Strong Science for anti-cancer soy

Dr. Weeks’ Comment:    a superb article supporting the scientific merits of this whole food:  a fermented, non GMO soy product.

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Soy’s Cancer Protection Resembles Cancer Protection during Pregnancy

Running Title: Fermented Soy and Cancer

Uwe D Rohr*¹, Anca G Gocan¹, Doris Bachg², Adolf E Schindler³

Acceptance for Publication:  October 20, 2010

Ref.: Soy’s Cancer Protection Resembles Cancer Protection during Pregnancy

Hormone Molecular Biology and Clinical Investigation

Prof. Jorge R. Pasqualini

Editor-in-Chief

‘Hormone Molecular Biology and Clinical Investigation’

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Summary

It has been established that carrying a pregnancy to full term at an early age may protect against contracting cancer by up to 50%in later life. The trophoblast theory of cancer states that trophoblast and cancer tissue are very similar. New findings suggest that the loss of fetal cells during pregnancy resemble those cells responsible for causing metastasis in cancer. Fetal cells and spreading cancer cells are highly proliferative, and although less developed, they are similar to stem cells, exhibiting no or low hormone receptor expression, and require a hormone receptor independent mechanism for control. Control of membrane stability during pregnancy is of vital importance for a successful pregnancy and is mediated by androstenediol and 2-methoxy-Estradiol. 2-methoxy-Estradiol has no hormone receptor affinity and elicits strong anticancer effects particularly against cancer stem cells and fetal cells, for which currently no treatment has yet been established. There is a discussion whether pregnancy reduces cancer stem cells in the breast. Soy isoflavones a) are structurally similar to both hormones, and b) elicit strong anticancer effects and c) anti-angiogenesis via inhibition of NFkB, even in hormone receptor independent breast cancers seen in epidemiologic studies. The trophoblast theory of cancer may help to explain why soy baby nutrition formulas have no effect on baby physiology, besides the nutritional aspect, although soy elicits many effects on the adult immune system. To survive the immune system of the mother, the immune system of the fetus has to be separated, otherwise the reduction of the immune system in the mother, a necessary feature for the blastocyst to grow, would immediately reduce the immunity for the fetus and endanger its survival. Like a fetus, newly born babies show immune insensitive to Th1 and Th2 cytokines, which are nessary and crucial for regulating the immune system of the mother, thus raising the risk of the baby of developing allergies and neurodermitis. Gene expression studies in vitro as well as in circulating tumor cells from patients consumimg a fermented soy product support the anti-angiognetic as well as antiproliferative effects of soy.

Key words: Trophoblast, soy, isoflavones, curcumin, 2-methoxy-estradiol, androstenediol, Th1, Th2, cytokines, capillary, cancer, cachexia, depression, fermented soy, soy nutritional formula, breast cancer, fetus,

Introduction

While plant-based formulations have been used to treat cancer for centuries, current treatments usually involve poisonous mustard gas, chemotherapy, radiation, and targeted therapies[1]. Natural products have been the most significant source of drugs[2]. Their dominant role in cancer chemotherapeutics is clear with about 74% of anticancer compounds being either natural products or natural product-derived, mostly via inhibition of mitosis [2-4]. Numerous bioactive chemical compounds of plant origin influence not only mitosis, they are also known to increase apoptosis via modifying Nuclear factor kappa Beta (NF-kB) transcription factor and angiogenesis and may affect blood vessels in tumors and may reduce cancer growth as well (Fig. 1,2)[3-4].

NF-kB has a key role in many physiological processes such as innate and adaptive immune response, cell proliferation, cell death, and inflammation[5]. It has become clear that aberrant regulation of NF-kB and the signaling pathways that control its activity are involved in cancer development and progression and appear resistant to chemotherapyand radiotherapy as well[6]. Although the dominant role NF-kB plays has been recognized, its therapeutic use in cancer treatment has not yet been fully established[5,6].

The first generation approach to anti-angiogenesis beyond NF-kB mechanism via anti-VEGF-inhibition was aimed to “destroy and suppress” blood vessels in cancers[7,8]. This concept was less effective in clinical trials than anticipated [7,8]. Newer evaluations reveal that capillaries in cancer have lost their stability and are leaky and unstable so that the tumor can spread and grow[7,8].

Soy ingredients show strong effects on NF-kB (Fig. 1,2), and it was shown in several epidemiological meta-analysis that consumption of soy food, particularly soy isoflavones, protects against various types of cancers, particularly against breast cancer[9,10]. The majority of animal studies and in vitro cell culture studies support these observations[10-12]. However, a few animal experiments produced contradictory results and do not support a cancer protection hypothesis[13].

Although many investigations have been conducted, it is still not clear how soy or its ingredient soy isoflavones achieve anticancer activity. A recent very important epidemiologic finding claims that soy’s protection against cancer is independent from the Estrogen receptor (ER) status (Fig. 3), and irrespective of whether a breast cancer is ER-positive (ER+) or ER-negative (ER-) or whether patients are Oriental [14-16]or Caucasian women [17]. This finding clarifies earlier epidemiologic studies, which were not specifically designed to investigate this question. Authors of these new epidemiological findings seek a new explanation concerning soy cancer protection mechanisms beyond hormone receptors[15,17].

The finding of soy’s protection against ER- breast cancer is of tremendous clinical importance, because women with ER- breast cancer have a poor prognosis, suffer from more aggressive tumors, and currently no medical prevention has been established, leaving those women with the highest risk and currently without any medical protection[18].

A new model describing cancer origin, recurrence, and progression, suggests that cancer evolves from a small subfraction of cells, which are stem cell-like, less differentiated, and have no or only few hormone receptors (Fig. 4,5,6)[19,20]. Whereas differentiated cells show hormone receptor expression in allcells, cancer stem cells show low hormone receptor expression (Fig. 6)[21]. It was shown in a clinical study in Caucasian women diagnosed with breast cancer that the primary tumor exhibits 68% ER+ cancer cells whereas in the blood circulating tumor cells show only 35% ER+ expression in circulating cancer cells (Fig. 4,5)[22]. This clearly supports the assumption that current adjuvant therapy is restricted to ER+ breast cancer, because it is effective via blocking the classical ER, what is now coined as ER-a[23].

