**9. Promising plant based targets in the prevention, treatment and progression of breast cancer**

#### **9.1. Summarizing the individual and combined effects of the soy isoflavones, genistein and diadzein, on mammary tumor development, metastases and invasive breast cancer cells** *in vivo* **and** *in vitro*

Genistein, daidzein and glycitein are the main isoflavones present in soybean and soy-based foods [341] [342]. Out of these, genistein is the mostly studied and dominant isoflavone of soy against breast cancer and has progressed to phase II clinical trials [343]. Soy isoflavones acting upon breast cancer cells *in vitro* and *in vivo* have been studied extensively with varying results and the clinical implications specific to breast cancer have been discussed. Soy isoflavones are structurally similar to female androgen estrogen, and thus they are also known as phytoes‐ trogens [344] and may possibly be competing with the physiological estrogens. Genistein and daidazine (but not glycietein) possesses the ability to transactivate the estrogen receptors.

Utilizing cell based assays on MCF-7 human breast cancer cells, estrogenic agonist actions of soy isoflavones have been studied by Matsumura and co-workers whereby genistein and daidzein exert estrogen response in MCF-7 cells [345] via ER with higher affinity to ERβ1. Similarly, in hepatoma cells transfected with ER, genistein and daidazine bind to both ERα and ERβ but with more affinity to ERβ. Genistein is more potent compared to daidazine [346]. However, these phytoestrogens are 400-600 times less potent compared to 17-β estradiol [347].

*In vitro*, genestein is capable of identifying cells that specifically carry BRCA1 mutation and strongly inhibits the growth of BRCA1 mutant cells compared to cells expressing the wild-type BRCA1 protein [348]. The resistance shown by cells expressing wild type BRCA1 protein has been attributed to increased AKT and decreased p21 (WFA1/CIP1) protein levels [349].


**Table 4.** Plant Based Natural Products indicated in Breast Cancer

The history of Tamoxifen and its derivatives in the successful treatment of estrogen receptor (ER)+ve breast cancers are well documented. In the past Tamoxifen was successful in reducing breast cancer mortality rate in hormone receptor+ve breast cancer patients by up to a third and thus was the stronghold of endocrine treatment [323]. Clinical trials have indicated that aromatase inhibitors (AI) have improved efficacy compared with Tamoxifen for the treatment of post-menopausal hormone receptor+ve patients [324-327]. Additionally, the response rate for third generation AIs as first-line agents range from 30%–50% in ER+ve advanced breast cancer. [323]. Leading to these discoveries, and in an effort to capitalize on the advantages of both anti-aromatase and anti-estrogenic activity, many natural products have been tested for their ability to prevent and treat breast cancer, *in vivo* and *in vitro*. Table 4 summarizes plant based compounds that have been indicated for their potential as a prevention or treatment in breast cancer. This list is not exhaustive; however it does capture the extracts studied to date according to activity in cell lines and animal models and represents the most common types of breast cancer. In addition to those listed within Table 4, soy-based extracts, curcumin and piperine have been studied and we will discuss these in detail as the most promising plant

368 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

based targets in the prevention, treatment and progression of breast cancer.

**progression of breast cancer**

*vivo* **and** *in vitro*

**9. Promising plant based targets in the prevention, treatment and**

**9.1. Summarizing the individual and combined effects of the soy isoflavones, genistein and diadzein, on mammary tumor development, metastases and invasive breast cancer cells** *in*

Genistein, daidzein and glycitein are the main isoflavones present in soybean and soy-based foods [341] [342]. Out of these, genistein is the mostly studied and dominant isoflavone of soy against breast cancer and has progressed to phase II clinical trials [343]. Soy isoflavones acting upon breast cancer cells *in vitro* and *in vivo* have been studied extensively with varying results and the clinical implications specific to breast cancer have been discussed. Soy isoflavones are structurally similar to female androgen estrogen, and thus they are also known as phytoes‐ trogens [344] and may possibly be competing with the physiological estrogens. Genistein and daidazine (but not glycietein) possesses the ability to transactivate the estrogen receptors.

Utilizing cell based assays on MCF-7 human breast cancer cells, estrogenic agonist actions of soy isoflavones have been studied by Matsumura and co-workers whereby genistein and daidzein exert estrogen response in MCF-7 cells [345] via ER with higher affinity to ERβ1. Similarly, in hepatoma cells transfected with ER, genistein and daidazine bind to both ERα and ERβ but with more affinity to ERβ. Genistein is more potent compared to daidazine [346]. However, these phytoestrogens are 400-600 times less potent compared to 17-β estradiol [347]. *In vitro*, genestein is capable of identifying cells that specifically carry BRCA1 mutation and strongly inhibits the growth of BRCA1 mutant cells compared to cells expressing the wild-type BRCA1 protein [348]. The resistance shown by cells expressing wild type BRCA1 protein has been attributed to increased AKT and decreased p21 (WFA1/CIP1) protein levels [349]. Cyclooxygenase-2 (COX-2) expression, which is associated breast cancer risk [350], can also be inhibited by soy isoflavones [351]. Thus, it seems that soy isoflavones are capable of curtailing breast cancer risk factors.

