogen receptor observed in mammalian cells. These results confirm that the estrogen receptor produced in yeast is biologically active in a ligand
dependent manner (33).
Screening for steroids and related compounds in plant extracts
To screen for plant extracts that can stimulate the transcriptional activity of the
steroid responsive reporter gene, yeast cells were incubated overnight with 75 gl of plant
extract. The induction of lac Z was assayed after harvesting the cells by centrifugation. Among the 24 species of plants which were tested for steroid compounds, 7 species from 5
families activated the estrogen and progesterone receptors expresased in S. cerevisiae. The
results of these experiments are summarized in Table III and the photos of the active plants 27 are shown in Figures 9-14.
3000-
OW
#*-a 2000
0l PC ' 1000 -
0 u ...... - 0 5 10 15
Estradiol (nM)
Figure 8 - Transcriptional activity of hER reconstituted in yeast.Yeast cells transformed with a receptor
expression plasmid (YEPE1O) and a reporter plasmid (YRPE2) were grown overnight with 0.5, 1,2,5, 12
nM estradiol and 100 pM CuSO4. The level of 8-galactosidase produced in the yeast cells was measured and
the results expressed in Miller Units per mg of total protein. 28
TABLE Ill
Effect of plant extracts on the steroid receptor activation
Saccharomyces cerevisiae strains
Plant BJ3505(ER) EU5A(ER) BJ3505(PR)
Ambrosia sp. NT NT
Caryasp. NT NT
Citrus sp. NT
Croton sp. NT
Eustoma grandifLorum (Raf.) Shinners ++ NT +
Euphorbia dentata Engehn.&Gray - NT
E. bicolor + ++ +
Helianthus sp. NT
Ipomea sp. NT NT
Lupinus sp. NT
Lycopersicon esculentum Mill. ++ +
Maclurapomifera (Raf.) Scheind. +++ NT ++
Melothria sp. NT NT
Morus microphylla Buckl. +++ NT NT
Nicotianatabacum L. - + +
Nothoscordum sp. NT NT
Oenotherasp. NT NT
Plantago sp. NT NT
Polanisiadodecandra.(L.) DC + ++ + 29
TABLE III- continued
Saccharomyces cerevisiae Strains Plant BJ3505(ER) EU5A BJ3505(PR)
Salvia sp. NT NT
Solanum eleagnifolium Cav. NT
S. rostratumDun. NT
S. carolinense L. NT
Toxicodendron sp. NT NT
NT = not tested
- = no activity observed
+= activity
?= could not be properly tested
As shown in Table Ill, three S. cerevisiae strains were used to screen for steroid compounds in plant extracts: two estrogen strains, BJ3505 and EU5A, and a progesterone one, BJ3505. Maclura and Morus extracts showed the highest activity, while Polanisiaand
Euphorbia extracts showed very low activity with the BJ3505 strain. For this reason, the last two extracts were tested with the EU5A strain. Two extracts, from tomato and tobacco, showed no activity with the BJ3505 strain but were active with EU5A.
All the plant extracts showing activity with one of the estrogen strains were also positive for the progesterone strain BJ3505. 30
10%
Ar L,"m
~6~
Figure 9 - Texas mulberry, Morus microphylla Buckl. (Moraceae) is a small dioecious tree to 6 m. The picture shows the female tree bearing syncarps composed of numerous small, one-seeded drupes. 31
A
I ko
'41
be.
Figure 10 - Maclura pomifera (Raf.) Schneid.(Moraceae) is a dioecious tree with a milky sap, 18 m.high.
The tree has been widely planted in windbreaks. (A) Male tree with staminate flowers in peduncled axillary racemes of 2.5-3.75 cm long. (B) The osage orange fruit is a syncarp of one-seeded drupelets. 32
44
, 10* -4W
.9 ,0
V444
Figure 11 - Bluebell gentian (Eustorna grandiflorum (Raf.) Shinners, Gentianaceae).Usually a short-lived perennial to 70 cm with stems branched in the upper portion and containing a milky sap. It bears flowers to
6.25 cm high, to 10 cm across, varying from blue-purple prominently marked in center with darker purple, to white, or white tinged with purple or yellow. The flower is deeply cupped with 5 petals, united at base. 33
Figure 12 - Snow-on-the-prairie, Euphorbia bicolor Engeln. & Gray (Euphorbiaceae),is an annual plant of
30-90 cm high with solitary stem branched in the upper portion with the branches in two's or three's.