Cancer stem cells fail to respond to radiation or chemotherapy (Fig. 4)[24-26]. They leave the primary tumor and cause metastases (Fig. 5)[24-26]. Cancer stem cells have no or low hormone receptor expression. They do express, however, NF-kB [6,27] and may very well be silenced via inhibition of NF-kB.  The finding that soy isoflavones silence ER- breast cancer, which is more stem cell like and has no or low Estrogen receptor expression raises the question of its mechnism of action. NF-kB can be altered by soy isoflavones, it may therefore influence cancer stem cell via this mechanism as is discussed in this review.

It appears that cancer shares many properties with a trophoblast(Fig. 8)[28,29].After implantation of a fertilized egg in the uterus, the immunity of the mother has to be carefully balanced with the immunity of the trophoblast so that the immune system of the mother does not reject the trophoblast. As will be shown, regulating the immunity of the mother during pregnancy is correlated to hormones structurally similar to soy isoflavones, such as androstenediol and 2-methoxy-Estradiol, all of which are increased during pregnancy (Fig. 9)[30,31]. They are not only structurally similar (Fig. 1), they also exhibit strong anti-prolifeartive effects and have strong anti-angiogenesis effects[30-34]. Additionally, 2-methoxy-Estradiol, in contrast to its misleading name, has no affinity to any hormone receptor and elicits its antiproliferative effect via non hormonal receptors in stem cells, which are more aggressive than differentiated cancer cells (Fig. 7)[21]. The increase during pregnancy of these hormones may be necessary to reduce highly active fetal cells, which leave the uterus and nest in precancerous tissues of the mother, which may facilitate angiogenesis and cancer growth, because fetal cells are highly proliferative[35].

Full-term pregnancies are associated with long-term reductions in maternal risk of breast cancer, but the biological determinants of the protection are unknown and many factors may contribute [22]. Nevertheless, immunity modification by steroids produced by the mother during pregnancy share many similarities with soy isoflavones. Clinical trials and animal studies have shown maternal steroids to enhance immunity and to be strong anticancer agents against breast cancer [31-34].

The steroidal hormones mediate their immune modifying effects via Th1-cytokines, which may in return alter membrane permeability[30]. In particular, androstenediol has a very strong protective immune modifying effect via cytokines (Fig. 10,11,12,13)[36]. A clinical trial with breast cancer patients revealed a reduction of Th1-cytokines and a reduction of symptoms related to Th1-cytokines after consuming fermented soy (Fig. 18)[37,38]. It is now recognized that cytokines in the blood of cancer patients are released from immune cells within the tumor, increasing the surrounding capillary membrane permeability, leading to the spread of tumor cells and cytokines, and causing metastasis and discomfort (Fig. 15)[39-45]. The Th1-cytokines inthe blood are released from the tumor causing leaks in the blood capillariesor trophoblast membrane. This situation is similar to complications in pregnancy, and can result in possible miscarriage, premature birth, or other compliations during labor or birth (Fig. 11,12).

Although androstenediol and 2-methoxy-Estradiol elicit strong immunological effects in the mother and are present in the amniotic fluid, they have no effect on the fetus [31]. The necessary separation of the mother’s immunity and the trophoblast/fetus may help to explain why soy baby formulas have no effect on the immunity of the baby. The reduced immunity in babies may well be a remnant of fetal development. As the baby progresses through childhood, a stronger immunity system begins to be established. Thus, studies suggest that consumption of soy during early childhood begins to have an effect, protecting against cancer later in life.

Present studies suggest that it may be advantageous to include agents modifying NF-kB during cancer therapy, because they interfere with mitosis on the genetic molecular level (Fig. 23), and may protect the patient against the side effects of chemotherapy.

Breast Cancer Stem Cell Model

Breast cancer remains a significant public health problem despite advances in our understanding of the molecular and cellular events that underpin the disease[25,26,46]. Recently, a new model has been proposed. This model links breast cancer cells to stem cells and progenitors, an observation originally made in other cancer entities[25]. It hypothesizes that the tumors originated from a small population of undifferentiated cells, which have no or only small amounts of hormone receptors [47]. These cells can undergo self-renewal and are able to generate a large number of partially differentiated cells, which constitute the bulk of the tumor.

These cancer stem cells resemble adult stem and progenitor cells found in the normal breast, but are deregulated from their patterns of proliferation and differentiation[21]. They could originate from normal stem cells or from more differentiated progenitors and lose their normal growth restraints through a series of oncogenic mutations that deregulate a small number of central signaling pathways[46-47]. It was found that ER was expressed in circulating tumor cells (CTC) up to 35%, whereas ER expression was found in approximately 68 % of all cells at the primary site (Fig. 4,5)[48]. This reduces the possibility to limit cell growth of CTC through aromatase inhibitors or tamoxifen, because they reduce only cancer cells, which express ER [23].

Despite the fact that hormone recptors may have limited possibilities to regulate stem cells and cancer stem cells, they do respond to changes in NF-kB [6,49,50].

Breast cancer stem cells do not respond to chemotherapy, radiotherapy, or adjuvant therapy. Also, it is estimated that at the time of diagnosis cancer stem cells have have left the primary tumor in 40 % of all cases and are distributed in the body via blood, bone, or lymphatic system. Therfore targeted treatment of disseminated cancer stem cells is of vital importance, because surgical removal of the primary tumor will not reduce distributed tumor stem cells [25,26,46-47,49].

Full Term Pregnancy at Young Age causes Cancer Protection

By completing a pregnancy at a young age, a woman reduces the risk of breast cancer by up to 50% over her lifetime [51,52]. A similar protective effect has been observed in rodent models with an early pregnancy by using chemical carcinogens to induce cancer [52]. However, the mechanisms responsible for this protective effect remain unclear.

Cancer is predominately a disease of old age, however, every 20^th child is born already with leukemic cancer cells, although the child may not nessasarily develop the disease[53]. Therefore the  threat of cancer is not only a threat to the mother, but also to the child.