The influences of soy isoflavones on cell growth, cell cycle and apoptosis are all relevant to their effectiveness as chemopreventive agents for breast cancer. A number of studies have indicated the potential of genistein to inhibit proliferation of breast cancer cells in culture by causing cell cycle arrest and/or apoptosis. Genistein induces G2/M cell cycle arrest [352-354]. This effect was seen both in hormone sensitive and hormone independent cells [352]. Accord‐ ing to Li *et al*. [2008] G2/M cell cycle arrest occurs, via stable activation of ERK1/2 pathway [354].

Demarcation on the relative importance of cell adhesion, invasion and migration for primary tumor growth verses metastatic tumour growth is not clear. However, motility, migration and adhesion are more connected to metastasis which is undoubtedly the most life-threatening aspect of breast cancer. Thus, it is crucial to identify the effects of soy isoflavones on disease metastasis.

Microarray analysis of genistein treated HCC1395 cells, a cell line derived from an early stage primary breast cancer, has indicated up-regulation of genes that inhibit invasion and downregulation of genes that promote invasion [355]. Genistein enhances the adhesion of breast cancer cells [356, 357]. This may possibly be one method utilized by genistein to reduce metastasis.

A study by Vantyghem *et al* (2005) describes the ability of dietary genistein to affect metastasis in a post–surgical model in mice [358]. This test model mimics the clinical situation where primary tumors are surgically removed and therapeutic strategies are applied to prevent the growth of any cancer cells seeded to other locations prior to surgery. In this study, primary tumours were established by injecting human breast carcinoma cells, MDA-MB-435/HAL, into the mammary fat pad of nude mice. After 5 weeks, tumours were surgically removed and mice were maintained with a soy free diet or genistein supplemented diet. At the end of 5 weeks, a 10 fold reduction in percent lung metastasis in mice fed on a genistein supplemented diet was seen. In another study, as described by Zhang *et al.,* genistein has shown its ability to reduce the number and volume of osteolytic bone metastases in Balb/c(nu/nu) mice injected with MDA-MB-231 human breast cancer cells [359]. As there are clear indications of the ability of this compound to inhibit breast cancer metastasis, as described in the various animal studies, it would be useful to identify the associated molecular mechanisms. Although there are no conclusive findings, a number of different mechanisms have been suggested.

In studies focused on determining related mechanisms, scientists have given more attention to molecules that are overtly expressed in malignant breast tumours. For example, much attention has been invested into the actions of focal adhesion kinase (FAK), a tyrosine protein kinase. As described previously, cell motility is an integral part of metastasis and it is justifiable to investigate the components directly involve in cell motility. FAK has been designated as a regulator of cell migration and invasion [360]. Since over expression of FAK in human tumors occurs, it has been proposed as a potential therapeutic target [361]. Increased expression of FAK expression in invasive breast carcinomas is associated with an aggressive phenotype [362]. In a transgenic model of breast cancer, mammary epithelial specific disruption of FAK blocks transition of premalignant hyperplasias to carcinomas (and their subsequent metasta‐ Cyclooxygenase-2 (COX-2) expression, which is associated breast cancer risk [350], can also be inhibited by soy isoflavones [351]. Thus, it seems that soy isoflavones are capable of curtailing

370 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

The influences of soy isoflavones on cell growth, cell cycle and apoptosis are all relevant to their effectiveness as chemopreventive agents for breast cancer. A number of studies have indicated the potential of genistein to inhibit proliferation of breast cancer cells in culture by causing cell cycle arrest and/or apoptosis. Genistein induces G2/M cell cycle arrest [352-354]. This effect was seen both in hormone sensitive and hormone independent cells [352]. Accord‐ ing to Li *et al*. [2008] G2/M cell cycle arrest occurs, via stable activation of ERK1/2 pathway [354]. Demarcation on the relative importance of cell adhesion, invasion and migration for primary tumor growth verses metastatic tumour growth is not clear. However, motility, migration and adhesion are more connected to metastasis which is undoubtedly the most life-threatening aspect of breast cancer. Thus, it is crucial to identify the effects of soy isoflavones on disease

Microarray analysis of genistein treated HCC1395 cells, a cell line derived from an early stage primary breast cancer, has indicated up-regulation of genes that inhibit invasion and downregulation of genes that promote invasion [355]. Genistein enhances the adhesion of breast cancer cells [356, 357]. This may possibly be one method utilized by genistein to reduce