Leaves alternate, long, bordered with white. Flowers in terminal cluster, about 35 male and 1 female flower borne in a cup-shaped structure having 5 white, petal-like glands around the rim, the whole appearing as a five-petaled flower. The plant contains sticky, milky sap. 34
4*4
Figure 13 - Clammy-weed, Polanisiadodecandra (L.) DC (Cappariddaceae)is an upright annual plant, 60 cm high, its stems and leaves bearing gland-tipped sticky or gummy hairs. The leaf blade is divided into 3 leaflets with entire margins, Flowers are numerous, grouped in racemes; each flower to 1.88 cm long, white, with 4 petals, all spreaded upward, tapered and narrow in basal portion; stamens dark purple, numerous, long-exserted, unequal in length, pointed outward. 35
A
B
Figure 14 - (A) Tomato callus. Leaves of Lycopersicon esculentum Mill. cv. Improved Summertime, grown under greenhouse conditions, were cultured aseptically on Murashige and Skoog medium (39) and incubated in darkness to initiate callus. (B) Tobacco callus. Leaves from plantlets grown aseptically were used to initiate callus on M&S medium. 36
In another series of experiments, yeast cells were grown with varying amounts (12 100 gl) of those plant extracts which responded positively in the first testing.
Fig. 15 shows the activation of transcription by hER expressed in Saccharomyces cerevisiae BJ3505 strain in the presence of different amounts of Eustoma extract. The increase of transcriptional activity of the estrogen receptor is directly proportional with the amount of plant extract added to the cells. This suggests that the action of Eustoma extract on the ER activation is similar to that of estradiol.
500
400
300 DO
"' 200 pow
I0 100
04 0 25 50 75
Eustoma Extract (p1)
Figure 15 - Transcriptional activity induced by the ER expressed in S. cerevisiae strain BJ3505 in the presence of increasing amounts of Eustoma plant extract. The B-galactosidase activity is expressed in Miller
Units per mg of protein. 37
To test whether the transcriptional activity induced by a plant extract is dependent
on the receptor concentration or not, yeast cells were incubated with a constant amount of plant extract (100 gl) and different concentrations of CuSO4 ranging from 0 to 100 pM.
Fig. 16 shows that the transcriptional activity induced by Maclura fruit extract in these
conditions is independent of the amount of receptor in the cell. These results suggest that, even at the basal level of ER expression, the target sites for the estrogen receptor, the
estrogen response elements, are maximally occupied in the presence of Maclura fruit extract.
1200 1
1000 OC)
800
Dow PON 600
(0M 400
200
0- 0 5 10 50 100
CuSO4 (gM)
Figure 16 - Transcriptional activity induced by the ER expressed in S. cerevisiae strain BJ3505 in the presence of 100 1Maclura fruit extract at increasing concentrations of CuSO4. 38
Since Maclurapornifera is a dioecious plant, both female and male plants were
tested for induction of the transcriptional activity of the estrogen responsive reporter gene in
S. cerevisiae strain BJ3505. Branches from female and male trees were collected at the time of blossom (end of March and the beginning of April 1992) from plants grown in Denton,
TX. Forty g of fresh branches in 10 ml of 95% ethanol were used for the extracts. S. cerevisiae strain BJ3505-ER was grown overnight with increasing amounts of Maclura extracts from fruit and branches of female and male plants. The induction of B galactosidase activity was measured. The results (Fig. 17) show that Maclura male plant extract activated the transcription of lac Z at a lower than that from the female plant, which was approximately 3 times more active. There is practically no significant difference between the activities induced by the extracts from fruit and female plant at low amounts used (25-75 gl).
Extracts from two plants, Euphorbia sp.and Polanisia sp., induced low activities in S. cerevisiae strain BJ3505-ER (Table Ill). They were used with EU5A strain which has a mutation in ssn 6 gene coding for the repressor of estrogen receptor gene (McDonnell D.P. and Nawaz Z., personal communication). This mutation allows for more transcriptional activity from compounds which are active at a very low level with the strain BJ3505. Also the ER expressed from YEPE10 plasmid in strain BJ3505 carries 14 extra amino-acids at the amino terminus, a subclonal artifact. The strain EU5A was transformed with YEPKB1 receptor expression plasmid which generates an ER from which the extra amino acids were removed (33). The results of these experiments are summarized in Table IV. 39
1000
E Fruit 800 2 E Female Plant
0 3 M Male Plant 600
PC 400
200
01- 25 50 75 100
Maclura Extracts (gl)
Figure 17 - Transcriptional activity induced by Maclura plant extracts in S. cerevisiae strain BJ3505-ER: 1, fruit extract; 2, female plant extract; 3, male plant extract. 40
TABLE IV
Transcriptional activity (Miller Units) induced by Polanisiadodecandra and Euphorbia bicolor extracts in S. cerevisiae ER strains
Saccharomyces cerevisiae strains Plant extract (75 pl) BJ3505 EU5A
Polanisiadodecandra 87 407
Euphorbiabicolor 100 526
Table IV and Fig. 18 indicate that the transcriptional activities induced by Euphorbia and Polanisiaextracts in EU5A strain are about 5 times higher than those obtained with BJ3505 strain. S. cerevisiae strain EU5A proved to be efficient in detecting steroid compounds in plant extracts which showed low or questionable activity with the strain BJ3505.