Despite the fact that pregnancy protects against many cancers, such as breast cancer, it is now more and more recognized that pregnancy generates a pro-cancerous environment as well[54,55]. The Italian physicianBernardino Ramazziniwas the first one to describe how catholic nuns suffer from high rates of breast cancer [54,55]. Contradicting observation illustrated how some cancers, such as neuro-fibroma or melanoma for instance, grow rapidly in young mothers during pregnancy[21,35].

Many similarities between cancer and trophoblasts continue to be observed [28,29]. Cancer tissue manipulates its own environment to receive nutrients, oxygen, and alters barrier functions so it can create space to grow.

Pregnancy increases breast tissue differentiation, a feature that protects against cancer. New investigations illustrate how two hormones, androstenediol [30] and 2-methoxy-Estradiol [21] are elevated during pregnancy and show strong anti-proliferative (Fig. 7) and anti-angiogenetic effects, stabilizing capillary membranes and at the same time act anti-proliferative particularly against cancer stem cells believed to cause and spread cancer [28,46].

Plant Substances with Anti-angiogenesis and Anti-proliferation Properties

Plant growth is silenced in times of environmental stress, e.g. in winter or in dry season[56,57]. Plant derived compounds show strong anti-proliferative effects and anti-angiogenesis (Fig. 1, 16). Because most cancers are caused by deregulation of as many as 500 different genes, agents that target multiple gene products are needed for prevention and treatment of cancer beyond mitosis[58]. Curcumin, e.g., is a diferuloylmethane derived from the Indian spice, turmeric (popularly called “curry powder”) that has been shown to interfere with multiple cell signaling pathways, including cell cycle (cyclin D1 and cyclin E), apoptosis (activation of caspases and down-regulation of antiapoptotic gene products), proliferation (HER-2, EGFR, and AP-1), survival (PI3K/AKT pathway), invasion (MMP-9 and adhesion molecules), angiogenesis (VEGF), metastasis (CXCR-4) and inflammation (NF-kB, TNF-a, IL-6, IL-1, COX-2, and 5-LOX), and activate the antiproliferative Estrogen Recepetor beta [59-61].It is reported that the activity of curcumin against leukemia and lymphoma, gastrointestinal cancers, genitourinary cancers, breast cancer, ovarian cancer, head and neck squamous cell carcinoma, lung cancer, melanoma, neurological cancers, and sarcoma, reflects its ability to affect multiple targets[59,60].

Many molecular anticancer effects of soy isoflavones are similar to curcumin, particular anti-angiogenetic and anti-proliferative via inhibiting NF-kB. Of the close to 10.000 published isoflavone studies in peer-reviewed publications, almost one fifth pertain to its antitumor capabilities and describe its mechanism of action in normal and malignant cells, animal models, in-vitro experiments, or phase I/II clinical trials[62]. The soy isoflavone genistein and daidzein, which are abundant in plants, particularly in soybeans, reduce chronic inflammation and cancer in Asian [63-66] as well as in western populations [67-69]. Migration studies have shown that after successive generations, breast cancer incidence in Asian women becomes similar to that of Western women[70]. Furthermore, an increasing incidence of breast cancer among Japanese women parallels the Westernization of the Japanese diet [71]. These new findings contradict the assumption that soy may protect only Asian women against breast cancer due to a genetic susceptibility. Only recent epidemiological studies show that soy isoflavones are effective in first line as well as in second line prevention of breast cancer [17,70]. A very important finding is that soy is equally effective protect against ER+ as well as ER- breast cancer (Fig. 3). The finding that soy isoflavones protect against ER-negative tumor supports the assumption that they are similar to 2-methoxy-Estradiol to reduce cancer cell growth in stem cell like cancers who have no or reduced hormone receptors and are more proliferative and are more aggressive than ER+ tumors.

Trophoblast-Theory of Cancer

It was the British developmental biologist,John Beard(1858-1924), who was first to suggest that to understand anti-cancer treatment, the similarity between tumors and trophoblast needs to be discussed[28,29]. Trophoblasts provide protection by completely surrounding the embryo, while carrying nutrients and oxygen from the mother’s blood to that of the developing fetus. The word trophoblast means “original feeding tissue” and was named after the Dutch embryologist Ambrosius Hubrecht (1853-1915)[28, 29].In the early part of the twentieth century, scientists began to notice a remarkable similarity between trophoblastic cells and cancer. It was said, that if you mixed up microscope slides of both trophoblasts and cancer, you could not tell the difference[28, 29].Both, tumor tissue and trophoblast, are highly proliferative, migratory, and invasive with an almost limitless ability to perpetuate themselves.

The main difference between cancer and trophoblasts is that trophoblast`s growth is a natural self-contained process, limited to the environment of the uterus. In rare cases, however trophoblats can escape and the result is chorio-angio-carcinoma, a highly malignant form of cancer (Fig. 8)[28,29].

The cancer-like growth of a trophoblast is kept under control by a cascade of hormonal and cytokine signals[74]. New investigations show that this process is actively supported in the first trimester of pregnancy by a decrease in androstenediol and increases in Th1-cytokine (e.g. TNF-a, IL-6)(Fig. 9,10,11) [30]. Generally, in pregnancy a direct correlation exists between androstendiol and Th1-cytokines (Fig. 10) [30].

A reduction of the DHEA-metabolite androstenediol in the first trimester reduces immunity in pregnancy, so that the fragile fetus is not rejected by mothers immune system (Fig. 9) [30]. It is interesting to note that fatique and discomfort in the beginning of the pregnancy may be similar to cancer patients, because they are caused by similar Th1-cytokines.

This adaptation process, reduction of immunity in the first trimester, is of vital importance because it also increases capillary permeability in the uterus feeding the fetus. On the other side, more dramatic increases of permeability increase the risk that the fetus will suffer a miscarriage in the first trimester. This process is reversed in the second and third trimester of pregnancy: Androstenediol and 2-methoxy-estradiol are increased, anti-angiognesis, anti-proliferation as well as Th1-cytokines in the mother are decreased (Fig. 9)[30].

Thirty years ago, Schindler detected high amounts of androstenediol in the umbilical cord of pregnant women [75,76], supporting the evidence that androstenediol is stabilizing the membranes[30]. Interestingly, umbilical cells are presently used for screening of plant-derived compounds exhibiting anti-angiogenesis[1]. It is also of interest that at that same time Thijsen detected the anti-proliferative effects of androstanediol working against breast cancer, revealing that anti-angiogenesis is mostly related to anti-proliferation [77].

