| Summary: | 碩士 === 國立臺灣大學 === 臨床醫學研究所 === 91 === 七、論文英文簡述 (Summary):
Introduction:
Phytoestrogens include isoflavones from soy, lignans from flax and fiber-rich vegetables, and coumestrol from alfalfa sprouts. The major isoflavones are genistein and daidzein. These compounds and their precursors have all been isolated in human urine, serum, feces, semen, and bile. The lignans and isoflavones have weak estrogenic and antiestrogenic activity due to estrogen receptor affinity. The lignans and isoflavones have been shown to have antiviral, anticarcinogenic, bacteriocidal, and antifungal effects, while isoflavones also possess antioxidant, antimutagenic, anti-inflammatory, and antiproliferative properties.
The mean daily intake of isoflavones was 1.2mg in USA, which is lower than the 200mg/day reported for Japanese. Among women from Asian populations, characterized by the consumption of large quantities of soy products, serum levels of testosterone and estradiol have been found to be 20-50% lower than in Western women and inversely related to the consumption of soy products. Recent studies have provided strong evidence that high serum levels of testosterone and estradiol increase the risk of developing breast cancer in postmenopausal women. The major source of estrogen in postmenopausal women is aromatization of androstenedione to estrone in peripheral tissue such as adipose.
Recent publications have highlighted the difference between countries in the incidence of many diseases, including coronary heart disease, breast cancer, endometrial cancer, ovarian cancer and prostate cancer, as well as that of menopausal symptoms. These have been attributed to racial characteristics, diet, and life-style. The role of diet, particularly the content of phytoestrogen is attracting interest.
Phytoestrogens are among the dietary factors affording protection against cancer and heart disease. The majority of the evidence regarding the effects of phytoestrogens in humans is epidemiologic. It has been suggested that phytoestrogen may contribute to prevention of estrogen—dependent cancers by some of the same mechanisms as pharmaceutical antiestrogens.
According to studies by Strom et al, an inverse association between coumestrol and daidzein and prostate cancer risk was shown, a positive association was found between campesterol and stigmasterol and risk of prostate cancer. Decreased prostate cancer risk has been shown in Adventist men who consumed high amounts of beans, lentils, peas, and dried fruits rich in flavonoids and among Japanese men in Hawaii who consumed much rice and tofu, a soybean product containing isoflavones.
Many chemicals such as pesticides, insecticide, fungicide, diethylstilbestrol, and PCB were found to have hormone-like effect. Exposure to such chemicals may be responsible for adverse effects in both humans and wildlife. These chemicals were so-called endocrine-disrupting chemicals. The endocrine effects of these chemicals are believed to be due to their ability to: (1) mimic the effect of endogenous hormones, (2) antagonize the effect of endogenous hormones, (3) disrupt the synthesis and metabolism of endogenous hormones, and (4) disrupt the synthesis and metabolism of hormone receptors.
Diverse animal models and assays have been used to measure estrogenicity. The proliferative effect of estrogen on the female genital tract has remained the hallmark of estrogen action. This requires measuring the increase of mitotic activity in tissues of the female genital tract after estrogen administration. This approach is not suitable for large-scale screening of suspected chemicals and an equally reliable, easy and rapid method would be preferable, such as that using established estrogen-sensitive cells in culture to measure the proliferative effect of xenoestrogens.
The E-SCREEN assay was developed to fulfill these requirements. (C. Sonnenschein-1995) Quantitative bioassays using cells in culture have been developed to screen large number of chemicals for estrogenic activity in addition to E-SCREEN assay. They are based on estrogen-induced gene expression, both of endogenous genes and reporter genes. Induction of reporter genes under the control of estrogen-responsive elements have been proposed to assess estrogenicity; however, elevated basal expression in the absence of estrogen often occurs and this may raise concern about the reliability of these assays. The MCF7 cell proliferation assay may also be used to assess the antiestrogenic properties of chemicals. A chemical is considered a true estrogen antagonist when (a) it does not affect cell number in the absence of estrogens, (b) it blocks the proliferative effects of estradiol, and (c) this block is reversed by merely increasing the dose of the estradiol while keeping constant the dose of the antagonist.