To test for progesteronic compounds, the S. cerevisiae BJ3505-PR was transformed with the progesterone receptor expression plasmid YEPhPR1 and the progesterone responsive reporter plasmid PC3G2. These transformed yeast cells were grown with different plant extracts (Table III) and the transcriptional activity of PR was measured. Among the plant extracts tested for progesteronic compounds, only those with a positive effect on transcriptional activity of the reporter gene in ER strains were also able to activate the progesterone receptor. However, the transcriptional activities induced by the plant extracts in the strain BJ3505-PR were low compared with those of the same extracts in ER strains (Table V). 41
A
800
600
I
96
Us 400
vw 200-
u0 0 40 80 120
Euphorbia extract (pl)
B
500
400 0
300 PC U1'W Urib 200
100
0-9 40 80 120 p olanisia Extract (il)
Figure 18 - Transcriptional activity induced by increasing amounts of (A) Euphorbia bicolor extract and (B)
Polanisiadodecandra extract in S. cerevisiae strain EU5A. 42
TABLE V
Transcriptional activity of plant extracts in S. cerevisiae BJ3505-PR
Plant extract (75 gl) Transcriptional activity (Miller Units)
Polanisiadodecandra 110
Euphorbiabicolor 105
Eustona grandiflorum 257
Maclurapomzfera 264
Morus microphylla 102
Nicotiana tabacum (leaf extract) 227
Lycopersicon esculentum (fruit extract) 128
Fig. 19 shows that the transcriptional activities induced by Eustoma and Maclura extracts in S. cerevisiae strain BJ3505-PR increase in direct proportion to the amount of plant extract used.
To screen for plant extracts that can compete with estradiol and inhibit the transcriptional activity of the estrogen responsive reporter gene, extracts from Maclura fruit and Eustoma leaves were used. S. cerevisiae strain BJ3505 cells were incubated overnight with a constant amount of estradiol (10-6 M). The next day, 75 gI of plant extract were added to the culture and the cells were allowed to grow for another 6-7 hours prior to harvesting and assaying for B-galactosidase activity.
Table VI shows that both Maclura and Eustoma extracts reduced the transcriptional activity induced by estradiol with 55 and 32%, respectively. 43
A
200
40
100
PC
0- ,0 25 50 75 100 150 Eustoma Extract (gi)
B
400
U
0 U 300 tic
200 U) 0 'C U) 0 U 0 100. 0 Co Co 0- 0 25 50 75 100 150 Maclura Extract (gl)
Figure 19 - Transcriptional activity induced by increasing amounts of (A) Eustoma extract and (B) Maclura
fruit extract in S. cerevisiae BJ3505-PR. 44
TABLE VI
Effect of Eustoma and Maclura fruit extract on the transcriptional activity induced by estradiol in S. cerevisiae strain BJ3505-ER
Treatment Transcriptional activity (MU) Inhibition
Estradiol (10-6 M) (E) 2145
E + Eustoma extract 1467 32%
E + Maclura extract 956 55%
To test whether the inhibition of transcriptional activity increases with progressively increasing amounts of plant extract, the BJ3505-ER strain cells incubated overnight with
10-6 M estradiol were grown an additional 6-7 hours with Maclura fruit extract in amounts varying from 12 to 100 jl.
Fig. 20 shows that the transcriptional activity induced by estradiol decreased with progressively increasing amounts of plant extract. The percentage of inhibition was calculated and is presented in Table VII.
