In mice fed a diet supplemented with red clover isoflavones the prostatic epithelium displays a significant increase in the production of estrogen receptor β and the adhesion protein E-cadherin but a decrease in transforming growth factor β1. These proteins are estrogenically-induced markers of proliferation, maintenance of histological architecture, preservation of cell phenotype and reduction of the potential for neoplastic and metastatic transformation. This study suggests that red clover isoflavones represent a non-toxic dietary treatment for prostatic hyperplasia and a reduction in the potential for neoplastic transformation.
Both benign prostatic hyperplasia (BPH) and prostate cancer are processes of excessive and uncontrolled growth.1 BPH is a very common condition in middle aged men and has also been observed in mature rats.2,3 BPH is currently thought not to be a precursor of prostate cancer, but the two conditions are often found together. In the Western world, the incidence of prostate cancer is increasing at an alarming rate, having more than doubled in the past 5 y. In Australia, prostate cancer has overtaken lung cancer as the biggest cancer killer of men4 with 4150 new cases and 817 deaths (1995 data) in NSW alone. This disease is characterised by a striking racial variation. It is most prevalent in African-Americans, intermediate in Caucasians, slightly lower in Latinos and has the lowest incidence in Asians. For instance, in the West the annual incidence of prostate cancer is 91.2 per 100 000 people (age standardised) but in China it is only 1.8 per 100 000.5 United Nations data from 59 countries have shown that diet, not racial predisposition, is the primary influencing factor. Western-style diets contain increased fat, low calcium and vitamin D which contribute to the induction of epithelial cell hyperproliferation.6 Asian diets however, contain a high proportion of legumes which confer health benefits including a lower incidence of cancer and cardiovascular disease.4,7,8,9 The reason for this is thought to be that legumes are a rich source of the isoflavonic phytoestrogens: daidzein, genistein, formononetin and biochanin and produce bioactive metabolites such as equol. Isoflavones inhibit cellular proliferation, protect against reactive oxygen species and loss of cell adhesion in a variety of prostate carcinoma cell lines.10,11 These compounds are converted by intestinal bacteria into weakly estrogenic diphenols which act as weak estrogens, influencing androgen and estrogen production, altering metabolism, protein synthesis and growth factor expression. They also retard some of the most important characteristics of cancer cell function including proliferation, de-differentiation, loss of cell adhesion, tumour angiogenesis, apoptosis and metastatic invasion.4,12
Hormone-related cancers, namely breast, endometrium, ovary, prostate, testis, thyroid and osteosarcoma, share a unique mechanism of carcinogenesis. Endogenous and exogenous hormones drive cell proliferation, and therefore the opportunity for the accumulation of random genetic errors.13 While the androgen-dependence of the prostate gland has long been accepted, the participation of estrogen in BPH has only recently been recognised. Early hyperplasia is particularly sensitive to estrogenic stimulation.14 In the castrated dog, estradiol also causes marked dose-dependent hyperplasia of the prostate.15
Estrogens exert their actions via specific nuclear receptors that are members of the steroid/thyroid receptor superfamily of transcription factors.16 After ligand binding, these receptors become transcriptionally active in the nucleus.17 Estrogen receptor alpha (ERα) is not expressed anywhere in the prostate.18 Instead, in normal human and rat prostate epithelial cells, estrogenic action is signalled exclusively by ERβ which regulates growth-inhibition.19 The ERα and ERβ receptor subtypes are regulated by separate mechanisms and have distinct biological roles.20 Based on this information, it would seem logical to use exogenous estrogen as a treatment for BPH and prostate cancer. The use of synthetic estrogen (DES) in the treatment of advanced prostate cancer was pioneered by Charles Huggins in the early 1940s.21 Recent investigations have demonstrated that DES exerts growth-inhibitory effects on prostatic cancer cells via mitotic arrest or apoptosis.22 Unfortunately, because of serious side effects such as feminisation, exacerbation of heart failure, vascular complications, gynecomastia and impotence, this treatment has lost favour.23