A study by Vantyghem *et al* (2005) describes the ability of dietary genistein to affect metastasis in a post–surgical model in mice [358]. This test model mimics the clinical situation where primary tumors are surgically removed and therapeutic strategies are applied to prevent the growth of any cancer cells seeded to other locations prior to surgery. In this study, primary tumours were established by injecting human breast carcinoma cells, MDA-MB-435/HAL, into the mammary fat pad of nude mice. After 5 weeks, tumours were surgically removed and mice were maintained with a soy free diet or genistein supplemented diet. At the end of 5 weeks, a 10 fold reduction in percent lung metastasis in mice fed on a genistein supplemented diet was seen. In another study, as described by Zhang *et al.,* genistein has shown its ability to reduce the number and volume of osteolytic bone metastases in Balb/c(nu/nu) mice injected with MDA-MB-231 human breast cancer cells [359]. As there are clear indications of the ability of this compound to inhibit breast cancer metastasis, as described in the various animal studies, it would be useful to identify the associated molecular mechanisms. Although there are no

In studies focused on determining related mechanisms, scientists have given more attention to molecules that are overtly expressed in malignant breast tumours. For example, much attention has been invested into the actions of focal adhesion kinase (FAK), a tyrosine protein kinase. As described previously, cell motility is an integral part of metastasis and it is justifiable to investigate the components directly involve in cell motility. FAK has been designated as a regulator of cell migration and invasion [360]. Since over expression of FAK in human tumors occurs, it has been proposed as a potential therapeutic target [361]. Increased expression of FAK expression in invasive breast carcinomas is associated with an aggressive phenotype [362]. In a transgenic model of breast cancer, mammary epithelial specific disruption of FAK blocks transition of premalignant hyperplasias to carcinomas (and their subsequent metasta‐

conclusive findings, a number of different mechanisms have been suggested.

breast cancer risk factors.

metastasis.

metastasis.

sis) indicating direct involvement in mammary tumor progression [363]. Further, attenuation of FAK function dramatically increased apoptosis in breast cancer cells [364]. Disruption of FAK signaling by expressing the N-terminal domain FAK in human breast carcinoma cells has led to rounding, detachment and apoptosis [365]. To gain insight into the influence of genistein and daidazine in this important pathway in breast cancer, *in vitro* and *in vivo* studies have been conducted. According to an *in vitro* study, the soy isoflavones genistein, daidzein and 17β estradiol increased the number of focal adhesions and FAK activity in ERα +ve (T47D cells) as well as in ERα -ve (MDA-MB-231) breast cancer cells indicating possible involvement of novel signaling pathways and independent of estrogen receptors. Authors of this study suggested a progressive role (to metastasis) for soy isoflavones in the activation of multiple FAK regulated signaling pathways relevant to breast cancer [366], however the mechanism was not investi‐ gated. The studies of Mitra and co-workers may possibly explain the mechanism of FAK in breast cancer metastasis [367]. According to this study, reduced FAK activity or expression blocked 4T1 breast cancer cell invasion through matrigel and the blocking was associated with a 2-3 fold reduction in the expression of urokinase plasminogen activator (uPA) [367]. uPA is a serine protease that cleaves extracellulaer matrix and stimulate plasminogen to plasmin. Cancer cells are known to digest the ECM via substances like uPA and matrix metalloproteases (MMPs) as a means of invading surrounding tissue. This idea is supported by the fact that breast cancer patients with higher level of MMP-9 in tissue is associated with lymph node metastasis; thus, MMP-9 levels in serum, tumour tissue and urine are used as prognostic markers [368]. Furthermore, a study on the role of membrane-type 1 matrix metalloproteinase (MT1-MMP) *in vitro* and in SCID mice reports that the down regulation of mammary cancer cell MT1-MMP has no effect on primary tumour growth and lymph node metastasis, but reduces the occurrence of lung metastasis [369]. Interestingly, uPA secretion from mammary carcinoma cells can be influenced by genistein. This property of genistein has been shown *in vitro* and *in vivo* and the implication on tumour angiogenesis has been studied using F3II mammary carcinoma cells in culture as well as in a syngeneic mouse model. Accordingly, noncytotoxic concentrations of genistein (0.1-50µM) significantly reduced motility in F3II mam‐ mary carcinoma cells and inhibited the secretion of uPA from cell monolayers. Once F3II cells were implanted in syngeneic mice receiving a treatment of genistein (10mg/kg/day), anti antiangiogenic effects were evident [357].

