JP2006176427A - Estrone 3-sulfate transporter activity inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an estrone 3-sulfate transporter activity inhibitor or breast cancer cell proliferation inhibitor or breast cancer curative, with no need of being taken in cells, good in drug delivery tendency and extremely slight in side effects. <P>SOLUTION: The estrone 3-sulfate transporter activity inhibitor or breast cancer cell proliferation inhibitor or breast cancer curative contains, as active ingredient, one or more components selected from bromosulfophthalein, dehydroepiandrosterone sulfate, dehydroepiandrosterone and taurocholic acid. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an inhibitor of transporter activity of an estrone-3-sulfate (estrone-3-sulfate) transporter, a breast cancer cell growth inhibitor, and a breast cancer therapeutic agent.

  Membrane proteins that transport substances inside and outside the cell are called transporters. When a specific molecule binds to a transporter embedded in a lipid bilayer, the conformation changes, and the substance is taken up or transported. It is known that In recent years, such transporters, for example, organic anion transporters such as OAT1 that transport organic anionic substances, organic cation transporters such as OCT1 that transport organic cationic substances, peptide transporters such as PEPT1 that transport peptide substances, etc. These genes have been isolated and identified one after another. Some of these transporter genes are ubiquitous in normal tissues and organs throughout the body, but others are known to be localized in specific tissues and organs such as kidney, liver and brain.

On the other hand, estrogens are so-called female hormones that cause estrus in female animals, and are naturally occurring estrone (E ₁ ), estradiol (E ₂ ), estriol (E ₃ ), and estetrol (E ₄ ). Are classified into synthetic estrogens having the same biological activity. With the exception of some synthetic forms such as stilbestrol, estrogens have a steroid structure. The biological action of estrone 0.1 μg is defined as 1 IU (international unit), which is used as a unit of estrogen. The source of secretion is mainly ovarian follicles and corpus luteum, but it is also secreted from the fetal placental system, adrenal gland, testis, etc. during pregnancy. The secretion of estrogen from the ovaries is governed by gonadotropins secreted from the anterior pituitary gland (downward regulation), but conversely, estrogen also has a feedback effect on the pituitary system (ascending regulation). It is said that the sexual cycle is established by the mutual relationship between the two.

  Furthermore, estrogen acts through its receptor (receptor), and estrogen acts not only on the target tissue, but also on the whole brain, anterior pituitary gland, genitals and mammary gland, and the main physiological functions are endometrium Such as growth of uterine muscle, development of secondary sexual characteristics, mediation of establishment of menstrual cycle, induction of maternal changes during pregnancy, promotion of growth secretion of mammary ducts. Estrogens are metabolized mainly in the liver and become conjugated estrogens such as estrone trisulfate and are excreted in the urine. Clinically, it is used for treatment of amenorrhea and menstrual abnormalities, artificial movement of menstruation, menopause, hormone therapy for prostate cancer and breast cancer, osteoporosis, and the like.

  Breast cancer has an abnormal operation of the estrogen receptor system from the time of carcinogenesis, and continues to grow estrogen-dependently at an early stage, but gradually grows out of this control. This suggests that estrogen and its receptor are deeply involved in the development and progression of breast cancer, but the mechanism is still unclear. Breast cancer is a malignant tumor that arises from the peripheral ducts and acinar epithelium of the epithelial tissue of the mammary gland, and is the leading cancer death among women in the West. Also in Japan, the death rate from breast cancer is on the rise due to recent westernization of eating habits and changes in lifestyle. Prevalence age is in the 40s, risk factors include history of breast cancer, family history, unmarried, elderly first birth, early menarche and late menopause, obesity, radiation exposure, high fat diet, history of benign breast disease, etc. it can. Lymph node metastasis is more frequent in the axilla, subclavian and parasternal, and the number of metastases correlates with prognosis. Hematogenous metastases are common in bone, lung, and liver. The main symptom is a breast mass, surface irregularity, hardness, unclear borders and low mobility.

  2/3 of breast cancers are estrogen dependent (Non-Patent Document 1), that is, cell growth is controlled by estrogen. The biologically active form of estrogen is estradiol, which is synthesized via two main pathways (Non-patent Documents 2 and 3). One is the aromatase pathway in which aromatase converts androgen to estrogen, and the other is the sulfatase pathway in which steroid sulfatase converts estrone 3 sulfate to estrone. In patient breast cancer tissues, the activity of sulfatase is 50 to 200 times that of aromatase, so the sulfatase pathway is a major source of estrone trisulfate-derived bioactive estrogens (Non-Patent Document 3, Non-Patent Documents). 4). In addition, the circulating plasma concentration of estrone trisulfate is about 10 to 20 times that of unconjugated estrogen, and the half-life of estrone trisulfate is longer than that of estradiol (Non-Patent Documents 5 to 7). Therefore, estrone trisulfate is believed to play an important role in the progression of breast cancer as a reservoir of active estrogens, even though estrone trisulfate itself does not exhibit much biological activity.

  Stimulation of breast cancer cell growth by estrone trisulfate involves the following series of processes: uptake of estrone trisulfate into cells, desulfation by estrogen sulfatase, estrone to estradiol by type I 17β-hydroxysteroid dehydrogenase Conversion, nuclear estrogen receptor binding, and gene transcription control (Non-Patent Documents 8 to 11). We have examined the enzymes and receptors involved in the response of breast cancer cells to estrone trisulfate, but the transport mechanism through which ligands such as estrone trisulfate cross the cell membrane is unknown. The logP value of estrone trisulfate is reported to be 1.4, which is significantly lower than that of estrone (4.4) and estradiol (3.9), so that estrone and estradiol are diffusible. Nevertheless, it will not be easy to cross the cell membrane by diffusion. (Non-patent document 12). Estrone 3 sulfate was detected in an intact form in the cytoplasmic fraction of MCF-7 cells after incubation with estrone 3 sulfate (Non-patent Document 13), and showed an estrogenic action (Non-patent Document 4, Non-patent document 14, Non-patent document 15). Therefore, a specific transporter seems to be involved in the supply of estrone trisulfate to breast cancer cells exhibiting hormone-dependent growth.

  Among the members of the organic anion transporter (OAT, SLC22A) (Non-Patent Document 16) family and the organic anion transport peptide (OATP, SLC21A, SLCO) (Non-patent Document 17, Non-Patent Document 18) family, estrone trisulfate is transported. It is already known that there is something to do. They are expressed primarily in the liver, kidney, brain, intestine, and other sites, but no expression in breast cancer cells has been reported. Their Km values for estrone trisulfate are in the range of 0.05 μM to 59 μM. In women, plasma levels of estrone trisulfate vary in the range of 1-10 nM (19), so these or other transporters are the first step in hormone-dependent growth. It appears to play a role in the uptake of cancer into cancer cells.