In the last series of experiments testing for the inhibition of transcriptional activity by plant extract, the cells were grown overnight in different dilutions of estradiol. The next day, a constant amount of plant extract (75 gl) was added and the cells were allowed to grow for additional 6-7 hours. Fig. 21 presents the results of these experiments with Maclura fruit and Eustoma plant extracts. At low levels of estradiol (10-12 M), the plant extract is more effective in activating transcription than the hormone. As the amount of estradiol increases, the transcription through it is restored, estradiol being able to displace the plant extract and activate the receptor. The displacement of Eustoma extract is more efficient than that of Macluraextract suggesting that the latter competes more efficiently 45
3000
2000
1000
0 0 12 25 50 75 100
Estradiol (10-6 M) + Macura Extract (l)
Figure 20 - Transcriptional activity induced by estradiol in S. cerevisiae strain BJ3505-ER in the presence
of increasing amounts of Maclura fruit extract.
TABLE VII
Effect of increasing amounts of Maclura fruit extract on the transcriptional activity induced by estradiol in S. cerevisiae strainBJ3505-ER
Treatment Transcriptional Activity (MU) Inhibition (%)
Estradiol (E) 2433
E + 12 pglextract 2050 16
E + 25 g1 extract 1688 31
E + 50 p extract 1513 38
E + 75 extract 1363 44
E + 100 lextract 1163 52 46
A
3000
Estradiol 2000 E+Eustoma Extract
1000
0 f--I-I-I- -14 -12 -10 -8 -6 -4 -2 10 10 10 10 10 10 10 Estradiol Concentration (M)
B
3000
Estradlol 2000
1000-
E+Maclura Extract
na 12 -0 8 -6 - -104 10 14 10 12 10 18 10- 10-2 Estradiol Concentration (M)
Figure 21 - Transcriptional activity induced by different dilutions of estradiol in S. cerevisiae BJ3505-ER in
the presence of (A) Eustoma extract and (B) Maclura fruit extract. 47 with estradiol for the hormone-binding site of the estrogen receptor. In this experiment,
Maclura fruit extract reduces the transcriptional activity induced by estradiol by 75%. To test the transcriptional activity induced by plants in tissue culture, calli of tobacco and tomato obtained on Murashige and Skoog medium (39) were used. Both tobacco and tomato calli extracts were unable to activate the transcription of ER in S. cerevisiae strain BJ3505-ER but were active in EU5A (Table II).These extracts were also able to activate the transcription of the reporter gene through the progesterone receptor expressed in BJ3505-PR (Table III).
Two types of callus were used: one grown on a complete M & S medium, the other grown on a M & S medium deprived of boron. Extracts from the respective plants were also tested along with those obtained from calli. There were no significant differences among the transcriptional activities induced by extracts from tobacco plant and calli.
With tomato extracts, there was no significant difference between the complete M &
S medium and the extract from callus grown on medium lacking boron. The extract from the green tomato fruit was twice as active, and that from callus grown on a complete M&S medium approximately four times more active compared with M & S medium alone (Fig.
22). 48
1500 1 - M&S Medium 2 2 - Tomato Callus (Control) 3 - Tomato Callus (- Boron) 4 Tomato Fruit
Now 1000
4 omi
-p
PC 500 1I 3
0- 75 75 75 75
Extracts (75 il)
Figure 22 - Transcriptional activities induced by tomato plant and callus extracts in S. cerevisiae strain
EU5A-ER. C = extracts obtained from calli grown on complete M&S medium, and -B = extracts obtained from calli grown on M&S medium without boron. M&S medium alone was also tested. CHAPTER 4
DISCUSSION
The steroid responsive transcriptional unit which has been reconstituted in Saccharomyces cerevisiae (33) proved to be effective and easy to manipulate in screening for steroid compounds in plant extracts. Although yeast is not a natural host for the human estrogen receptor (hER) and for the human progesterone receptor (hPR), data indicate that the regulation of these receptors in yeast reflects their regulation in animal systems.The transcriptional activation function of the steroid receptors is directly regulated by
(hormonal) ligands in the yeast system as well as in animal systems suggesting that conserved mechanisms are involved (46).
The plant extracts which had a positive effect on the ER and PR activation (Table
III), may contain compounds that are able to bind to the steroid receptors expressed in yeast and elicit agonistic (Fig 15-19) or antagonistic actions (Fig 20-21). Mixed agonism antagonism is very common among the antisteroid compounds (12).