These studies indicate the effectiveness of genistein to inhibit angiogenesis and metastasis by inhibiting proteolytic substances such as uPA. In this respect, our attempt to further explore the value of soy isoflavones in modulating metastasis enabled us to review some important findings. Studies by Shao *et al*, [1998] reported that genistein inhibited the invasion of MCF-7 and MDA-MB-231 cells *in vitro* and the inhibition was characterized by down regulation of MMP-9 (matrix mettaloproteinase-9) and up regulation of TIMP-1 (tissue inhibitor of metta‐ loproteinase-1) [370]. The same effects were seen in nude mouse xenografts of MCF-7 and MDA-MB-231 cells [371]. Furthermore, in MDA-MB-231 xenografts, genistein inhibited tumour growth, stimulated apoptosis, regulated p21 WAF1/CIP1 expression, inhibited angiogenesis with reduced vessel density and decreased the levels of vascular endothelial growth factor and transforming growth factor β1 [370]. Further studies by the same authors reported that genistein inhibits both constitutive and epidermal growth factor stimulated invasion in ER- human breast carcinoma cells as characterized by up regulation of TIMP-1 as well as other trypsin inhibitors like protease nexin-II (PN-II) and alpha 1-antitrypsin (alpha 1- AT) [371]. Kousidou *et al* [2005], examining normal mammary cells (MCF-12A), low invasive (ER+ve) MCF-7 cells and high invasive MDA-MB-231 (ER-ve) cells (in parallel) showed differences in the effect of genistein on highly invasive and low invasive cells. Accordingly, all cell types expressed genes of MMP-2, MMP-9, membrane-type matrix metalloproteinase (MT-1, MT-2, MT-3), MMP and TIMP-1, -2 and -3. However, once genistein was added, down regulation of all MMP genes in highly invasive cells and down regulation of many genes in low invasive MCF-7 cells was observed [372].

Based on the above findings, genistein has a role in reducing metastasis and this appears to arise from its ability to suppress uPA and MMPs thereby invading barriers with no direct effect on the capacity of cell mobility. According to the literature, expression of uPA and MMPs is regulated by NFk-B [373] [307]. Therefore it would be worthwhile to review any association between genistein and the NF-kB pathway.

#### *9.1.1. Genistein may act via inhibition of the NF-ĸB pathway*

The possible connection between NF-κB and breast cancer has been extensively studied [374]. Using a doxycycline-inducible new mouse model to inhibit NF-κB activity, specifically within the mammary epithelium at the time of tumor development, Connelly *et al* (2011) indicated the active contribution of NF-κB in mammary tumor progression [301]. In this model, inhibi‐ tion of NF-κB activity showed an increase in tumor latency and a decrease in tumor burden [301]. Specifically, soy isoflavones inhibited the tumors by suppressing the NF-κB pathway. Furthermore genistein potentiates the activity of a number of NF-κB mediated chemothera‐ peutic agents by increasing apoptosis in various cancer cells, including MDA-MB-231 breast cancer cells [375]. In the same cell line, genistein induces G2/M cell cycle arrest via stable activation of ERK1/2 pathway [354],[376]. Furthermore, the MDA-MB-231 cell line has the ability to selectively block NF-κB transactivation of IL-6, a cytokine that is known for estrogen independent tumorigenesis activity [377]. Inhibition of proteosome activity by genistein in MCF-7 breast cancer cells has also been associated with NF-κB inhibition [378]. This property of genistein is particularly important for ER deficient breast cancer as constitutive NF-ĸB and Mitogen- and Stress- Activated Protein Kinase-1 (MAPK) /MSK activity are linked with aggressiveness and the metastasis.

It is clear that almost all of the studies that show beneficial effects of soy isoflavones utilized genistein. However, within the natural products of soy and soy food not only genistein, but daidzein and glycitein are present. A recent study testing genistein, daidazine and glycitein separately has indicated interesting results. According to this study, only genistein induced apoptosis in MCF-7 breast carcinoma cells whereas daidzein caused a slight cell-stimulating effect in the absence of E2; thus, the authors pointed toward the possible risk of breast cancer in postmenopausal women who take soy supplements [379]. This statement is important as a number of soy supplements available in the market contain high levels of daidzein [380, 381].

An animal study using nude mice and MDA-MB-435 breast cancer cells reported the individual and combined soy isoflavones exerting differential effects on metastatic cancer progression [382]. As described in this study, daidazine increased mammary tumour growth by 38% while genistein decrease tumor growth by 33%. Moreover, the combined isoflavones increased metastasis to all the organs examined, although no effect on primary tumour growth was noted. These results have led authors to include the consumption of soy foods as a cause of increased breast cancer metastasis. Also, a number of studies by Ju *et al* [383-385] have cited enhanced growth effects of soy components in ER+ve breast cancers. However, with these studies conducted in immune compromised mice, the relevance of these findings have been criticized [386], especially as pre-treatment with genistein has shown to been protective against mammary tumors [387]. More recently, the antiproliferative activity of both genistein and quercetin has been indicated in the prevention and treatment of HER2-overexpressing breast cancer via inhibition of NFκB signaling [388]. In this study, these specific phytoestrogens inhibited proliferation in MCF-7 cell lines accompanied by an increase in intrinsic apoptotic indicators, induction of the extrinsic apoptosis pathway (up-regulating p53), a reduction in the phosphorylation level of IκBα, and negated the nuclear translocation and subsequent phosphorylation of nuclear p65 [388].