  Recently, Pizzagalli et al. Have suggested the expression of OATP-B, a steroid sulfate transporter in human mammary gland (Non-patent Document 20). However, OATP-B mRNA was not detected in estrogen-dependent MCF-7 cells and T-47D cells, whereas OATP-D and OATP-E mRNA were found in these cells (non-patented). Reference 21). OATP-D and OATP-E are involved in the transport of estrone 3 sulfate in cancer cells because they exhibit transport activity against estrone 3 sulfate when the gene is transiently expressed in HEK293 cells (Non-patent Document 22). It seems that However, transporter-mediated uptake of estrone trisulfate in estrogen-dependent cells has not been investigated so far, but uptake in other tissues such as liver, placenta and brain has been reported. In order to fully understand the growth mechanism of breast cancer cells, the mechanism of estrogen supply to such cells must be elucidated.

N Engl J Med 302: 78-90, 1980 J Clin Endocrinol Metab 57: 1125-1128, 1983 Breast Cancer Res Treat 7: 35-44, 1986 Ann N Y Acad Sci 464: 126-137, 1986 J Clin Endocrinol Metab 81: 1460-1464, 1996 J Biol Chem 236: 1043-1050, 1961 J Clin Invest 51: 1020-1033, 1972 Endocrinology 138: 863-870, 1997 Mol Endocrinol 11: 77-86, 1997 Endocrinology 123: 1281-1287, 1988 Breast Cancer 9: 296-302, 2002 J Clin Endocrinol Metab 59: 1128-1132, 1984 Endocrinology 106: 1079-1086, 1980 J Clin Endocrinol Metab 81: 1460-1464, 1996 J Steroid Biochem Mol Biol 73: 225-235, 2000 Pflugers Arch 440: 337-350, 2000 Biochim Biophys Acta 1609: 1-18, 2003 Pflugers Arch 447: 653-665, 2003 Hormone Res 27: 61-68, 1987 J Clin Endocrinol Metab 88: 3902-3912, 2003 J Clin Endocrinol Metab 88: 3902-3912, 2003 Pharm Res 18: 1262-1269, 2001

  As shown in FIG. 1, the following methods (blocks 1 to 4) are known for inhibiting breast cancer cell growth, which blocks upstream signal transduction due to binding of estrogen and its receptor in breast cancer cells. .

Block 1 (inhibition of estrogen synthesis from androgen)
Fadrozole: After menopause, it selectively inhibits aromatase, which acts as a rate-limiting enzyme for estrogen synthesis, preventing the conversion of androgen to estrogen, thereby reducing the in vivo estrogen concentration and suppressing breast cancer growth.

Block 2 (competitive inhibition at estrogen receptor)
Tamoxifen, toremifene: Estrogen receptors such as breast cancer tissues are competitively inhibited with estrogen and exert anticancer activity by exhibiting antiestrogenic activity.

Block 3 (Inhibition of estrogen synthesis from estrone sulfate)
Conjugated estrogen is activated by estrogen sulfatase in the cell and promotes cancer cell growth. Inhibits this deconjugating enzyme.

Block 4 (cytotoxic effect using antibody to membrane protein)
Herceptin: Some breast cancer cells highly express HER2 protein. Utilizing the binding between HER2 and an antibody, it exerts an antitumor effect due to an antibody-dependent cytotoxic effect.

  In the method of blocks 1 to 4 above in inhibiting the growth of breast cancer cells, if an aromatase inhibitor, an estrogen sulfatase inhibitor, or an estrogen competitive inhibitor such as tamoxifen or toremifene is not taken into the breast cancer cell, an antitumor effect is obtained. Since it is not exerted, it cannot be said that there are no problems in terms of drug delivery and in terms of side effects when incorporated into cells. An object of the present invention is to provide an inhibitor of transporter activity of an estrone trisulfate transporter, a breast cancer cell growth inhibitor, or a breast cancer therapeutic agent that does not need to be taken into cells, has excellent drug delivery properties, and has very few side effects. There is.

The present inventors have intensively studied to solve the above problems, and examined the following block 5 (see FIG. 1) as a method for inhibiting the growth of breast cancer cells.
Block 5 (inhibition of transporter activity)
An anti-tumor effect appears by inhibiting intracellular uptake of estrone trisulfate, which is a source of estrogen, against estrogen-sensitive breast cancer cells.

  When the human breast cancer cultured cell MCF-7 cell line is cultured in the presence of estrone 3 sulfate and bromosulfophtalein (BSP) as a test substance, the estrone 3 sulfate transporter expressed on the cell surface. It was found that the uptake of estrone 3 sulfate through the cell through the cell was inhibited and the growth of the MCF-7 cell line was suppressed, and the present invention was completed.

  That is, the present invention effectively uses (1) one or more components selected from bromosulfophthalein, dehydroepiandrosterone sulfate, dehydroepiandrosterone, taurocholic acid, p-aminohippuric acid, tetraethylammonium, probenecid and benzylpenicillin. An inhibitor of transporter activity of the estrone trisulfate transporter as an ingredient, (2) an inhibitor of transporter activity of an estrone trisulfate transporter containing bromosulfophthalein as an active ingredient, and (3) bromosulfo Effective with one or more ingredients selected from phthalein, dehydroepiandrosterone sulfate, dehydroepiandrosterone, taurocholic acid, p-aminohippuric acid, tetraethylammonium, probenecid and benzylpenicillin Breast cancer cell growth inhibitor, (4) breast cancer cell growth inhibitor containing bromosulfophthalein as an active ingredient, (5) bromosulfophthalein, dehydroepiandrosterone sulfate, dehydroepiandrosterone, Breast cancer therapeutic agent comprising one or more components selected from taurocholic acid, p-aminohippuric acid, tetraethylammonium, probenecid and benzylpenicillin as an active ingredient, and (6) a breast cancer therapeutic agent comprising bromosulfophthalein as an active ingredient. .

  According to the present invention, unlike an aromatase inhibitor, an estrogen sulfatase inhibitor, or an estrogen competitive inhibitor such as tamoxifen or toremifene, it does not need to be taken into cells, has excellent drug delivery properties, and has very few side effects. Porter inhibitors, breast cancer cell growth inhibitors, and breast cancer therapeutic agents can be provided.

  Examples of inhibitors of transporter activity of the estrone trisulfate transporter of the present invention, breast cancer cell growth inhibitors and breast cancer therapeutic agents include bromosulfophthalein, dehydroepiandrosterone sulfate, dehydroepiandrosterone, taurocholic acid. Among them, bromosulfophthalein can be preferably exemplified. Moreover, these can be used individually by 1 type or in combination of 2 or more types.