Transforming growth factor beta 1 (TGFβ1) is a potent inhibitor of proliferation and is expressed as a response to the excessive growth that is characteristic of both BPH and prostate cancer.24,25,26 It is found in the hyperplasic basal prostatic epithelium of middle aged men and is also increased in prostate cancer.27,28 Estrogen receptor-mediated mechanisms are known to regulate both TGFα and TGFβ1 expression.29 Androgens also regulate TGFβ1 expression. Estrogen (and phytoestrogens) are also thought to cause an ‘anti-androgenic effect’ which reduces TGFβ1 expression.25,26,30
E-cadherin is a calcium-dependent cell adhesion molecule that plays a crucial role in maintaining tissue architecture. This protein anchors cells to their surrounding extracellular matrix by expressing immediately adjacent to the plasma membrane.31 E-cadherin is known to be a suppressor of de-differentiation, neoplastic change,32,33 phenotypic transformation and tumour progression.34 In both prostate cancer and transitional cell neoplasia of the urinary bladder, loss of E-cadherin has also been associated with invasion and metastasis.35,36 Estrogen modulates E-cadherin levels in vivo and may promote breast, uterine and ovarian cancer by down-regulating E-cadherin levels in these tissues.37
This study tests the hypothesis that isoflavones have a similar anti-proliferative and anti-cancer activity to that of estrogen, but without the undesirable side effects. Three hormonally-modulated proteins that play a role in both BPH and cancer, ERβ, TGFβ1 and E-cadherin, were selected as markers for this study.
Materials and methods
Fifteen male Wistar rats approximately 12 weeks of age were divided at random into three groups of five. The animals were housed in plastic cages under a controlled 12 h:12 h light:dark cycle and given food and tap water ad libitum. The first group was fed a diet containing 5% of a proprietary red clover isoflavone extract (Novogen Ltd) red clover, providing a rich source of all four important dietary isoflavones, namely formononetin biochanin, genistein and daidzein. The second group was fed a diet containing no isoflavones (by exclusion of soya and alfalfa from the diet formulation), while the third was fed a diet of normal rat pellets.
At the end of 14 months, the animals were killed with an intraperitoneal injection of 0.3 ml sodium pentabarbitone (Nembutal, Boehringer, Ingelheim, Germany). Approved ethical guidelines were followed. The entire prostate was removed in each case, cut in half and placed in 4% paraformaldehyde for 6 h. At the end of this period the specimens were washed in buffer, dehydrated, cleared in xylene, infiltrated in paraffin wax at 58°C and embedded. The entire cross-sectional area of each embedded prostate was sectioned at a thickness of 7–10 µm and placed on ‘Superfrost plus’ microscope slides (Selby, Sydney, Australia).
Each block representing one half of the prostate from each rat was sectioned twice, at different depths. The first series of sections were taken near the surface of one paraffin block and the second series approximately 10 sections (100 µm) into the block. Ten acini were selected at random from sections taken from each series. This process was repeated with the other block, resulting in a total of 40 determinations of labelling intensity per rat. As there were five rats in each group (‘n’), this equalled (40 determinations per rat multiplied by five rats) 200 determinations per treatment group. The acini to be photographed were chosen at random from those present on the slides, which averaged 249±90 acini per slide. Random selection was carried out as follows: using the 10× objective, the acinus nearest to the center of the each field of view (FOV) was identified and then photographed using the 25× objective. Replacing the 10× objective, the stage was moved one entire FOV in the X-axis and the procedure repeated. At the lateral margins of the tissue section, the stage was moved one FOV in the Y-axis and then moved in the opposite direction along the X-axis, repeating the procedure. This process was continued in a chequerboard pattern until 10 acini had been photographed. This system ensured coverage of most of the section on each slide.