C. Sonnenschein also developed A-SCREEN assay using LNCaP cell line. To date, no androgen agonists have been found among environmental chemicals. Several chemicals such as vinclozolin and DDE were verified to be androgen antagonists. No reference is available about the topic of androgenicity and anti-androgenicity of isoflavone. In 1996, the Safe Drinking Water Act and the Food Quality Protection Act required that the U.S. Environmental Protection Agency develop screening and testing programs for endocrine-disrupting chemicals. To address these issues, the EPA formed a multi-stakeholder Endocrine Disruptor Screening and Testing Advisory Committee to make recommendations on how to address this legislative mandate. In 1998, the EDSTAC completed a final report recommending a 2-tiered screening and testing program designed to identify and characterize chemicals that had anti-estrogenic, anti-androgenic and anti-thyroidal activities. Hartig and Wilson et al reported several new androgen receptor gene expression assays in 2001.
Androgens and the androgen receptor (AR) play an important role in the growth of prostate cancer and normal prostate. Androgen ablation has been the cornerstone of treatment in patients with locally advanced or metastatic prostate cancer. Deprivation of androgen will cause marked shrinkage of prostate cancer. However, hormone-insensitive cancer cells will survive and prosper later. The mechanisms responsible for androgen independence remain unclear. Immunohistochemical staining showed AR expression may contribute to the response to hormone therapy. Several studies have also postulated that the alterations of AR functions by mutations of this gene may be associated with a poor response to antiandrogen. However, all the documented results cannot rule out the contribution of cellular environment and non-AR factors to the alteration of androgen activity.
The androgen receptor (AR) functions as a ligand activated transcription factor that may play critical roles in prostate cancer growth and sexual development. It has been known that the androgen receptor regulates androgen target genes by binding to androgen response elements with the potential involvement of coactivator or corepressor. Several coactivators were found in the George Whipple Laboratory such as ARA70, ARA55 and ARA54. There are increasing evidences to show that the AR cofactors play an important role in AR function.
The discovery of transcriptional interference of steroid receptors (SR) provided the concept of the existence of transcriptional cofactors that mediate SR function. Katzenellenbogen et al proposed a new tripartite system (ligand-receptor-cofactor) to explain the molecular interactions of steroid receptors that may define the potency and biological character of steroid hormones. The wild-type estrogen receptor and its mutants have either agonist or antagonist response to different antiestrogens in different cell environments. Additionally, the differing responses to estrogen and antiestrogens do not seem to be the result of a change in the estrogen receptor expression level or its binding affinity for ligands. Their data support the hypothesis that different cells may provide different cell contexts, like cofactors, which may be able to interact with the estrogen-estrogen receptor or antiestrogen-estrogen receptor complexes, and subsequently activate or inhibit the transcriptional activity.
Shuyuan Yeh et al in the George Whipple Laboratory reported that ARA70, a ligand-dependent AR-associated protein, was isolated by yeast two-hybrid system in 1996. Sequence of ARA70 was confirmed by dideoxy method from positive clones isolated from yeast two-hybrid system and RACE-PCR. DU145 cells were co-transfected with ARA70 and AR under eukaryotic promoter control. Addition of DHT (dihydrotestosterone) results in a 6-fold increase of AR activity. This transcriptional activity can be increased to 58-fold by the co-transfection of ARA70 cDNAs in a dose-dependent manner. They reported the cloning of ARA55 using a yeast two-hybrid system in 1999. Transient transfection assay in DU145 cells demonstrated that ARA55 could enhance AR transcriptional activity in the presence of 1nM DHT. ARA55 is more general to SRs whereas ARA70 is more specific to AR. ARA55 has a lesser effect than ARA70 on AR-mediated transactivation in the presence of estradiol and hydroxyflutamide.