The results presented in this study and in others (25-29) suggest that phytoestrogens are processed in the nucleus in a manner analogous to the estradiol bound receptor. Moreover, in the presence of estradiol they attenuate its action (29). It is known that antiestrogens produce a mixture of estrogenic and antiestrogenic actions (28). The active substances in the plant extracts tested can activate the transcriptional activity of the estrogen responsive reporter gene by themselves acting as partial agonists, and at the same time they are able to inhibit the transcriptional activity induced through estradiol. They can inhibit the activation of ER induced by estradiol either by competing with it for the hormone-binding site of the receptor or by displacing estradiol from the receptor. The
49 50
fact that once bound to antagonistic effect of these phytoestrogens may be the result of the the receptor they function by establishing receptor-DNA complexes that are transcriptionally nonproductive (46). The transcriptional activity induced by estradiol decreases progressively in the presence of increasing amounts of Maclura fruit extract (Fig. 20 and Table VII). Previously, it has been shown that nafoxidine, a synthetic antiestrogen (Fig. 6), competes with estradiol in a concentration-dependent manner and reduces the estrogen receptor activation (33).
The antiestrogen in Maclura fruit extract is a competitive inhibitor of estradiol gl of activity (Fig. 21) and has an agonistic activity by itself in the yeast system. With 100 28 fruit extract and female plant extract used (Fig. 17), the transcriptional activities were and 36% respectively of that obtained with estradiol. Although the inhibition of estradiol The activity is relatively inefficient with Eustoma extract, it is also competitive in nature. extract relative inefficiency can be due to a lower affinity of the phytoestrogen in Eustoma active for the estrogen receptor than that in Maclura extract. Further studies with purified
compounds from these extracts will establish their levels of affinities for the estrogen receptor.
In the ER expression system, the CUPI promoter exhibited a substantial basal level
of expression which can be further induced by adding copper to the growth medium. This even when way, ER levels can be increased approximately 6- to 10-fold (46). However, the ER expression is increased by copper, the level of lac Z transcription in the presence of
Maclura fruit extract remained practically unchanged (Fig. 16). This result suggests that
even under a basal level of ER expression the target sites on the reporter plasmid are
maximally occupied in the presence of the active ligand in Maclura extract. This is in
agreement with the results obtained with estradiol and nafoxidine (33). 51
The relatively lower levels of transcription in the presence of Maclura extract
(around 1,000 MU) compared with the transcriptional activity through estradiol (2,300
MU), as with the other plant extracts tested, may be due to a lower transactivation potential of the ER bound to compounds in the plant extracts or it may be due to a lower affinity of compounds for ER than that of estradiol. In other words, when the target EREs appear to be saturated with receptor bound to phytoestrogen, these phytoestrogen-receptor complexes elicit a lower level of trascription than estradiol-receptor complexes. The same plant extracts tested were active in both S. cerevisiae strains BJ3505-ER and -PR, but the transcriptional activity induced in the PR strain was low. It seems that the progesteronic compounds in the plant extracts had lower affinities for the PR than the estrogenic compounds have for ER. It is known that progestagens are precursors for estrogens and androgens in mammals (9). Moreover, pregnenolone and progesterone have been isolated from different species of plants and shown that they are precursors to other steroids in these organisms (10). Thus, the presence of both progesteronic and estrogenic compounds in the same plants tested in the S. cerevisiae system can be explained unless these active principles are non-steroidal compounds. Further studies implying their isolating and structural elucidation will reveal their steroidal or non-steroidal nature.
Sixty percent of human breast tumors which contain ER also contain PR. This indicates that the PR systhesis is mediated by the ER system (47). Both receptors play a role in the progression of breast cancer (47). It is known that phytoestrogens interact with the ER of human breast cancer cells in culture and, therefore, may affect estrogen-mediated events in these cells (29). Since phytoestrogens form nonproductive receptor complexes at target gene promoters by trapping the receptor in a variety of allosteric forms that are less active or even inactive, they may be used as pharmacologic antagonists of steroid hormones in breast cancer cells (29). 52
The fact that Maclura male extract exhibited a lower activity from ER than that of the extract from the female plant, suggests that the phytoestrogens have a definite biological role in the female plant. Steroid (sex) hormones do occur in higher plants. However, these hormones are associated with the endocrinology of higher animals. Yet, it is possible that at the molecular level there appear to be some interactions between plant chromosomes and steroid compounds that are analogous to those between animal chromosomes and steroid hormones (14). It may be possible that the phytoestrogens of Maclura have a role in the differentiation of plant sex and in the chemical regulation of plant reproduction. They may activate intracellular receptors similar to or even identical with the steroid receptors found in animal cells, and in a similar manner to that observed for the latter receptors.