The sections were de-waxed in two changes of fresh Histoclear for 10 min each and then rehydrated. Antigen retrieval was achieved by a 30 min incubation in Target Retrieval Solution, DAKO (DAKO, Carpenteria, CA, USA), at 95°C in a 2 l beaker using metal slide racks. The slides were allowed to cool to room temperature by placing the 2 l beaker in a water bath at room temperature. To block non-specific binding, the sections were incubated for 5 min in 1% hydrogen peroxide in 1% bovine serum albumin in phosphate buffered saline (PBS), and washed in PBS for 3×5 min. They were then incubated with 5% horse serum for 5 min. At this stage they were not washed. Instead the horse serum was drained off and replaced by the primary antibody.
The slides were then immunolabelled using an avidin-HRP-tyramide amplification protocol using a TSA-indirect tyramide signal amplification kit (NEN Life Science, Boston, MA, USA). Sections from each of the five blocks from each treatment group were incubated with the following antibodies respectively: chicken anti-human TGFβ1, (R&D Systems, Minneapolis, MN, USA), mouse anti-human E-cadherin (Zymed Laboratories, San Fransisco, CA, UAS), and rabbit anti-human estrogen receptor β (Affinity Bioreagents, Golden, CO, USA). Each antibody was used at a concentration of 1:100 with PBS for 30 min.
The sections were first incubated in TNB Block buffer (a supplied kit component) for 30 min at room temperature. The slides were drained and incubated with primary antibody diluted 1:100 with TNB Block. They were then washed for 3×5 min with TNT buffer (supplied) and incubated with secondary antibody diluted 1:100 with TNB Block. After a 3×5 min wash in TNT buffer, the sections were incubated with biotinyl tyramide working solution (supplied) for 10 min at room temperature. Following this step, they were washed for a further 3×5 min with TNT buffer and incubated with streptavidin-HRP working solution (supplied) for 30 min at room temperature. The slides were then washed three times in PBS for 10 min each, visualised using a 0.05% solution of diamino benzidine (DAB) for 10 min, washed, dried and mounted in Entellin mounting medium (Merck).
Tissue that was previously known to stain positively for each respective antibody was used as a positive control. Negative controls for each labelling parameter were established by incubation with normal chicken, mouse or rabbit pre-immune serum (1:25 dilution) in bovine serum albumin (BSA) in PBS and also by omission of the primary antibody. In each case, this procedure resulted in no labelling. The sections were also incubated with a monoclonal antibody of the IGM isotype, which does not react with any known rat protein (Silenus), as IGM aggregates may sometimes cause non-specific labelling. This procedure also resulted in no apparent labelling.
Antigen retrieval and signal enhancement controls
Tissue from each rat in each group was also labelled with a routine immunoperoxidase (IPX) protocol only, an antigen retrieval (AR) protocol followed by IPX, a tyramide signal enhancement (TSA) protocol (that included IPX labelling) and an AR plus a TSA protocol.
One microgram of human TGFβ1 (Sigma, MO, USA) was mixed with 30 µl of distilled water. This was thoroughly mixed with 30 µl of a 1 in 50 dilution of TGFβ1 antibody, and incubated with the prostate tissue sections as previously described. No labelling resulted. Estrogen receptor β and E-cadherin are not available commercially as proteins.