Inflammatory Th1-cytokines have profound effects on endothelial function not only in embryology (Fig. 11), but also in regulation of vascular tone, permeability, and leukocyte diapedesis in many diseases[78-80]: E.g. during times of overwhelming sepsis, these inflammatory mediators trigger septic shock, a syndrome associated with endothelial cell failure and death [78]. Likewise, “successful” tumors also appear to have the capacity to manipulate endothelial function, as they promote their own spread and become vascularized [81,82].

The endothelium regulates these dynamic interactions with the environment through intracellular signaling cascades. As is the case in many other tissues, JNK- and NF-κB-dependent pathways play an important role during the endothelial response to inflammatory stress [83-86]. In resting cells, NF-κB (i.e., RelA/p65 and NFkB1/p50) associates with IκBα, a negative regulator, forming an inactive complex. Upon stimulation with an appropriate ligand (e.g., TNF-α, IL-1, IL-6), IκBα serines 32 and 36 become phosphorylated, and the protein is targeted for degradation. This releases the p65:p50. Moreover, these studies have revealed the critical role this pathway plays in antagonizing apoptosis[87]. Gene-targeting studies, which have validated some observations about NF-κB function, have been hampered by redundancy within the family of NF-κB transcription factors and embryonic lethal phenotypes [87-90].

During pregnancy, cells from the embryo can permeate the trophoblast layer and combine with pre-existing epithelial cancer cells of melanoma, breast or colon cancer cells[35]. 8% of all cancers are melanoma, and receive fetal cells during pregnancy, which can be identified in 68% of all melanoma after pregnancies [35]. Lost fetal cells in the epithelium of the mother have high potential to mediate angiogenesis, so that pre-existing epithelial cancers can grow. They can stay in epithelial layers up to 40 years after birth and are capable of feeding preexisting tumors at any time[35]. This was demonstrated by chimeric cells in the epithelial cells of the mothers, which expressed the male phenotype of their sons[35].

Anti-angiogenic effects are of vital importance to stabilize trophoblasts, umbilical cords, as well as chimeric cells around existing epithelial cancers. For this reason, Th1cytokinesplay an important role during birth or miscarriage, a ruptured of the uterus membrane, and even as regards heart infarction and stroke (Fig. 11,12)[74].

Anti-Angiogenesis of Soy

As for cases involving in vitro, it has been reported, that soy isoflavones reduce angiogenesis by reduction of cytokines like TNF-a and IL-6, via the silencing of the MAP-Kinase/NFkB cascade of immune cells (Fig. 2)[91] Tumor angiogenesis plays a pivotal role in the complex, multistep nature of cancer growth and spread. Angiogenesis is intimately involved in metastasis at the site of entry of tumor cells into the vasculature, as well as at the site of eventual metastasis [92]. In this regard, a relationship between tumor cell invasion and tumor-induced angiogenesis is described, with cooperative functions of both processes during tissue breakdown and cell migration [92]. Adlercreutz discovered 25 years ago that soy has tremendous anti-angiogenesis and membrane stabilizing affects [91]. Micro-array analysis of gene expression of prostate cancer cells confirmed that soy isoflavones affect more than 1000 genes, many affect capillary membrane stabilization and angiogenesis [92]. Many genes governing angiogenesis resemble gene expression necessary in uterus rupture of pregnant women, like down regulation of MMP-9 [92]. Surprisingly, we found in our own in vitro gene expression studies with fermented soy that although docetaxel is an anticancer drug, it caused up-regulation of MMP-9 and down-regulation of ostoprotegrin, which could promote cancer cell growth in the bone environment[92].

The anti-angiogenesis marker KI67 was increased in biopsies from tumors in breast cancer patients consuming fermented soy(Fig.  14).

Today the local tumor compartment is seen as a functional unit composed of two distinctly different cells: cancer cells and immune cells (Fig. 1,16)[39-45](Fig. 5). Immune cells and a network of pro- and anti-inflammatory cytokines collaborate in the development and progression of cancer[45,100,101]. Cytokines from immune cells as well as cancer cells can leave the local tumor compartment into the peripheral blood stream and spread the disease as well as increase cancer symptoms (Fig. 5,6). Cytokine profiles (particularly IL-6 or TNF-a) of various cancers in the blood might even prove to be prognostic of cancer outcome [93-100].

There are two different ways to detect efflux of cytokines from tumor sites: first, one may detect cytokine levels in the systemic compartment, and second, one may detect typical symptoms of cancer patients particularly at a later stage of cancer development (Fig. 15). Efflux of cytokines from the local tumor compartment are associated with fatigue, depression and cognitive impairment, and can affect quality of life before, during and after treatment (Fig. 15)[39]. Tumor cytokines, despite their large molecular weight, can pass the blood brain barrier (Fig. 15).

Chemotherapy frequently increases side effects in cancer patients and increases cachexia (Fig. 17). In a clinical study, during the course of chemotherapy, fermented soy suppressed cachexia (Fig. 17)[38].Cachexia in cancer patients is directly related to cytokines in the blood (Fig. 15) and increased NF-kB activity of immune cells(Fig. )[102-110]. Cachexia accompanies numerous diseases, (Aids, cancer, rheumatic arthritis) and is a major cause of weight loss, increased mortality, and affects more than 5 million people in the United States alone [109].30-60% of people with cancer suffer from fatigue, cachexia, and that a subset of patients, especially women with breast cancer, suffer also from cognitive impairment during and after treatment [111-113]. Cachexia,fatigue and cognitive decline have a negative impact on the quality of life and such symptoms can persist for at least 10 years in some breast cancer survivors [113]. Soy isoflavones reduced TNF-a in the systemic blood compartment (Fig. 18)[38]. Our results are corroborated by in vitro studies showing that isoflavones reduce TNF-a release in immune cells [89].