Miyamoto et al in the George Whipple Laboratory reported that antiandrogens could promote the interaction between AR and its coactivator in a dose-dependent manner. They tested hydroxyflutamide, bicalutamide, cyproterone acetate, and RU58841. A mammalian two-hybrid assay was used. The addition of 10μM antiandrogens can induce the CAT activity 3-6 fold over the basal level in the presence of ARA70. To test the agonist activity of these antiandrogens in conditions close to the maximal androgen ablation therapy, they set up a transient transfection system. The DU145 cells were cultured with charcoal-stripped serum in the presence of 0.1, 1, 10μM antiandrogen. AR transcriptional activity could be induced 5-7 fold when AR was expressed with 1nM DHT. ARA70 can further enhance the transcriptional activities of these AR to 28-45 fold in the presence of 1nM DHT. Upon the transfection of wild-type AR in the absence of ARA70, only marginal inductions were detected in the presence of these antiandrogens. The addition of ARA70, at the ratio of 1:3 to AR, further enhanced the AR transcriptional activity to 5-12 fold.
Clinical data indicate that hydroxyflutamide can activate the AR-targeted prostatic specific antigen gene and that serum levels of the prostatic specific antigen can decrease after discontinuation of hydroxyflutamide. This phenomenon is known as flutamide withdrawal syndrome. According to studies of Miyamoto, the agonist activity of antiandrogens might occur with the proper interaction of AR and ARA70 in DU145 cells.
Shuyuan Yeh et al reported that ARA70 could induce AR transcriptional activity > 30-fold in the presence of 10nM 17β-estradiol (E2), but not diethylstilbestrol (DES). Using a yeast two-hybrid system under a serially diluted concentration of different hormones, they found that 50nM E2 could induce an interaction between ARA70 and GAL4DBD-AR, but DES and other nonandrogenic steroids were unable to induce any interaction. Using DU145 cells, MMTV-CAT assay showed that ARA70 could induce AR transcriptional activity > 30-fold in the presence of 10nM 17β-estradiol (E2). Only AR, but not ER, PR, or GR, can significantly induce MMTV-CAT activity in the presence of 1-10nM E2 and ARA70. As there is no estrogen response element in MMTV promoter-CAT reporter, E2 cannot activate ER activity. Testosterone/dihydrotestosterone may not be the only ligands for the AR. E2 represents another important natural ligand for AR that may play an essential role for the AR function. As several estrogenic chemicals may play a role in the disruption of normal endocrine functions in humans and other animals, it is interesting to know whether any estrogenic endocrine-disrupting chemicals also have some androgenic activity that may contribute to the disruption of the endocrine system.
Wilson et al developed a novel stable cell line, MDA-kb2, for screening of androgen agonist and antagonists and to characterize its specificity and sensitivity to endocrine-disrupting chemicals. MMTV-luciferase was used as reporter gene. Hartig et al developed two androgen receptor assays using adenoviral transduction of MMTV-Luc reporter and/or hAR for endocrine screening. In their report 17β-estradiol (E2) was showed to be AR agonist, hydroxyflutamide acted as AR agonist in high concentration but as AR antagonist in low concentration.
Isoflavones are shown to compete with estradiol for the estrogen receptor and have weak estrogenic and antiestrogenic activity. No literature was reported about the topic of androgenicity and anti-androgenicity of isoflavone. To date, no androgen agonists have been found among environmental chemicals. According to the description above, we want to know whether any phytoestrogen has some androgenic activity in the presence of different AR cofactors. We set up a screening model to test the ability of inducing AR transcriptional activity by various isoflavone. PC3 cell culture and transfection with pSG5-AR and AR cofactors were used in our model. Dual-luciferase reporter assay was used for comparison of effect by addition of different test substances. Our purpose is to make an effective and reliable model for screening androgenicity and anti-androgenicity of phytoestrogen and other substance. If positive results are obtained it will be a new area for more research in endocrinology and will help in prevention of prostate cancer.
