Johannes Huber1 and Andrea R. Genazzani2
(1)
Department of Gynecologic Endocrinology and Reproductive Medicine, Medical University Vienna, Vienna, Austria
(2)
Division of Gynecology and Obstetrics, University of Pisa, Pisa, Italy
Johannes Huber
Email: johannes.huber@meduniwien.ac.at
Isoflavones have emerged as one of the plant components studied most thoroughly by medical researchers in recent decades; in the scientific discussion, the wide variety of their effects draws attention to ever-new aspects that explain the protective effect of this group of substances that have been part of the human diet for millennia. The most recent aspects include the connections between bones, calcium and endoplasmic reticulum, the anticipatory effect of oestradiol in this cell compartment and the significance isoflavones have in this biological response pattern.
22.1 Endoplasmic Reticulum, Bones and Isoflavones
The endoplasmic reticulum can be described as the small bone of the cell – its functions are similar, and it performs on a small scale what the bones do for the body overall: it stores calcium and is controlled by the sexual steroids.
22.1.1 The Biophysical Background
As elements of the first two groups of the periodic table, alkali metals have only a weakly bound s electron that they easily surrender; alkaline earth metals are strong reducing agents and are particularly suitable for the flow of electrons that forms the basis of biological reactions – and that is why they are basically involved in a variety of signal responses that have been characteristic of cellular life since the beginning of evolution. ‘In the beginning, there was calcium’, as one repeatedly hears biophysicists point out – and this should be borne in mind when considering bones, bone loss and hence the loss of calcium, from all angles. Clinically speaking, the risk of fracture is undoubtedly the most important parameter, yet concern for bones is addressed on other higher intellectual levels where osteoporosis is concerned.
22.1.2 Why Oestradiol Protects the Bone
Indeed, the protection oestrogen provides in the bones is just a side effect of a higher consideration by evolution: to reproduce, mammals require high quantities of calcium, for ovulation, for menstruation and particularly for lactation; that is why oestrogen stores this important element of the second main group in the periodic table in the bone, where it is kept available for the reproductive events mentioned above. Immediately before it is suddenly required – in the preovulatory, premenstrual and immediately post-partum settings – there is a drop in the levels of the oestrogen that previously stored calcium in the bones’ ‘vault’. This mobilises calcium from storage, making it available as the important transmitter substance in reproduction. If, however, oestrogen levels fail to rise again – as is the case in menopause – the bones empty themselves of their reserves, depositing calcium in other organs instead, in the breast, for instance, where, among other things, they stimulate breast stem cells to proliferate, and in the worst case, forming calcium deposits that typically give rise to a suspicion of a malignancy.
The endoplasmic reticulum has an important role to play in this carousel of molecular-physiological processes: like the bone, it stores calcium in the cell but with the purpose of unlocking it at the moment of the highest surge of oestrogen. This triggers countless reactions in the cell that promote the survival of organism and species alike. Among other things, calcium stimulates a mechanism known to biologists as the ‘unfolding protein response’: the endoplasmic reticulum controls the proteins formed therein and ensures its correct folding; this prevents the cell from entering apoptosis.
And this is where we encounter a loss of precision that evolution was not able to eliminate: malignant cells benefit from the same general screening programme, a programme that strengthens them in their survival strategy. The situation is similar to the genetic programmes of wound healing that guarantee an organism’s survival. A malignancy ‘abuses’ this programme, though, by continuously activating the genes involved in wound healing. The situation is similar in the case of the unfolding protein response: what survive are not just the healthy cells but, unfortunately, the mutated cells as well.