Some immune stimulating Th2-cytokines, including interferon-α and IL2, have been used in cancer treatment; however, they can cause fatigue, depression, and cachexia and other symptoms [114-116]. Adjuvant and neo-adjuvant treatment of women with breast cancer with paclitaxel also increased serum levels of IL6, IL8 and IL10, and these changes correlated with joint pain and flulike symptoms[117]. Other anticancer drugs known to increase inflammation-causing cytokines are etoposide[118],cisplatin[119], and bleomycin [120].

Anti-Proliferative Effects of Soy Isoflavone via NF-kB on Circulating Tumor Cells

Clearly, the boundaries between regular proliferation, hyperplasia, and cancer, in endometrium, are difficult to define, since they share many similar mechanisms on the genetic level (Fig. 10,11)[121]. These mechanisms are controlled by natural hormones via hormone receptors and hormone receptor independent mechanisms [121]. It has been reported that soy isoflavones reduce not only cytokines, like TNF-a and IL-6, via silencing of the MAP-Kinase/NFkB cascade of immune cells,[89,122]. NF-kB is silenced in cancer cells as well [89]. Our own in vitro experiments showed strong anti-proliferative effectsby fermented soy, kinetically, as well as on the molecular oncology level, in breast-, colon-, lung-, prostate- and liver cancer cell lines (Tab. 1)[72].

For the first time ever, results of in vitro gene expression studies in cancer cell lines of a plant based formulation (fermented soy) are corroborated by in vivo investigations with circulating tumor cells (CTC) after extracted form blood (Table 1 and 2). Highly sensitive and specific immuno-cytochemical and molecular assays now enable the detection and characterization CTC at the single cell level in bone marrow or peripheral blood, providing insights into the first crucial steps of the metastatic cascade [6,123]. CTC, tumor cells leaving the local tumor compartment into bone marrow or peripheral blood, are of utmost clinical importance for the establishment of distant metastasis during the metastatic cascade [123-133]. The advantage of CTC diagnosis over cancer cells from a local tumor compartment is first, an easy detection, second, reduces the risk of local efflux of cytokines into the periphery by the needle, and third, they consist of cell forms from which metastasis may develop. Prospect and limitation of this method is discussed in the literature [48,123].

2-methoxy-Estradiol expressed similar anticancer properties like soy isoflavones on the genetic level, like reducing NF-KB (Fig. 19, 20) [134-136].

Fifty percent of all cancer patients show no sign of tumor suppressor gene p53 expression. Therefore the increase of tumor suppressor factor p53 by soy (Fig. 21) may help to explain soy’s prevention of breast cancer, which was viewed as a first and second line defense [17,70].Soy isoflavones ability to reduce NF-kB is not limited to cancer symptoms. Patients suffering from pollen allergy or frequent flu infections reported complete remission of these symptoms [38]. It was reported in the literature that intracellular release of specific cytokines in asthma or other allergic reactions is linked to increased c-Jun Kinase, p38 MAP-Kinase, and NF-kB in local immune cells [137-139]. First generation antihistamines like diphenhydramine and chlorpheniramine reverse cytokine afforded eosinophil survival by enhancing apoptosis[139]. It may therefore be concluded that first generation antihistamines and soy isoflavone share similar immune modulating responses.

It is particularly important that activated NF-kB mediates the chemo-resistance of anticancer drugs, as this is a major problem in cancer therapy [140-148]. There is a clinical report, where a prolonged stabilization of platinum resistant ovarian cancer was obtained when consuming fermented soy [149]. In the future, plant extracts, like fermented soy, may help to avoid chemo-sensitization. Curcumin [143], silibinin [147]and Hibiscus [150] may also improve the cancer therapy if used in combination with anti-cancer drugs by silencing NF-kB.

Our clinical results may also explain why tamoxifen and isoflavones do not interfere with breast cancer protection, as was seen in a large epidemiologic trial, where the combination of tamoxifen with consumption of soy isoflavones reduced the risk of recurrence risk of breast cancer by more than 60% when compared to tamoxifen alone [70]. Tamoxifen acts via blocking ER-a. Also, Tamoxifen acts on the G1-phase of the cell cycle, whereas isoflavones act on G2-phase (Fig. 23)[155]. Therefore our investigation supports the observation that a combination adjuvant therapy in breast cancer patients may reduce a relapse of breast cancer.

Babies Immune System does not respond to Soy

Soy based nutrition formulas for babies have been in use for more than 100 years [151]. A council of pediatricians have judged their use as safe [151] and a recent literature review of the US National Institute of Environmental Health Sciences did not reveal anything other than a nutritional effect, without any effect on the immunity or any other physiological effect in babies [152]. There is a theoretical concern that ingredients like soy isoflavones may have negative effects in babies, particularly causing cancer in female babies or feminizing in boy babies[152].

The authors of this review have learned that baby soy formulas have a high soy isoflavone contend, exceeding that of natural dietary consumption in Asian food up to 20 times (calculated at 160 to 960 mg/day for an 80 kg adult male) [152]. The question is, why does soy express measurable effects in adults, however no effects in babies. The trophoblast theory may help to answer this important question.

As can be seen in Fig. 22 androstenedol, which is correlated to immunity (Fig. 13), is increased only during childhood and adrenarche and is negligible in babies [164,165]. The reader of this review is encouraged to read the review by Loria et al, describing the relationship between androstenediol, cytokines and immunity [36], becausee it becomes apparent that the immunity of babies is simly too low to respond to soy isoflaovnones via this pathway. The neonate is born with a distinct immune system that is biased against the production of T-helper cell 1 (Th1) cytokines[153]. Birth imposes a great challenge on the neonatal immune system, which is confronted with an outside world rich in foreign antigens. Exposure to these antigens shapes the developing neonatal immune system and it takes several years until immunity is fully developed[153]. It takes several years inducing Th1 or Th2 polarized responses that may extend beyond the neonatal age and counteract or promote allergic sensitization [153]. The immune system of a mother and the fetus have to be separated and may be a necessity to survive the first three-months within the mother. Therefore, soy does not provide any protective effect against allergies or neurodermitis in babies, due to diminished Th1 response: We saw however a rather impressive redcution of allergies in cancer patients after consumption of a fermented soy formulation[38].