#### *9.1.2. Sources of soy*

invasion in ER- human breast carcinoma cells as characterized by up regulation of TIMP-1 as well as other trypsin inhibitors like protease nexin-II (PN-II) and alpha 1-antitrypsin (alpha 1- AT) [371]. Kousidou *et al* [2005], examining normal mammary cells (MCF-12A), low invasive (ER+ve) MCF-7 cells and high invasive MDA-MB-231 (ER-ve) cells (in parallel) showed differences in the effect of genistein on highly invasive and low invasive cells. Accordingly, all cell types expressed genes of MMP-2, MMP-9, membrane-type matrix metalloproteinase (MT-1, MT-2, MT-3), MMP and TIMP-1, -2 and -3. However, once genistein was added, down regulation of all MMP genes in highly invasive cells and down regulation of many genes in

Based on the above findings, genistein has a role in reducing metastasis and this appears to arise from its ability to suppress uPA and MMPs thereby invading barriers with no direct effect on the capacity of cell mobility. According to the literature, expression of uPA and MMPs is regulated by NFk-B [373] [307]. Therefore it would be worthwhile to review any association

The possible connection between NF-κB and breast cancer has been extensively studied [374]. Using a doxycycline-inducible new mouse model to inhibit NF-κB activity, specifically within the mammary epithelium at the time of tumor development, Connelly *et al* (2011) indicated the active contribution of NF-κB in mammary tumor progression [301]. In this model, inhibi‐ tion of NF-κB activity showed an increase in tumor latency and a decrease in tumor burden [301]. Specifically, soy isoflavones inhibited the tumors by suppressing the NF-κB pathway. Furthermore genistein potentiates the activity of a number of NF-κB mediated chemothera‐ peutic agents by increasing apoptosis in various cancer cells, including MDA-MB-231 breast cancer cells [375]. In the same cell line, genistein induces G2/M cell cycle arrest via stable activation of ERK1/2 pathway [354],[376]. Furthermore, the MDA-MB-231 cell line has the ability to selectively block NF-κB transactivation of IL-6, a cytokine that is known for estrogen independent tumorigenesis activity [377]. Inhibition of proteosome activity by genistein in MCF-7 breast cancer cells has also been associated with NF-κB inhibition [378]. This property of genistein is particularly important for ER deficient breast cancer as constitutive NF-ĸB and Mitogen- and Stress- Activated Protein Kinase-1 (MAPK) /MSK activity are linked with

It is clear that almost all of the studies that show beneficial effects of soy isoflavones utilized genistein. However, within the natural products of soy and soy food not only genistein, but daidzein and glycitein are present. A recent study testing genistein, daidazine and glycitein separately has indicated interesting results. According to this study, only genistein induced apoptosis in MCF-7 breast carcinoma cells whereas daidzein caused a slight cell-stimulating effect in the absence of E2; thus, the authors pointed toward the possible risk of breast cancer in postmenopausal women who take soy supplements [379]. This statement is important as a number of soy supplements available in the market contain high levels of daidzein [380, 381]. An animal study using nude mice and MDA-MB-435 breast cancer cells reported the individual and combined soy isoflavones exerting differential effects on metastatic cancer progression

low invasive MCF-7 cells was observed [372].

between genistein and the NF-kB pathway.

aggressiveness and the metastasis.

*9.1.1. Genistein may act via inhibition of the NF-ĸB pathway*

372 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Soybeans can be considered as the richest source of isoflavones in the human diet [389, 390], and are available in fermented and non-fermented forms. Fresh green soy beans, whole dry soybeans, whole-fat soy flour, soy milk and soymilk products such as tofu, okara and yuba are non-fermented while soy sauce, temphe, miso and natto are fermented products [391]. Additionally, products such as soy dairy substitutes, soy cheese, soy yogurt, and soy burgers seem to be popular in Western countries. The isoflavone content in various soy-based food products greatly differ. Other than soy food products, soy supplemented (categorized as a class of complementary medicine) nutraceuticals are widely consumed by Western commun‐ ities. The quality and standard of these supplements are questionable. For instance, a survey carried out in the Eastern Washington Region of U.S.A. tested 13 products (7 tablet and 6 capsule formulations) by HPLC and showed that only 4 of the 13 products contained the minimum of 90% isoflavone content claimed on the label and variations in composition over time were noted [392]. Interestingly, a recent review shows that overall, most commercially available nutraceuticals are poor in quality [393].