  The inhibitor of transporter activity of the estrone 3 sulfate transporter of the present invention (hereinafter sometimes referred to as “the present inhibitor”) is important in identifying the estrone 3 sulfate transporter. Candidate transporters of estrone 3 sulfate transporter include SLC transporter, OAT1, OAT2, OAT3, OAT4, OATP-A, OATP-B, OATP-C, OATP-D, OATP-E, OATP-F, OATP- 8, NTCP, MRPs, BCRP and the like can be preferably cited. As an identification method, cells expressing the candidate estrone 3 sulfate transporter on the cell surface are cultured, and the uptake of the candidate estrone 3 sulfate into the cultured cells is measured in the presence of the inhibitor. Methods for measuring and evaluating the degree of inhibition of the transporter activity of estrone 3 sulfate into cells and cell membrane fractions isolated from cells that express the candidate estrone 3 sulfate transporter on the cell surface A method of measuring and evaluating the degree of inhibition of the specific binding of estrone 3 sulfate to the cell membrane fraction by contacting with an inhibitor, or from a cell expressing a candidate estrone 3 sulfate transporter to the cell membrane A fraction is isolated, a vesicle is produced from the cell membrane fraction to form an isolated cell membrane vesicle, and estrone trisulfate is added to the isolated cell membrane vesicle. Contacting the present inhibitor may be a method in which the degree of inhibition of uptake or uptake of estrone 3-sulfate into vesicles by the present inhibitor is measured and evaluated. Cell culture in the presence of the above estrone trisulfate and the inhibitor is carried out using a growth medium for the cells used, and at least significant cell growth is observed under normal cell culture conditions (in the absence of the inhibitor). The contact with the cell membrane fraction of the estrone 3 sulfate and the inhibitor, or the contact with the isolated cell membrane vesicle of the estrone 3 sulfate and the inhibitor, The incubation can be performed by incubation in a growth medium or an appropriate buffer.

  As a method for measuring and evaluating the degree of inhibition of the transporter activity of estrone 3 sulfate into cells by the inhibitor, the method for measuring and evaluating the concentration of estrone 3 sulfate in cells or the degree of cell proliferation is measured. -List methods of evaluation. When using human breast cancer cell lines such as MCF-7 cell line, KPL-1, MKL-F or cell lines derived from these cell lines, which will be described later, a method for measuring and evaluating the inhibitor concentration in the cells, A method for measuring and evaluating the degree of cell proliferation can be used, but when a transformed cell expressing a candidate estrone trisulfate transporter is used, a method for measuring and evaluating the inhibitor concentration in the cell can be used. Can be used. In addition, the candidate estrone 3 sulfate transporter gene is expressed in Xenopus oocytes, etc., and changes in membrane potential caused by transport of substrate through the transporter are detected using two microelectrodes (current measurement) It can also be measured and evaluated by electrophysiological methods.

  Examples of a method for isolating a cell membrane fraction from cells expressing the estrone 3 sulfate transporter on the cell surface include the method of F. Pietri-Rouxel et al. (Eur. J. Biochem., 247, 1174-1179, 1997). Conventionally known methods can be used, and methods for preparing vesicles from cell membrane fractions to make isolated cell membrane vesicles include JE Lever et al. (J. Biol. Chem., 252, 1990-1997 (1977). A conventionally known method such as the method of)) can be used. In addition, as a method for measuring the degree of inhibition of specific binding of estrone 3 sulfate to the cell membrane fraction by the present inhibitor, the equilibrium dissociation constant KD is measured from the steady state binding inhibition using radiolabeled estrone 3 sulfate. The method can be exemplified, and as a method for measuring the amount of estrone 3 sulfate incorporated into isolated cell membrane vesicles by the present inhibitor or the degree of inhibition of the amount incorporated, I. Tamai et al. (Biochim. Biophys. Acta , 1512, 273-284 (2001)), and the like can be used as a method for measuring intracellular estrone trisulfate.

  As a cell that expresses the estrone 3 sulfate transporter on the cell surface, a transformed cell that expresses a candidate estrone 3 sulfate transporter can be used. The SLC transporter, OAT1, OAT2, OAT3, OAT4, OATP-A, OATP-B, OATP-C, OATP-D, OATP-E, OATP-F, OATP-8, used for the production of such transformed cells The origin of the gene encoding NTCP, MRPs, BCRP is not particularly limited, and examples thereof include humans, dogs, cows, horses, goats, sheep, monkeys, pigs, rabbits, rats and mice. However, human origin is preferable.

  A method for expressing a gene or cDNA encoding the above transporter in a cell is not particularly limited, but Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other methods described in many standard laboratory manuals such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection By introducing a transporter gene into a host cell by microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection, etc. Expressed However, it is preferable to introduce an expression vector containing a transporter gene into the host cell, and in particular, the transporter can be expressed in a cell by infecting the host cell with an expression vector containing the transporter gene. More preferred.

  Examples of the expression vector include adenoviral vectors (Science, 252, 431-434, 1991) used for transient expression in all cells including non-dividing cells (other than blood cells), and in dividing cells. Retrovirus vectors used for long-term expression (Microbiology and Immunology, 158, 1-23, 1992) and adeno-associated virus vectors used for long-term expression (Curr. Top. Microbiol. Immunol., 158, 97-129, 1992), virus vectors such as SV40 and vaccinia virus vectors, as well as liposomes, and the like. It is not something. Among these, an adenovirus vector capable of expressing genes in cells with high efficiency is particularly preferable. In addition, the transporter gene can be introduced into these expression vectors by a conventional method. For example, an expression vector can be constructed by inserting a transporter gene or the like downstream of an appropriate promoter in these expression vectors. it can. In addition to the transporter gene and marker gene, the expression vector is an IRES (ribosome binding site in the mRNA) for normalizing the expression level of the transporter per cell, as well as a control sequence that regulates the expression of enhancers, terminators, etc. May be included.

  Examples of host cells that express the transporter include insect cells such as Drosophila S2, Spodoptera Sf9, Vero cells, HeLa cells, CHO cells, WI-38 cells, BHK cells, COS-7 cells, MDCK cells, C127 cells, and HKG cells. , Human kidney cell lines, CV-1 cells, LLC-MK2 cells, MDBK cells, MRC-5 cells, Caco-2 cells, HT29 cells, human lymphoblasts, Xenopus and other oocytes, and their dhfr deficiency Strains, HGPRT-deficient strains, ouabain resistant strains, and the like. Specifically, CHO-K1 (Chinese hamster ovary cell: ATCC CCL61), BHK (hamster kidney cell: ATCC CCL10), COS-7 (CV-1 Origin, SV-40 cell: ATCC CRL1651), Vero cell (Africa) Examples thereof include midisal kidney cells (ATCC CCL81) and human lymphoblast cells (IM-9, ATCC CCL159).