Soy isoflavones have no breast cancer protective effects later in life when consumed by babies. The cancer protective effect begins in adrenarche[12]. Timing of soy consumption is important [12]: As can be seen in Fig. 22, androstenediol increases significantly only in childhood. Many hormones are necessary to elicit a complete immune protection in children, but androstenediol blood concentration seems to be a specificly important hormone for vaccination success [36].

One may argue, that the mother’s androstendiol concentration will diffuse into the amniotic fluid and may elicit effects on the fetus. However, any effect of androstediol on the fetus can be excluded, because the fetus HPA axis (androstendiol is a hormone metabolized by the adrenals) and the HPA axis of the mother are completely separated and cannot interfere with each other, as was shown in animal experiments[166].

Therefore it can clearly be excluded that fluctuations of hormones, which are affecting the mother’s immunity during pregnancy, like androstendiol [166], 2-methox-Estradiol [31] and Th1/Th2 cytokines [153], will not affect the fetus. On the contrary, pediatricians wish that babies would exhibit sensistivity to Th1/Th2 cytokines, which would reduce their risk of suffering from allergies and neurodermitis[153]. However, there seems to be a price to pay for surviving in the womb as a fetus.

This corroborates that soy may need a developed immune system, which babies have not yet developed.

Also, soy isoflavones are coined as “phytoestrogens”, leaving a false impression on their pharmacologically efficacy. As was earlier explained, soy may not even need hormone receptors to elicit their pharmacological effects. Also, it is reported that the affinity of soy isoflavones for the ER-ß is as least 20-30 times greater than their affinity to the ER-a, the classical estrogen receptor. If they would exhibit significant ER-a activity[154], then perhaps growth in babies could be held back, due to close of the growth plates, a condition that has never been observed. There are other important clinical observations, which support that soy has no classical estrogenic effects: As explained above, soy isoflavones are not recognized by the liver during the first pass metabolism as an estrogen, like estradiol in contraception or in HRT. The difference between ER-ß and ER-a is evident when investigating androstenediol, which is structurally similar to soy isoflavones. Both share that they are ER-ß -agonistic and not ER-a -agonistic[155]. ER-ß may be an antiproliferative hormone receptor with strong cancer protective effect[155]. Androstenediol, which increases significantly during adrenarche in both sexes, elicits no estrogenic effects on boys or girls, but, is however, responsibe for the strong bone growth during puberty without producing any feminizing effect[164,165]. The later increase in bone mass during puberty by estradiol and testosterone increases bone stability, while humans in adrenarche have the highest risk of suffering from broken bones[164,165]. It may be interesting to note that soy isoflavones is assumed to be bone protetctive. The effect of androstendiol on bone growth proves, you do not need estrogen or testosterone for bone growth. There may be a general misconception about soy isoflavones physiologic effect, which may not be mediated via the cancerous, highly proliferative ER-a.

Discussion

It has been shown that soy substantially effects and alters gene expression as related to cancer in circulating tumor cells in cancer patients. There are too few patients to make general conclusions. Our results, however, are corroborated by in vitro experiments in cancer cell lines and literature. Although epidemiological evidence is mounting that soy may be beneficial in cancer protection, the mechanism of action is still not fully understood. For example, it was found that soy isoflavone protects women with ER-negative tumors in Oriental[14-16] and Caucasian women [17] for which no prevention has yet been established. ER-negative cells are less differentiated cancer cells, have virtually no hormone receptors (Fig. 6), and do react poorly to chemotherapy and radiation. Therefore, this paper suggests a new mechanism of action, which is based on the “trophoblast theory” as well as the evolving field of cancer stem cell theory.

Soy contains many anticancer ingedients besides soy isoflavones[73]. Soy isoflavones have strong anticancer activities themselves: they interfere with those steroids, which are effective in pregnancy to contain highly proliferative cells, because they are structurally similar to androstendiol and 2-methoxy-estradiol (Fig. 1). These hormones increase apoptosis by increasing NFkB. The barrier function of the tropoblast has to be decreased to allow nutrients and oxygen to permeate freely to feed the embryo (Fig. 9). This increases the risk that cells from the trophoblast overcome the barrier and may seed in the mother and can be found up to 40 years after pregnancy. [35]. These cells are highly proliferative and may help preexisting epithelial cancer cells, eg in skin and grow to become Melanoma, one of the few cancers, which is increased by pregnancy [35] besides fibroma [21]. During pregnancy, a small number of fetal cells enter the maternal circulation (Fig. 8)[35]. These cells persist and then migrate to various maternal tissues where they may engraft and differentiate, particularly ifthere is organ damage, adopting the phenotype of the host organ. Although pregnancy decreases breast cancer risk, it is a concern that young pregnant women suffer from increased melanoma risk, although melanoma normally occurs in older persons [35]. Cells from the embryo are capable of inducing angiogenesis around cells they engulf, so that preexisting tumors may get blood supply for their growth providing “fuel” for tumor growth. This may explain why melanoma can be detected in 18 year olds after pregnancy [161].

Therefore, hormones are needed, slowing down cell activity entering the blood circulation of the mother. Hormones slow down cells from embryo and must have two capabilities: anti-angiogenesis as well as increase of apoptosis to reduce their activity. However, these highly proliferative cells, although they adopt the phenotype of their host environment, are not differentiated and may be considered stem cell like and have no or insignificant amount of hormone receptors. Therefore, a “hormone receptor independent mechanism” is necessary to reduce their activity, where pre-existing tumor cells with the highest malignancy may be down regulated via “non-hormone-receptor-mechanisms” [21]. There is developing evidence that hormones like 2-methoxy-Estradiol are not only effective in pregnancy, they are also effective to reduce rheumatoid inflammation [162,163] as well as cancer in humans [160]. 2-Methoxy-Estradiol has no affinity to hormone receptors, but is however, highly effective in slowing stem cell-like tumors (Fig. 7)[21].

More than 30 years ago, Judah Folkman developed the idea that inhibition of VEGF (vascular endothelial growth factor) by targeting therapy that may lead to tumor containment by anti-angiogenesis of blood capillaries [81]. Clinical efficiency of VEGF inhibition was modest in mono-therapy[7,8] and reduction of capillaries in tumors may cause failures of chemotherapy to work[7,8]. Jain re-investigated this concept and came to a different conclusion [7,8]. On one side, blood flow reduction in tumors may be an important mechanism for cancer control, however an important shift in anti-angiogenesis research is necessary: The old idea “reduction” of capillary formation (destroy and kill) has to be replaced with stabilizing abnormal, leaky existing vessels in cancerous tissues and make them less leaky [7,8].