Materials and Methods:
PC3 cell culture was co-transfected with pSG5-AR and AR cofactors plasmids. Dual-luciferase reporter assay was used for comparison of effect by addition of different test substances.
Androgen and antiandrogens: DHT, 17β estradiol, genistein, daidzein, biochanin were purchased from Sigma.
Plasmids: pSG5-AR, pSG5-ARA70, pSG5-ARA55, MMTV-Luciferase are constructed as description on reference by Dr Chang, when he was in George Whipple laboratory for cancer research of Rochester University. pRL-TK, pRL-CMV were purchased from Promega Inc.
Cell culture and transfection: Human prostate cancer PC-3 cells were maintained in Dulbecco,s minimum essential medium containing with 1% penicillin-streptomycin, and5% fetal calf serum. About 4x105 cells were plated on 12 well dish 24hour before transfection. pSG5-AR, MMTV-Luc, pRL-CMV were mixed with serum-free DMEM. Each 4 wells was set as one transfection unit, each unit has 6μg DNA for superfect kit. Transfections were performed using SuperFectâTransfection Reagent (QIAGEN). Two hours after transfection, the medium was changed again, and the cells were treated with DHT, 17β estradiol, or one of the isoflavone (genistein, daidzein, or biochanin) for 24 hours. Then the cells were harvested and whole cell extracts were used for Dual-Luciferase reporter assay. At least three independence experiments were carried out in each case.
Study design: We demonstrate the effect of inducing AR transcriptional activity in presence of10-5 ~10-7M DHT with different AR cofactors, various AR: AR cofactor ratio. In addition to DHT, serial concentration of 17β estradiol, genistein, daidzein, and biochanin were all tested. Different AR cofactors, various AR: AR cofactor ratio were compared in assessing the effect of inducing AR transcriptional activity in presence of 10-4 ~10-6M of these reagents.
Results:
Negative control group revealed reliability of the system of assay. Positive control group revealed effectiveness of DHT and validity of Dual-Luciferase reporter assay.
AR transcriptional activity could be induced about 9 fold when AR was expressed with 10-7M DHT, ARA55 can enhance the transcriptional activity to 11-19 fold in the presence of 10-7M DHT. AR transcriptional activity could be induced about 17 fold when AR was expressed with 10-5M DHT, ARA55 can enhance the transcriptional activity to 23-40 fold in the presence of 10-5M DHT by various AR: AR cofactor ratio.
AR transcriptional activity could be induced about 23 fold when AR was expressed with 10-5M 17β estradiol, ARA55 can enhance the transcriptional activity to 38-50 fold in the presence of 10-5M 17β estradiol by various AR: AR cofactor ratio. However, no enhancements of AR transcriptional activity were found in serial concentration of genistein, daidzein, and biochanin. The only exception is 10-4M daidzein, ARA55 can enhance the transcriptional activity up to 6 fold.
ARA70 failed to achieve comparable results as ARA55 did in test of DHT, 17β estradiol, or any one of the isoflavone (genistein, daidzein, or biochanin). The only exception is 10-4M daidzein, ARA70 can also enhance the transcriptional activity up to 6 fold.
Conclusion:
Our result revealed that the model using PC-3 cell, transient transfection, Dual Luciferaseâ Reporter assay is an effective and reliable model. ARA55 could enhance the AR transcriptional activity induced by DHT and 17β estradiol. In tests of several isoflavones, the only significant result attained is that both ARA55 and ARA70 can enhance the AR transcriptional activity up to 6 fold when 10-4M daidzein was tested. This finding implies that more study is deserved for daidzein.
Key words: Androgen receptor, Androgen receptor cofactor, Phytoestrogen
|
|---|