  Since the transporter is a membrane protein expressed on the surface of the cell membrane, neutralizing antibodies against the estrone trisulfate transporter may selectively inhibit the target from outside the cell. From this, the inhibitor inhibits estrone trisulfate trans Once the porter can be identified, neutralizing antibodies against the estrone trisulfate transporter can be generated. Specific antibodies that can be specifically recognized include monoclonal antibodies, polyclonal antibodies, single chain antibodies, humanized antibodies, chimeric antibodies, bifunctional antibodies that can simultaneously recognize two epitopes, and the like. These antibodies are produced by administering cells expressing the estrone trisulfate transporter on the membrane surface and cell membrane fractions thereof to animals (preferably other than humans) using conventional protocols. The preparation involves the hybridoma method (Nature 256, 495-497, 1975), the trioma method, the human B cell hybridoma method (Immunology Today 4, 72, 1983) and EBV resulting in antibodies produced by continuous cell line cultures. -Any method such as a hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) can be used.

  The breast cancer cell growth inhibitor of the present invention can be used for identification of an estrone trisulfate transporter and as a breast cancer therapeutic agent because it can inhibit the growth of breast cancer cells. Examples of the identification method include a method of culturing cells expressing the candidate estrone trisulfate transporter on the cell surface in the presence of the breast cancer cell growth inhibitor of the present invention, and measuring and evaluating the degree of cell proliferation. it can.

  The breast cancer therapeutic agent of the present invention can also be used as a preventive agent for breast cancer, and when used as a pharmaceutical therapeutic agent, a normal pharmaceutically acceptable carrier, binder, stabilizer, excipient, Various preparation ingredients such as a diluent, a pH buffer, a disintegrant, a solubilizer, a solubilizer, and an isotonic agent can be added. These therapeutic agents can be administered orally or parenterally, and can be administered orally, for example, in the form of powders, granules, tablets, capsules, syrups, suspensions, etc., or For example, a dosage form such as a solution, emulsion, suspension or the like can be administered parenterally by injection. When administered parenterally, it can be administered directly locally. In the case of preparations for oral administration, various conventional organic or inorganic carrier substances are used as pharmacologically acceptable carriers. For example, tablets include excipients such as lactose and starch, talc, and magnesium stearate. Lubricants such as hydroxypropylcellulose and polyvinylpyrrolidone, disintegrants such as carboxymethylcellulose, etc. can be blended. Solvents such as physiological saline alcohol, polyethylene glycol, propylene Solubilizing agents such as glycol, suspending agents such as stearyltriethanolamine, sodium lauryl sulfate, lecithin, isotonic agents such as glycerin and D-mannitol, buffering agents such as phosphate, acetate and citrate Etc. can be blended. If necessary, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be blended. For preparations for parenteral administration, water-soluble solvents such as distilled water and physiological saline, solubilizing agents such as sodium salicylate, isotonic agents such as sodium chloride, glycerin and D-mannitol, human serum albumin, etc. Stabilizers, preservatives such as methylparaben, and narcotics such as benzyl alcohol can be blended. The dosage of the therapeutic agent can be appropriately selected depending on the patient's weight and age, dosage form, symptoms, etc. For example, when administered to an adult, the active ingredient is usually about 0.001 to 500 mg as a single dose, preferably It is desirable to administer 1 to 50 mg once to 3 times a day.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

It was examined whether or not the suppression of the cell growth promoting effect of estrone trisulfate on MCF-7 cells (ATCC-HTB22) was achieved by suppressing intracellular accumulation of estrone trisulfate. MCF-7 cells were seeded in a 96-well plate at a concentration of 8 × 10 ⁴ cells / cm ² and cultured for 1 day, and then 10 μM to 10 μM of various concentrations of estrone trisulfate (manufactured by Sigma Chemicals) and 100 μM of bromosulfo. Phthalein (BSP: manufactured by Sigma Chemicals) and media containing various concentrations of estradiol (estradiol: manufactured by Sigma Chemicals) at 1 pM to 10 nM and 100 μM BSP were added, followed by further culturing for 3 days. Thereafter, the amount of cells was measured. The result of the cell growth promotion effect by estrone trisulfate and estradiol is shown in FIG.

  As is clear from FIG. 2, a cell growth promoting effect was observed in the presence of estrone 3 sulfate alone and estradiol alone as positive controls (open column). On the other hand, culturing in the presence of 100 μM BSP simultaneously with estrone trisulfate or estradiol shows an effect of promoting cell growth under low conditions of estrone trisulfate, which is caused by BSP itself or its metabolite. Estrogenic activity is considered. This effect was relatively low as the estrogen concentration increased. Interestingly, when 10 μM estrone trisulfate was added, the cell growth effect by BSP was inhibited, while the effect by addition of estradiol did not change. Therefore, the intracellular uptake of estrone trisulfate was inhibited by BSP, and the highly lipophilic estradiol was not inhibited. Therefore, it was considered that the cell proliferation effect was not changed. From the above, the importance of the estrone trisulfate uptake transporter in cell growth was suggested, and the possibility of inhibiting cell growth by inhibiting the transporter was shown.

  It was investigated whether induction of ERE (estrogen response element) activity by estrone trisulfate in MCF-7 cells (ATCC-HTB22) can be suppressed by BSP. MCF-7 cells were transiently transfected with the pERE-TA-SEAP vector (purchased from BD Biosciences) shown in FIG. This pERE-TA-SEAP vector is a SEAP (Secreted Alkaline phospatase) protein expression vector having an ERE sequence upstream of a DNA sequence to which estrogen binds. When estrogen is present, SEAP protein synthesis is promoted via ERE, SEAP activity is increased. SEAP is an enzyme that cleaves a phosphate bond. The substrate of this enzyme is MU-P (methyl umbelliferone phosphate), and the resulting MU (methyl umbelliferone) is quantified to measure SEAP activity.

pERE-TA-SEAP vector (purchased from BD Biosciences) MCF-7 cells transfected according to the kit manual were seeded in a 96-well plate at a concentration of 8 × 10 ⁴ cells / cm ² and cultured for 1 day. ^After adding estrone 3 sulfuric acid (manufactured by Sigma Chemicals) at various concentrations of ⁻⁹ to 10 ⁻⁶ M and further culturing for 8 hours, SEAP activity was measured. The results are shown in FIG. 4 (left). As a result, MCF-7 cells transiently transfected with the pERE-TA-SEAP vector showed SEAP activity depending on the concentration of added estrone 3 sulfate. Next, MCF-7 cells transfected according to the manual of the pERE-TA-SEAP vector (purchased from BD Biosciences) kit were seeded in a 96-well plate at a concentration of 8 × 10 ⁴ cells / cm ² and cultured for 1 day. Thereafter, a medium containing estrone trisulfate at various concentrations of 20 nM, 50 nM, and 100 nM and a medium containing or not containing 100 μM bromosulfophthalein (BSP: manufactured by Sigma Chemicals) were added, and further cultured for 8 hours. SEAP activity was measured. The results are shown in FIG. 4 (right). As a result, MCF-7 cells transiently transfected with the pERE-TA-SEAP vector suppressed the SEAP activity induced by the addition of estrone 3 sulfate by the addition of 100 μM BSP.