22.2 The Importance of Oestrogen Receptors to the ‘Unfolding Protein Response’
This ‘unfolding protein response’ is also triggered by calcium released from the endoplasmic reticulum when oestradiol enters the cell – before it can dock onto the nuclear receptor (Fig. 22.1). Here, the female reproductive hormone takes an ‘obedience-in-advance’ approach, launching the repair programme even before the cell reports any damage. This protects premalignant cell structures, preventing their elimination by the immune system. This is referred to as the ‘anticipatory’ effect of oestrogen and is mediated by the receptor alpha. The second docking site for oestrogen, the oestrogen receptor beta, applies an approach comparable to yin and yang in an attempt to blunt the effect of oestradiol. Recently, this is the model used to explain the chemopreventive effect of all the substances that stimulate oestrogen receptor beta, above all the isoflavones. This is how isoflavones reduce cell protection in carcinoma cells – a newly identified mechanism that can help explain the safety of isoflavones and their epidemiologically confirmed, chemopreventive effect.
Fig. 22.1
Endoplasmatic reticulum and gene expression
22.3 Epidemiological Data That Demonstrate the Safety of Isoflavones
Alongside a large number of experimental facts that suggest the chemoprotective effect of isoflavones, there is also a wealth of epidemiological data demonstrating with a high level of medical evidence the protective effects of isoflavones vis-à-vis hormone-dependent malignancies:
· A publication by the European Prospective Investigation Group appeared in Breast Cancer Research and Treatment in April 2013. In this work, at least 334,000 women between the ages of 35 and 70 years were evaluated in 10 European countries. The work showed that there is no risk whatsoever associated or linked with isoflavones as far as breast cancer is concerned [1].
· Another study (Boucher et al.) was published in the IJC (International Journal of Cancer). There, around 3000 cases were compared with 3000 controls; in this study, yet another aspect emerged as well. Isoflavones possess a protective effect with regard to the development of breast cancer [2].
· As early as 2007, a working group in the Netherlands (Verheus et al. 2007) published prospectively collected data in the Journal of Clinical Oncology. A group of 200 women showed that the measurable level of isoflavone in the blood correlated directly with protection from breast cancer. The higher the serum level of isoflavone, the lower the likelihood of developing breast cancer. The study was carried out on the basis of a scrupulous methodology [3].
· Also published in Journal of Clinical Oncology was a publication from Israel and Hong Kong that included 24,000 women and substantiated a familiar finding: high levels of isoflavones in the blood are associated with a markedly low incidence of breast cancer [4].
· A work by Magee, P.J., and Rowland, I., that appeared in January 2013 posed the provocative question: is it legitimate to use soya following a malignancy? The authors answer the questions with: Yes – it is legitimate, and there is no interference with tamoxifen or with an aromatase inhibitor such as anastrozole [5].
· In the ‘Shanghai Breast Cancer Survival Study’, Prof. Shu of Vanderbilt University (USA) observed 5043 women for 5 years following breast cancer therapy and who were given a daily dosage between 30 mg and 70 mg of isoflavones and 11 g of soya proteins. Clinical data demonstrated a significant improvement in the 5-year survival rate, a significantly lower rate of recurrence and a synergism in combination with tamoxifen [6].
· A follow-up study tracked 524 breast cancer patients who had undergone surgical treatment. All were treated with adjuvant anastrozole, and 85 % of the patients were ER+ and PR+. The average follow-up period was 5.1 years. A statistically significant reduction in the risk of recurrence was observed in the most vulnerable subgroup of postmenopausal women [7].
· DKFZ has also published a work online that found that among postmenopausal patients with breast cancer, not only the mortality rate but also the risk of developing metastases or secondary tumours were up to 40 % lower among patients with diets rich in soya [8].
Even in the past, it was demonstrated several times over that components of soya and red clover had no negative effect on hormone-dependent malignancies. The two publications by the groups from the Netherlands and Israel, however, go a step further and corroborate the suspicion that, on the contrary, there is a high likelihood that isoflavones have a protective effect on the development of hormone-dependent malignancies.
Still, some time ago, professional societies such as the Austrian Society for Reproductive Medicine and Endocrinology already stated their positions on the matter [9]. They made it clear in no uncertain terms that the breast-cancer-preventive effects of isoflavones had been demonstrated with a relatively high level of evidence.
This is not just limited to breast cancer, however. As a meta-analysis (KORMA) Study Group [10] showed, this finding applies to hormone-dependent ovarian and endometrial cancers in the gynaecological area as well; the protective effects of isoflavones have been presented here too.