Labelling intensity quantification
Actual levels of antigen were not quantified in this study, but rather relative differences in labelling intensity using a standardised protocol. Differences in relative labelling density were measured using previously published methods.3,38,39,40 In short, sections to be compared were labelled at the same time, using a single protocol. A Leica DC 200 digital camera using Leica ‘DC Image’ capture software was mounted on a Letiz Diaplan research microscope. The microscope illumination was set on 5 volts and was not altered. The exposure compensation option of the camera was deactivated and the resolution set at 1798×1438 ppi. These precautions ensured that all the images were taken under the same conditions and could therefore be validly compared. The images were saved in the TIFF image format, transferred to a Macintosh G4 computer (Macintosh, Eupertino, CA, USA), and opened without alteration directly into the NIH Image 1.6 application (written by Wayne Rasband and available on the Internet at http://rsb.info.nih.gov/nih-image/). This resulted in image of each acinus that occupied approximately half of the area of the computer monitor screen. Using the freehand selection tool, the prostatic epithelium (only) of each acinus was selected and the labelling intensity of the image was analysed, resulting in a mean and standard deviation value. Epithelial selection reproducibility was tested by outlining a single defined area of epithelium 10 times, resulting in a variation coefficient of only 1.0%. The Alternative Welch t-test (two tailed P value) was chosen for analysis of the data because it does not rely on the assumption that the sampled populations have equal standard deviations. It is more conservative than Student's t-test and results in a higher P-value and wider confidence interval. The null hypothesis was that the two population means were equal.
The prostatic stroma did not label using any of the antibodies. The label for TGFβ1 appeared to be punctal in nature and located in the basal epithelium (Figure 1a). The most dense label was present in the phytoestrogen-free treatment group (Figure 1a), the least intense label was present in the red-clover treatment group (Figure 1b), while the normal rat cubes (Figure 1c) showed a slightly greater intensity than the red clover group. Quantification of these labelling densities revealed that the mean labeling intensity of TGFβ1 was 160.4±32.8 in the group fed an isoflavone-free diet, 82.9±17.1 in the group fed normal rat cubes and 44.9±9.5 in the group fed a diet supplemented with 5% red clover extract. The units given are arbitrary, in that they represent pixel intensity values as assigned by the NIH Image application. These data represent a 3.6-fold decrease (P<0.0016) caused by a diet of 5% red clover, compared to an isoflavone-free diet. There was also a 1.9-fold TGFβ1 decrease (P<0.0034) in animals fed normal rat cubes, compared to those fed a isoflavone-free diet. Similarly, a 1.8-fold decrease (P<0.0049) was observed in red clover-treated animals compared to those fed normal rat cubes.
The label for anti-estrogen receptor β (ERβ) also appeared to be punctal in nature and located in the basal epithelium (Figure 1a). The least intense label was present in the isoflavone-free treatment group (Figure 2a). The most dense label was seen in the red clover treatment group (Figure 2b). The normal rat pellet treatment group label (Figure 2c) appeared to be of a similar intensity to that of the isoflavone-free treatment group. Not only was the labelling intensity different between the groups, but the location aswell. In the red clover group the label was nuclear whereas in the normal and isoflavone-free groups the label was cytoplasmic, the nuclei being devoid of label. Quantification of these labelling densities revealed that the mean labelling intensity of anti-ERβ was 15.8±9.0 in the isoflavone-free group, 27.9±17.3 in the group fed normal rat cubes and 158.7±66.5 in the group treated with 5% red clover. These data represent a labelling intensity increase of 9.9-fold (P<0.0089) resulting from a diet of 5% red clover, compared to an isoflavone-free diet. There was also a 1.7-fold increase, although not significant (P<0.2146), in ERβ in animals fed normal rat cubes compared to those fed an isoflavone-free diet. A significant increase (P<0.0131) of 5.7-fold was observed in red clover-treated animals compared with those fed normal rat cubes.
The label for the calcium-mediated, cell adhesion protein E-cadherin was located immediately adjacent to the plasma membrane of the prostate epithelial cells, forming an interconnecting meshwork. The least intense label was that of the isoflavone-free treatment group (Figure 3a). The most intense label, resulting in the most intense cell outlines, was seen in the red clover diet group (Figure 3b). While the normal rat pellet diet group appeared to have a slightly greater labelling intensity than that of the red clover group, statistical analysis demonstrated that this difference was insignificant (Figure 3c). Quantification of these labelling densities revealed that the mean labelling intensity for anti-E-cadherin was 9.9±5.8 in the phytoestrogen-free group, 15.5±13.3 in the group fed normal rat cubes and 53.9±10.5 in the group treated with 5% red clover. These data indicate an increase of 5.4-fold (P<0.0002) resulting from a diet of 5% red clover, compared to an isoflavone-free diet. Similarly, there was a 3.5-fold increase (P<0.0015) in E-cadherin levels in red clover-treated animals compared to those fed normal rat cubes. There was an 1.6-fold increase (not significant, P<0.4276) between the E-cadherin levels resulting from a diet of normal rat cubes compared to a phytoestrogen-free diet.