It was investigated whether estrogen-dependent cell growth by estrone trisulfate in MCF-7 cells (ATCC-HTB22) can be specifically suppressed by estrone 3-sulfate transporter by BSP. Unlike estrone trisulfate, estrone is taken into cells without using an estrone trisulfate transporter, so that it was expected that BSP cannot suppress estrone-dependent cell growth (see FIG. 5). MCF-7 cells were seeded in a 96-well plate at a concentration of 8 × 10 ³ cells / 0.32 cm ² and cultured for 1 day, and then estrone (manufactured by Sigma Chemicals) at various concentrations of 10 ⁻¹² to 10 ⁻⁷ M Then, media containing 0 μM, 30 μM, and 100 μM bromosulfophthalein (BSP: Sigma Chemicals) were added, and the cells were further cultured for 2 days, and then cell proliferation was measured. The results are shown in FIG. 6 (left). As a result, even when 30 μM or 100 μM BSP was added, estrone-dependent cell growth could not be suppressed. Next, it was confirmed that estrogen-dependent cell proliferation can be specifically suppressed by estrone trisulfate transporter by BSP. After inoculating MCF-7 cells in a 96-well plate at a concentration of 8 × 10 ³ cells / 0.32 cm ² and culturing for 1 day, various concentrations of estrone 3 sulfate of 10 ⁻⁹ to 10 ⁻⁵ M, 0 μM, 30 μM , 100 μM BSP-containing medium was added, and after further culturing for 2 days, cell proliferation was measured. The results are shown in FIG. 6 (right). As a result, it was confirmed that the addition of 30 μM and 100 μM BSP can suppress cell growth dependent on estrone 3 sulfate.

1. Materials and methods (materials)
[ ³ H] Estrone 3 sulfate, ammonia salt (1702.0 GBq / mmol) and [ ³ H] dehydroepiandrosterone sulfate (DHEAS, 2926.7 GBq / mmol) were purchased from PerkinElmer Life Science Products, Inc. T-47D cells were provided by Professor Takuma Sasaki of Kanazawa University Cancer Institute. Fetal calf serum (FCS) was obtained from Invitrogen Corp. All other reagents were purchased from Sigma Chemicals and Wako Pure Chemical Industries.

(T-47D cell proliferation assay)
T-47D cells were placed in RPMI1640 medium (manufactured by Sigma Chemicals) containing phenol red and 10% FCS, and grown as usual in a humidified incubator at 37 ° C. and 5% CO ₂ . For proliferation assays, FCS was incubated with 0.5% dextran charcoal (DCC) overnight at 4 ° C., after which the medium was removed and the DCC was filtered (0.2 μm) to remove steroid hormones. This process was repeated three times. Thereafter, T-47D cells were seeded at a density of 8,000 cells / well in a 96-well plate containing a phenol red-free RPMI 1640 medium containing 2.5% DCC-treated FCS. One day after sowing, estradiol or estrone trisulfate was added in water or in dimethyl sulfoxide (DMSO, 0.1%) at graded concentrations from the stock solution. The negative control was solvent only. After seeding, the cells were trypsinized for a predetermined number of days, and the number of cells was counted.

(Inhibition of estrone 3 sulfate uptake activity)
Transport experiments were performed as described in the literature (Tamai et al., 2001). After culturing T-47D cells in a 15 cm dish, the cells were harvested and 125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl ₂ , 1.2 mM KH _2. Suspended in transport medium containing PO ₄ , 1.2 mM MgSO ₄ and 25 mM Hepes adjusted to pH 7.4. Solutions containing cell suspension and radiolabeled test compound in transport medium were individually incubated at 37 ° C. for 20 minutes and transport was initiated by mixing them. When appropriate, remove 150 μL of the mixture and transport by centrifugal filtration through a layer of silicone oil (SH550, Toray Dow Corning Co.) mixture and 1.03 density liquid paraffin (Wako Pure Chemical Industries). Cells were separated from the medium. Each cell pellet was solubilized in 3N KOH and neutralized with HCl. Thereafter, the associated radioactivity was measured with a liquid scintillation counter using Clearsol-1 (manufactured by Nacalai Tesque) as the liquid scintillation fluid.