Stabilizing and de-stabilizing membranes in cancer and pregnancy is very similar (Fig. 8): As was outlined, cytokines created by tumor cells, make tumor vessels leaky to oxygen, nutrients, and most of all cancer cells and cytokines cause cachexia, depression, and vomiting, are similar to cytokines, which are increased in first trimenonm of pregnancy [74]: Th1 cytokines are increased in the first trimenon of pregnancy, and many women feel a reduction in their well being (Fig. 9)[74]. Capillary stability and a decreased permeability are of vital importance for a successful pregnancy (Fig. 11) and may serve as a model for cancer “containment” as well. Hormones modifying capillary permeability show not only anti-angiogenesis but also anti-proliferative properties. Anti-angiogenesis is combined with anti-proliferative activity and mediated by NFkB, like two sides of a coin. Therefore, anti-angiogenesis in vivo is always combined with apoptosis via NFkB.

A circadian rhythm of estradiol uptake was detected in transdermal delivery in postmenopausal women, although estradiol was delivered constantly by zero order release kinetics [156,157]. We assumed already at that time that estradiol uptake, naturally trough capillaries in the ovary and transdermally by skin capillaries, are influenced by the circadian rhythms of Th1-cytokines[157]. Clinicians can observe circadian rhythms of Th1-cytokines in their patients suffering from fever, fatigue is increased in the afternoon, a common side effect of Th1-cytokines.

Almost 30 years ago, Schindler discovered high levels of androstenediol in capillaries of umbilical veins of pregnant women [75,76]. Androstenediol is structurally and pharmacologically similar to soy isoflavones (Fig. 1). Interestingly, the umbilical vein cell model is presently used to screen for anti-angiogenesis drugs in cancer treatment [35].

The textbook knowledge of anti-angiogenesis effect of soy [91] is correlated to a decrease of Th1- cytokines (Fig. 18). Increased Th1 cytokines increases the risk of miscarriage (rupture of the uterus membrane). There are now reports that miscarriage risk can be lowered by plant formulations, reversing Th1/Th2 dysbalance [158].  Increased Th1/Th2 cytokine balance alter barrier functions in many diseases, not only in miscarriage, but also for heart infarction, stroke, cancer (Fig. 11)[74].

Soy isoflavones are structurally similar to androstenediol and 2-methoxy-Estradiol (Fig. 1). Soy increases 2-methoxy-Estradiol by about 40% in humans, due to the fact that they compete for the same enzyme [159]. Therefore in the in vivo situation it may be difficult to distinguish between the effect of soy isoflavones and 2-methoxy-Estradiol. 2-methoxy-Estradiol is currently in clinical trials to treat breast cancer[160].Colchicin and 2-methoxy-Estradiol have anti-mitosis activity (Fig. 1). Besides apoptosis, anti-angiogenesis, a chemotherapy-like mechanism is also used to protect the mother. Therefore, a combination of chemotherapy with soy formulation should be tested in the future, because mechanistically, anti-mitosis, apoptosis and angiogenesis do not interfere with each other. This has to be corroborated in larger trials.

Outlook

Besides cancer protection of soy products, hundreds of million people suffer from non-cancerous conditions of abnormal vasculature [7,8]. Blood vessel repair beyond cancer may be a key component of the therapeutic arsenal for those diseases [7,8]. Our studies may lead to other application besides cancer, like stroke, heart infarction and mental diseases. Our findings need to be corroborated by larger studies in cancer therapy. The trophoblast theory of cancer may not only explain some of the cancer protecting effects of soy, it may also explain, why soy has no hormonal effects on babies consuming soy nutritional formula, and also explain some pharmacological effects like bone protection.

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Fig. 1: Structural comparison of natural hormones with plant derived substances. Three anticancer mechanisms are possible, anti-angiognesis, anti-mitosis, and anti-proliferative.

Fig. 2: Schematic depiction of relationship between cancer and inflammation. Both are related via MAP-Kinase / NFkB Cascade. Silencing via Estrogen receptor beta (ER-ß) by soy isoflavone results in decreased cytokines like Interleukin 6 (IL-6) and an increased apoptosis, resulting into reduced inflammation and cancer. The increase of IL-6 results in increased cachexia (appetite loss) and increased cancer risk

Fig. 3: Correlation of daily soy consumption and breast cancer recurrence after 4 years.  Taken from ref. [16].

Fig. 4: Schematic depiction of invasive breast cancer: Primary breast cancer consists of two type of cancer cells. Cancer stem cells have only low estrogen Receptor expression, whereas primary cancer consists of cancer cells expressing 62% Estrogen Receptor. Chemo-, radio-, or adjuvant therapy cannot reduce cancer stem cells, because they have virtually Estrogen receptor expression. Therefore cancer recurrence is difficult to predict.

Fig. 5: Tumor spread is facilitated via cancer stem cells, which have very low Estrogen receptor expression and may not respond to Chemo-, Radio-, or adjuvant therapy.

Fig. 6: Hormone receptor distribution in stem cells, cancer stem cells, and normal fully differentiated Schwann Cells [21].

Fig. 7: In vitro cell growth in different neuro tumor cell lines with different Estrogen receptor expression (ER). 2methoxy-Estradiol (2ME2) inibits tumor growth independently from the Estrogen receptor expression. Taken form ref [21].

Fig.: 8: Schematic of the similarity between a trophoblast, chorio-angio-carcininoma and a normal tumor. The trophoblast looses fetal cells, simillar to tumors, which loose cancer stem cells, which can metastasise.  Th1-cytokines (TNF-alpha, IL6) can modify capillary permability.

Fig. 9: Schematic of the relationship between androstenediol, 2-methoxyestradiol, and Th1-cytokines. The risk to loose a baby in the first trimenon is caused by the increase of Th1-cytokines. The increase is necessary to reduce the immunity of the mother so that the trophoblast can befed with nutrients and O₂. The increase of Th1 cytokines can cause vomiting, fatique and discomfort of the mother, which is normally reduced if the pregnancy is continued. For date see ref.[14].