The controls in which the primary antibody was omitted from the IPX protocol were virtually transparent and difficult to distinguish from the glass of the microscope slide. The haematoxylin and eosin-stained slides of the prostatic epithelium were similar in appearance for each group. The control sections in which pre-immune serum was substituted for the primary antibody in an antigen retrieval (AR) IPX protocol, were not labelled. The addition of Tyramide Signal Amplification (TSA) to the AR IPX pre-immune serum controls also resulted in no labelling. Routine IPX labelling using the relevant antibodies without AR or TSA also resulted in no label. The addition of TSA but with no AR also resulted in no label. The use of AR followed by IPX but no TSA did result in a faint but definite label. Maximal labelling was achieved by AR followed by IPX followed by TSA. This proved to be the case for both anti-TGFβ1 and anti-ERβ receptor. Anti-E-cadherin labelled satisfactorily with AR retrieval and IPX alone, but TSA enhancement improved the clarity and intensity of the label without any increase in non-specific background.
In the this study, a diet supplemented with red clover isoflavones caused ERβ receptor components to express as functional receptors in the nucleus. ERβ is known to inhibit excessive cell proliferation, a process that is characteristic of both BPH and prostate cancer. Both the isoflavone-free and normal rat pellet diets failed to cause expression of ERβ in the nucleus. In the rats fed normal rat pellets and an isoflavone-free diet, the ERβ label was entirely cytoplasmic indicating that the ERβ receptor components had not been activated.
Similarly, in animals fed a diet containing 5% red clover extract the increased labelling intensity of the adhesion protein E-cadherin was observed along the plasma membranes of all prostatic epithelial cells. This indicates an increase in the expression of cell adhesion proteins between adjacent cells resulting in the preservation of histological architecture, prostatic epithelial phenotype and a reduction of the potential for neoplastic transformation.
A diet supplemented with red clover isoflavones (and to a lesser extent a normal rat cube diet containing low isoflavone levels) resulted in a significant reduction in TGFβ1 expression in the basal prostatic epithelium, compared to an isoflavone-free diet. This finding suggests that isoflavones in the diet decrease pathological proliferation of the prostatic epithelium and reduce the need for the (anti-proliferative) expression of TGFβ1. It has been reported that dietary genistein can down-regulate epidermal growth factor (EGF) which also has an anti-proliferative effect in the rat prostate. No apparent adverse toxicity was observed in the host.41 Another study has shown that genistein induces prostate cancer cell adhesion (thereby reducing metastatic potential) and growth inhibition (thereby counteracting hyperplasia) by acting as a protein-tyrosine kinase inhibitor.42
This study demonstrates that, in the prostatic epithelium, red clover isoflavones in the diet produce a significant increase in the production of ERβ and the adhesion protein E-cadherin but a decrease in the label for TGFβ1. These proteins are estrogenically-modulated markers for proliferation, maintenance of histological architecture, preservation of cell phenotype and reduction of the potential for neoplastic and metastatic transformation. This study suggests that dietary supplementation with red clover isoflavones may provide a non-toxic dietary treatment for prostatic hyperplasia with a concomitant reduction in the potential for development of prostate cancer.
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Slater, M., Brown, D. & Husband, A. In the prostatic epithelium, dietary isoflavones from red clover significantly increase estrogen receptor β and E-cadherin expression but decrease transforming growth factor β1. Prostate Cancer Prostatic Dis 5, 16–21 (2002). https://doi.org/10.1038/sj.pcan.4500546
- prostate cancer
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