(Reverse transcription PCR method)
The expression of OATP and OAT transporter in T-47D cells was examined by RT-PCR. Single-stranded cDNA was constructed using oligo (dT) primers (Invitrogen Corp.). The following specific primers were used.
OATP-A:
(F) 5'-AAACAAGCTGCCCACATAGG-3 '(SEQ ID NO: 1)
(R) 3'-CAGCAAGACAAGCTGACAGA-5 '(SEQ ID NO: 2)
OATP-B:
(F) 5′-CCTGCCGCTCTTCTTTATCGG-3 ′ (SEQ ID NO: 3)
(R) 3'-ACCAGATGGCTGCACGTTG-5 '(SEQ ID NO: 4)
OATP-C:
(F) 5'-CACTTGGAGGCACCTCACA-3 '(SEQ ID NO: 5)
(R) 3′-ACAAGCCCAAGTAGACCCTT-5 ′ (SEQ ID NO: 6)
OATP-D:
(F) 5′-CAGGCCATGCTCTCCGAAA-3 ′ (SEQ ID NO: 7)
(R) 3'-AGCCACCACTGCAATCTCC-5 '(SEQ ID NO: 8)
OATP-E:
(F) 5′-CCCTGGGAATCCAGTGGATTG-3 ′ (SEQ ID NO: 9)
(R) 3′-AGCAGGCTATGGCAAAGAAGAG-5 ′ (SEQ ID NO: 10)
OATP-F:
(F) 5′-GGAAATTCCTCAGGCATAGTGG-3 ′ (SEQ ID NO: 11)
(R) 3′-CTGGGATTCCTGCAAGAACTC-5 ′ (SEQ ID NO: 12)
OATP8:
(F) 5′-GGGAATCATAACCATTCCTACGG-3 ′ (SEQ ID NO: 13)
(R) 3′-GAGGATTTGCATCCTGCTAGAC-5 ′ (SEQ ID NO: 14)
OAT1:
(F) 5′-CTGATGGCTTCTCACAACAC-3 ′ (SEQ ID NO: 15)
(R) 3′-CCGACTCAATGAAGAACCAG-5 ′ (SEQ ID NO: 16)
OAT2:
(F) 5′-GCTGGTTTTACCATCATCGT-3 ′ (SEQ ID NO: 17)
(R) 3′-GACTCAGGCCGTAATAGGAG-5 ′ (SEQ ID NO: 18)
OAT3:
(F) 5′-AAGTGACCTGTTCCGGATAC-3 ′ (SEQ ID NO: 19)
(R) 3′-CCATACCTGTTTGCCTGATG-5 ′ (SEQ ID NO: 20)
OAT4:
(F) 5′-GGCGTTATCTCCATTGCTTC-3 ′ (SEQ ID NO: 21)
(R) 3′-GAGATTGGAACCCAGTCTCT-5 ′ (SEQ ID NO: 22)
In the presence of deoxynucleotide and tag polymerase (manufactured by Takara Bio Inc.), the final elongation was 94 seconds at 30 ° C., 58 ° C. (OATP) or 56 ° C. (OAT) for 30 seconds, and 72 ° C. for 30 seconds. The reaction was carried out by repeating a cycle of 10 minutes at 72 ° C. 35 times. PCR products were analyzed by 1% agarose gel (w / v) electrophoresis and such gels were stained with ethidium bromide to visualize the bands.

(Analysis method)
Using bovine serum albumin (Bradford, 1976) as a standard substance and BioRad protein assay kit (manufactured by Hercules), the cellular protein content was measured according to the method of Bradford. To estimate the kinetic parameters of saturable transport, the uptake rate (v) was applied to the following equation by nonlinear least squares regression analysis using KaleidaGraph ^™ (Synergy).
v = Vmax ^* s / (Km + s)
Here, v and s are the uptake rate and the substrate concentration, respectively, and Km and Vmax are the half-saturated concentration (Michaelis constant) and the maximum transport rate, respectively. All data are mean ± S. E. M.M. Expressed in Statistical analysis was performed by Student's t test using P <0.05 as a criterion for significance. The cell to medium ratio was obtained by dividing the amount of cell uptake by the concentration of the test compound in the uptake medium.

2. Results (Estradiol and estrone 3 sulfate effects on T-47D cell proliferation)
In order to elucidate the effect of estrone trisulfate on the proliferation of T-47D cells, such cells were treated with estradiol and estrone trisulfate. Consistent with previous studies using estrogen-dependent MCF-7 cells (Billich et al., 2000), proliferation of T-47D cells was compared to 1 pM estradiol and 100 nM compared to cells treated with solvent alone. Of estrone 3 sulfate (Fig. 3A). When evaluated 6 days after sowing, estrogen-dependent stimulation increased in a concentration-dependent manner (FIG. 3B). By non-linear regression analysis, the 50% effective concentration (EC ₅₀ ) values of estradiol and estrone trisulfate were estimated to be about 22.5 fM and 17.1 nM, respectively.

(Incorporation of estrone 3 sulfate by T-47D cells)
FIG. 4 shows the time course of [ ³ H] estrone 3 sulfate (7.1 nM) uptake by T-47D cells in the presence or absence of unlabeled 1 mM estrone 3 sulfate. [ ³ H] estrone 3 sulfate uptake increased linearly over 20 minutes. In the presence of unlabeled 1 mM estrone 3 sulfate, no significant increase in [ ³ H] estrone 3 sulfate incorporation was observed for 35 minutes (FIG. 4). This suggests that estrone 3 sulfate is rarely taken up by T-47D cells by simple diffusion. Therefore, in the following experiment, uptake of estrone trisulfate by T-47D cells was analyzed kinetically over 10 minutes. In addition, the apparent uptake after subtraction of uptake in the presence of unlabeled estrone trisulfate (unsaturated uptake) was analyzed to take into account adsorption to the cell membrane and mere diffusion.

In order to characterize the transport mechanism of estrone trisulfate, we investigated by substituting Na ⁺ with various cations (FIG. 5). The Na ^+, K +, Li ^+, or N- methylglucamine ⁺ and when ^substituted, the uptake ^[3 H] estrone 3-sulfate, was equivalent to that of Na ⁺ presence. This suggests a Na ⁺ -independent transport mechanism.
In order to obtain kinetic parameters of estrone 3 sulfate uptake by T-47D cells, the concentration dependence of estrone 3 sulfate uptake was examined. As shown in FIG. 6, the uptake of estrone 3 sulfate after subtracting the value in the presence of 1 mM estrone 3 sulfate was saturable. The Eadie-Hofstee plot analysis yielded a single line, suggesting that a single saturable mechanism is kinetically involved. In nonlinear regression analysis, the values of Km and Vmax (mean ± SEM) were 3.9 ± 0.78 μM and 576.3 ± 32.9 pmol / mg protein / 10 min, respectively.

(Inhibition of estrone 3 sulfate uptake activity)
In order to characterize the substrate specificity of the transport system, the inhibitory effects of hormones, their conjugated metabolites, and various organic compounds on the uptake of [ ³ H] estrone 3 sulfate by T-47D cells were examined. The results are shown in (Table 1). In Table 1, the control (%) is shown as a ratio when the uptake of estrone 3 sulfate in the control is 100%, and each value is expressed as mean ± SEM (n = 4), ^* P <0 .05 compared to control (Student 'test).

As a result, unlabeled estrone 3-sulfate itself showed the strongest inhibitory action (34.7% and 6.6%, respectively, for 5 μM and 50 μM controls). The inhibitory effect of DHEAS was significant (44.8% compared to 50 μM control), but weaker than that of estrone trisulfate. Estradiol-17β-glucuronide, a glucuronide conjugate, had no effect at 50 μM. All of the unconjugated steroid hormones significantly inhibited [ ³ H] estrone trisulfate uptake. However, their affinity was lower than that of sulfate conjugates, estrone trisulfate and DHEAS. Of the other organic compounds examined, bromosulfophthalein and taurocholic acid showed strong inhibitory activity, and the inhibitory action at 100 μM was 18.9% and 69.3% of the control, respectively. Among other organic compounds, p-aminohippuric acid, tetraethylammonium, probenecid and benzylpenicillin showed an inhibitory action although they were slightly weaker than bromosulfophthalein. On the other hand, salicylic acid did not show any action.