22.4 The Complexity of Evolution
Evolution knew how to enlist the same substances for different tasks, thus significantly expanding its reach. Endocrinology is no exception to this principle. For instance, a hormone can be converted into various other hormones; the best known example is testosterone, which is converted to oestradiol through the workings of aromatase. On the other hand, with a variety of receptors available for a single hormone, this also enables physiological diversification. There are two receptors for oestrogen in higher mammals: oestrogen receptor alpha (ER-α) and oestrogen receptor beta (ER-ß). Their influence is similar to that of a yin and yang system.
22.4.1 The two Oestrogen Receptors Can Be Selectively Activated
The oestrogen receptor familiar to researchers for longer is known as ‘ER-alpha’. In 1995, J.-Ă. Gustafsson discovered another oestrogen receptor, the ER-beta (ER-ß). Both are localised within the nucleus. Such nuclear receptors can be activated by low-molecular-weight substances such as oestrogens and structurally similar molecules, e.g. isoflavones, or by synthetic active ingredients. A precondition to activation of the receptors is that these substances pass through the membranes of their target cells, continuing through the cytoplasm of the cells to the nuclei, where they bond covalently with the receptors in the nuclei. They then form a ligand-receptor complex. Once activated by a ligand, ER-α causes ‘cellular activity in the broadest sense’, including proliferation and inflammation; this can result in an increased risk of mutations and the oncogenesis to which this can lead.
ER-ß, on the other hand, once activated by a ligand, provides for ‘calming’, for anti-proliferative and anti-inflammatory effects and hence for an option to protect against cancer through targeted activation of this receptor using suitable substances. This dimension also gives rise to treatment possibilities of the kind already seen with the oestrogen receptor-beta agonist tamoxifen.
22.4.2 Does ER-ß Provide Protection from Hormone-Dependent Cancers?
When it encounters ER-alpha, 17-beta oestradiol (E2) can stimulate mitosis and promote inflammation and angiogenesis. If, however, ER-beta predominates in the tissues, then the same stimulant, oestrogen, inhibits mitosis and has anti-inflammatory and antiangiogenic effects. Thus, the same substance can have two different effects, depending on the receptor constellation in the tissues.
The best evidence for the chemopreventive effect of the oestrogen receptor beta is provided by tamoxifen, referred to above, which prevents the tissue against further proliferation via the selective effect on the beta receptor in the breast.
Soya isoflavones are natural ER-ß agonists. They selectively stimulate ER-ß and promote ER-ß expression in tissues as well.
22.4.3 Isoflavones Intervene in Paracrinology
There are not only the different receptors ER-ß and ER-α, but there are also different metabolites of E2. Today, we know that the oestradiol, progesterone and testosterone are only precursors, i.e. precursor steroids. From these precursors, each organ can then assemble the hormone it needs in its own local situation.
The metabolism of oestradiol was presented for the first time at the major cancer congress (Conference of the National Cancer Institute) in Bethesda, Maryland, in 1998. It is evident that the original oestradiol can be hydroxylated at a different place in the molecule; this diversifies the effectiveness of the hormone in different directions.
The human body accomplishes this with other hormones in different organs as well. This has long been known and accepted for testosterone. In the prostate, it is hydroxylated further into dihydrotestosterone, which ultimately exerts the stimulative effect on the prostate gland. Progesterone, too, is transformed into other metabolites in the brain, such as allopregnanolone, which also has a highly differentiated task to perform. The situation with oestrogen is similar. It has three different highways available to it that it pursues following release from the ovary or following oral administration (Fig. 22.2).