#### *9.1.3. Clinical studies*

Although there have been a large number of studies carried out to evaluate a possible soybreast cancer link, evidence is inconclusive. The two main theories tested involve the effect of soy isoflavone consumption in risk of breast cancer incidence and its effect on recurrence. The largest population based cohort study, including 5,042 female breast cancer survivors, shows that soy food consumption is significantly associated with decreased risk of death and recurrence [394]. Another cohort of 1,954 female breast cancer survivors, who consumed soy isoflavones at the levels comparable to the Asian population, while undergoing tamoxifen therapy showed a reduction in the risk of cancer recurrence and no interference in the efficacy of tamoxifen [395]. Further studies have indicated that soy intake prior to cancer diagnosis is unrelated to disease-free breast cancer survival and that the association between soy protein intake and breast cancer survival does not differ according to the presence of other risk factors such as ER/PR status, tumor stage, age at diagnosis, body mass index (BMI), waist to hip ratio (WHR), or stage of menopause [396]. No variations were noted in the soy-survival association of indicated polymorphisms in ERα and ERβ indicating that soyfoods do not have an adverse effect on breast cancer survival. A recent meta-analysis by Dong and Qin (4 studies of breast cancer recurrence and 14 studies of breast cancer incidence) revealed that the consumption of soy isoflavone is inversely associated with risk of breast cancer incidence [397]. However, the protective effect is only observed among studies conducted in Asian populations, unlike those reported in Western populations [397]. One of the previous meta-analysis studies by Wu *et al* [2008] show a similar trend. Accordingly, in Asian populations a higher intake of soy isoflavones, as compared with lower intake, is associated with 29% reduction in the risk of developing breast cancer [398]. Hence, the consumption of soy food at levels similar to those consumed by Asian populations may have protective effects. However, there is evidence in the literature to show possible adverse effects of soy due to its known stimulatory effect on the premenopausal female breast as indicated by increased secretion of breast fluid, the appearance of hyperplastic epithelial cells and elevated levels of plasma estradiol [399]. Some animal studies support the idea of related disadvantages of consuming soy isoflavones. In overiectomized athymic nude mice, physiological concentrations of dietary genistein stimu‐ lates the growth of estrogen dependent MCF-7 tumors in a dose dependent manner [383, 400]. Furthermore, the same test model showed that dietary genistein reverses the inhibitory effect of tamoxifen on the growth of MCF-7 tumors [384].

#### **9.2. The role of curcumin and piperine in breast cancer prevention and its effects on normal human breast stem cell renewal and signaling**

Curcumin is a plant derived polyphenol which gives rise to the yellow colour in the spice, tumeric. This pigment is obtained from the plant *Curcuma longa* and has been noted to have power against cancer. In their review of the mechanisms of cell cycle regulation by curcumin, Gaurisankar and Das have named it as a multiple edged sword [401] because of its ability to regulate the cell cycle as well as apoptosis. Distorted cell cycle regulation and programmed cell death/apoptosis are characteristic features of cancer and curcumin has been shown to target both mechanisms. The ability of curcumin to inhibit telomerase activity [402] and to disrupt mitotic spindle structure causing [403] micronucleation in MCF-7 breast carcinoma cells has been reported. Also, curcumin is known to induce anti-proliferative activity via the decreased expression of cyclin D1 and CDK-4 in MCF-7 breast carcinoma cells [404] and can induce apoptosis through p53 dependent Bax induction [405]. Curcumin is able to disrupt breast tumor growth, but also to inhibit metastasis.

As with genestein, curcumin has been shown to mediate its anti-cancer effects via regulation of the NF-κΒ signaling pathway. In the nude mouse model, curcumin suppresses the pacli‐ taxel-induced NF-κΒ pathway resulting in the inhibition of lung metastasis of human breast cancer [406]. The modification of NF-kB signaling eventually leads to pro-apoptotic events and perhaps inhibition of ECM breakdown. Curcumin induced apoptosis in MDA-MB-231 cells *in vitro* is associated with IkB and p65 phosphorylation and hence reduced activation of NFkB [407]. This leads to reduced expression on MMPs, diminished invasion through a reconstituted basement membrane and a lower number of metastases in immunodeficient mice injected with tumor cells via intra cardiac route [407]. The high level of of MMP-3 expression noted in MDA-MB-231 invasive breast carcinoma cells is not evident on MCF-7 non-invasive breast cancer cells, implicating its importance in invasion and metastasis. The possibility of using the major forms of curcuminoids, curcumin, demethoxycurcumin, and bisdemethoxycurcumin (all of which are found in turmeric powder) as MMP-3 inhibitors to modulate MMP-3 expression has been suggested [408]. According to Chiu and Su (2009), curcumin inhibits proliferation by increasing the Bax to Bcl-2 ratio while inhibiting the migration via decreasing NF-kB p65 expression in breast cancer MDA-MB-231 cells [409]. Utilizing microarray gene expression analysis on MDA-MB-231 breast cancer cells, Bachmeier *et al.* demonstrated the ability of curcumin to downregulate inflammatory cytokines CXCL-1 and -2 via suppression of NF-κB translocation. Moreover, silencing CXCL-1 and -2 resulted in a downregulation of several metastasis promoting genes [410].