  In postmenopausal women, androgens and their sulfate conjugates play an important role in breast cancer progression as a source of active estrogens. DHEAS is a major precursor (J Clin Endocrinol Metab 54: 22-26., 1982) because it is present at high concentrations (approximately 1 μM) in many postmenopausal patients. Therefore, it was examined whether DHEAS is transported via a specific transport mechanism. DHEAS uptake increased linearly over 30 minutes and decreased significantly in the presence of 5 mM unlabeled DHEAS (data not shown). Uptake of DHEAS after subtraction of uptake in the presence of 5 mM unlabeled DHEAS (non-specific uptake) was saturable (FIG. 7). In the Ediedie-Hofstee analysis of DHEAS, a single straight line was obtained as in the case of estrone trisulfate (FIG. 7, inset), and the values of Km and Vmax were calculated to be 62.5 ± 21. 1 μM and 1893 ± 315.7 pmol / mg protein / 10 min.

  To identify transporters that mediate uptake of estrone 3 sulfate and DHEAS in T-47D cells, the expression of OATP and OAT transporters that could transport estrone 3 sulfate was measured by RT-PCR analysis. By using specific primers, among OATP and OAT tested, OATP-D and OATP-E expression was detected in T-47D cells (FIG. 8), but no band was found in PCR without RT reaction. Not detected (data not shown). Thus, OATP-D and OATP-E may be candidate substances that mediate uptake of estrone 3 sulfate and DHEAS in estrogen-dependent breast cancer cells.

3. Discussion Estrone 3 sulfate is the major circulating estrogen and has been implicated in the progression of estrogen-dependent breast cancer (J Steroid Biochem 34: 155-163, 1989). Within tumor cells, it is desulfated by steroid sulfatase and subsequently converted to bioactive estrogens. Because steroid sulfatase has been detected in the cytoplasm of breast cancer cells (Breast Cancer 6: 331-337, 1999), estrone 3 sulfate must have been transported into the cell across the cell membrane prior to desulfation. . In view of the hydrophilicity of estrone trisulfate, internalization of estrone trisulfate appears to be mediated by a specific transport mechanism. In contrast, hydrophobic unconjugated steroid hormones seem to simply diffuse into cells (J Clin Endocrinol Metab 59: 1128-1132, 1984). Therefore, in this study, we examined the presence or absence of a specific transport system that mediates uptake of estrone trisulfate and DHEAS into T-47D breast cancer cells, which are regarded as model cells exhibiting estrogen-dependent growth.

  First, estrogen dependence of cell proliferation was confirmed. Treatment with estrone 3 sulfate or estradiol increased the proliferation of T-47D cells, resulting in an EC50 value of 17.1 nM for estrone 3 sulfate. This value is close to the physiological plasma concentration of estrone trisulfate. Since estrone trisulfate itself induces a low direct biological response at the estrogen receptor (Endocrinology 138: 863-870, 1997), such a result is that estrone trisulfate is internalized across the cell membrane and subsequently cells It is suggested that it is converted into active unconjugated estrogens. Thus, this study focuses on the uptake of estrogen sulfate conjugates by T-47D cells as a first step in elucidating estrogen activity.

Time course of estrone 3 sulfate uptake by T-47D cells suggests that uptake is mediated by specific transporters, as uptake is significantly reduced in the presence of high concentrations of estrone 3 sulfate (1 mM). (FIG. 4). Specific uptake of estrone trisulfate is not affected by replacing Na ⁺ with Li ⁺ , K ⁺ , or N-methylglucamine ⁺ (FIG. 5). OATP and OAT family members are candidates for Na ⁺ -independent estrone 3 sulfate uptake transporters. The Km value of estrone 3 sulfate uptake by T-47D cells was calculated to be 3.9 ± 0.78 μM (FIG. 6). This is consistent with the known Km value range of OATP and OAT from 0.05 μM to 59 μM (Pharm Res 18: 1262-1269, 2001; J Biol Chem 274: 13675-13680, 1999; J Biol Chem 275: 4507 -4512, 2000).

  To further characterize the transport mechanism and identify the transporters involved, inhibition of estrone trisulfate uptake by T-47D cells was studied. The inhibitory action of sulfate conjugates of steroid hormones such as estrone trisulfate and DHEAS was more potent than that of unconjugated steroid hormones, whereas the glucuronide conjugate had no inhibitory effect. Thus, the sulfate moiety may provide high affinity for the transporter, while the glucuronide moiety may be hardly recognized. Since sulfate conjugates have lower ionization constant (pKa) values compared to unconjugated steroids or glucuronide conjugates, these results suggest that anionic moieties are essential for transporter substrate recognition. ing. Such plasma transporters have an effective effect on the supply of estrogen to breast tumors because the plasma concentrations of steroid hormone sulfate conjugates are high compared to unconjugated hormones or their glucuronide conjugates. It should be. Furthermore, DHEAS uptake is also saturated in T-47D cells, and the Km value calculated is 62.5 ± 21.1 μM, which is from the value of estrone 3 sulfate (3.9 μM, FIGS. 6 and 7). Is also quite expensive. The plasma concentrations of estrone 3 sulfate and DHEAS are about 1-10 nM and 1 μM, respectively (Hormone Res 27: 61-68, 1987; J Clin Endocrinol Metab 54: 22-26, 1982), so breast cancer cells are saturated. It may be effective to capture them without.

Some members of OATP, OAT3, and OAT4 can transport steroid hormone sulfate conjugates (Biochim Biophys Acta 1609: 1-18, 2003, Pflugers Arch 447: 653-665, 2003; Breast Cancer 6: 331- 337, 1999; J Biol Chem 274: 13675-13680, 1999). Among them, OATP-B and OAT4 can recognize sulfate conjugates with Km values of 9.04 μM and 1.01 μM, respectively, while having no affinity for steroid hormone glucuronide conjugates. (Pharm Res 18: 1262-1269, 2001; J Biol Chem 275: 4507-4512, 2000). Since the uptake of [ ³ H] estrone trisulfate was not inhibited by glucuronide conjugates in T-47D cells, the functional attributes of OATP-B or OAT4 are related to T-47D cells with respect to substrate recognition of steroid conjugates. Consistent with the uptake mechanism in On the other hand, because OATP-A, OATP-C, OATP8, and OAT3 transport both sulfate and glucuronide conjugates, these transporters transport estrone 3 sulfate in T-47D cells. (Biochim Biophys Acta 1609: 1-18, 2003, Pflugers Arch 447: 653-665, 2003; J Biol Chem 274: 13675-13680, 1999). To gain insight into the key transporter properties, we examined the expression of various human OATP and OAT by RT-PCR analysis (FIG. 8). Expression was detected only in OATP-D and OATP-E, which was consistent with a previous report (J Clin Endocrinol Metab 88: 3902-3912, 2003). OATP-D and OATP-E showed transport activity against estrone 3 sulfate when expressed transiently in HEK293 cells (Biochem Biophys Res Commun 273: 251-260, 2000), so in T-47D cells. It may be involved in the uptake of estrone trisulfate through the cell membrane. However, because their functional characteristics are not well understood, transporters involved in the uptake of estrone 3 sulfate by T-47D cells could not be identified in this study. BCRP (ABCG2) and OSTalpha-OSTbeta have also been reported to be able to accept estrone 3 sulfate as a substrate (Breast Cancer 9: 296-302, 2002; Proc Natl Acad Sci USA 95: 15665-15670, 1998; Biochem Biophys Res Commun 298: 41-452002; Mol Pharmacol 64: 610-618, 2003; J Biol Chem 278: 27473-27482, 2003). Therefore, transporters other than OATP and OAT may be involved in the uptake of estrone 3 sulfate in T-47D cells.