Correlation of Cytokines/Androstenediol

During Pregnancy

Fig. 10: Relationship between the DHEA metabolite androstenediol and Th1-cytokine (INF-gamma) and Th2 cytokine (IL-4) in pregnancy. Taken from ref. [30].

Fig. 11: Molecular relationship between inflammatory Th1 cytokines, and rupture of the cervical membrane during pregnancy. It may be noticed that the rupture of capillary membrane in stroke and heart infarction follows the same path[74].

Fig. 12: Th1/Th2 cytokine balance in physiological pregnancy and in gestational diseases[74].

Fig. 13: Androstenediol (metabolite from DHEA) and Androstanediol (metabolite from dehydrotestosterone) regulate simultaneously cellular anti-infective (anti-viral and anti-bacterial) and anti-inflammatory response in contrast to cortisones, which regulate only inflammation. Taken from ref. [36].

Fig. 14: Histology of a breast cancer sample of 38y old women. Upper row was taken before consumption and lower row was taken 14 days later. “H&E” stand for hematoxylin and eosin. Hematoxylin and eosin stain is used for routine tissue preparation frequently. Eosin is an acid aniline dye. It will bind to and stain basic structures (or negatively charged structures), such as cationic amino groups on proteins. It stains them pink. It can be interpreted as DNA activity and the loss of color as a reduced DNA activity. F8 as a vascular marker discussed  by Folkman 1996.KI67 is a common anti-angiogenesis marker. Coutesy of Drs.Karen McCarron and Regina Chorsky.

Fig. 15:A conceptual model of cytokines in cancer:Tumor and immune cells are sources ofcytokines, which support the growth of cancer and lead to psychobehavioural symptoms (fatigue, depression, and cognitive impairment), drugtoxicity, drug resistance, anorexia and cachexia, pain, and cancer recurrence and progression. Genetic background, cancer treatment and psychological distress may corroborate the production of cytokines. In cancer survivors, hyperactive immune cells might be the major source of cytokines in psychobehavorial symptoms[39].

Fig. 16: Schematic how anti-angiogenic compounds may stabiles membranes around a tumor and contains its ingredients. If it is mediated via NFkB, then Th1 cytokine release and proliferation may also bedecreased.

Fig. 17: Influence of daily consumption of a fermented soy formulation on appetite loss (cachexia) in cancer patients under chemotherapy compared to a group receiving placebo solution containing casein. Study was conducted doubly blind. Taken from ref. [37].

Fig. 18: TNF-alpha blood levels before, during and after daily consumption of 125 mg soy isoflavone of 12 postmenopausal women receiving no other medication[38].

Fig. 19: Schematic description of the relationship between Apoptosis, NFkB and BAX, BCL₂.

Fig. 20: Schematic description of the relationship between cell cycle inhibitor p21 and p53 and apoptosis.

Fig. 21: Individual relative gene expression of Tumor suppressor factor p21 in circulating tumor cells of treatment resistant patients suffering from ovarian-, prostate-, or breast cancer. Taken from ref.[38].

Fig. 22: Relationship between androstenediol and and age in boys (l) and girls (m). Taken from ref. [164,165].

Fig. 23: Schematic description of critical steps of cell cycle. Plants inhibit cell cycle in the G2 phase and in the M-phase (mitosis) like the natural hormone 2-methoxyestradiol, which is increased in, whereas Tamoxifen acts in the G1 phase[70].

Table 1: Gene Expression Changes by fermented Soy.

in vitro cell culture experiments compared to from blood extracted in vivo human cancer patients circulating tumor cells after soy consumption

Gene	Function	Breast CA		Prostate CA		Ovarian CA	Liver CA	Lung CA
		In Vivo in Blood circulating Human Cancer Cells (CTC)	In vitro Cell culture BT474	In Vivo in Blood circulating Human Cancer Cells (CTC)	In vitro Cell culture (LNCAP)	In Vivo in Blood circulating Human Cancer Cells (CTC)	In Vivo Human CTC (HepG2)	In vitro Cell culture (SW480)
BAX	Apoptosis	–	Up	Up	Up	Up	Up	Up
Bcl2	Apoptosis		Down	down	down	down	down	down
C-myc		–	Down	down	down	down	down	down
ER-a	Proliferative Estrogen Receptor	–	–	–	–	–	–	–
ER-ß	Anti-proliferative Estrogen Receptor	–	–	Up	Up	Up	up	up
Telomerase	Anti-caner marker	Up	Up	Up	Up	Up	up	up
Cell cycle inhibitor p21	Inhibits cell proliferation	Up	Up	Up	Up	Up	up	Up
VEGF	Angiogenesis marker	–	–	Up	Up	Up	up	Up
MMP-9	Marker indicating membrane stability	Down	Down	Down	down	Down	down	Down

Table 1: Summarizing in vitro and in vivo effects of fermented soy of gene expression changes. In vivo results were taken from ref. [38].In vitro result were taken from ref. [72]. Results from fermented soy experiments are compared to gene expression changes by 2-methoxy-estradiol (Results were taken form the literature). BT474: human breast cancer cell line; HepG2 : human liver cancer cell line ; LNCaP: human prostate carcinoma cell line; SW480:  humancolorectal adenocarcinoma cell line, MMP-9: matrix metallo proteinase; CTC: circulating tumor cells; VEGF: vascular endothlial factor.

Table 2 :

In vitro, in cancer cell cultures
In vitro cell culture CA cell line	Breast CA BT474	Prostate CA LNCaP	Liver CA HepG2	Lung CA SW480
Decrease of Bcl2/Bax Ratio	-94%	-52%	-64%	-52%

In vivo, from human cancer patients with in blood circulating tumor cells (CTC)
In vivo, determined in extracted human cancer patient after 3 month soy consumption	Breast CA	Prostate CA	Ovarian CA
Decrease of Bcl2/Bax Ratio	-20%	-94%	-80%

Table 2: Comparison of Bcl₂/BAX ratio. In vivo results were taken form ref. [38]. In vitro result were taken from ref. [72].Bcl₂/BAX-ratio is a surrogate marker for NF-kB.