Interestingly, curcumin can interfere with estrogen-mimicking pesticides such as endosul‐ fane, DDT and chlordane [411].

#### *9.2.1. Sources of curcumin*

unrelated to disease-free breast cancer survival and that the association between soy protein intake and breast cancer survival does not differ according to the presence of other risk factors such as ER/PR status, tumor stage, age at diagnosis, body mass index (BMI), waist to hip ratio (WHR), or stage of menopause [396]. No variations were noted in the soy-survival association of indicated polymorphisms in ERα and ERβ indicating that soyfoods do not have an adverse effect on breast cancer survival. A recent meta-analysis by Dong and Qin (4 studies of breast cancer recurrence and 14 studies of breast cancer incidence) revealed that the consumption of soy isoflavone is inversely associated with risk of breast cancer incidence [397]. However, the protective effect is only observed among studies conducted in Asian populations, unlike those reported in Western populations [397]. One of the previous meta-analysis studies by Wu *et al* [2008] show a similar trend. Accordingly, in Asian populations a higher intake of soy isoflavones, as compared with lower intake, is associated with 29% reduction in the risk of developing breast cancer [398]. Hence, the consumption of soy food at levels similar to those consumed by Asian populations may have protective effects. However, there is evidence in the literature to show possible adverse effects of soy due to its known stimulatory effect on the premenopausal female breast as indicated by increased secretion of breast fluid, the appearance of hyperplastic epithelial cells and elevated levels of plasma estradiol [399]. Some animal studies support the idea of related disadvantages of consuming soy isoflavones. In overiectomized athymic nude mice, physiological concentrations of dietary genistein stimu‐ lates the growth of estrogen dependent MCF-7 tumors in a dose dependent manner [383, 400]. Furthermore, the same test model showed that dietary genistein reverses the inhibitory

**9.2. The role of curcumin and piperine in breast cancer prevention and its effects on normal**

Curcumin is a plant derived polyphenol which gives rise to the yellow colour in the spice, tumeric. This pigment is obtained from the plant *Curcuma longa* and has been noted to have power against cancer. In their review of the mechanisms of cell cycle regulation by curcumin, Gaurisankar and Das have named it as a multiple edged sword [401] because of its ability to regulate the cell cycle as well as apoptosis. Distorted cell cycle regulation and programmed cell death/apoptosis are characteristic features of cancer and curcumin has been shown to target both mechanisms. The ability of curcumin to inhibit telomerase activity [402] and to disrupt mitotic spindle structure causing [403] micronucleation in MCF-7 breast carcinoma cells has been reported. Also, curcumin is known to induce anti-proliferative activity via the decreased expression of cyclin D1 and CDK-4 in MCF-7 breast carcinoma cells [404] and can induce apoptosis through p53 dependent Bax induction [405]. Curcumin is able to disrupt

As with genestein, curcumin has been shown to mediate its anti-cancer effects via regulation of the NF-κΒ signaling pathway. In the nude mouse model, curcumin suppresses the pacli‐ taxel-induced NF-κΒ pathway resulting in the inhibition of lung metastasis of human breast cancer [406]. The modification of NF-kB signaling eventually leads to pro-apoptotic events and perhaps inhibition of ECM breakdown. Curcumin induced apoptosis in MDA-MB-231 cells

effect of tamoxifen on the growth of MCF-7 tumors [384].

374 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

**human breast stem cell renewal and signaling**

breast tumor growth, but also to inhibit metastasis.

Curcumin is generally considered to be the most active component and the principal curcumi‐ noid found in tumeric [411]. The spice tumeric is commonly used in curries and contains 2-8% of this active ingredient [412], however a supplement form is also available.