  The above example clearly demonstrated that estrone 3 sulfate is taken up by estrogen-dependent T-47D cells via a specific transport mechanism. This is the first demonstration that the major circulating estrogens, estrone trisulfate and DHEAS are supplied to estrogen-dependent breast cancer cells via specific transporters. Although further research is required to identify transporters in T-47D cells, such transporter molecules would represent new molecular targets for the treatment of estrogen-dependent breast cancer.

It is a schematic diagram which shows the molecular target of breast cancer. It is a figure which shows the uptake | capture to the breast cancer cell of the estrone 3 sulfate by BSP. It is the model which shows the induction | guidance | derivation of ERE (estrogen response element) activity by the estrone trisulfate taken in by the breast cancer cell, and the schematic diagram of the pERE-TA-SEAP vector used for a reporter gene assay. It is a figure which shows the effect of the estrone 3 sulfate on the ERE induction SEAP (Secreted Alkaline phospatase) activity in the MCF-7 cell transiently transfected by pERE-TA-SEAP vector. It is a figure which shows the schematic diagram of an estrogen dependence cell proliferation assay, and its time schedule. It is a figure which shows the effect of the bromo sulfophthalein on the estrogen dependent cell proliferation in MCF-7 cell. It is a figure which shows the time-dependent change (a) and density | concentration dependence (b) of T-47D breast cancer cell proliferation stimulation by an estrogen. (A) T-47D cells were seeded at a density of 8,000 cells / well and cultured. 24 hours after seeding (1st day), estradiol (1 pM, ▲), estrone trisulfate (100 nM, ●), DMSO (0.1%, △) or water (◯) was added. After a predetermined number of days, T-47D cells were trypsinized and counted. (B) T-47D cells in the presence of various concentrations of estradiol (▲) or estrone trisulfate (●) having a concentration range of 10 ⁻¹⁶ to 10 ⁻¹¹ M or 10 ⁻¹¹ to 10 ⁻⁶ M, respectively. Allowed to grow. The number of cells was counted 6 days after seeding. Each value is mean ± S. E. M.M. (N = 3). It is a figure which shows the time-dependent change of [< ³ > H] estrone 3 sulfate uptake by T-47D cell. The cultured T-47D cells were cultured at 37 ° C. in a medium (O) containing [ ³ H] estrone trisulfate (7.1 nM) and further containing 1 mM unlabeled estrone trisulfate (●). Incubated for minutes. Each value is mean ± S. E. M.M. (N = 4). If error bars are not displayed, they are smaller than the symbol. It is a figure which shows the effect | action of the extracellular cation with respect to the uptake of [< ³ > H] estrone trisulfate by a T-47D cell. The uptake rate of [ ³ H] estrone trisulfate (7.1 nM) was measured over 10 minutes in the presence or absence of extracellular Na ⁺ . Extracellular Na ⁺ was replaced with K ⁺ , Li ⁺ , or N-methylglucamine ⁺ (NMG ⁺ ). Each value is mean ± S. E. M.M. (N = 4). It is a figure which shows the density | concentration dependence of the estrone 3 sulfate uptake | capture by a T-47D cell. The uptake of various concentrations of estrone trisulfate from 5.1 nM to 50 μM was measured at 37 ° C. for 10 minutes. After subtracting uptake in the presence of excess unlabeled estrone trisulfate (1 mM), saturable uptake was obtained and used to evaluate rate constants by nonlinear least squares analysis. Inset shows Eadie-Hofstee plot of saturable uptake of estrone 3 sulfate. Each value is mean ± S. E. M.M. (N = 4). If error bars are not displayed, they are smaller than the symbol. It is a figure which shows the density | concentration dependence of DHEAS uptake | capture by a T-47D cell. Uptake of DHEAS at various concentrations from 10.3 nM to 100 μM was measured at 37 ° C. for 10 minutes. After subtracting uptake in the presence of excess unlabeled DHEAS (5 mM), saturating uptake was obtained and used to evaluate rate constants by non-linear least squares analysis. Inset shows Eadie-Hofstee plot of DHEAS saturable uptake. Each value is mean ± S. E. M.M. (N = 4). If error bars are not displayed, they are smaller than the symbol. It is a figure which shows the expression of OATP and OAT transporter in T-47D cell. RT-PCR analysis was performed using mRNA obtained from T-47D cells. Reactions were performed as described in Materials and Methods. Arrows indicate specific bands for OATP-D and OATP-E.

Claims (6)

Estrone trisulfate transporter containing as an active ingredient one or more components selected from bromosulfophthalein, dehydroepiandrosterone sulfate, dehydroepiandrosterone, taurocholic acid, p-aminohippuric acid, tetraethylammonium, probenecid and benzylpenicillin Inhibitors of transporter activity. Inhibitor of transporter activity of estrone trisulfate transporter containing bromosulfophthalein as an active ingredient. Breast cancer cell growth inhibitor comprising as an active ingredient one or more components selected from bromosulfophthalein, dehydroepiandrosterone sulfate, dehydroepiandrosterone, taurocholic acid, p-aminohippuric acid, tetraethylammonium, probenecid and benzylpenicillin . A breast cancer cell growth inhibitor comprising bromosulfophthalein as an active ingredient. A therapeutic agent for breast cancer comprising as an active ingredient at least one component selected from bromosulfophthalein, dehydroepiandrosterone sulfate, dehydroepiandrosterone, taurocholic acid, p-aminohippuric acid, tetraethylammonium, probenecid and benzylpenicillin. A breast cancer therapeutic agent containing bromosulfophthalein as an active ingredient.