Fig. 22.2
Oestradiol metabolism
22.4.4 Soya Isoflavones Influence the Metabolism of Oestrogen
17-beta oestradiol can be converted either into the 16-hydroxy-oestrone, 4-hydroxy-oestrone or 2-hydroxy-oestrone. These hydroxylated oestrogens are completely different hormones compared to the parent compound. Decisive, then, is the different further processing of hormones in the tissue. A mitosis-enhancing and angiogenic effect is ascribed to 4-OH-E2 and 16-OH-E2. 2-OH-E2 and, on the other hand, demonstrates antimitotic and angiostatic (vascular formation-inhibiting) properties as part of a yin-yang mechanism. So the functions it performs are diametrically opposed. If the level of 4-OH-E2 is too high, dangerous depurinated adducts (molecules) are formed, and these place a strain on the breast.
The conversion occurs via the CYP systems, meaning via CYP enzymes that themselves are subject to a high degree of genetic variation.
And this is where isoflavones also come in; as a result, e.g. through soya extract, CYP 1A1 can stimulate the hydroxylation of E2 in the 2-OH E2 position. In addition, with soya isoflavones, one can also inhibit CYP1B1, thereby reducing hydroxylation in the direction of the straining hydroxylation positions 4 and 16. Whether by nature, a woman tends to metabolise in one direction or another is a function of a polymorphism, meaning different genetic variations.
There are studies that clearly show that soya proteins and isoflavones give rise to protective effects at the level of the enzyme modulation:
· Work by Hooper et al. 2009 and Teas et al. 2009 demonstrated that soya isoflavones give rise to a trend towards reduction of oestrone and a shift in oestrogen metabolites towards the protective 2-OH-E2 [11, 12].
· Prof. Jeffrey Tice of the University of California reported on the results of a systematic analysis of published data. With regard to the isoflavones, his working group compiled seven randomised studies involving a total of 1602 women and an observation period of up to three years; from these, the effect of administration of isoflavone could be read based on the density of the breast tissue; based on the state of current knowledge, this is a function of the hydroxylated oestrogen metabolites at positions 4 and 16. In these studies, the daily doses of isoflavones ranged between 40 and 120 mg. In none of the studies was there an increase seen in breast tissue density under the influence of isoflavones.
· A meta-analysis by Hooper et al. focusing on isoflavones and circulating hormone levels clearly shows no significant changes in hormone levels and no cancer-promoting effects among premenopausal and postmenopausal women [13].
22.5 Further Oncopreventive Mechanisms of Action by Isoflavones
22.5.1 VEGF (Vascular Endothelial Growth Factor)
The soya isoflavone genistein is an inhibitor for VEGF (vascular endothelial growth factor) synthesis. There are two different routes by which this is accomplished. Because genistein inhibits the transcription factor 1-α, less VEGF is formed. In addition, it inhibits signal transmission of the angiotensin II receptor Type 1 to VEGF by inhibiting ER1/2 kinases. This suppresses angiogenesis, which is dependent upon growth factor VEGF [14] [15].
22.5.2 The RANK Ligand NF-kB
Malignancy development is linked to an increase in NF-kB (nuclear factor kappa-light-chain-enhancer of activated B-cells). NF-kB is a transcription factor that induces the reading of several antiapoptotic and proliferative genes, thereby inhibiting apoptosis.
Chemotherapy increases MAP kinases and hence NF-kB. This seems to play a role in the development of chemoresistance as well.
A low NF-kB means less development of resistance – even during chemotherapy, it prevents the emergence of a cachexia, promotes apoptosis, prevents angiogenesis and affects the immune response.
Soya isoflavones inhibit NF-kB, and this makes them natural NF-kB inhibitors.
In addition to this, they are significant to vitamin D3 levels. Soya extract keeps the concentration of vitamin D3 high, and through reduction of NF-kB, it also has a positive and protective effect on the osteoporosis development.
MMPs (matrix metalloproteinases) are responsible for cachexia in tumour patients; both NF-kB and MMPs are reduced by soya extracts. The protein Myo-D is responsible for the structure and repair of muscle fibre. A high NF-kB suppresses Myo-D. Muscles are responsible for movement, for the generation of numerous hormones formed in the muscle and for the synthesis of glutamine. The muscle is the largest producer of glutamine. With soya extract, then, one can do something to combat cachexia and tumour necrosis [16–19].
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