#### *9.2.2. Clinical trials*

Little data is available on the pharmacokinetics and metabolism of curcumin in humans. Doselimiting toxicity is not reported and high oral doses of curcumin (up to 12g/day) have been tested [220, 413, 414]. In a phase one clinical trial involving individuals with non-invasive cancer and pre-cancerous conditions, oral dosing of 4g, 6g and 8g of curcumin yielded peak serum concentrations of 0.51 +/- 0.11microM, 0.63 +/- 0.06 microM, and 1.77 +/- 1.87 microM, respectively. Peak serum concentrations of curcumin are seen 1-2 hours after oral intake and this gradually declines within 12 hours [220]. In another phase I clinical trial involving 15 patients with advanced colorectal cancer, 3.6g of curcumin daily for up to 4 months was welltolerated [413]. Another study examined the pharmacokinetics of 450mg-3600mg curcumin (daily for 1 week) in twelve patients with hepatic metastatic disease from primary colorectal adenocarcinomas. Using a high-performance liquid chromatography assay, low nanomolar levels of the parent compound and its glucuronide and sulphate conjugates were found in the peripheral or portal circulation; despite its absence in liver tissue, trace levels of products of its metabolites were detected [415]

Due to the poor bioavailability of curcumin systemically, high priority has been given to study its potential against colorectal cancers. A very recent publication on a phase IIa clinical trial involving men and women 40 years of age or over and smokers that carry 8 or more colorectal aberrant crypt foci (ACF) indicates that oral dosing of curcumin (4g per day for 30 days) significantly reduces colorectal ACF, a biomarker of colon carcinogenesis. [416] The reported anti-carcinogenic effect of curcumin is not associated with increased levels of curcumin in local tissue but increased levels of conjugate concentrations in suggesting that curcumin may mediate its effects by cuccumin conjugates delivered systemically. The same study showed that the presence of curcumin conjugates in plasma and tissue prior to treatment (believed to be originated from the normal diet of the studied population) were accompanied by a steady increase of curcumin conjugates following the month-long daily dosing [416]. A study examining the pharmacokinetics of curcumin at the concentrations of 10g and 12g in twelve healthy volunteers indicates comparable results. Accordingly, a single dose of orally admin‐ istered curcumin resulted in the detection of conjugates, glucuronides and sulfates in plasma in all subjects while free curcumin was evident in only one subject [414]. Even though curcumin conjugates and other breakdown products have not been assessed for their anticarcinogenic properties [416], these findings shed some light on the potential of curcumin as a treatment of all cancers, including those of colorectal origin. This may offer a likely explanation of how continuous exposure to small quantities of curcumin via normal diets protects Asian women from breast cancer.

A phase I dose escalation trial of combined effects of docetaxel and curcumin in patients with advanced and metastatic breast cancer was published very recently. This study involved 14 patients and demonstrated the feasibility, safety and tolerability of a combination of curcumin with a standard dose of docetaxel which warrants further investigation and progression to a Phase II clinical trial [417]. Similarly, the curcumin inhibiting effects of chemotherapy induced apoptosis in models of human breast cancer have been identified [418].

#### *9.2.3. Curcumins' ability to destroy cancer stem cells*

The properties of CSC's are connected with major signaling pathways. The signaling pathways active in mammary stem cells are shown to be Wnt/β catenin, Hh and Notch [60],[51],[185], [206],[269].

A recent study by Karkarala *et al.* has demonstrated the potent inhibitory effect of curcumin and piperine on Wnt/β-catenin signaling in primary human breast epithelial cells [419]. In this study, inhibition of Wnt signaling pathway was shown to affect breast stem cell renewal by inhibiting the mammosphere formation. According to the authors, curcumin and piperine (separately and in combination) inhibited breast stem cell self-renewal; however toxicity to differentiated cells was not reported. The plasma concentration of curcumin in people taking high oral doses has been shown to be very low due to many reasons such as metabolism of the compound in the intestine and the liver, as reviewed by Burgos-Moron *et al.* [420].

The lack of bioavailability of curcumin was known as a potential disadvantage for years and various strategies have been investigated to overcome the problem. One such strategy has been the use of piperine in combination of curcumin. Accordingly, concomitant administration of piperine and curcumin tends to increase the bioavailability (up to 2000%) compared to administration of curcumin alone in an experimental group of people [421]. This finding could well be a possibility as piperine has been shown to inhibit P-glycoproteins and CYP3A4 expressed in enterocytes where the bioavailability of many orally ingested compounds are determined [422]. Alternatively, increasing the solubility of curcumin by heat as means of increasing the bio availability has been suggested [423]. This method is easily achievable and a well-cooked curry with tumeric and piperine could be a tasty way of obtaining the goodness of these natural compounds.

Based on the ability of curcumin and piperine to inhibit CSC's as described above, curcumin has great potential as a possible therapeutic agent against breast cancer. The majority of the breast cancer patients have tumors that respond to the naturally occurring hormone, estrogen. Therefore, most of the currently available drugs known to be effective against breast cancer can prevent the action of estrogen and are thus referred to as selective estrogen receptor modulators (SERM). Unfortunately, there is a cohort of patients whose tumours do not express estrogen receptor. SERMs are of no use for this group with ER -ve breast cancers. The potential of curcumin and piperine to suppress the self-renewal of stem cells could prove beneficial in ER +ve as well as ER -ve breast cancer patients.