The results obtained by testing callus extracts showed that it can be possible with some species to obtain secondary metabolites, phytoestrogens in this case, in larger amounts in tissue culture than from the field-grown plant. The extract from tomato callus grown on a complete M & S medium was approximately twice as active as the extract obtained from the green tomato fruit (Fig. 22). It was shown that by depriving the plant tissue culture medium of certain elements the synthesis of secondary metabolites can be increased even more (48). In the present study, the lack of boron in the M&S medium not only that did not increase the synthesis of phytoestrogens in tomato callus, but lowered the synthesis below the level of these compounds in the field-grown plant.
The Murashige and Skoog medium used to initiate callus from tomato leaves, by itself, induced lac Z activity in the yeast system. This activity may be due to the ring structure of the growth regulators (NAA and BA) in the medium used to initiate callus.
These substances might have induced the transcriptional activity of the reporter gene by themselves since it is known that some simple biringed compounds are active estrogens
(28), or the induction of transcription might have occurred following their metabolizing in the yeast cell, which can be true with the plant extracts as well. Yet, the level of 53 transcription induced by the extract obtained from the tomato callus grown on a complete M & S medium is approximately four times higher than that induced by the medium alone.
Moreover, it is known that lack of boron in the medium triggers the synthesis of high levels of auxins (hyperauxiny) (49) as well as of phenolic compoundsin plant cells. Therefore, the transcriptional activity of the reporter gene in the yeast system was not solely induced by the growth regulators in the medium, since the extract obtained from callus grown on medium deprived of boron had the same level of activity as the M&S medium tested alone.
These results suggest that some other and more active estrogenic compound in the callus is the inducer. In addition, these findings also suggest that boron may be implied somehow in the biosynthesis pathways of these phytoestrogens. To convert sterols in steryl glucoside compounds for instance, UDP-glucose is required, or it is known that deficiency of boron affects its synthesis in the cells (22, 50).
The first lot of plants used for tests were all collected from the same area in the field. All of them are Angiosperms and among them only one, Nothoscordum bivalve (L.)
Britt, Liliaceae, is a monocot, the other being dicots (Table I). It could be interesting to test Gymnosperms as well as more monocots and lower plants.
Some families are represented by more than one species (Table I) and in some cases the extracts were made from different parts of the same plant (Table II). The results showed that the induction of transcriptional activity of the steroid reporter gene in yeast system varies more with the family, genus, species and plant sex, but less with the plant part used for extraction. However, this conclusion can not be generalized since the present discussion involved limited experiments.
In the Moraceae family, both plants tested, Maclura sp. and Morus sp., were positive. This is not the case with Euphorbiaceaeand Solanaceae families in which one
species from three tested and two species from five, respectively, proved to contain active 54
compounds. Moreover, it can be stated that the activation of the yeast system is species
specific with Euphorbia since only E. bicolor induced the transcriptional activity of lac Z.
It is difficult to draw a general conclusion concerning the part of the plant used for
cellular extraction, due to a limited sampling. However, the observation can be made that
the plants showing activity with the yeast system possess either a milky and/or sticky sap or sticky or gummy hairs. This suggests that the estrogenic and progesteronic compounds
in the extracts from these plants may be found in the sap and hair cells. If this is the case, these substances are activily synthesized in the plant cells and therefore have a definite metabolic role for these species
Some of the plant extracts (rag-weed, red-seeded plantain, poison-ivy) exhibit various degrees of inhibitory action on yeast cell growth and multiplication. The most inhibitory activity was shown by the orange-peel extract even at very low amounts added to the culture medium. This is due to the fact that the extracts used for testing were total cell extracts, containing all the metabolites in the cells. Some plants, like poison-ivy for instance, are known to contain substances toxic to higher organisms. Because of the inhibitory action on yeast culture, the ethanol extract from orange-peel could not be properly tested in the yeast system (Table Ill).
Some of the plants which did not show activity when tested as an ethanolic extract in the yeast system, but perhaps they do contain compounds that can activate steroid receptors. The host system used for screening, and the solvent used for extraction, both can influence the outcome. It has been shown in the case of Medicago sativa,
Papillionaceae,tested in an animal system that the ether extract was estrogenic and the chloroform extract was antiestrogenic (30).
The present study is just a part of a search for tumor inhibitors of plant origin, and for the first time the newly developed Saccharomyces cerevisiae system was used in screening for steroid compounds in plants. Further work could result in the elucidation of a 55
active compounds in the plant extracts tested possible function as tumor inhibitors for the active principles here. Further studies are needed to isolate and structural elucidate of the tumor cell systems. showing antiestrogenic properties and their testing in REFERENCES
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